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SUMMARY TECHNICAL REPT)RT rt)F DIVISION 9. NDRC VOLUME 1 CHEMICAL WARFARE AGENTS, 234 270 AND RELATED CHEMICAL PROBLEMS Paris l-II OFFICE OF SCIENTIFIC RESEARCH AND DEVELOPMEN VANNEVAR BUSH. DIRECTOR NATIONAL DEFENSE RESEARCH COMMITTEE JAMES B. CONANT. CHAIRMAN DIVISION 9 W. R. KIltNER, CHIEF WASHINGTON, D. C., 1046 SUMMARY TECHNICAL REPORT OF THE NATIONAL DEFENSE RESEARCH COMMITTEE Tliis document contains information affecting the, national defense of the United States within the meaning of the Espionage Act, 50 U. S. C., 31 and 32, as amended. Its transmission or the revelation of its contents in any manner to an unauthorized jicrson is prohibited by law. This volume is classified SECRET in accordance with security regulations of the War and Navy Departments because certain chapters contain materials which was SECRET at the date of printing. Other chapters may have had a lower classification or none. The reader is advised to consult the War and Navy agencies listed on the reverse of this page for the current classification of any material. Manuscript and illustrations for this volume were prepared for publication by the Summary Reports Croup of the Columbia University Division of War Research under contract OEMsr-1131 with the Office of Scientific Research and Development. This vol- ume was printed and bound by the Columbia University Press. Distribution of the Summary Technical Report of NDRC has been made by the War and Navy Departments. Inquiries concerning the availability and distribution of the Summary Technical Report volumes and microfilmed and other reference material should l>c addressed to the War Department Library, Room 1A-522, The Pentagon, Washington 25, D. C., or to the Office of Naval Re- search, Navy Department, Attention: Reports and Documents Section, Washington 25, D, C. Copy No. 30 This volume, like the seventy others of the Summary Technical Report of NDRC, has been written, edited, and printed under great pressure. Inevitably there are errors which have slipped past Division readers and proofreaders. There may be errors of fact not known at time of printing. The author has not 1 >een able to follow through his writing to the final page proof. Please report errors to: JOINT RESEARCH AND DEVELOPMENT BOARD PROGRAMS DIVISION (STR ERRATA) WASHINGTON 23, D. C. A master errata sheet will be compiled from these reports and sent to recipients of the volume. Your help will make this book more useful to other readers and will be of great value in preparing any revisions. NATIONAL DEFENSE RESEARCH COMMITTEE Richard C. Tolman, Vice Chairman James B. Conan t, Chairman Roger Adams Frank H. Jewett Karl T. Compton Army Representative 1 Navy Representative ’ Commissioner of Patents3 Irvin Stewart, Executive Secretary M rmy representatives in order of service: •Xiwy representatives in order of service: Maj Gen. G. V. Strong Maj. Gen. R, C. Moore Maj, Gen. C. C. Williams Brig. Gen. W. A. Wood, Jr. Col. L. A. Denson Col. P. R. Faymonville Brig, Gen. E. A. Rcgnicr Col. M. M. Irvine Rear Adm. II. G. Bowen Capt. Lybrand P. Smith Rear Adm. .1. A. Purer Rear Adm. A. IE Van Keuren Commodore 11. A. Schade Commissioners of Pakula in order of-service Col. E. A. Routheau Conway P. Coe Casper W. Corns NOTES OX THE ORGANIZATION OF XDHC The duties of the National Defense Research Committee were (1) to recommend to the Director of OSRD suitable projects and research programs on the instrumentalities of warfare, together with contract facilities for carrying out these projects and programs, and (2) to administer the tech- nical and scientific work of the contracts. More sjiecifically, XDRC functioned by initiating research projects on re- quests from the Army or the Navy, or on requests from an allied government transmitted through the Liaison Office of OSRD, or on its own considered initiative as a result of the experience of its members. Proposals prepared by the Division, Panel, or Committee for research contracts for performance of the work involved in such projects were first reviewed by NDRC, and if approved, recommended to the Director of OSRD. Epon approval of a projiosal by the Director, a contract permitting maximum flexibility of scientific effort was arranged. The business aspects of flic contract, including such matters as materials, clearances, vouchers, patents, priorities, legal matters, and administra- tion of patent matters were handled by the Executive Sec- retary of OSRD. Originally NDRC administered its work through five divisions, each headed by one of flic NDRC • memlicrs. These were: Division A — Armor and Ordnance Division R — Bombs, Fuels, Gases, &• Chemical Problems Division C — Communication and Transportation Division D — Detection, Controls, and Instruments Division E — Patents and Inventions In a reorganization in the fall of 1042, twenty-three ad- ministrative divisions, panels, or committees were created, each with a chief selected on the basis of his outstanding work in the particular field. The NDRC members then be- came a reviewing and advisory group to the Director of OSRD. The final organization was as follows: Division 1 — Ballistic Research Division 2 — Effects of Impact and Explosion Division 3 - Rocket Ordnance Division 4 Ordnance Accessories Division 5 New Missiles Division 0 Sub-Surface Warfare Division 7 — Kire Control Division 8 — Explosives Division 9 — Chemistry Division 10 — Absorlicnts and Aerosols Division 11 —Chemical Engineering Division 12 — Transportation Division 13 — Electrical Communication Division 14 — Radar Division 15— Radio Coordination Division Ifi — Optics and Camouflage Division 17 — Physics Division IS— War Metallurgy Division 19 — Miscellaneous Applied Mathematics Panel Applied Psychology Panel Committee on Propagation Tropical Deterioration Administrative Committee SECRET NDRC FOREWORD As events of the years preceding 1940 revealed l more and more clearly the seriousness of the world situation, many scientists in this country came to realize the need of organizing scientific research for service in a national emergency. Recommendations which they made to the White House were given careful and sympathetic attention, and as a result the National Defense Research Committee (NDRC) was formed by Executive Order of the President in the summer of 1940. The members of NDRC, appointed by the President, were instructed to supplement the work of the Army and the Navy in the development of the instrumentalities of war. A year later, upon the establishment of the Office of Scientific Research and 1 development (OSR D), N D R C became one of its uni t s. The Summary Technical Report of NDRC is a conscientious effort on the part of NDRC to summa- rize its work and to present it in a useful anil perma- nent form. It comprises some seventy volumes broken into groups corresponding to the NDRC Divisions, Panels, and Committees. The Summary Technical Report of each Division, Panel, or Committee is an integral survey of the work of that group. The first volume of each group’s report contains a summary of the report, stating the prob- lems presented and the philosophy of attacking them, and summarizing the results of the research, develop- ment, and training activities undertaken. Some vol- umes may be “state of the art” treatises covering subjects to which various research groups have con- tributed information. Others may contain descrip- tions of devices developed in the laboratories. A master index of all these divisional, panel, and com- mittee reports which together constitute the Sum- mary Technical Report of NDRC is contained in a separate volume, which also includes the index of a microfilm record of pertinent technical laboratory re- ports and reference material. Some of the NDRC-sponsored researches which had been declassified by the end of 1945 were of sufficient popular interest that it was found desirable to report them in the form of monographs, such as the series on radar by Division 14 and the mono- graph on sampling inspection by the Applied Math- ematics Panel. Since the material treated in them is not duplicated in the Summary Technical Report of NDRC, the monographs are; an important part of the story of these aspects of NDRC research. In contrast to the information on radar, which is of widespread interest and much of which is released to the public, the research on subsurface warfare is largely classified and is of general interest to a more restricted group. As a consequence, the report of Di- vision (> is found almost entirely in its Summary Technical Report, which runs to over 20 volumes. The extent of the work of a division cannot therefore 1m‘ judged solely by the number of volumes devoted to it in the Summary Technical Report of NDRC: account must be taken of (he monographs and avail- able reports published elsewhere. Under the leadership of Walter R. Kirner as Chief, Division 9 conducted a broad program of research in (he field of chemical warfare, hot h for offense and de- fense. Its principal responsibility was to ensure that this country would be prepared, should the enemy resort to the employment of poison gas as an offensive weapon. The staff of the Division prepared somelwo thou- sand chemical compounds, ami tested them for tox- icity and vcsicancy at a central laboratory. During the course of this program, a number of new chemical warfare agents were discovered which were potential deadly weapons. For defense, the Division contrib- uted to the development of methods and equipment for detecting and protecting against chemical agents, in vapor form or dissolved in water; important work was done in the development of an improved type of impregnated clothing. The Division also worked with the Committee on Medical Research in the problem of new ant i-malarial agents, insecticides, and roden- ticides. The Summary Technical Report of Division 9, prepared under the direction of the Division Chief and authorized by him for publication, is a record of this work, a great deal of which constituted an insur- ance policy against a threat which did not material- ize. We can be thankful therefore, and we are grate- ful to the staff of Division 9 for its vital contributions in the field of chemical research. Vannkvar Bush, Director Office of Scientific Research and Development J. B. Coxaxt, Chairman National Defense Research Committee V FOREWORD Division 9, also known as the Chemistry Division, specialized mainly in chemical warfare prob- lems. lis activities were concerned with problems of l>oth offense and defense. A large part of the program involved a search for new candidate chemical warfare agents particularly of the so-called persistent types. Nearly two thousand of such compounds were pre- pared. The most promising candidates were carried through the pilot plant so as to_secure engineering data on their preparation and also to provide suf- ficient material for further evaluation in the labora- tory and in the field. The Division maintained a large central laboratory in which the candidate compounds were screened for toxicity and vesicancy. As a result of this program a number of new chemical warfare agents were discovered possessing the necessary tox- icity and other desirable properties. In addition, cer- tain improvements were suggested for the synthesis of some of the agents which had previously been standardized for chemical warfare use. On the defensive side, Division 9 made important contributions to the development of methods and equipment for the detection and analysis of chemical warfare agents in the vapor form or dissolved in water. Procedures were also devised for the removal of such agents from water. A great deal of effort was expended in the development of protective clothing. Division 9 investigators discovered stabilizers for the chemically-impregnated clothing manufactured by the Chemical Warfare Service, which greatly ex- tended its storage life. A new, so-called “aqueous im- pregnation process” for protective clothing was de- veloped which avoided shipment of large quantities of organic solvents to war theatres. Several kits were devised with which an individual or group of soldiers could impregnate their clothing in the event of an emergency. An intensive search was made for substitute im- pregnites to replace the one adopted by the Army. Certain of these new compounds, particularly several first prepared by Naval Research Laboratory inves- tigators, proved of outstanding value for use in pro- tective ointments. These agents were incorporated into the protective ointments standardized, man- ufactured, and distributed by the Arm}’ and Navy to personnel throughout the world, after their efficacy for this purpose had been discovered by Division 9 investigators and methods developed for improving their synthesis and compounding them into oint- ments. A new approach to the problem of protective clothing was undertaken by Division 9 in its work on carbon-impregnated clothing following a lead fur- nished by the British. Three different methods of impregnating carbon into cloth were successfully de- veloped, two of which show unusual promise. The advantage possessed by carbon-impregnated fabrics over chemically-impregnated fabrics is that the for- mer will protect the wearer against all known persis- tent agents whereas the latter is limited to protection against agents of the mustard-gas ty|*». Division t) also carried on an extensive research program on the physiological mechanism of action of chemical warfare agents. The goal of this program was the discovery of effective methods of therapy to 1m> used against gases which might be used by the enemy. While (his did not result successfully in the case of mustard gas7 a much clearer understanding was leached as to the mechanism by which this agent produces cell injury and vesication. The discovery of BAT by the British has made available a very ef- fective antidote against vesication by arsenical war gases of the Lewisite type. Toward the end of the war, when the effectiveness of the flame thrower was demonstrated in attacks on Japanese entrenched in caves and pill boxes, this program was extended to include a basic study of the physiological mechanism of action of heat on animals. Division 9 personnel participated in (he field eval- uation of chemical warfare agents at all of the Chem- ical Warfare Service proving grounds. Practically all of the analytical work done at the Dug way Proving Ground Mobile Field Unit Installation, Bushnell, Florida, was performed by Division 9 men on loan from its various contractors. Much of tin- analytical equipment used in these field tests was developed by Division 9 investigators. The important discovery made during these experiments under sub-tropical conditions was the considerably enhanced activity of mustard gas at high temperatures and high hu- midity. When it became evident that chemical warfare would not be init iated by the enemy or by the Allies, Division 9 shifted its emphasis from chemical war- fare problems to other urgent chemical problems. It cooperated with the Committee on Medical Research SECRET. Ml viii FOUtvUOHO in a search for new, effective anti-malarial agents, insecticide formulations, insect repellents, and ro- dentieides. The discovery of the new rodenticide “1080” was made jointly by investigators of Di- vision 9 and the Fish and Wildlife Service, Depart- ment of the Interior. Division 9, NDRC, was created in December 1912, at the time the Office of Scientific Research and De- velopment was organized and the NDRC reorgan- ized. Its predecessor Sections in Division B weie first Sections A-2, A-3, and A t and later Sections B-3 and B 1. The early organization of the scien- tific work of these Sections had been most effectively carried out by Drs. Roger Adams, H. S. Gasser, W. ( . Johnson, and C. S. Marvel. Their leadership was one of the important factors which contributed to the successful solution of many of the problems assigned to Division 9 by the Army and Navy. The other important factor was the ability, industry, and en- thusiasm of the official investigators and their asso- ciates and assistants in university and industrial laboratories in attacking the problems which were, in turn, assigned to them. Generous credit should also be given to the Division Members and Technical Aides whorra tried out the scientific administration of the many contracts,which were tinder the aegis of Division 9. It is a pleasu a to gratefully acknowledge here the assistance loyally rendered by all of these men in the laboratory accomplishments described in detail in the Division 9 Summary Technical Report, presented herewith. Particular expressions of gratitude are due the authors of the chapters which constitute the volumes summarizing the work of Division 9. Because of the policy adopted by this Division to summarizecrit- ically not only the work of its own investigators, but also the contemporary work done in American Serv- ice laboratories and in the laboratories of our Allies, the task of writing was made considerably more difficult. However, it is believed that this overall summary will greatly add to the value of the volumes by giving as complete a picture as possible of present knowledge on each subject. Finally, special acknowledgment is made of the outstanding work done by Dr. Birdsey Renshaw, the editor of the Division 9 Summary Technical Report . He organized the report, coordinated the efforts of the authors of the respective chapters, wrote all or part of a considerable number of the chapters, and carefully edited each chapter as it was completed. This work has required his full time attention for well over a year during which his own desires to carry on laboratory work had to Ik' postponed. However, now. that the task is completed he will, I am sure, derive a great dealdf satisfaction from having done it so well. W. R. Kirneb Chief, Division 9 SECRET . ■ * y i PREFACE IT WAS thk consensus in Division 9 that (he value of its Summary Technical Report, requested as a supplement to the numerous detailed reports already prepared and issued, would bo greatly enhanced if an attempt were made not only' to summarize (he Divi- sion’s work but, in addition, to review critically' the information av ailable from other sources and relating to the subjects on which the Division had undertaken investigations. 'This seemed particularly' desirable lie- cause in most phases of the work the contributions of (he Division and those of other agencies in the United States and British Commonwealth of Nations supple- mented and reciprocally influenced each other. Fur- thermore, the data on most of the pertinent subjects are scattered in numerous reports of various origins which in the future will lie difficult to locate and evaluate. It seemed worthwhile, therefore, also to include a fairly complete Bibliography. This undertaking has been pursued, for the most part subsequent to the defeat of Japan, by men who had actively participator! in the work of the Divi- sion. Most of the authors were burdened with other duties and have made considerable personal sacri- fices to write the summaries. Nevertheless, they have attempted conscientiously to present in useful form the significant facts and concepts that emerged rel- ative to their subjects during World War II. In so far as this aim has been attained, the reader will no doubt lie willing to overlook stylistic and editorial heterogeneities. Unfortunately, it is inevitable that occasional omissions and errors must creep into a rapid compilation and assessment of as much mate- rial as is included in this Report. These the reader will accept with less equanimity, and for ( hem the authors and the Editor ask forgiveness. By the time the Division closed in 1916 practically all of its work had been presented in detail in OSRD Formal Reports. These, therefore, comprise most of the references to the* Division’s work that are given in the chapters and reproduced in microfilm. Most of the numerous informal and miscellaneous.-reports issued during the war were of an interim nature. They have been referred to and microfilmed only' when they appeared to possess permanent value or in- cluded material not incorporated in OSRD Formal Reports. Among the informal reports that may ap- propriately Ik* made a part of the accessible perma- nent record are the Section 9:4:1 (formerly B4-A) Informal Reports on Toxicity of Chemical Warfare Agents, and the Section 9:5:1 (formerly B6-C) In- formal Reports on Physiological Mechanisms of Chem- ical Warfare Agents. Both of these series are included in tola in microfilm. Birdsey Renshaw Editor SEQtETv, IX CONTENTS“ CHAPTER PART I PACE PREPARATION AND EVALUATION OF POTENTIAL CHEMICAL WARFARE AGENTS 1 Resume of Agent Assessments 13 2 Hydrogen Cyanide and Cyanogen Chloride 7 .'3 Phosgene . . . 17 I Disulfur Decafluoride . — 24 5 Mustard Cl as and Other Sulfur Mustards 30 0 Nitrogen Mustards . ... 59 7 Arsenieals S3 8 Aliphatic Nitrosocarbamat.es and Related Compounds . . 115 9 Fluorophosphates and Other Phosphorus-Containing Com- pounds . . . . . 131 10 Methyl Fluoroacetate and Related Compounds .... 150 11 (’adrnium. Selenium, and the Carbonyls of Iron and Nickel 173 12 Ricin 179 _13 Aromatic Carbamates . . — 204 14 Miscellaneous Compounds Prepared or Examined as Candidate Chemical Warfare Agents . —. . . . . . 246 SPEC IA L PHYSIOLOGICAL AND TON I COLOGIC A L S TV DIES PART II 15 The Assessment of Particulates as Chemical Warfare Agents 267 16 Apparatus and Techniques Utilized in Toxicological. Studies on Chemical Warfare Agents . . 278 17 Physiological Mechanisms Concerned in the Production of Casualties by Exposure to Heat ........ 303 18 Miscellaneous Toxicological Studies ........ 382 • For facility of handling, this Summary Technical Report of Division !•, Volume 1, has been hound and published in two sections. The first section contains Part I and Part 11 of Volume 9 1. Parts 111 VI are found in the second section, together with the Glossary, Bibliography, OSRD Appointees, Contract Xumbers, Project Numbers, and Index, all of which are applicable to I loth sections of Volume 0-1. SECRET XI PART I PREPARATION AND EJALUATION OF POTENTIAL CHEMICAL WARFARE AGENTS SECRET Chapter 1 RESUME OF AGENT ASSESSMENTS By Stanford Moore and liirdscy Renshaw 1.1 INTRODUCTION Reviews of the information on tin* principal standard and potential chemical warfare agents are presented in Chapters 2 through 11. Summaries of the data on the standard agents — mustard gas, phosgene, hydrogen cyanide, and cyanogen chloride are accompanied by reviews of other potential chemical warfare agents that were investigated during World War II. The reviews and bibliog- raphies are not limited to data obtained by the National Defense Research Committee So far as possible all available information lias been considered. The chapters deal primarily with the laboratory data on the chemical and toxicological properties of the agents. The technical aspects of the use of chem- ical warfare agents in the field have recently been summarized by the Project Coordination Staff.' In the discussions presented in this volume it is assumed (hat under certain conditions the use of each of the standard agents would be more effective from a military point of view than a similar expenditure of munitions charged with high explosive.2 1 he experi- mental agents are assessed relative to the standard chemical agents on this basis. It has not been possible U fake into consideration the recent development of the atomic bomb as n high-explosive weapon, the highly toxic radioactive gases encountered in the course of the research leading to the production of the atomic bomb, or the military potentialities of bacteriological warfare. For this reason the assessments made in the following chapters are limited in scope and cannot be considered complete in the broadest sense. 1.2 MAJOR [RENDS SINCE WORLD WAR I Mi sTAiin Gas Vapor From the laboratory and field test data obtained by the United States and the United Kingdom there developed during World War II a growing realization of the effectiveness of mustard gas vapor as a po- tential offensive weapon, particularly in tropical climates, in addition to its well-defined role as a de- fensive weapon. Emphasis in the field testing was placed on the thorough assessment of vapor dosages, as well as on the contact hazard presented by the contamination of various types of terrain with the liquid agent. It was demonstrated 1 that in hot weather relatively moderate expenditures of muni- tions would yield severely incapacitating vapor dos- ages within less than an hour, although the time of onset of the incapacitating symptoms was from 12 to 24 hours after the brief exposure period. The effects were optimal on heavily vegetated tropical terrain of the type characteristic of some of tlie combat areas in the war against Japan. Arsenical Vesicants At the close of World War I lew isite gained almost, legendary fame as a potential vesicant agent. More recently thorough assessment of the arsenical vesi- cants has shown lewisite to possess few if any advan- tages over mustard gas and to have several properties which greatly reduce its efficiency. Its marked sus- ceptibility to hydrolysis, for example, lowers the vapor return from contaminated terrain and de- creases the effectiveness of the vesicant through clothing. The development of British anti-lewisite [BAL] as an antidote in arsenical poisoning also affected the assessment. Lewisite and related arseni- cal vesicants have, of recent date, received little con- sideration in the United States as potential offensive agents. Sternutators and Lacrimators Interest in sternutators and lacrimators has mark- edly decreased since World War I. Particulate filters effective against, sternutators are now standard equipment in the gas masks of all countries. In so far as harassment by chemical agents is a legitimate military objective, there is a tendency to prefer the use of agents that are potentially lethal, particularly since British field trials under simulated combat con- ditions have demonstrated that the effectiveness of sternutators as harassing agents is limited. Use of Nonpersistent Agents As a means of attaining casualties by use of non- persistent gas against troops equipped with masks, secret SlMMUtV OK \SSESSM ENTS OK COM POINDS eided to put into production for use in high explosive- chemical shell and homhs. The tendency of AC to flash in some munitions is a disadvantage which has not been overcome. In the I hi i ted States cyanogen chloride (CK) was second choice to AC in the task cited in the preceding paragraph and was also considered for the special task of penetrating early World War II models of Japanese and German canisters under highly favor- able conditions of terrain and meteorology. Phosgene Phosgene (CG) was the principal uonpersistent gas of World War I and was the standard non per- sistent chemical filling for United States bombs and mortar shell at the outbreak of World War II. When delayed physiological effects are acceptable, CG has been considered (he most economical standard non- persistent agent for the production of casualties by attainment of effective dosages in less time than is required to mask and for the production of casualties among unprotected personnel. Disuleur Decaeluoride The difficulty and expense of manufacture of disulfur decafluoride (Z) on a large scale have pre- vented it from being seriously considered as a com- petitor for CG. The agent possesses the important advantage of relative lack of odor, and its physical properties are satisfactory. However, its toxicity shows considerable species variation ami the lethal dosage for man cannot at present be estimated with a degree of accuracy sufficient for a close toxicologi- cal comparison with CG. Sulfur Mustards No competitors for 6f.s(/J-chloroethyl) sulfide (H) were developed in the course of the study of a wide variety of analogs after World War 1 and during World War II. The search for a more volatile (i.e., less persistent) vesicant agent led only to compounds with markedly inferior toxicological potency. The high vesicancy of some of the less volatile analogs was studied quantitatively, however, and found to be sufficient to merit the addition to H of I,2-6t.s(j3-chlo- roethylthio)ethane (Q) or 6/s(/3-ehloroethylthioefhyl) ether (T) when long persistency of contact hazard on terrain or materiel is desired. Nitrogen Mustards The principal item of military significance that emerged from the study of the nit rogen mustards was the possible use of fm(j3-chIoroe(hyl)amine (IIN3) as a filling for high explosive-chemical shell. For most other purposes where a vesicant agent could be em- ployed to advantage 11 was shown to have properties superior to those of the nitrogen mustards. Arskmcals The thorough assessment oL the arsenical vesi- cants indicates (hat lewisite (L) and analogs are in general inferior to the sulfur mustards. Subsequent to the introduct ion of silver impregnation for canister fillings arsine has not seriously been considered as a potential agent. Relatively little military value is currently attached to the use of irritant arsenical smokes. A LI PM A TIC NITROSOUA RBAM ATES Studies on methyl N-(0-chloroethyl)-N-nitmso- carbamate (KB-16) and related structures showed this series of compounds to possess a high degree of toxicity. As an eye-injurant KB-16 is approximately as potent as II and is less readily detected by odor. As a vesicant, however, it is markedly inferior to H. It has also been found to be insufficiently stable for storage in munitions. Fluohophosphaths and Trilons In 1911 British investigators initiated studies on the fluorophosphates as potential war gases. The most effective members of the series studied by the United States and the United Kingdom were di- methyl fluorophosphate (PF-1) and diisopropyl fluorophosphate (PF-3). PF-1 and FF-3 do not pos- sess sufficiently high toxicities by inhalation to have an advantage over the standard chemical warfare agents. Limited data are available on the related agents (Trilons) discovered by the Germans during World War II. The member of the series which they placed in production was ethyl dimethylamidoeyano- phosphate (MCE). Difficulty in synthesis prevented the Germans from producing isopropyl methane- fl 11 orophosj ihona te (MFI), which appa ren t ly pi assesses superior chemical and toxicological properties. It is clear that the Trilons are more toxic than PF-1 or PF-3. They are quick-killing agents possessing a powerful parasympathomimetic action. The Trilons would seem to be the one new group of chemical agents discovered during World War II that merit a position among the standard agents. SECRET 6 RESl'ME OK AGENT ASSESSMENTS Fltokoacetatks and Related Comcot nds Reports that methyl fluoroaectate (MFA) is highly toxic were received from Polish investigators by the British in 1912. This class of compounds received careful chemical and toxicological study in the United Kingdom and the United States. The wide species variation in toxicity pointed to the need fora reason- ably accurate estimate of the lethal dose for the human species before an assessment could be made. If these agents had proved to be as toxic for man as for some animal species they would have been in a class with the Trilons. However, data becoming available in 1911 made it possible to estimate that the toxicity for man is low and to conclude that M FA and related compounds do not possess the general utility of the currently standardized gases. They re- main a subject of military concern because of their possible use as food or wafer poisons. In addition, as a by-product of the chemical warfare research, so- dium Huoroacetate has been demonstrated to have many practical properties as a rodenticide. ('admicu Oxide; .Metal Carboxyls; Cadmium is considered a promising material for addition to incendiary munitions if toxicity as well as fire is desired. The highly toxic cadmium oxide which results from the combustion of the incendiary mixes is odorless and relatively non irritating. Iron and nickel carbonyls have been considered as possible additions to flame thrower fuels if increased toxicity of the combustion products is an objective. Ricin Kic-in (W) is a protein of very high toxicity. The absence of odor and the complexify of the detection problem in the field would render it more insidious than any standard United States or British chemical warfare agent. As a result of progress made during World W ar II on the preparation of ricin. it is poten- tially available in relatively large quantities. Im- proved munitions for its dispersal as a particulate cloud are required before the toxicity of the agent can be adequately utilized. Comparison of the ef- fectiveness of the initial dust cloud of ricin with that of the initial cloud from the German Trilons would be indicated. The casualty-producing effects from exposure to ricin, however, are delayed, like those of phosgene {Masoning, whereas incapacitation and death from the Trilons is produced relativ ely rapidly. Aromatic Carbamates Research by the United Kingdom and the United States during World War II has made available in pilot plant quantities a series of crystalline aromatic carbamates possessing extremely high toxicity. By subcutaneous injection the most effective members of the series arc comparable in potency with the German Trilons. For some purposes the fact that they are crystalline solids is advantageous. For dis- persion by high explosive-chemical shell (he Trilons have the advantage of being liquids. The possible utilization of aromatic, carbamates may rest prima- rily on the availability of suitable munitions. SECRET Chapter 2 HYDROGEN CYANIDE AND CYANOGEN CHLORIDE" Stanford Moore and Marshall Gates 2.1 INTRODUCTION Auklativkia large amount of open and classified literature has aeeumulated on the properties of hydrocyanic acid and cyanogen chloride and on their behavior when Migrated from munitions. To survey the complete subject is beyond the scope of (his chap- ter. The sections which follow are designed to show the relationship of the data obtained by NDKC Di- vision 9 to the body of knowledge on these agents. Hydrmwanic acid (AC) was adopted by the United States as a standard filling for frangible grenades in July 1942 and as a substitute standard filling for 1,000-lh bombs in Octoler 1943. From the toxico- logical standpoint this agent was one of the standards for comparison in the evaluation of potential new gas warfare agents under investigation by NDRC during World War II. Cyanogen chloride (CK) was standardized as a quick-acting nonpersistent gas filling for 1,000-lb and 500 1b bombs in October 1943 and for 4.2-ineh mortar shell in July 1945. For the task of production of immediate deaths by the attainment of effective dosages in less time than is required for masking, AC is considered from the toxicological standpoint to In1 the l>est agent avail- able.41 43 Its competitors are the almost equally quick-acting but much less volatile fluorophosphates and related compounds, one of which was put into production by Germany (Chapter 9). The tendency of AC to flash in some standard munitions is a dis- advantage which has not been overcome. The primary interest in CK arose from the demon- stration by NDRC Division 10 that CK showed greater promise for the penetration of World War II models of Japanese and German canisters than any other readily available agent. The protection af- forded by Japanese canisters in particular was so low that there was promise of attaining lethal pene- tration of enemy masks from moderate expenditures on jungle terrain or heavily wooded areas. CK can !>e satisfactorily stabilized for storage and Is non- inflammable. It is also second choice to AC in the task cited in the preceding paragraph. In this chapter reference will he made to (be pri- mary chemical and toxicological basis on which the standardization of those agents rested, 2.2 PREPARATION AND PROPERTIES 2.2.1 Preparation Hydrocyanic acid (AC) has long been available on a commercial scale. The cost of production at the present time is about fine times that for phosgene. Cyanogen chloride' (CK) was produced by the French in World War I and on a small scale in (lie United States prior to World War II, Larger scale produc- tion, by a process based upon the chlorination of aqueous hydrocyanic acid, was undertaken in 1944 at the plant in Azusa, California. 2.2.2 Physical Properties The physical properties which have the most direct bearing on the effectiveness of these agents as war gases are the following: AC CK Density (liquid) g/ml at 20 C 0.69 1.19 boiling point, C 25.7 12.6 Freezing point, C —13.4 —7.0 Heat of vaporization, cal/g 210 135 Vapor density, relat ivc to air 0.93 2,0 The liquid density affects the amount of agent which can be loaded per munition; the freezing point is of importance in cold weather or on high-altitude bombing missions; the boiling point influences the rate of evaporation of the liquid agent ; the heat of vaporization also influences the rate of evaporation and determines the cooling of the air produced by evaporation of the agent from a functioned munition. The cooling effect in turn influences the stability of the gas cloud produced, since the cooler layer of air tends to remain longer near ground level. Prior to the performance of adequate field studies on these agents it was considered that, since AC has a lower vapor density than that of air, the persistence of its clouds should be much less than the persistence of those ’ Based on information available to Division 9 of the Na- tional Defense Research Committee [N DHC] as of November I, 1945. SECRET 8 IIVDKOGEN CYANIDE \ND CYANOGEN Clfl.ORIUE from CK and CG, which are heavier than air. Actu- ally, clouds of AC and CK show essentially the same persistence and downwind travel.41 Considered in conjunction with the cooling effect arising from the latent heat of vaporization and with the meteoro- logical factors governing the turbulence of the air, the data show that the vapor density of the gas does not play so important a role as was first thought. The boiling points of AC and CK are sufficiently low so that the agents vaporize completely within a few seconds after dispersal in droplet form from bursting munitions. 2.2.:t Stability of VC Pure AC is unstable on storage, ultimately decom- posing with explosive violence, lint the presence of small amounts of mineral acids, particularly phos- phoric acid, produces marked stabilization. Present Chemical Warfare Service specifications call for the addition of 0.07 per cent orthophosphorie acid and 0.3 per cent sulfur dioxide as stabilizers, and surveil- lance tests have demonstrated that AC stabilized according to these specifications and charged into clean bombs is stable when held at 150 F for 60 to 90 days.20 35 41 Powdered copper, used by the Japanese as a stabilizer in AC munitions, has also shown prom- ise as a stabilizer, particularly in (he presence of steel, which gradually exhausts the phosphoric acid sta- bilizer.sk-,,°'r _ The tendency of AC to inflame on functioning of munitions constitutes the principal disadvantage of AC as a chemical warfare agent. In field trials with 500-lb and 1,000-Ib bombs an appreciable percentage of the munitions flashed in some instances. Extensive efforts have been made to overcome or minimize this tendency both by changing the bursters and by al- tering the composition of the charging. The addition of aliphatic hydrocarbons in (-CT range originally appeared promising 8, 2 ikn and led to field tests Oil them and also on gasoline as Hash-inhibit ing dil- uents. The early trials indicated40 that flashing could be reducer! by the addition of 5 per cent of 70-octane gasoline to AC in M47A2 100-lb bombs, and in larger bombs, but subsequent work indicated (hat the addition of gasoline to AC does not satisfac- torily prevent flashing if the mixture is allowed to stand for 20 days or longer after (lie addition of the gasoline.21,1 No solution of the problem of flashing of bombs charged with AC which would keep the per cent flashing consistently below 10 per cent, for ex- ample, has been obtained. The fact that in some series of trials no flashing is encountered indicates that the problem may not be an insoluble one. 2.2.t Stability of (IK Pure CK. and also the CK produced technically in this country, may be kept in glass for long periods even at elevated temperatures.'4 a0 M Stability be- comes a problem only on storage in metals. Investi- gations have proceeded along three lines; (1) the determination of the effect of storage in contact with metals, particularly steel, on Vue stability of C'K. (2) determination of the effects of (he impurities en- countered in technical CK on its storage life in steel, and (3) minimisation of the effects of contact with inyjUd and of impurities by the addition of a stabilizer. The chemical change involved in the polymeriza- tion of CK is largely"one of trimerization to cyanuric chloride 54 5 8 50 but other reactions, the products of which were undetected until recently, also occur to a minor extent.5 It was recognized many years ago51 54 that acids, particularly hydrochloric acid, promote the polymerization to a marked degree. The addition of 2 per cent of hydrochloric acid is sufficient to pro- duce explosive polymerization,17 but the amount of hydrochloric acid likely to be present in technical CK is harmful only if appreciable water is also present.51 8f The presence of chlorine and hydrogen cyanide were also held to lie deleterious by early workers.58 53 Chlorine is still regarded as harmful,8® 47 although there is some evidence that it is less critical than formerly supposed. However, it presents no problem since it is well controlled in the manufacturing proc- ess. The amount of AC present can be varied within wide limits without affecting the stability of CK.4 5" Work on the stabilization of CK was undertaken in 1942 by NDRC Division 10. The early work was confined to experiments on pure CK stored in glass at room temperatures.1 The harmful effects of chlo- rine and gross quantities of acid were observed, and it was noted that cyanuric chloride had no effect on the rate of polymerization. Later work, however, has shown that this is (me only in the absence of steel **•'* and also that water, observed in this early work to have little effect even in gross quantities, is actually very harmful to CK stored in steel,4 Mag- nesium oxide effectively stabilizes CK against the harmful effect of chlorine. Canadian workers have observed that magnesium oxide also protects against excess acidity48 but it does not appear to meet the SECRET PR HP V R ATI ON AM) PROPERTIES requirements for a good stabilizer.47 A number of other substances were examined for their effects on the stability of CK, but the surveillance tests, done at room temperature o\er relatively short periods fi.e., 2 to 6 weeks), were not sufficiently rigorous to give information useful in predicting the stability of munitions charged CK. In 1933 the study of the stabilization of CK was intensified by NDRC Division 10. and much funda- mental chemistry relating to CK, in particular to those reactions taking place during the storage of male CK, was elucidated. A detailed description of this work is given else- where.1' The more important results are enumerated here. 1. Although the earlier work showing the general deleterious effect of acids and of chlorine on the sta- bility of CK was confirmed,6" the special position of water, particularly in the presence of steel, was recog- nized.6" 1 Thus the stability of CK in the presence of steel decreases with increasing original water con- tent, w hereas no such correlation is shown w ith acid or with hydrogen cyanide.**" Hydrogen chloride, AC, and ammonium chloride all cause little polymeriza- tion if moisture is absent.6" 1 2. A number of stabilizers were proposed and tested. They were suggested before the importance of iron salts and of water was realized, and the ra- tionale for most of them was removal of acid. Propy- lene oxide, first proposed as a stabilizer by the American Cyanamid Corporation,17 and ethylene oxide absorb acid even when dry 6,1 and were at first believed to be promising stabilizers. Later work showed them to be definitely harmful.4 Dimethyl- eyanamide, an end product of the reaction of CK with trimethylamine, a reaction analogous to von Braun’s “bromeyan” degradation, absorbs two moles of acid.6* It was suggested for trial as a stabilizer by the fact that no polymerization occurs during its forma- tion from CK and trimethylamine.57 However, the stabilizing effect appears to be due primarily to the production of a coating on the steel, and. if the sam- ple is agitated during surveillance, no stabilization results.4 Likewise, it was possible to demonstrate a stabilizing effect due to the formation of the complex (HCN)2-(HCI)j only if a considerable excess of AC was present, and then the stabilization was slight.61' Parallel to this work and supplementing it, the Chemical Warfare Service carried on surveillance tests in munitions charged CK, originally on CK produced by the American Cyanamid Corporation at Warners. New Jersey, and later on CK produced at Azusa, California, as well. The results of these surveillance tests, which were carried out for 30, (30, and 90 days and ultimately for longer periods at 65 C, in general confirmed the conclusions reached in laboratory scale tests as to the effects produced by the usual impurities in technical CK. (Jood quality CK was shown to have adequate stability when charged into clean munitions,17-1*-19 These surveil- lance tests also afforded realistic opportunities for the evaluation of stabilizers developed in laboratory scale tests. The NDRC Division 9 group which undertook a search for stabilizers for CK during 1911 observed the Inmcficial effects of a number of inorganic sub- stances including sodium pyrophosphate, calcium oxide, and potassium fluoride. In the interest of ex- pediency it proved necessary to carry out surveil- lance tests on these stabilizers at 100 (' and 125 (’.** The applicability of the results of such accelerated tests to stability at lower temperatures was a matter of detailed study. All groups engaged in laboratory scale surveillance tests on CK ultimately adopted these temperatures. The rate of polymerization of CK increases by the usual factor of 2 per 10-degree rise in temperature (determinations have given 1,7 to 2.5) and the character of the polymerization ap- pears to be the same at 125 C as at 75 C.8b However, ammonium chloride in CK produces acid in surveil- lance at 100 C but not at 65 C,12,1 and it is possible ♦ hat surveillance at higher temperatures may lead to an underestimate of stability at lower, temper- atures.1-’"cf The effectiveness of sodium pyrophosphate, the best of the above-mentioned group, and of calcium oxide is much greater than that of organic stabilizers such as ethylene and propylene oxides and the dial- kylcyanamides, which in spite of early promise were shown to be definitely deleterious.*" Marked im- provement in the storage qualities of even poor grade CK is obtained w ith sodium pyrophosphate. For CK of average quality, 2 percent sodium pyrophosphate is adequate, although 5 per cent has been recom- mended and adopted to make certain that poor batches receive adequate stabilization.4 With this concentration of stabilizer, CK of any grade has Ixdter keeping qualities in the presence of steel than has the same CK stored in glass but unstabilized.4 Poor quality CK has a short life even in glass, how- b S(H* the Summary Technical Report of Division 10. SECRET 10 HYDROGEN CYANIDE AND CYANOGEN CHLORIDE Table 1. Surveillance of CK (Azusa Lot 080) for 00 days at 05 C.a Xa.PO; Acidity Soluble Munition (%) Density as MCI residue 1 run ILO IICX M7S bond) 0 Polymerized after 00 days M78 bomb 5 1.204 0.017 0.00 0.001 0.05! 1.94 ever, and to Ik* suitable for charging into munitions CK should have a solidification time in glass at 125 C of at least 10 days.4 v '■* This implies low values for soluble iron and for water content. An example of the behavior of actual munitions charged CK when stabilized with sodium pyrophos- phate is given in Table l.~ The stability of CK samples which have already been stored in steel without a stabilizer is markedly improved by the addition of 5 js-r cent sodium pyro- phosphate, and CK thus treated has at least as long a life in steel as when stored in glass without a stabilizer. *■ Then* appears to be some correlation between the stability of a sample of CK and its soluble iron con- tent.8* A corresponding correlation ladween water- content and stability appears at low values of water content but is much less marked for higher values, the critical value being about 0.2 per cent water.8* However, the susceptibility to stabilization by sodium pyrophosphate is strikingly dependent on water content, as illustrated by the data of Table 2.4 Although only limited data arc available, the pres- ence in CK of potassium pyrophosphate and pre- sumably of sodium pyrophosphate appears to have little effect on its content of water, iron, acid, or hydrogen cyanide, although the rate of t rimer forma- tion is reduced. Over a 9-day period less than 0.002 per cent potassium pyrophosphate dissolves in CK.7 Since the stabilizing action of sodium pyrophos- phate is probably a surface phenomenon the particle size is important. “Kiln run” or “granular” sodium pyrophosphate is unsuitable, and it is necessary to include in the specifications a clause, easily met by the commercial “powder” grade, specifying the proper screening characteristics. A suitable set of specifications, readily met by commercial material, has been suggested.*5 Little or no heat effect is produced when 5 j>er cent sodium pyrophosphate is added to ('K with as much as 0.3 per cent water content, whereas the addition of calcium oxide to similar material may produce a rise in temperature of 4 C. The addition of calcium oxide to CK of higher water content may produce dangerous heat effects.4 511 The recommendations made-in 1943,7,l8as to the limits of impurities which should be allowed in Cl\ for charging chemical warfare munitions (i.e.. H(’l, 0.005 per cent; water, 0.005 per cent; IICN, 0.02 per cent; and chlorine, none) proved to be unnecessarily st ringent. The present XL S. Army specifications for CK stored in 1-ton containers call for water, 0.5 per cent ; hydrogen cyanide, 3 per cent; soluble residue, 0.02 per cent; chlorine, 0.005 per cent; acid (as HC'I), 0,024 per cent; iron, 0.02 per cent; and CK assay, 90 per cent minimum.37 Five per cent sodium pyrophosphate is added to the container prior to the addition of the CK. The specifications for 4.2-inch mortar shell charged CK also require the addition of 5 per cent sodium pyro- phosphate to the shell prior to charging.36 CK is noninflammable and there is thus no prob- lem of ignition of the charging by bursting munitions as in the case of AC. Table 2. Effect of water on stabilization of CK by sodium pyropho: spliate.4 Water Days for complete Stabilization Conditions content solidification in days CK 0.03% 40, 40 CK + 0.3 g steel (eontroll 7, 7 CK + 0.3 g steel -f 5% N.id’.O; >209, >209 >202, >202 CK 0.23% II, 12 CK + 0.3 g steel (control) 7, 7 CK 4- 0.3 g steel -j- 5% NaT/); 89, 89 82, 82 CK 0.53% 8, 12 CK 4- 0.3 g steel (control) 3, 0 CK + 0.3 g steel + 5% XajPjOr 15, 15 9. 9 SECRET TOXICOLOGY 2.2.5 Detection anil Analysis Chemical methods for the detection of AC and CK arc outlined in Chapter 34. Methods of analysis have been developed for the assay of plant run products with special reference to the impurities which may lower the stability of the agents on storage. The dis- cussion of titrimeters in Chapter 37 includes the de- scription of continuous recording instruments for AC and CK utilizing potent iometricy titrations. NDRC Division 10 has developed recording instru- ments utilizing conductivity measurements and those instruments have been widely employed in the de- termination of concentrations of AC ami CK in the field test programs. AC is detectable by odor at about 0.03 g 1 (i.e., 30 mg I)13 on the average, although some men may lack almost completely the ability to detect the odor of cyanide. CK is readily detectable at about 0.02 mg 1 both by its immediate laerimatory effect and its irritant effect on the nasal passages.14 At concen- trations as low as 0.002 mg 1 the eye irritation is noticeable by some observers in less than 3 minutes. 2.2.6 Canister Penetration The dosages of AC and CK required for lethal penetration of captured German and Japanese can- isters 1213 (1941 42 models) are about the same but the advantage lies with CK because of the higher dosages attained in the field.* The higher liquid density of CK, which permits heavier bomb load- ings, contributes to the higher dosages obtained per bomb. Lethal penetration of Japanese canisters by CK was obtained at dosages of 50,000 to 200,000 mg min m3 at normal breathing rates. U. S. canisters filled with ASC charcoal give protection against sev- eral times these dosages. Field trials have demon- strated that dosages of CK in the range of 50,000 to 200,000 can be attained by feasible munition ex- penditures under favorable conditions of terrain and meteorology.43 2.3 TOXICOLOGY _ 2.3.1 Toxicity For the production of casualties among men prior to adjustment of the mask, the toxicity of the agent when breathed for 30 seconds, or perhaps 2 minutes in the case of sleeping troops, is the important char- acteristic. Against personnel without gas masks (e.g., in city populations) the toxicity of the agent over longer periods is of importance. The toxicity over longer periods also comes into consideration in study of the physiological action of the concentrations pen- etrating canisters. Ideally, (he toxicity for man is the information de- sired. The data to he summarized in this chapter are based primarily on measurements of the action of the gases on various animal species and conclusions on the values of the agents are drawn therefrom. It should he noted, however, that in 1914 a thorough analysis of the problem of the indirect estimation of toxicities of AC, CK, and CG for man was made jointly by the Toxicological Research Laboratory of the Medical Division, Chemical Warfare Service, and (he NDRC-University of Chicago Toxicity Laboratory.4" For each of the three agents research programs were drawn up designed to provide in- directly the answer to the question of the concentra- tions required to cause death in man. Such an approach in the case of cyanide appeared particularly promising since certain basic data on the effect of cyanide on man are known from the open literature on the clinical use of sodium cyanide. The work on these programs is still in progress. The detailed considerations of the probable effects of the three agents on man have led to a fuller under- standing of the action of these nonpersistent gases and to more adequate interpretations of munitions trials. From the summary of the above-mentioned status report the sections on AC and CK are quoted to give the principles which enter into the attempt to make an indirect determination of the toxicities for man. The purpose of this review was to define the laboratory ex- periments still required to reduce to a minimum (he error in estimate of" I he concentrations of AC, CK, and CG required to cause death in man at different times of exposure. A consideration of the tasks proposed for AC, CK, and CO discloses that concentrations of each agent lethal to man in 0.5, 1, 2, 10, 30, and 60 minutes are desired. AC is known to l>e detoxified by animals and man. CK is detoxified by animals and so is presumably detoxified by man. AC is detoxified by man at a rate of ea. 0.017 mg/kg/min when injected'1 slowly.5* This does not differ markedly from the rates at which it is detoxified by lower animals.55 CK is detoxified by the rabbit, dog, and goat at rates of 0.03 0.06; 0.02 0.01; and 0-03-0.1 mg kg min rcs[K-etively depending on the rate of inject ion.17•34*-h Arguing by analogy with AC, man presumably will detoxify CK at a rate within these limits (0.02 0.1 mg kg min). d The experiments were made with XaCX, which upon in- jection into the tissues at about p\\ 7 yields HCX almost quantitatively. c See the Summary Technical Report of X DRC Division 10. SECRET 12 IIVDKOCEN CVAM IDE \M> C> A'.OGEN CHLORIDE We have estimated that the LDM of AC' for man is ea. l.l mg kg. This estimate rests on: a. The I.Dm of AO for six species of animals sf t> which indicates that the various s|xeies are not different in suscep- tibility, thus implying that man will fall in the same range. b. Amounts of cyanide found in the tissues of humans com- mitting suicide by taking cyanides.1- ’1 c. The fact that the of AC bears an apparently con- stant ratio to the rate of detoxication of AC in various species. The /.DCs of CK for the rabbit,dog and goat are 3.15, 3.30, and 2.07 mg kg respectively.*7 By analogy with AC man Ls probably equally susceptible. Such evidence as is available,indicates 1 that AC is equally toxie by inhalation or intravenous inject ion.*h It is held in this review that this is also true for CK. The minute volumes of different species of animals are st im- ulated differently by AC, 7-fold in the dog, 2 3-fold in the rabbit, and 1.5-fold in the guinea pig.sh These volumes are suf- ficient to allow inhalation of an amount of AC approximately equal to the /.//«, of AC for the several species. Man’s respiration is stimulated 7 to 10-fold by intrave- nously injected AC in single doses of ea. 0.055 mg kg or more (estimated from reference 56).* The duration of such stimula- tion Ls ca. 20 seconds.*1 When infused slowly the [xrceut stim- ulation is less (2-3-fold) but is longer maintained. If CK is not more toxic by inhalation than by ini ravenous injection, then it too must stimulate the respiration of some species to allow the inhalation of a lethal dose. CK is known to stimulate the respiration of the dog (after causing apnea be- cause of its irritancy)."* Xo satisfactory evidence is available to indicate that dif- ferences in the susceptibility of various species to AC and CK cannot lie explained on the basis of difference in minute vol- umes in the presence of the gas, and the value of the intra- venously injected LDM.‘ It is suggested that the toxicity of agents like AC and CK can be deserilxd as a first approximation by the formula VaC - Dt = K in which 1’ = total volume of air breathed in I kg a = the fraction of inhaled gas absorbed C = concentration in mg 1 D = rate of detoxication in mg kg 'min 'Intravenous /.DCs of AC in mg kg (unanesthetized animals): dog 1.34, cal 0.81, monkey 1-30, rabbit 0.66, guinea pig 1.43. rat 0.81, and mouse 0.90. Kor anesthetized goals an /,/>.. of 0.66 has lieen obtained.17 There are indications that the intravenous LDms for anesthetized and unanesthetized animals are the same in some species -• but may not lx iden- tical in others.®' i hater confirmed for dogs by the development of apparatus for direct measurement but found not to hold for where the I.Dm, by way of the lung proves to tx about half of the intravenous /.Die. • This value fur the respiratory stimulating dosage of in- jected XaCX has been checked in more recent studies on measurement of the vehxity of blood flow in man.17 h Ixsser stimulation but longer duration have Ixen olsxrved in legal executions with AC (reference 16 and later unpublished data). i Six exception in Note e. I ~ time in minutes from first entrance of the sul>- stsince into the Itody (roughly, the exjiosure time) K — the lethal dose in mg kg The source of greatest doubt in estimating concent rati..ns of At' and CK required to cause death .in man from the above e<)uation is the question of man’s minute volume in the pres- ence of the substance. On tlu* above basis, if a 70-kg man breathed 25 1 of air during a 1-minute exposure to AC, the volume breathed per kilogram would Ik* 25 70 — 0.36 1. If 70 per cent of the inhaled AC is absorbed by the lungs, as experimentally determined for the «log,3!w the equation ean be solved for the necessary lethal concentration of AC: 0.36 X 0.7C - 0.017 X I = 1.1 and C — 1.4 mg 1. In this instance for the 1-minute exposure the L{Ct)M would be 4,100 mg min m3. As has been pointed out, however, a meaningful estimate of man’s minute volume in the presence of the gas is not readily made. It is upon earlier calculations of this type by British investigators that the internationally agreed esti- mates of 5,000 and 11.000 mg min in’ for the L{€l)u's of AC and CK are baser!.44 As guides for use in mu- nitions assessments the estimates have proved useful. An alternative approach for use in determination of munitions requirements, developed from these data, places the emphasis on attainment of (he mini- mum respiratory stimulating dosage rather than on the L(Ct)iu- This approach is based upon the fact that upon detection of AC a man will try to hold his breath long enough to adjust the mask. If the amount of gas which he inhales in the first breath is sufficient to stimulate the respiration, the probability of his receiving a lethal dose prior to adjustment of the mask is increased. For a sedentary individual the volume inhaled per breath is about 0.6 1. If the first breath is to insure stimulation of respiration it should yield absorption of about 0.055 X 70 = 3.9 mg of AC per 70-kg man. If the absorption coefficient is 0.7, the concentration of gas to lx* aimed at in the gas cloud would lx* 3.9 (0.6 X 0.7) = 9.3 mg I. It can he seen from this analysis that the predic- tion of the toxicity of AC for man, if feasible, is a problem requiring knowledge of the mechanism of the action of AC, its inherent toxicity per kilogram of laxly weight in different species, its influence on the rate and volume of respiration, the per cent of SECRET TOXICOLOGY 13 T.vbi.k 3. L(Cl)u's of AC for different species. Exposure time (mg min/ra") or suggested value where number of A — anal. cone. Xuinlier of animals Species (min) animals is small X — nom. cone. used Reference Mouse 1 400 A 25 II 3 500 A 44 2 i !K10 A 44 2 1 000 A 50 11 2 1,300 A 05 6a 2 1,280 A 200 15 2 1,100 N 100 Of 2 1,280 X 100 tlf 2 1,320 X 80 Of 3 1,100 A 30 1 1 R) 2.300 A 300 12 30 o,25Q_ X 0g 30 5,000 A 180 i5 Rat S 800 A 24 2 1 1,550 X 76 6h 2 2,200 A 100 ■ — 23 3 1,800— A IS 11 Guinea pig 1 J 2,500 A 28 2 1 2,100 X 60 Oh - Rabbit 1 850 X 32 He — 10 3,200 A 21 it Cat 1 8.50 X 30 6e Monkey 1 1,700 X 10 6c Dor 3 800 A 30 n 1 700 X 24 tie 1 700 A 20 11 3 1,000 A 26 It Goal 3 1,300 A 20 31 - 2 2,200 A 18 24 the gas which is absorbed by the lungs, and its rate of detoxication. The outlay of research required to gain an approach to the actual toxicity for the human species is an effort which has been attempted only for a few agents of primary importance. In general the screening of possible new war gases has rested on the routine determination of the L(Cl)M’s for vari- ous animal species. Most of the toxicological conclu- sions on new agents have had to rest on comparison of animal s, combined with qualitative evalu- ation of the effects of the different agents on man. The data on the L(Cl)m values of AC and CK for different species are summarized in Tables 3 and 4. Where the number of animals used in the determina- tion is small, the L(Cl)M is only an approximation. In these tables the values which have a fairly sub- stantial experimental basis have been included. The tables include World War 1 and World War II data. A fuller tabulation is given elsewhere.40 A comparison of the L(Cl)-,o’s of AC and CK for different species is given in Table 5 for exposure times of 1 to 2 minutes. Since the density of the gas governs the weight of filling per bomb of given vol- ume, the last eolunin in the table represents the effi- eieney in terms of munition chargings. From Table 5 it is seen that for most of the species AC is several times as effective as (Tv in short exposure periods. For long exposures the ratio of the toxicities ap- proaches a value close to the inverse ratio of the cyanide radical contents of the two gases. The course of respiration of goats during exposures to AC and CK has been studied in detail at Dugway Proving Ground. During exposure to AC for 2 min- utes, respiration was normal for the first few sec- onds.21" At concentrations above 2.5 mg/1 the time of onset of stimulation of respiration was 10 to 20 seconds after the start of the exposure. The dura- tion of stimulation was 20 to 100 seconds and was followed by a depression of respiration setting in at 40 to 150 seconds. In similar experiments with CK at concentrations above 2 mg/1 respiration is irregu- lar and depressed during the first 30 to SO seconds.21' The breath is not actually held, however, as in the case of goats exposed to phosgene (CG).2,,,•S, Fol- SECRET 14 HYDROGEN CVAMUE AND CYANOGEN CHLORIDE Table 4. L(Ct)M's of CK for different species. L(Cl)u (mg min m3) Exposure time or suggested value where number of .1 = anal. cone. Numlier of animals Species (min) animals is small AT = nom. cone. used Reference Mouse 1 3,000 A 50 44 1 4,550 X 160 6b 3,010 A 1 4,200 A 29 44 2 5,000 N 120 lif 2 5,300 N 100 3 2 0,200 A 180 14 2 3,600 A 70 44 3 4,200 A j 40 44 10 7,900 n/ 200 3 10 7,500 - X 160 14 30 13,500 N ISO 3 30 13,800 . A 140 14 Hat 1 13,000 X 35 3 2 9,100 A 30 44 3 5,400 A 20 44 75 0.300 A 18 10 30 9,000 V 16 10 Guinea pig 1 15,000 X 35 3 . 2 7,000 A - 30 44 25 5,500 A 16 10 7} 9,000 A 13 10 30 17,000 A 13 10 Rabbit 1 13.000 X 10 3 2 S,000 A 24 44 — 7i 6,000 A 23 10 30 17,000 ~ A 20 10 Cat 1 6,000 X 10 3 Monkey 1 1,400 X 20 3 Dog 1 3,800 X 26 3 3 4,200 X 18 3 75 1,500 A 26 10 10 5,000 N 26 3 30 6,000 X 14 3 Goat 2 3,600 A 30 25 lowing the period of depression, there is a stimulated phase of 40 to 90 seconds’ duration and this result is in accord with the conversion of CK to AC in the body, as described in Section 2.3.3. There follows a variable period during which the respiration again falls below the basal rate. W ith both AC and CK 1 his later period of depression is followefl by com- plete respiratory paralysis, usually within a few minutes, in those animals receiving lethal dosages and by gradual return to normal in those receiving sublethal quantities of the agents. Since CK yields AC in the body, a 50/50 mixture Table 5. Comparison of /.(f'/)M’s of AC and CK for short exposure times. Exposure time /-(CO*, of CK L(Cl)io of CK Density of AC Species (min) n n )w of AC UCl)M of AC X Density ot CK Goat 2 1.6 0.9 Monkey I 2.6 1.5 Mouse 1 4.0 2.3 Dog 1 5.4 3.1 Cat 1 7.0 4.1 Guinea pig 1 7.1 4.1 Rat 1 8.4 4.9 Rabbit I 15.3 8.9 SECRET TOXICOLOGY 15 of AC and CK should show a toxicity which is a function of the combined effect of the cyanide con- tributions from both agents. In a series of determina- tions of the toxicities for mice of AC dilutee! with increasing percentages of CK, the 2-minute was 1,250 for pure AC, 1,880 for 50 50 AC CK, ami 5,400 for pure CK.6( Calculated on the basis of cya- nide radical content the values are, respectively, 1,200, 1,300, and 2,300. Part of the lower effective- ness of CK on a cyanide content basis is to be ac- counted for by the tendency of the irritant vapors of CK to depress resp*ration during the first part of the exposure period. That there is reinforcement between the two agents is evidenced by the fact that the 50/50 mixture is essentially as toxic as pure AC on a cya- nide radical basis. AC vapor is absorbed slowly through the skin, experiments on laxly exposure during which the animals breathed uneontarninated air indicated that mice were killed at 10-minute Cl's of about 200,000 mg min nv\ eats at 500,000, and dogs at 1,000,000.-’ The order of the sensitivity in these tests is to be ex- pected on the basis of an increase in the required Cl with decrease in the ratio of body surface to body weight. The results serve to indicate that for man (he required Ct is of such a magnitude that it does have significance in the consideration of AC as chemical warfare agent. The irritancy of CK vapor to the human skin has been noted by personnel engaged in field tests with this agent. In a series of chamber experiments under controlled conditions groups of men wearing masks were exposed to several concentrations of CK.4* Un- der hot and humid conditions the irritation, primarily in the genital region, became severe at 2.0 mg/1. Under temperate conditions similar effects were ob- tained at alx»ut 3.6 mg/1. The irritation, although severe, is transient and does not persist after re- moval from the CK-contabling atmosphere. The LT)m's of AC and CK by intravenous injection have already been cited. When administered orally or by stomach tube, the rate of absorption of the agents from the digestive tract affects the LDm-m In orally administered doses of NaCN sufficient to kill dogs within less than 10 minutes, as much as three- fourths of the administered cyanide is found in the gastrointestinal tract after death. In dosages yield- ing later deaths the per cent absorption is higher. Calculated on the basis of absorbed HCN, the l.D„, orally administered is not significantly different from the intravenous value. The LDm's for ocular and for nasal administration of AC in cats arc also found to be close to the intravenous value.*" The I.DM of CK administered in aqueous solution by stomach tube has been found to be approximately 6 mg kg with deaths occurring at about 30 minutes.26 2.3.2 Pathology In animals dying from acute cyanide poisoning pathological examination shows evidence of marked tissue anoxia. Hemorrhages are most apparent in the thymus glands.2 Pathological studies from World War I and from World War II indicate that residual lesions from AC are significant only in the ease of animals receiving an exposure in a narrow range just below the minimal lethal dose, resulting in irrevers- ible injury to (he more susceptible nervous tissue but failing to cause acute respiratory paralysis and death of the animal.4* Nearly all animals recovering from sublethal doses do not experience significant tissue anoxia and are free from any demonstrable after- effects. In the animals showing residual neurological damage the principal pathological changes are noted in the cerebrum and the cerebellum.- 2* Residual paralysis following CK exposures is sim- ilar to (hat obtained with AC and is observed more frequently in dogs than in other species.*' In addition CK has a local irritant effect on lung tissue. In occa- sional instances the lung irritation can lead to pul- monary edema,2844 which may lx* important from the therapeutic standpoint. From the offensive standpoint the pathological results show that the immediate paralyzant effect of CK greatly over- shadows its other effects.44 Mice appear to l>e more resistant than dogs to lung irritation from CK. Alice surviving .'15 exposures to nearly lethal dosages of CK showed no gross pathological lesions in the lungs.6*1 2.3.3 Physiological Mechanism Hydrocyanic acid exerts its lethal action through inhibition of cellular respiration producing an as- phyxia leading to death. The agent causes a tem- porary increase in respiratory volume as a result of action on the carotid laxly; if the carotid body is re- moved, only respiratory* depression is obtained. The therapeutic value of met hemoglobin is a result of its strong affinity for cyanide, in competition with cyto- chrome oxidase, yielding a nonionized and nontoxic combination. In extension of the basic information on the meeh- SECRET HYDROGEN CYVMDE AM) CYANOGEN CHLORIDE an ism of action of AC, the competition for cyanide between methemoglobin and cytochrome oxidase has been demonstrated in vivo.Kh The therapeutic value of methemoglobinemia induced by nitrates and by p-aminopropiophenone has been thoroughly studied by Medical Division. Chemical Warfare Service, and by the Office of Scientific Research and Development [OSRD] Committee on Medical Re- search. Cyanide is detoxified by gradual excretion as thiocyanate and the therapeutic value of thiosul- fate has l>een further investigated. From the prac- tical standpoint the deaths from exposure to gaseous AC are rapid and under battlefield conditions there would be few opportunities to apply therapeutic procedures of this type in time to be of value. The production of methemoglobinemia among large groups of troops as a protective measure prior to possible exposure has been considered but is not a practical procedure in the field and would reduce the efficiency of all troops thus treated.30 It has been demonstrated that cyanogen chloride is converted to hydrocyanic acid in the body and exerts its lethal effect as AC. Some of its other toxi- cological properties such as irritancy to the nasal passages and local action on lung tissue are char- acteristics of CK itself. The similarities in the actions of CK and AC were pointed out by British investi- gators4* in the reporting of the conversion of CK to AC in whole blood in a matter of seconds. Blood serum alone does not accomplish the conversion. The reaction is not a simple one and proceeds in two stages.46 CK reacts with hemoglobin to give a com- pound which in the presence of reduced glutathione yields AC. In connection with the mechanism of the first stage it has been demonstrated that CK is capable of reacting with both amino groups and sulfhydryl groups of amino acids and pro- teins.9 Following the British work the observations were extended to additional animal species, to blood in vitro and in vivo, and to human blood.39* The eon ver- sion is not quantitative. The maximum conversion by human red cells was SO per cent and the average was considerably lower than this value. About 75 per cent conversion has been found In vivo in the rabbit.32” No free CK is present, however. The fate of the frac- tion which does not appear as 11CN has not been determined. Although a slow combination of CK with met- hemoglobin can be demonstrated when (he agent is present in great excess, the slowness of this combi- nation comparer! with the rate of the conversion reaction to AC makes it unlikely that the combi- nation of CK with iron compounds such as met- hemoglobin has any significance in vivu.m> Induced methemoglobinemia is effective therajM'utically against CIv32***11 in agreement with the data on conversion to AC. 2 t K VALIDATION \S WAR CASKS For the task of production of immediate deaths by tlie attainment of effective dosages in less time than is required for masking, AC is considered from the toxicological standpoint to be the best agent avail- able.414* Its competitors are the ahnost HiTiVdlv quick-acting but very much less volatile Huorophos- phates and analogs, one of which was put into pro- duction by the Germans (Chapter 9). The tendency of AC to Hash in some standard munitions is a dis- advantage which has not been overcome. For the task of penetration of World War II mod- els of German and Japanese canisters under favorable conditions of terrain and meteorology, CK was the most suitable agent. It can lie satisfactorily stabi- lized and is noninffammable. It is also second choice to AC in the task cited in (he preceding paragraph. SECRET Chapter 3 PHOSGENE* By Stanford Moon and Marshall (laics 3.1 INTRODUCTION Phosgkxk (CG) was the principal nonpersistent gas of World War I and was the standard non- persistent gas filling for United States bombs and mortar shell at the outbreak of World War II. Throughout World War II the stocks of CG were maintained at a much higher level than those of AC and CK. which were later standardized as quick- acting nonpersistent agents. When delayed physiolog- ical effects are acceptable, CG has been considered the most economical standard nonpersistent agent for the production of casualties by-attainment of effective dosages in less time than is required to mask or for production of casualties among unpro- tected personnels0 This chapter summarizes primarily the recent ad- vances in the information on the toxicological action of CG. Data on the chemistry and toxicology of diphosgene and carbonyl ehlorofiuorido are also in- cluded. These two agents parallel CG iu toxic action but have not been considered to possess any major advantages over CG as chemical warfare agents. 3.2 PREPARATION AND PROPERTIES 3.2.1 Preparation and Stability CG has been prepared industrially for many years by the direct combination of carbon monoxide and chlorine under the catalytic influence of activated carlxm. The product is stable and long experience has shown that CG can be stored indefinitely in iron and steel containers in (he absence of moisture. 3.2.2 Physical Properties The physical properties of CG which have the most direct bearing on the use of this gas as a war- fare agent are listed below (see Chapter 2 for proper- ties of AC and (T\). Density (liquid) g/ml at 20 C 1.38 Boiling point, C 8.3 Freezing point, C — 104 Heat of vaporization, eal/g 00 Vapor density, relative to air 3.5 CG possesses several advantages over AC and CK in its physical properties. It has a higher liquid den- sity and a lower boiling point than AC or CK. The freezing point of CG is so low that the agent will re- main liquid at any temperature to be encountered in operations. From the standpoint of gas cloud be- havior, the effect of the lower heat of vaporization of CG is offset in part by its greater vapor density. Field (rials show little difference between the travel of CG clouds and that of CK clouds.*1 3.2.3 Detection and Analysis Chemical methods for the detection and analysis of CG are outlined in Chapter 31 of this volume.1* The median detectable concentration by odor is given as 0.006 mg I.2* 3.2.4 Canister Penetration The dosage of CG required for lethal penetration of the more recent models of German, Japanese, or Allied canisters is 500,000 to 1,000.000 mg min m* under conditions favorable for penetration and as much as twice these figures for humidified canisters at, low breathing rates.31 In general, dosages in this range are beyond those which can be obtained over a significant portion of a target area by feasible ex- penditures. Thus both Allied and enemy canisters provide good protection against this agent. 3.3 TOXICOLOGY 3.3.1 Toxicity — CG exerts its lethal action by injury of the lung tissue in contrast to AC and CK, which'are systemic poisons. The rate of detoxification is so low that it comes into consideration only in the study of de- fensive measures against long exposures to trace con- centrations in manufacturing plants. An approach to the problem of the toxicity of CG for man differs from that applied to the systemic poisons. The dif- ferences in the susceptibility of the various species to CG have been attributed 2* to the follow ing factors which govern the extent of injury to the lung tissue: • Based on information available to Division 0 of the National Defense Research Committee [XDKC] as of Decemlier 1, 1945. b See also the Summary Technical Report of Division 10. SECRET 18 18 PHOSGENE TaBI.E 1. /.(Cl)iu’s of CCi I for different species. Species I!xpostire time (min) L(CiU (mg min m3) or suggested value where number of animals is small .1 — anal. cone. \ = nom. cone. Xumlier of animals used Reference Mouse 1 3,450 X 240 9 1 0,300* N 3(K) 9 2 4,700 A ISO 22 3 1,950 X 200 9 10 1,800 N 220 9 10 3,800 A 100 20 20 2,000 X - . 2 SO 9 30 3.4(H) A 160 22 Rat 1 6,500 X 24 9 30 1,400 X 32 17 Guinea pin 1 2,800 X 28 9 — 30 1,300-2,200 X 30 17 Rabbit 30 1,000 X 30 17 Monkey 1 600-1,000 X 13 9 5 625 A 21 39 10 750 A 25 39 30 1,000 — X 14 17 Dog 0.5 8,100 A 28 18 — 1 8,400 A 28 18 - I 7,000 X 9 9 - 3 4,500 A 29 18 5 4,500 X 16 9 5 4,250 A 29 18 20 4,200 X 19 9 Goal 2 4,600 A 14 28 2 6,500 A 72 27 Horse ca. 10 ea. 10,000 A 23 34 * Determination on Jaekwm strain of mice. Other OSKD data are on Car worth male mice. 1. Differences in the amount breathed during ex- posure. — 2. Differences in the depth of inhalation and the size and shape of the upper respiratory tract, leading to a relatively greater absorption of agent in the upper respiratory tracts of the smaller sj»ecies. 3. Minor differences in tissue susceptibility. 4. Differences in resistance to death from the pro- longed anoxia resulting from pulmonary edema, such differences being known to be present even within a given species with variations in nutritional state and general health of the animals. The factors involved in the toxic action of CG are such that the differences in the sensitivities of dif- ferent species to this agent are more difficult to in- terpret than in the study of AC and CK. L(Cl)M measurements for different species are summarized in Table I. The relative />(C<)w’s of CG, AC, and CK arc given in Table 2. The data show (he effect of a depression of respira- tion during short exposures to CG.15 The l-minute L{Ct)m’s are in most cases several times the 10- or 30-minute figures. In the case of the goat it was not possible to determine the L(Ct)hl, for a 30-second ex- posure time since the animals frequently held their breath during the complete period in the presence of high concentrations.27 The depression of respiration in this sjjecies was actually characterized by breath holding and was more marked than the action of CK, which under similar conditions induced depressed respiration but not complete cessation. This reflex respiratory inhibition evoked by CG has also been st udied in detail in the dog.9 At concentrations higher than 7 mg 1 the first inhalation of the phosgenized atmosphere caused total cessation of respiration, with the lungs in (he deflated phase. The duration of this apnea averaged 2fi seconds. For exposure periods of 1 minute the average reduction of lung output was fi‘2 per cent. If the vagus nerves were cut prior to ex- posure, no reflex inhibition of breathing was ol>- tained. In the case of the monkey the /.(( t).vi data give no indication of breath holding during a 1-min- ute exposure (Table 1). The monkey is by far the most sensitive species tested with C G and the lethal concentration (0.6 to 1.0 mg/1) in this case appears to be below the level capable of inducing the reflex SECRET TOXICOLOGY 19 T \ m e 2. Comparison of L(Ct)so*s of CG, AC, and CK. Species Kx|w»sure time (min) UClh,, AC UCtho AC „ Density CG* Urt),.. CG L(Cth« CG X Density AC urty.. ck lAct):.,, CG /.(CfhoCK Density CG Urt)i« CG Density CK Mouse 1 0.26 0.52 1.3 1.5 30 1.6 3.2 4.0 4.6 Hat I 0.24 0.4S 2.0 2.3 30 6.4 7.4 Guinea pig 1 075 1.50 5.4 6 3 Monkey I 1.7 3.4 4.4 5.1 Dor 1 0.10 0.20 0.45 0.52 3 0.22 0.44 0.03 1.1 30 1.4 1.6 Goat 2 0.34 0.68 0.55 0 64 * Calculated for rom|xirisoi\ on a }>cr bomb baaia. 19 action. In the dog little or no apnea was produced by exposures to 0.65 and 1.2 mg 1 of CG.® It was shown following World War 1 19 that in tracheotomized dogs inhaled CG was 60 per cent al>- sorlied during 7b£-, 10-, 15-, and 30-minute expo- sures. Based on theCG absorbed, the LDm was about 0.74 mg kg. This value, when compared with the f lho of 0.95 mg kg for absorbed AC in the dog (see Chapter 2), would indicate that for this species CG is intrinsically more toxic than AC. On the other hand, the approximate 30-minute L(Ct)ao’s of AC and CK for the dog, which involve close to the same volume breathed, indicate that AC is slightly more effective on an LD-M basis than is CG for this species. But it is striking to note in Table 2 that on the basis of the dosages to which dogs are exposed (not the air- sorbed dosage given above) AC is ten times as toxic as CG in short exposure periods (I minute). This result is a function of the manyfold increase in mi- nute volume induced by AC coupled with the depres- sion of respiration produced by CG. These figures illustrate the im|x>rtance of respiratory volume in any attempt to estimate the toxicity of CG for man. With a possible exception in the case of AC in the dog. for longer exposure periods such as 30 minutes CG is more toxic than AC or CK. The L{Ct)iU’s of the cyanide agents increase as a result of the detoxi- fication rate and the differences in respiratory vol- ume decrease in significance as the t irne is lengthened. The variations in sensitivity to CG within a given species are evidenced by the data on mice in Table 1. Two strains of mice were exposed under the same conditions in the same laboratory and gave widely differing L(CV)S0’s. It is not certain whether the sensi- tivity is purely a function of strain. The Jackson mice were tough, scrawny, and extremely active and the Carworth mice were relatively fat and glossy, averaging 2 to 4 g heavier than the Jackson strain. From several sources there is evidence that within a given strain animals that have been deprived of food or water for a period before exposure are more re- sistant to CG.,®*-b-M,‘-" To cite one example. Car- worth mice placed on a restricted diet leading to a weight loss of about 15 per cent were gassed for 10 minutes along with a group of control animals al- lowed to fotwl nrl libitum. Mortality in the restricted group was only 4 '30 compared with 13/29 in the controls.® An abrupt change in environmental tem- perature was found to l>e another factor which af- fected the resistance of mice to CG.® Abnormal post exposure temperatures led to increased sensitivity. Effects of these types, whether they be due to de- hydration, fasting, strain, or temperature, probably account for (he apparent discrepancies among mouse L{CtU determinations from different laboratories (Table 1). The comparison of the L(Cl)i0'n of CG. AC, and CK in Table 2 shows that for short exposures CG is only one-fifth as effective as AC against the dog and is more than three times as effective as AC against the monkey. There is no evidence which permits establishment of the relative L(Ct)so for man. For the calculation of munition expenditures the Allies have employed the value of 3,200 mg min nr1 for CG in comparison with 5.000 for AC and 11,000 for CK. Taking into consideration the relative liquid densi- ties of the three agents, (he toxicity estimates which have been used on a i>er bomb basis are in the order of 1 3/4. It will he noted that the ratios of these values are similar to the ratios given for the monkey in Table 2. An estimate of the relative values of the toxicities of CG and the cyanide agents is only a part of the picture. The relative suitabilities of the three agents SECRET 20 PIIOSC EXE in the field would also be a function of additional im- portant considerations. CG kills only after a delay of a number of hours, whereas AC and CK were stand- ardized as “quick-acting” nonpersistent agents. CG also produces serious casualties in sublethal dosages and individuals may require hospitalization for sev- eral weeks prior to recovery. AC and CK.in general produce no extended disability if the quantity in- haled is less than the lethal dosage. Injurious sub- lethal dosages of CG may be received prior to masking or after masking in the presem-e of high concentrations of CG if hurried adjustment of the facepiece leads to the presence of small leaks. For the production of casualties by attainment of effective dosages in less t ime than is required to mask, the toxicological data point to the working hypothe- sis that CG is the preferable agent if delayed effects are acceptable.*0 This estimate takes into considera- tion both deaths and disablement of troops from sublethal dosages. If the tactical situation requires immediate deaths, the choice lies only with AC and CK. For harassment CG is considered the most satis- factory nonpersistent agent in view of its disabling action in sublethal amounts. Studies have been made of the effects of breathing low concentrations of CG for long periods to deter- mine the health hazards from trace concentrations which might be encountered in manufacturing oper- ations. British investigators exposed animals to a concentration of 0.0044 mg 1 (1 1,000,000) for 5 hours on 5 successive days. Microscopic findings showed that all the animals were affected by CG to a degree likely to give rise in man to serious clinical symptoms.32 The experiments were repeated at a CG concentration of 0.0009 (1 5.000,000). Evidence of slight pulmonary edema and bronchitis was observed even at this low concentration.33 It was concluded (hat at this concentration the limiting level of safety has approximately been reached. It will l>e noted that 0.0009 mg 1 is below the median detectable con- centration by odor measured with the osmoseojx'.2' This does not necessarily mean that dangerous con- centrations of CG may be undetectable by odor. The osmoscojje values are useful for laboratory com- parison of the detectability of different gases. It is known, however, that in free air, where an average low concentration is actually present in instantane- ous peaks and valleys of concentration, a person is aware of the presence of mustard gas. for example, at average concentrat ions much below tlie osmoscope value. 3.3.2 Pathology The basic data on Ibe pathology of phosgene poi- soning were reported following World War I.47 The subject was reviewed in 1943 23 with reference to the observations made since that time, including studies by the Medical Division, Chemical Warfare Service, and the Committee on Medical Research [T'MR] of the Office of Scientific Research and Development [OSRD], The most recent ('MR data on the pa- thology are summarized elsewhere.12' 4l In man and experimental animals the factor initiating the patho- logical changes in the lungs is bronehiolar injury. Classed patients die most frequently in-the second half of the first day in pulmonary edema, with or without peripheral circulatory failure. Residual ef- fects of phosgene poisoning in human subjects have been summarized by the Medical Division.26 3.3.3 Physiological Mechanism anil Therapy CG injures lung tissues by virtue of combination with cell constituents. With the simpler amino acids, for example, the reaction product is an amino acid ureide, 0 = C(NHCHRCOOH)2. Investigations by the Committee on Medical Research have shown that CG is capable of reacting under physiological condi- tions with -NH2, -SH, and Oil groups of amino acids, peptides, and proteins.12'•1314' The reactivity of CG towards proteins produces in enzymes and hormones irreversible inhibitions of their enzymatic and hormonal activities.1314* Experiments with CG containing radioactive carbon 16 have established the fact that a significant percentage of the inhaled agent is bound locally in the lung tissue, paralleling the re- sults on the tissue fixation of radioactive mustard (see Chapters 22 and 23). The early theory that CG might exert its toxic action by virtue of the hydrochloric acid liberated intracellularly on hydrolysis has been shown to be untenable.14* Part of the evidence is the parallelism between the toxic action of ketene (CH2 = C = 0) and CG. Ketene kills animals with the same clinical picture of lung edema and with the same histological injury to (he lungs as is produced by CG. The lACt)-, (As of ketene for different species are of the same order of magnitude as those of CG.1" Ketene, how- ever, produces no mineral acid on hydrolysis. Also hexamethylenetetramine serves as a prophylactic agent against ketene as well as against CG, the pro- phylactic action resulting from competition for the toxic agents between the hexamethylenetetramine SECRET Dl PHOSGENE 21 and the amino or other reactive groups of the cell const ituents.14* The extensive studies on the treatment of CG casu- alties have been reviewed ls'22-24 and summarized.25 Agents such as hexamethylenetetramine are effective if administered prior to exposure but have no prac- tical application to the soldier in the field. The knowledge on the general treatment of pulmonary edema has been extended through studies on the ap- plication of oxygen therapy under positive pressure in the case of (’G casualties. In general no procedures have been found which have therapeutic value spe- cifically effective against phosgene poisoning as dis- tinguished from pulmonary edema of different etiology. 3.1 FA M l \TK)N ()l CG VS V W \R (LAS When delayed physiological effects are acceptable, CG has been considered the most economical stand- ard nonpersistent agent for the production of casual- ties _ by attainment of effective dosages in less time than is required to mask or for production of casual- ties among unprotected personnel.30 3.5 DI PHOSGENE Trichloromethyl chloroformate, diphosgene (DP), was used as a combat gas by the Germans in World \\ ar I. The fact that I)P could be filled into ordinary HF shell by the simple expedient of cementing the joints was of importance to the Germans.45 With the development of special shell and bombs for plant filling with CG the interest in DP became less. For the task of production of lethal dosages in 30 seconds or 1 minute the more volatile CG was preferable. Interest in DP was temporarily renewed in World War II when it was shown that DP could be rapidly converted catalytically to CG in shell and a study was made of whether plant fillingvvitli DP followed by conversion to CG in the munition would be a useful procedure. The advantages of this method did not appear to offset the increased production costs for DP and the modifications of standard munitions re- quired in some instances. The more recent data on the chemistry and toxicology of DP are included in the following brief summary of work on this subject. DP boils at 127.5 128 C at atmospheric pressure and freezes at —57 C.46 Its specific gravity is 1.945 at 20 C.2 and its vapor density relative to that of air is 6.9. DP reacts with aniline in aqueous solutions or in benzene to give quantitative yields of carbanilide. This reaction may he used for defect ion and analy- sis.4" DP was first prepared l»y Hentschel in 1887 42 by the chlorination of methyl formate in direct sun light. Methyl chloroformate was employed as a starting material hy the Germans in World War f. Thorough studies hy the French 40-4’41 indicated that the photochlorination of methyl formate proceeds with increasing difficulty as the number of hydrogen atoms replaced increases, and that the last stage is slow unless adequate illumination rich in the shorter wavelengths is used. It is possible to conduct the later stages of (he chlorination at higher tempera- tures, although the French workers recommended a temperature not exceeding 90 C. since decomposition of the product to (G becomes appreciable at higher temperatures. The direct chlorination of liquid methyl formate leads to considerable charring and may become vio- lent. To avoid undue loss of the volatile methyl formate, very efficient cooling of the by-product gas must l>e maintained. To minimize these difficulties, chlorination can lx* carried out in dilute solution, diphosgene itself being a convenient solvent.2 Using a laboratory batch process employing this solvent and internal illumination from low-pressure dis- charge tubes, methyl formate can be converted into excellent quality DP in yields of 92 per cent on the ester and 90 per cent on the chlorine.2 In (his process it is necessary to carry out the final stages of chlorin- ation at S0(' because of the low velocity of the hist chlorination step. This temperature appears to be about the maximum which can be used without pro- hibitive losses of DP through conversion to CG.2 A continuous process based on the above method has been operated on a pilot plant scale.7 A total of about 1,000 lb of DP was produced in SO per cent yield on the ester, 55 per cent on chlorine, by this pilot plant, which consisted essentially of two 5-1 re- actors in series, each reactor being illuminated by a well containing a 200-w projection lamp. Liquid methyl formate and gaseous chlorine were introduced into DP in the first reactor, which was operated at 50 55 C and received 80 per cent of the chlorine in- put. The overflow from this reactor was led to (he second reactor, which was maintained at 75-80 C and which received 20 percent of the chlorine input. The product overflowed from the second reactor and was cooled and scrubbed with dry air to remove IK 'I and Cl,. All stages of the reaction are exothermic, and the capacity of the unit appeared to be limited SECRET 22 PHOSGENE by the rate at which heat could l>e dissipated from the first reactor, in which most of the chlorination occurred. Hough estimates indicate that 121 kcal must be removed per mole of methyl formate used. A program to determine design factors for a large- scale plant was not completed.7 A continuous two-stage process has been developed by the i ’anadiansfor the pnotochlorination of methyl formate to DP. It is similar to the above process in that DP is used as a diluent but different from it in that chlorine concentrations approaching saturation are used, better provision for the dissipa- tion of heat during the initial stages is provided, and the charge is allowed to remain considerably longer in the second stage. In agreement with earlier work, the reaction was found to have a high temperature coefficient and to be promoted by fight of wavelength shorter than 5300 A. The violet mercury fine of com- mercial “blue neon” discharge tubes proved to lie highly effective as a light source. The process gives DP in 85 per cent yield based on methyl formate, and has been operated on a pilot plant scale to yield 2 11:> per hour of good quality DP in a yield of 87.5 per cent on methyl formate and 08.4 per cent on chlorine.3* _ Commercial chlorine contains some substance, possibly oxygen, which markedly inhibits the chlo- rination of methyl formate.2 36 iH Venting of the chlorine cylinders used removes this impurity, and satisfactory chlorination results thereafter, although the loss of chlorine may lie considerable.** Decomposition of DP to C’G can be effected by heat. The reaction is accelerated by activated carbon and some metallic halides.4"41 43 Work carried out in this country in 1941 indicated that in addition to the known thermal and contact-catalyzed decompo- sition of DP to CG. this decomposition was remark- ably susceptible to catalysis by organic bases.1 •4 A number of liquid amines, e.g., pyridine, produce a violent and almost instantaneous conversion. The suggestion was made that bombs could be charged with DP and stored as such, the catalytic conversion being made to proceed rapidly by the use of pyridine as catalyst after (he release of (he bomb. A prelimi- nary model of a device by which this might be ac- complished was designed and constructed.1 However, the production of a bomb incorporating such a de- vice' presents several difficulties, and the advantages were not considered sufficient to merit further re- search and development. Solid amines, such as Michler’s ketone, produce a slower decomposition, the rate of which can he in part controlled by con- trolling the proportion of catalyst, its rate of disso- lution. and the temperature. It was suggested that gas shell could he charged with DP, which is simpler than filling with low-boiling CG, a pellet of catalyst containing Michler’s ketone added, and the shell closed, after which conversion to CG would take place. Laboratory experiments indicated that this conversion would l>e substantially quantitative and complete within an hour. In attempting to work out the details incident to the use of solid catalysts for the conversion of DP to CG in shell, it was soon discovered that tempera- ture effects play a critical role in the course'of the re- action. If the catalyst pellet disintegrates rapidly, the strongly exothermic dissociation of DP quickly warms the liquid to a certain critical temperature, after which the decomposition proceeds very rapidly and to completion. If, however, the pellet disinte- grates slowly, or the temperature and heat capacity of the bomb are such that this temperature is not reached, the conversion is never complete and is usu- ally not over 20 per cent. Small-scale experiments in glass vessels are thus valueless for the prediction of the behavior of DP in actual munitions on the addi- tion of dissociation catalysts. Experiments made with a metal-bomb whose weigh( and void closely approximated those of a 105-mm shell indicate that a catalyst pellet composed of 2 parts Michler’s ketone, 5 parts p-dichlorobenzene, 1 part paraffin wax, 4 parts red lead when used in an amount equal to 1 to 1.5 parts per thousand of DP will produce satisfactory conversion. Under these conditions, maximum temperatures of 42-43 C, starting with the bomb and contents at 25 C. are reached, and momentary pressure surges of 250-275 psi, dropping to 30-35 psi after 24 hours, are observed.4 The func- tion of the p-dichlorobenzene and the paraffin in the catalyst is to prevent too rapid disintegration of the pellet; that of the red lead is to increase the density so that the pellet will not float on the DP. — The phenomena which occur after the addition of such a pellet to a sample of pure DP are striking. Michler’s ketone forms with ( G a deep blue addition compound. On addition of the pellet a faint layer of blue appears on the surface of the DP, and the red pellet turns almost black. After several minutes the pellet begins to disintegrate rapidly, and the liquid rapidly becomes colored an intense blue. During this period there is a slight evolution of gas, which sud- denlv becomes violent and continues until the SECRET <; A RBO> VI, CMLOROFI.LOKIDE 23 liquid has almost completely boiled away. The start of the violent ebullition, which can l»e determined on duplicate samples within 20 seconds, begins when the sample reaches 34 C and under controlled con- ditions can l>e used as a criterion of purity for Dp i.- Mssi The presence of hydrogen chloride in the DP increases its “ebullition time” considerably. When liquid DP is decomposed in this way in an open vessel, insufficient beat is produced to over- come the latent beat of vaporization of the CG pro- duced, and, in spite of an initial rise in temperature, the temperature of the liquid eventually falls below the boiling point of CG. If pyridine in amounts equal to 10 11 pel’ cent of the weight of DP is used to cata- lyze the decomposition, calorimetric experiments in- dicate that the heat produced is approximately equiv- alent to that required to overcome the latent heat of the phosgene prodnc«,d.*Hb Several rough experiments indicated that it may Ik- passible to produce instan- taneous clouds without cooling the immediate at- mosphere by the simultaneous mixing and bursting of DP plus 10 1 i percent pyridine/1* In view of the important contribution of the local inversion pro- duced by the cooling effect, which aids in the attain- ment of high dosages w ith nonpersistent agents, it »s doubtful whether the production of a gas cloud with no fall in temperature would have advantages at air temperatures above the boiling point of CG. In aqueous solution DP yields 2 moles of CG/* Much of the work on the mechanism of action of CG (see Section 3.3.3) has for convenience Ix-eri car- ried out by the addition of DP to aqueous solutions or suspensions of cell constituents. The pathology of DP poisoning is the same as that of CG/7 The data on the toxicity of DP indicate that the IACt)-.u s do not differ markedly from those for CG -* but the de- terminations are not adequate for a close differen- tiation. For the mouse a recent determination (140 animals) gives a 10-minute L(Ct);,n of 3,600 mg min in1 for DP- to Ik1 compared with values of 1.800 and of 3,800 for CG (Table 1). 3.6 CARBONYL CH LOROFLl OKI I )F The fluorine analog of CG, carbonyl fluoride, is of a low order of toxicity compared with CG 37 and has not been considered as a potential war gas. The com- pound in which only one of the chlorines is replaced by fluorine, however, has merited special study.' Carbonyl eblorofluoride has been prepared in yields approximating 25 per rent by beat ins ('G with a tenfold excess of anhydrous hydrogen fluoride at 125-145 C in a pressure vessel. Small amounts of antimony pentafluoride promote the reaction and with this catalyst lower temperatures can In- used. The products of the reaction are hydrogen chloride, carbonyl fluoride, and carbonyl chlorofluoride, the last of which can be separated by distillation.4 ft has also l>een prepares! in low yield by passing CG over calcium fluoride at temperatures from 150 to 325 C and at pressures from 7 to 79 cm of mercury. From the amount of uncondensed gas produced if appears that the ratio of carbonyl chlorofluoride to carbonyl fluoride produced is greater than 1 at temperatures below 270 C. The maximum conversion (0 per cent) was obtained at 200 C." Carbonyl chlorofluoride islTgas boiling at — 42 C. Its melting point is — 138 ( V’ Its odor closely resem- bles that of phosgene, various observers living in dis- agreement as to whether the two are distinguishable by odor.*-* It reacts readily with solid sodium hy- droxide or with soda lime, shows no tendency to etch glass4 and undergoes no loss in toxicity on storage in copper for 13 months at ordinary temperatures,8 although storage in a tank lined with polymerized shellac led to decomposition/ The protect ion against carbonyl eblorofluoride afforded by whetlerite charcoal is of the same order as that against CG/ For most species the toxicity of carbonyl eh loro- fluoride is very close to that of CG. For the mouse the 10-mimite l,{Ct)M (220 animals) is 1,200 mg min ms * compared with 1,800 for CG in determina- tions on the same strain of mice by the same labora- tory, For the rat. guinea pig, and dog approximate determinations give 10-minute values of <2,700, <2,700, and <6.000 s to be compared with CG values of 1.400 (30 minutes), 1,300-2,200 (30 min- utes), and 4.200 (20 minutes). CG is apparently much more toxic than carbonyl chlorofluoride for the rabbit, for which the L{Cl):,„ of CG is <1.000 (30 minutes) anil that of the fluorine compound is ap- proximately 7,000. Carbonyl chlorofluoride yields a symptomatology and pathology corresponding to that following phosgene poisoning/ Any advantage which carbonyl eblorofluoride might have over CG would rest upon its lower boil- ing point, which might make it more effective in the production of crash concentrations’. However, at the present rime the procedures for its preparation are not satisfactory for large-scale work/ c Additional compounds related to CG but of minor interest arc included in Chapter It. SECRET Chapter 1 msi LFUR DECA FLUOR IDE* By Birifsey Rcnshnir and Marshall Gales t.l INTRODUCTION DisixrrR DKCAKbroHiDE (S-Fl0) is a dense, highly volatile liquid whose comparatively (xlorless and nonirritating vapor is a lung-injurant similar in mode of action to phosgene; for some species it is at least as toxic as phosgene. It thus presents attractive features as a potential non persistent agent, and its synthesis has been carefully investigated by Cana- dian researchers, by XDRC Division 10. and by the Chemical Warfare Service. The synthetic methods developed to date require the use of elemental flu- orine and give maximum yields of only about 30 per cent. The consequent difficulty and expense of its production in quantity at the present time have pre- elm led serious consideration of its adoption as a standard agent. The stability and other physical and chemical properties of disulfur deeafluoride appear well suited to its effective dispersal from chemical munitions. The gas mask canister can be expected to protect against it approximately as well as against phosgene. Consequently, if its large-scale production were lo become feasible in the future, its merits as a non- persistent agent would be determined in large part by its toxicological properties, in particular by its toxic- ity for man, and by whatever advantages would accrue to a non persistent agent which, at least in the pure state and at moderate concentrations, is odor- less and nonirritating. At present there are available no data upon which might be based an estimate of the incapacitating and lethal doses for man. If, as is the ease for several of the smaller mammalian species, disulfur deeafluoride should prove to be as toxic as or somewhat more toxic than phosgene, it would have some advantages over currently standardized nonpersistent agents. If. as appears to hold fur tlie monkey, it should prove to be only one-tenth as toxic as phosgene, its large-scale use in warfare could hardly merit consideration. 1.2 SYNTHKSIS \M) PHom.KTIKS'1 t.2.1 Synthesis Disulfur deeafluoride was discovered in 1934 by Denbigh am! W hy t!aw-(5ray,’’ It occurs as ;t hy- produet formet! in small (|Uantities (1 per cent) dur- ing the synthesis of sulfur hexafluoride from sulfur and elemental fluorine. In spite of exienshe studies during World War II, its preparation in quantity re- mains difficult .and exjiensive;12 ■’5■*’ it has not yet been possible lo prepare it except hy the use of ele- mental fluorine' or to increase the yield based on fluorine altove 30 to 34 per cent in spite of a thorough investigation of the reaction ettriditions.12 9-s -l|i 35,1 g The proeedures which have given the Ix'st yields uti- lize the reaction of elemental fluorine, either pure or diluted with 5 to 30 parts of nitrogen, with solid roll sulfur, either pure or diluted with potassium or sodium fluoride. Adequate cooling of the reaction vessel is essential and either (he fluorine or the sulfur must lx? diluted; oxygen and moisture must be ex- eluded. Failure has attended attempts to convert sulfur hexafluoride, the principal product of the re- action, to disulfur deeafluoride by a variety of meth- ods which include its passage through an electric are and its reaction with hydrogen sulfide or with molten liotassium.*** 1.2.2 Physical and Chemical Properties Disulfur deeafluoride is a colorless, mobile liquid boiling at 30.1C; it solidifies at low temperatures and melts at — 53 C.1 Its liquid density is 2.00 at 20 C, its vapor density approximately nine times (hat of air.'--3Sf The vapor pressure, which has been precisely determined as a function of temperature, is 235 mm at 0C and 075 mm at 25 C."’12 It is virtually insoluble in water (<0.005 per cent by '• For a more romplHc review the reader is referred to the .Summary Technical Report of XDRC Division 10, r The production of elemental fluorine has lieen the subject of intensive investigation by several XDWC groups. It now appears possible to produce it in quantity at relatively low cost. The reader may consult the Summary Technical Re- port of N DRC Division 10 for a review of this work. • bused on information available to Division 9 of (lie Na- tional Defense Research Committee [XDRC] as of August I, 194r>. SFCRFT SYNTHESIS AM) PROPERTIES 25 weight), in 0.9 per cent sodium chloride, and in 0.19/ phosphate buffer at pH 7.4. but soluble in various common organic solvents;21517 it is somewhat soluble in olive oil, with which it reacts to a limited extent.2 The thermodynamic properties of disulfur tleca- Huoride have been evaluated using both thermo- chemical data and statistical considerations, and from these the gas-phase equilibria of a number of possible reactions of the sulfur fluorides have been calculated.-6 v,u 31 Disulfur decafluoridc does not react with such agents as strong alkalis or acids, phosphorus pent- oxide, or common solvents; fluorine has no action upon it at temperatures up to its decomposition point (100 210 U), but chlorine attacks it to yield a slightly volatile liquid.12 33,1 On prolonged contact with aqueous solutions, disulfur decafluoridc appears to produce acid, but this conclusion must be regarded as tentative because the tests were made with a commerieal preparation which may have contained small quantities of hy- drolyzable impurities.1517 On activated carbon, disulfur decafluoridc is cata- lytically decomposed to sulfur hexafluoride and sulfur tetrafluoride, the latter presumably decomposing further to sulfur difluoride and sulfur hexafluoride; nearly one-half of the weight of the original material appeal's as the hexafluoride.2 12 Thermal decompo- sition, which is slow at 200 C but rapid at 300 C, appeal's to involve similar reactions and gives sulfur hexafluoride as the principal product.2 12 The ability of disulfur decafluoridc to act as an oxidizing agent is an important property which may lie involved in its physiological mechanism of action (see the following section) and which has been uti- lized in the development of methods for its detection and analysis. 1.2.3 Detection and Analysis The comparative inertness of disulfur decafluoridc limits the number of methods available for detection and analysis. Its oxidizing ability and its decompo- sition on charcoal have been utilized. A number of easily oxidizable substances, includ- ing several oxidation-reduction indicators, have been examined as detectors for disulfur deeafluoride.7-37 Of these p-phenylenediamine and Os (/>-di methyl- aminophenvDmcthane appear to be the most suita- ble. A quantitative colorimetric procedure using the former has been developed for the analysis of cham- ber air in toxicitv determinations.19 Disulfur decafluoridc may be detected in air in concentrations as low as 10 gg 1 by passing the air through charcoal and then filtering a fluoride ion indicator (e.g.. thorium or zirconium alizarin sul- fonate) through the charcoal. For use in detector tubes, however, charcoal decomposition followed by recognition of fluoride ion by standard methods is less satisfactory than the use of oxidation-reduction indicators.7" The reaction of disulfur decafluoridc with either sodium or potassium iodide to produce iodine can lie used for both qualitative and quantitative analy- ses.7-24 M“ The reaction is suitable for determina- tions of concentration in gassing chambers and for analyses of canister effluents. Acetone bubblers, pre- ceded by alkali scrubbers to remove hydrolyzable sulfur fluorides, are customarily employed.- The detection and analysis of disulfur decafluoridc are reviewer! in more detail in Chapters 3 t and 37. 1.2.1 Stability . Disulfur decafluoridc reacts slightly with iron at oo C, but the reaction is sharply terminated, possibly by the formation of a protective coating. There is no reason to believe that the material cannot be stored satisfactorily in mild steel containers.2*12 That disulfur decafluoridc possesses sufficient sta- bility to be dispersed without decomposition from explosive munitions is suggested by the results of the one test for which data are available.23 A 75-mm shell was exploded in a large chamber; subsequent chemical analyses and toxicological bioassays of the chamber air demonstrated that at least 82 per cent of the dispersed agent was recoverable as such. 1.2.3 Canister Penetration The protection afforded by the modern gas mask canister against disulfur decafluoridc is reviewed in detail d and the conclusion reached that it is com- parable to that afforded against phosgene. As an ex- ample, no disulfur decafluoridc appeared in the efflu- ent for more than 130 minutes when the United States M 10AI canister was tested in the Intermittent Mow Canister Testing Apparatus K2 against 5 to 6 mg 1 of the agent.21 However, small amounts of sulfur hexafluoride, which is odorless and relatively innocuous,5" 1-1 l6“ penetrate charcoals almost im- mediately, and subsequently odorous and irritating substances (sulfur dioxide, hydrogen fluoride, or J St* (he Summary Technical Hoporl ofXDHC Division 10. SIX II FT 26 nisi I FI R DECAFLUOU1DE lli iony I fluoride) appear in the effluent in concentra- tions which progressively increase to attain intol- erable and potentially dangerous levels before lexicologically significant amounts of disulfur deca- fiuoride K are passed.3’"-2*’**-** Addition of soda lime removes these decomposition "products and thereby materially increases the dosage against which char- coal affords useful protection.*-" 24 4.3 TOXICOLOGY Oisulfur deeaHuoride is to l>e viewed as a non- persistent/ lung-injurant producing casualties qualita- tively -imilar in character and time course1 to those caused by the inhalation of phosgene. At flu1 concen- trations which have been tested, it doe’s not produce lacrimal ion or skin irritation.15 17 22 In marked con- trast with phosgene and cyanogen chloride, for whit’h the median detectable concentrations are less than 0.01 mg I,20 pure samples are odorless and non- irritating to the respiratory tract when breathed briefly at concentrations of at least 0.2 mg I." Other observers have ascribed to presumably impure preparations an odor similar to that of sulfur di- oxide,151' 29 39 and on several occasions the sniffing of a commercially prepared sample, which had a sul- furotis odor, was followed by the development of mild nasal irritation of several hours’ duration.1 1517 It remains to l>e determined whether impurities were responsible for (he odor and irritation described in the latter observations, and whether or not the pure material is odorless at concentrations higher than those which have lieen tested. t.3.1 Toxicity for Animals .Most of the available toxicity data for disulfur deeaHuoride are summarized in Table d. Included for the sake of comparison are the most nearly compa- rable data for phosgene (see also Chapter 3). By and large, the data do not substantiate a currently prev- alent impression that disulfur deeaHuoride is dis- tinctly (he more toxic. Although this impression is probably correct for the mouse, rat, and goat, partic- ularly at short exposure times, the opposite relation holds for the guinea pig and monkey. The dis- crepancy is striking in the ease of the monkey; the data, unfortunately limited in number, suggest that for this species disulfur decalluoride is only about one-tenth as toxic as phosgene. Information bearing upon the effect of exposure time on the L(Ct)iu of disulfur deeaHuoride and upon its rate of detoxification in the body is scanty. The data (Table 1) indicate that the /,(Cl)50 of disulfur decafluoride does not vary significantly with exposure time over the range 1 to 30 minutes. Tints, this rela- tively odorless compound does not exhibit the in- creased L(Ct);,„'s at short (I-minute) exposures which characterize phosgene and which have been associated with an inhibition of respiration due to sensory irritation (see Chapter 3), and it does not appear to lie detoxified at a rate comparable to that for hydrogen cyanide (see Chapter 2). On the other hand, the results of a limited number of experiments suggest that animals can tolerate two to four expo- sures at 24-hour intervals to vapor dosages each of which is of nearly lethal magnitude.n A striking feature of toxicity data for disulfur deca- lluoride is the narrow range of concentration be- tween that causing no deaths and that producing 100 per cent mortality; with phosgene, on the other hand, the dose-mortality curve is spread widely on the dose axis.32 As a result the curves for disulfur deeaHuoride and phosgene may cross, the latter com- pound killing more animals at relatively low dosages, the former, more at higher dosages. In spite of the steepness of the dose-mortality curve, however, disulfur deeaHuoride in siiblethal doses does produce pulmonary pathological changes which in man might be of clinical and military significance.®0 t.3.2 Symptomatology Animals utilized for toxicity determinations have, in general, been exposed to dosages of less than three times the and to concentrations of less than n mg 1. Under these conditions no obvious changes in respiration or other indications of sensory irrita- tion are apparent 11 22 32 and the animals appear normal upon return to their cages;" practically all fatalities occur between 1 hour and 2 days after ex- posure. the majority occurring between 3 and 20 hours." 22 2• There seems to be a fairly definite in- verse correlation between dosage and time for death.’1 ■' In a typical ease culminating in death after 3 to 6 hours, the animal liegins to appear quiet and depressed after I to 2 hours, its respiration be- ■ Thus, an Interesting situation, which might or might not have military significance, could conceivably arise during ex- posures to high dosages of disulfur deeafluoride; Masked troops upon inhaling the irritating hut not dangerous sub- stances in the canister effluent might suppose their protection inadequate; ii|K>ti removing their masks, they would 1h- ex- posed to the much less odorous and irritating but vastly more toxic disulfur deeafluoride. SKCR KT Taiii.k 1. Toxicity of disulfur decafluoride. Included ire 1 be most nearly coni|>arable data for phosgene. nless otherwise noted, the /.((’/),,,’s are based on nominal concent rations and 10- or 15-day observation jieriocU. Disulfur decafluoride Phosgene Exposure Estimated X umber Estimated Xuml)er time Uriho of Time of Refer- «o)» of Time of Refer- Species (min) (mg min m5' animals death once (mg min nr' animals death enc<» Mouse 1 2,200 UK) 16b 3,450 240 4 hr 3 days* 14 It) 1,960 UK) 16b 1 „S(X) 220 14 1 1,400 2,000 40 11 - 10 1,310 150 I 48 hr 11 30 1,000-1,400 10 11 10 1,000 160 <24 hr 22 3,650) 109 83' ( in 2 .lavs .. 21 10 1,000 30 11 20 hr 27 30 1,620* 160 32 1,980* 175 32 Rat 1 2.300 s 30b 6,500 24 s‘40 hr 1 1 10 2,000 3,000 30 1-16 hr 11 20 1,8004* 16 IS Guinea 1 2,800 28 3 hr 2davs 14 P'K 10 0,000 i- 20 1 hr-9davs 11 10 1,000 6,000 15 4 hr 2 days 27 l.fXXlt 27 Rabbit 1 7,500' + S 3 hr-4 days 1 1 to 6,000 ± 10 5 IS hr 11 10 4,000 6,(XX) 6 7-21 hr 27 13,000f 27 v.*at 1 3,:«xi 7 < 1 day 14 10 4,500 + 10 50 min-12 hr 11 10 <4,000 4 6 8 hr 27 3,5(X)t 27 Don 1 5,(XX) 16 Kill 7,000 9 < 1 dav 1 1 10 1,000-6,000 10 6 20 hr II 20 1,200 20 6 20 hr 14 Goat 10 1,000-6,000 9 6 hr-15 days 27 8,5001 27 Monkey 1 600-1,000 13 3 15 hr 14 (Rhesus) 10 9.0001 10 4-51 hr I6b 1,200 10 14, 36 * A few diod after 3 tr» 10 days. f Analytical eoneen trillion. J Two-tlay observation |>eri«d. TOXICOLOGY 27 coming shallow and rapid; after an additional I to 2 hours, respiration becomes labored; cyanosis then soon sets in, to be .quickly followed by asphyxia! convulsions and death accompanied by a flow of colorless foamy fluid from the nose and mouth." In an experiment in which mice were exposed to a high concentration (45 mg I), symptoms and death occurred with striking rapidity. The animals immediately flattened out on their bellies and Iwgan gasping for breath. All died within 6 to 11 minutes after the beginning of the 10-minute exposure," 31h Pathology Disulfur decafluoride acts primarily as a pul- monary irritant, producing an anoxic death due to massive, fulminating pulmonary edema and hy- peremia. It differs from phosgene and chlorine in that it does not injure the columnar epithelium of the bronchi and bronchioles, (lie pathological changes being confined to the alveoli and the pulmonary con- nective tissue, and being more prominent in the hilar than in the peripheral portions of the lung." Some observers have also reported acute vesicular em- physema and, in a large proportion of animals, marked pleural effusion;17 inasmuch as the pleura show no evidence of acute inflammation, it has been suggested that the pleural fluid arises from the lym-~ phatics draining the edematous lungs.27 In some in- stances marked edema of the mediastinum, accom- panied by distention of the mediastinal lymphatics has been noted.27 In comparison with phosgene, disulfur decafluoride produces in dogs a more fulminating lung edema and hemoconcentration but less marked blood pressure or other circulatory changes.19 The hematocrit may rise slowly for some hours and (hen leap to high SECRET 28 DISI r.KI K WEC.VFLVOHIDE values. Plasma protein concentration arises progres- sively. There is no initial bradycardia such as that which occurs with phosgene, and. in contrast to poisoning by the latter agent, the pulse rate does not Iwcome greatly accelerated in the late stage's. Arterial and venous pressures are little altered. Respiration is increased more gradually than by phosgene but eventually rapid and shallow breathing is established. Arterial and venous oxygen concentrations fall slowly at first, and then rapidly to values incom- patible with life. No ext rapulmonary pathological changes other than congestion and fatty degenerative changes in the liver and kidney - effects presumably secondary to lung edema — have’been reported as sequelae of inhalation of the vapor:"19'27 35* in particular, the eyes, nasopharynx, trachea, and lymphoid tissue appeal- normal. Observations on the corneal circula- tion of gassed dogs revealed hemoconcen (ration but not the other changes (cell clumping, vessel spasm, and local transudation of fluid) which have been ob- served iTUphosgene poisoning.19 Dogs w ith one bron- chus-plugged during gassing and the other plugged subsequently to prevent edema fluid from pouring into the protected lung sometimes survived expo- sures to w hat would have been let hat dosages for normal dogs, and regularly lived longer than bilater- ally gassed animals; the protected lung remained grossly and microscopically normal.15 4.3.4 Physiological Mechanism The physiological mechanism of action of disulfur decafluoride has not been systematically investigated and definitive conclusions cannot be drawn at the present time. It is evident from the findings review ed above that the significant pathological changes con- sequent upon inhalation of the vapor are confined to the pulmonary tissues. That other tissues are sus- ceptible, however, and would be affected if sufficient quantifies of the agent reached them, is indicated by the'‘action on hemoglobin and by the lethal effects of infra peritonea I injections (see below). In evaluating the significance of the following iso- lated findings bearing on mechanism, and in planning future studies, the following facts set forth in the chemical section above may be recalled; (1) disulfur decafluoride acts as an oxidizing agent, and (2) its carbon-catalyzed decomposition in the presence of moisture involves the formation of sulfur hexafluoride and other substances among which may be. thionyl fluoride, hydrogen fluoride, and sulfur dioxide. As in the ease of water and salt solutions, addition with shaking of a plant-run sample of disulfur deca- fluoride to blood plasma resulted in (he slow pro- duction of acid; the experiment has not been repeated with the purified compound.1517 In the presence of the same plant-run sample, bromthymol blue in 0.01.1/ phosphate buffer at pH 7.4 failed irreversibly in about 1 day; the rate of fading was accelerated in the presence of dissolved sodium chloride and in more alkaline solutions.1517 It is rot known whether (his reaction was an oxidation of the indicator or merely an acceleration of the tendency of (nominated sulfonphthaleins in aqueous solution to form, the corresponding carbinols. Upon shaking whole blood with the plant-run di- sulfur decafluoride, the hemoglobin slowly darkened with the evolution of gas bubbles. Addition of an acetone or ether solution of the agent produced these effects immediately. Solutions of oxyhemoglobin ob- tained by hemolyzing and filtering blood behaved as did whole blood.1511 At pH 7.4 the absorption spec- trum of (he altered pigment resembled that of met- hemoglobin in general features but not in all details; over the wavelength band 370 to 700 mg it had no resemblance to the spectrum of oxyhemoglobin treated with sodium fluoride. Rapid local damage and death with hemoconcen- t rut ion follow tin* intraixuitoneal injection of disulfur decafluoride as liquid or vapor.1*-*8 In one experi- ment with a rat injected with the gas. death occurred w ith ext reme hemoconcentrat Ion after a latency of 2 hours; at autopsy the lungs were clear but the peritoneal cavity contained 3 ec of fluid; injection of this fluid into a second rat had no detectable effects.38 A single analysis of the pleural fluid accumulating after lethal gassing with disulfur decafluoride re- vealed a fluorine content of 2 mg 100 ml.35c Traces of sulfur hexafluoride were obtained from the reactions of disulfur decafluoride with olive oil and with pieces of excised rat lung;12 the reactions were sharply limited in extent. l.3..» Prophylaxis and Therapy The results of a single exploratory study’9 indicate that prophylactic inhalation of magnesium carbonate dusts and intramuscular injection of magnesium sulfate solutions may have limited value in prolong- ing and saving the lives of mice gassed with disulfur decafluoride. In(ra|>eritoneal injection of calcium chloride following exposure appeared to be harmful SECRET 29 Table 2. Physical projjerties of disulfur deeafluoride and of currently standardized non ipersistent agents. Property Disulfur deeafluoride Phosgene Hydrogen cyanide Cyanogen chloride Liquid density (g ml at 25 C) 2.0 1 .36 0.68 1.2 Vapor density (air = 1) 8.1 3.4 0.93 2.0 Boiling point, C 30.1 8.3 26 12.fi Melting point, (’ -53 -lot -13.4 — 7 Latent heal of evaporation, eal 'g 25 fit) 210 135 Vapor pressure, mm Ilg at 25 C 075 1,400 740 1,200 at -20 C 87 230 88 ISO ' Volatility, mg 1 at 25 C 9,000 1.060 at -20 G 1,400 i,460 145 680 EVALUATION AS V WAR OAS and the inhalation of calcium carbonate dusts prior to exposure was without clear effect. Prophylactic injections of hexamethylenetetramine, a chemical specific for phosgenet were without effect, and 2,3- dimercaptopropanol (HAL), a similar specific for arsenic and cadmium, was detrimental. Prophylactic intramuscular injections of pitressin did not alter the course of the poisoning; exercise immediately subse- quent to exposure was not harmful. t,t EVALUATION \S A WAR CAS Because of the difficulty and expense of manufac- ture on a large scale at the present time, it has not been practicable to make disulfur deeafluoride for use in World War II. The available information does not permit a clear decision as to whether it would possess greater general utility than currently stand- ardized agents if its production and use in quantity were to become feasible. At the present time it should be evaluated in comparison with phosgene, the standard agent to which it is most similar in physical and toxicological properties. In terms of current con- cepts of chemical warfare, the tentative conclusion seems justified that its use on the battlefield would not demonstrate it to be markedly auperior to phos- gene as a casualty-producing agent and might reveal it to be definitely inferior. The physical properties of disulfur deeafluoride are well suited to its dis|»ersion in high concent rations as a nonpersislent agent (Table 2). Moreover its high liquid density would permit significantly greater amounts to be carried in any given munition than is possible with phosgene, cyanogen chloride, or hydro- gen cyanide. Ho far as is known, its stability would suffice to permit its storage in currently available chemical munitions and its dispersal from them with- out destruction. Its insolubility in water and resist- ance to hydrolysis would give it an advantage over phosgene for use under (hose conditions of terrain and meteorology which permit clouds of “non- persistent” gases to exist for many minutes. The protection afforded by modern gas mask can- isters against disulfur deeafluoride, like that afforded against phosgene, is so good that with reasonable munitions expenditures one could not hope to set up dosages sufficiently huge to break the canister, ex- cept under very special circumstances. One would therefore expect that the bulk of the casualties to lie realized from its use would be among individuals who, because of lack of time, unawareness of the presence of the poison, or other reasons, would be exposed unmasked or imperfectly masked. Conse- quently, attention focuses on toxicological properties. To whatever the extent that relative lack of odor and of irritating properties are desirable in a non- persistent agent, disulfur deeafluoride (at least in the pure state) has an advantage over phosgene and cyanogen chloride. However, the critical toxicologi- cal data, namely the incapacitating and lethal dos- ages for man, are not available. If, as holds true for some animal species, it is as toxic or more toxic than phosgene, its use (assuming availability) in place of this agent would merit consideration. If, on the other hand, it is only one-tenth as toxic as phosgene (as appears to lie the case for the Rhesus monkey), its utility as an offensive agent would hardly merit its production for use in warfare. SECRKT Chapter 5 xMlSTAKD GAS AM) OTHER SI MTR MUSTARDS" By Marshall dales and Stanford Moore 5.1 INTRODl CTION A major cart of the activities of Division 9 of the National Defense Research Committee [NDRC] centered around the defensive and offen- sive problems presented by mustard gas and closely relates! vesicant agents. Chapters in Barts III, IV, and V deal in detail with the mechanism of action of agents in this class and means for protection and de- tection. The present chapter deals mainly with the methods for preparation of mustard gas and its analogs, a tabulation of (he compounds which have been preparer!, and the basic toxicological measure- ments on the more important members of the series Mustard gas (H) was the principal battle gas of the last year of World War I II was the agent which was manufactured and stocked in the largest tou- nagp for possible use in World War II. The more re- cent investigations on the subject of H have great ly extended the knowledge of the mechanism of action of the agent, the information on its behavior in mu- nitions under field conditions,113 and the means for protection against the vesicant action of the vapor and liquid forms of the agent. Two relatively nonvolatile vesicant agents have been studied in detail for possible use in mixtures with II. They are l,2-fri«(0-chloroethyllhio)ethane (Q) and 6ts(/J-chloroethylthioethyl) ether (T). These two agents have a higher vesicanev in contact with bare skin and greater persistence on terrain than H. but because of their low vajmr pressure they lack the ability to produce casualties by vapor action. HQ and HT mixtures have been prepared on a small scale. For special purposes the nitrogen mus- tards would have some uses is<-e Chapter b). Among the hundreds of com|K>unds that have been‘studied in the sulfur mustard series since II was first used in 1917. no agent has been found to have a more advan- tageous combination of toxicological, chemical, and physical properties than H. 3.1’ PRODUCTION PROCESSES FOR II. no. \ND IIT 5.2.1 General Methods The original laboratory methods of Guthrie27" and of V, Meyer®7* for the preparation of H1’ were both put to use on an industrial scale during World War 1. The Meyer process, applied to large-scale produc- tion by the German Dye Trust, consisted essentially of the chlorination of thiodiglycol hy hydrochloric acid. The thiodiglycol required was prepared from ethylene chlorohydrin hy the action of sodium sul- fide.-7* The English had begun erection of a plant for the manufacture of mustard gas from thiodiglycol and thionyl chloride in 1918, but apparently the plant did not come into production before the end of World War I. Other chlorinating agents, such as phosphorus trichloride and thionyl chloride, have been used, and other processes, notably the action of hydrogen sulfide on ethylene oxide, are now avail- aide for the preparation of thiodiglycol. The Guthrie process involves the interaction of ethylene and sulfur chlorides and has been formu- lated as follows: 2CH?- CH* + SCb —► S«'Il,(’Il,ri)2 (1) 2CTD--CH. f S,.(T. —► S(Cll..riI2CI)2 + S (2) although the course of the second reaction is more complex than required by this equation. Various modifications of this process provided all the mustard gas used by the Allies in World War 1, the three principal processes being; 1, The French process (Cattelain process). In (his process a 10 percent solution of sulfur dichloride in carbon tetrachloride was saturated with ethylene, and the dilute solution of mustard gas so obtained was stripped to a concentration of about 85 per cent. 2. The 00 C process. Dry ethylene was led into sulfur monochloride maintained at 55-00 C. Under these conditions about one-half of the excess sulfur * Based on information available to XDKC Division it as of Jan. 1, UMO. *’ Both Iiesprel7. and Ilirhe*7*- appear to have prepared bi>(a-ehloroethyl) sulfide Itefore Guthrie. SECRET PROD! CTIOV PROCESSES FOR H, IUt>, \Ml UT 31 remained in solution as polysulfides, whereas the other half separated on standing or on treatment with moist ammonia. 3. The Ldnnfttein process. In this process, also known as (he “30 C process," pure ethylene was led into a mixture of sulfur monoehloride and crude mustard gas maintained between 30 3-1 C. Under these conditions the precipitation of sulfur was mini- mized and charging operations were facilitated. This process had been investigated by Pope and his co- workers27i and was successfully used on a plant scale by the British firm of Levinstein, Ltd., and by the United Slates Chemical Warfare Service [CWSJ. The thiodiglycol method for the preparation of pure H was not used by the United States or Great Britain during World War II, although successful laboratory procedures for carrying out the "synthesis by both batch and continuous processes have been worked out.8 27 40 Apparently the Germans, as in 1917 18, relied principally on this method for their mustard stocks, although the thiodiglycol was ob- tained by the action of hydrogen sulfide, synthesized eatalytieally from hydrogen and sulfur vapor, on ethylene oxide, and the hydrogen chloride was ob- tained by burning hydrogen and chlorine.172"-24* Although the thiodiglycol process has uot l>een us

CVS VXD OTIiKR SILKL R MLSTVROS The process developed was considered suitable for scaling up in existing I. plants.17" Pure Q can be prepared by the photochemical ad- dition of 1.2-ethanedithiol to vinyl chloride, as de- scribed in Section 5.2.7. 5.2.2 Sulfur Dichloride Processes Ethylene and sulfur dichloride react according to the equation: SCI. + 2(.’;H4 —■> S(C.HiCl). and processes employing sulfur dichloride are not attended by the sulfur precipitation or the poly- sulfide formation characteristic of sulfur mono- chloride processes, which are now considered obsolete by the British. The reaction is much more rapid than that lie tween ethylene and sulfur monochloride, and adequate cooling is required. Sulfur dichloride- exists at ordinary temperatures as an equilibrium mixture with sulfur monochloride and chlorine, approximately in the proportion 85 10 5. The equilibrium is mobile at ordinary tem- peratures and it is consequently not possible to ob- tain pure sulfur dichloride by simple fractionation at atmospheric pressures, although at low temper- atures the rate of attainment of equilibrium is slow enough for fractionation at reduced pressures to be effective. The discovery by British investigators that the presence of phosphorus pentachloride markedly decreases the rate of attainment of equilibrium, how- ever, has allowed I he preparation in quantity of pure sulfur dichloride (99.5 per cent) by fractionation of the equilibrium mixture at atmospheric pressure in glass apparatus. Metals in contact with the dichlo- ride promote dissociation to a variable degree. Brass, one of the least active, when used in still construc- tion, allows production of sulfur dichloride of 98 98.5 per cent purity.2® The* type of sulfur dichloride used affords a basis for classification of the various British II processes. Thus; 1. HS (obsolete). In this process a 1 6 mixture of crude (equilibrium mixture) sulfur dichloride and carbon tetrachloride were treated with ethylene at 25 C. The process was continuous and the product was obtained by stripping off the carbon tetrachlo- ride under reduced pressure to a content of about 15 percent, this being sufficient to reduce the melting point of the mixture to less than 5 C. 2. HM and HB. HS produced by a modification of the procedure just described is snipped under 70- 100 min to a carbon tetrachloride content below I per cent and is then diluted with 7 10 per cent monochlorobenzene or benzene to form 11,M or HR. These have better pressure stability than HS. chieflv because of the thermal decomposition of unstable factors (principally trichloroniustard) during strip- ping. 3. I1M I), Him, and IK 'D, These processes utilize pure sulfur dichloride, prepared by continuous two- stage distillation of sulfur dichloride stabilized by phosphorus pentachloride. In the plant, the first distillation stage is carried out in a cupronickel col- umn to separate chlorine and sulfur dichloride from sulfur monochloride, and the second stage, which separates sulfur dichloride from chlorine, is carried out in glass. The continuous HMD reaction fakes place in nickel reactors with only enough solvent (monochlorobenzene) present to give the required freezing point to the product, which requires no stripping. The reaction has been run with benzene and carbon tetrachloride as solvents, giving IIBD and IK’I). A similar process for two-stage distillation of sta- bilized sulfur dichloride using brass columns has been developed, and sulfur dichloride produced in this way and containing 1 1.5 per cent sulfur monochlo- ride gives HMD B and HBD B when used in the II processes. Further identifying letters signify the type* of reactor used (nickel or cast iron).-'® Heating for short periods (A£-l hour) at 165-180 C imparts greatly improved pressure stability to HMD and HBD.-® Apparently the Germans had also built plants for the continuous production of sulfur dichloride mustard.17-* 3.2..1 Sulfur Monochloride Processes 1. South African (I)ESA) proves*. In this proc- ess controlled precipitation of the excess sulfur is achieved by using ethylene saturated with alcohol vapor. The gas is passed into a batch of sulfur mono- chloride held at 55 ( ’ and is recirculated after wash- ing and drying by brine cooling. A batch of 1.200 lb of sulfur monochloride requires 12 hours for reaction. After removal of the precipitated sulfur in a settler the H layer is stripped of a low-boiling fraction and then distilled at approximately 35 mm from a mild steel pot and condensed in lead. The pressure stabil- ity of the product is good.-® 2. The C1F.S Levinstein process. This process has been standard with the American Chemical Warfare SKC’HET PRODUCTION PROCESSES FOR II, HQ. AND 1IT Service since World War 1. and extensive studies on the stabilization, storage stability, purification, com- position. and behavior in the field of Levinstein mustard have been carried out in this country. Sulfur monochloride is added to a seed charge of II in mild steel reactors to give a concentration of about 25 per cent. Fthylene is then passed in and further monochloride is added to maintain its con- centration between 18 and 22 per cent. On comple- tion of the addition, ethylene is passed in until the sulfur monochloride content falls to less than 0.05 per cent. The excess sulfur is largely retained in solution in the form of polysulfides. Nine hours are required to complete a 6-ton batch. Brine cooling is necessary during the early part of the reaction to maintain the temperature at 85 ('. >.2.t The Composition of Levinstein H Most of the early work on Levinstein H or on sul- fur monochloride II, since many of the early experi- ments were carried out on “60 C mustard.” was concerned with accounting for the excess sulfur re- quired by the equation — 2(\.IL + Sat'l» —>- (CUT1-.H4)2S + s Among the theories proposed to explain the failure of this sulfur to precipitate completely were the following: 1. The excess sulfur is present in the mustard in colloidal solution “pseudo-solution,” 261 272 In sup- port of this, the fact that (he sulfur in 60 C II could be largely precipitated by heating to 100 (’ without changing the freezing point of the H was brought forward. Likewise, the precipitation of sulfur from 80 (’ II on dilution with alcohol appeared to support this hypothesis. 2. The sulfur is present as (he dispersed phase of a two-phase liquid-liquid dispersion.*** The difference in properties of various Levinstein samples was held to lie due to differences in the degree of dispersion. The precipitation of sulfur by the addition of ether without change in freezing point was attributed to the removal of the dispersed phase. Attempts to pre- pan- stable dispersions, however, failed. 3. The sulfur is present in the form of a loose com- pound with mustard itself.2*-1 As evidence for this point of view the insolubility of sulfur in H, together with the fact fhat sulfur dissolved in sulfur mono- chloride II by heating precipitates quantitatively on cooling, was presented. The sulfur cannot be com- bined with fefs(/3-chloroelhyl) sulfide itself, how- over, unless (lie complex is dissociable at room temperature.272 Other early evidence for compound formation (not necessarily with II itself) was the production of sulfuric acid by oxidation of industrial II2fifi and of sulfuric acid and a chlorine-containing alkane sulfonic acid by oxidation of a distillation residue of the approximate composition (CR'llsClLbS,, from 00 C H.2*4 A solid substance of the composition (t’K'HjC'HjJsSi which could be oxidized to sulfuric acid and 2-chIoroethanesulfonic acid was isolated by fractional distillation of 60 C 11 and it was shown that its formation was favored by lower temperature reaction of ethylene and sulfur monochloride.2"2 4. The sulfur is present partly in combination, parth as a colloidal dispersion or solution.2** This hypothesis was based upon the fact that only a part of the excess sulfur can be precipitated by heating, freezing, or treatment with moist ammonia. In a series of distillation studies on Levinstein mustard of current manufacture carried out at Kdge- wood Arsenal,100 the distillate obtained by the CWS specification assay for Levinstein H was subjected to fractional distillation and found to consist of pure bis(0-c h 1 o n>ethy 1) sulfide (78 percent) and a residue (20 per cent). Fractionation of the residue gave about 85 percent of fn#(/J-chlorocthyl) sulfide, 28 per cent of 6/s(/3-chloroethyl) disulfide (IIS2), and 20 per cent of residue, with about 15 per cent loss during the distillat ion. Tn order to minimize changes in composition oc- curring during distillation by ordinary methods, molecular distillation was resorted to. A preliminary distillation gave three fractions, the first of which was subsequently fractionated into three relatively volatile components, possibly chlorinated hydro- carbons. which contained no sulfur. The next two consisted essentially of bi*(J3-chloroethy 1) sulfide. The residue was rich in sulfur, containing 4.5 molec- ular proportions of sulfur to every I of chlorine. Repealed passage of crude Levinstein H through the molecular still at successively higher tempera- tures gave 6*’s(/3-chloroeth\4) sulfide fractions progres- sively richer in sulfur. This increase was attributed to the presence of increasing amounts of HS-» in the distillate. The unstable residue from these dis- tillations could be separated into two components, an acetone-insoluble fraction having a composition corresponding approximately to (C1(TI2CH2)jS12, and an acetone-soluble fraction of composition cor- responding to t(’l(’II3(TI-)..S4.;,. The acetone-insolu- SIX’RET 34 MUSTARD GAS AND OTI1KR SULFUR MUSTARDS Lie residue deposL xi sulfur when allowed to stand. Treatment with gaseous ammonia caused rapid pre- cipitation of sulfur, and the material remaining was found to have a composition corresponding closely to (CICH2CHj)jS&. Only slightly different results were obtained in similar distillation studies on Levinstein of 1037 manufacture and on Levinstein of 1018 manufacture. From these results and those of other investigators it was postulated that the chief impurities in Levin- stein mustard were polysulfides of variable compo- sition. The transient existence of a polysulfide of any definite composition was attributed to the. probable ability of the- CUsSSCH*—linkage easily to gain or lose sulfur atoms. It did not appear possible to distill Levinstein II without altering its composition. A review of the evidence available at the time of this work indicated that Levinstein mustard was composed of; 1. Cases, noncondensable at — 78 C; probably ethylene. 2. Chlorinated hydrocarbons; 1 per cent or less. 3. /3-Chloroethyl j8-chlorovinyl sulfide .as such or as trichlorodiethyl sulfide. 4. ’hloroethyl) sulfide; 60 to 70 per cent. 5. bis(J3~i 'hloroethyl) disulfide; free and as poly- sulfides. 6. Diethylene disulfide, partly free as monomer (dithiane) and polymer, and partly potential. 7. Sulfur, free and as Jjolysulfide The results of some experiments on methanol ex- traction of Levinstein H also led to the conclusion that polysulfides are present.14* Cold methanol ex- traction of a sample of American Levinstein H left an insoluble residue of low vesicancy with the ap- proximate composition (C1CH2CH2)2S9. The soluble portion, after stripping of solvent and removal of most of the II by freezing, was again fractionated by methanol extraction, yielding an insoluble fraction of the approximate composition (C1CII2CII2)2S* and a soluble fraction whose composition approached (ClCHjCIIt)jS* but which contained about 10 per cent II. In view of the now known lability of the higher j8-chloroethyl polysulfides, however, it may- be unsafe to assume that material which has been stripped of methanol by distillation is unaltered. In 1943 a theory of the formation and composition of Levinstein H which explained many of the avail- able data was proposed by workers in the CWS. The salient points of this theory were the following; "* 1. Sulfur monoehloride was supposed to be an equilibrium mixture of at least the following com- ponents; sulfur in solution, sulfur dichloride, and two isomeric forms of sulfur monoehloride. C1SSCI and CljS —► S. 2. Ethylene may react with all of these except sulfur. a. 2C'Hj =rn, + sci* —> cicii,cii2scii2cii.( ’i b. 2CH,=--CI1I + (’IjS—>S—> (ririi.,rii,.).s—> s c. 2CH2 CH, + cissci cicilcilsscilcilci Since the compound (CK 'ILCII2)2S—> S had not been isolated, it was postulated that it could have only a transient existence, decomposing into bis{0- chloroethyl) sulfide and sulfur. This “nascent sulfur” could then he taken up by the disulfide. CICH.CII.- SSCHjCHjCI. to form higher polysulfides. The num- ber of sulfur atoms in the polysulfide would depend upon the proportions in which the two forms of the disulfide were present. For example, if the ratio were three to one. the resulting mixture would contain 62 per cent by weight of fus(d-chloroethvl) sulfide, and the polysulfide would be a pentasulfide. -> S + (C1CH2CH2)2S2 —> 3(C1CH2CH2)2S + (CICH2CH2),S-, It was suggested that the ratio of CI»S—> S to CISSCI might be changed by altering conditions so as to increase the amount of C12S ——>■ S, which in turn would increase the amount of hfs(j8-chloroefhyl) sulfide and sulfur formed. This would account for tlie higher yield of 6n»(/3-chloroethyI) sulfide and for the precipitation of sulfur actually observed when the reaction is run at 60 C. Available analytical data on the sulfur-chlorine ratio in Levinstein mustard and on the relation be- tween freezing point and fi/.s(/3-chIoroelhyl) sulfide content of Levinstein mustard were used to extend and support the hypothesis. It was postulated that the principal impurities in Levinstein had the structures C1CH*CH,S-SCH.CH,C1. s s s s t t CICTIaC'IIjS- SCW'H-CI, I \ s s SECRET PKODICTIO'N PROCESSES FOR II, HQ. -VXD IIT 35 S S t t CICH-CILS—SCHsCTIjCI, I \ S 8 I s | etc. Excess sulfur could be stripped from impurities of this type without altering the mole fraction of im- purity and consequently the melting point should remain constant, as is actually observed. The bi.sijj- chloroethyl) sulfide content of Levinstein II as de- termined by distillation and as calculated from its freezing point was shown to be very nearly the same on the assumption that the molecular weight of the impurity is 319, corresponding to f»f«(j9-chloroethyl) hexasulftde (HS,;). If the equation 2ciT,=rHs + s2n. —> n(,H2rn2S(.'H2rii2n + s actually represents the Levinstein reaction, then it would appear that one atom of sulfur per molecule of II or about 16.8 per cent of the total product should lie precipitated. Actually it is possible to in- duce only about half of this amount of sulfur to pre- cipitate from Levinstein mustard. Analyses of fresh Levinstein H show that iLcontains approximately 37 per cent chlorine and 33 per cent sulfur, giving a sulfur-chlorine ratio of 11. It was postulated by the CWS workers that the 30 per cent impurity in Levinstein mustard was HS6, leading to a chlorine and sulfur content in the mixture of 37.9 per cent and 32.2 i>er cent, respectively. During aging or stripping HSe was assumed to lose sulfur until the more stable level HS.) was reached. S S t t CICIU 'HsS—SCilsCHsCl —> \ \ s s s s t t nC’H2C,H2S—SCHt(H?f’l + 2S This would represent a loss of sulfur amounting to 6 percent of the total weight of the product. It would have no effect on the mole fraction or on the freezing point but would raise the weight-fraction of bitv(/3- chloroethyl) sulfide to alsinl 0.75, with about 25 per cent of polysulfides still present. This mixture would contain 27.7 percent sulfur ami 10.4 percent chlo- rine, whirl) is in close agreement with the observed values for aged or stripped Levinstein mustard.*4 This hypothesis, which has become known as the Keid-Macy hypothesis, as to the formation of and thc> composition of Levinstein mustard, was a sig- nificant forward step, but it was based on scanty experimental evidence and although substantially correct as an overall view, required considerable modification in the light of later experimental data.121 The NI)R(’ Division 9 group which undertook a study of Levinstein mustard in May 1943 36 intro- duced a valuable technique for the quantitative re- moval of 6iA‘(£-chloroethyl) sulfide from Levinstein II without altering the composition of the polysulfide fraction. This removal was achieved merely by ex- haustive hydrolysis,35 and its success depends upon the fact that the polysulfides in Levinstein are stable toward water at room temperature, whereas bis(j3- chlqroethyl) sulfide is readily hydrolyzed. The prog- ress of the hydrolysis is followed by titration of the hydrogen or chloride ion produced. When the rate of hydrolysis becomes negligibly small, the non- hyd roly zed residue amounts to about 30 percent by weight of the original product. The composition of the dark, oily residues from different samples of Levinstein mustard is not the same but varies be- tween values corresponding to (CICTTjCHjJjSs (TIS«) and (C1CH2(’H2)2S9 (HS,). These residues deposit sulfur slowly. When treated with Cellosolve, in which the polysulfides are fairly soluble but in which sulfur has very limited solubility, sulfur is observed to separate in the form of fine crystals, and a small amount of dark gum remains insoluble. This gum has the composition of a high molecular weight polysulfide such as (ClCHtCHOaSi*. It invariably deposits sulfur within a few days and takes on (he appearance of crystalline sulfur. The soluble polysulfides can be recovered by wash- ing the Cellosolve solution with water until the Cello- solve is'completely removed, drying the resulting oil in ether solution, and removing the ether. A clear amber oil, containing slightly less sulfur and slightly more chlorine than a compound having the compo- sition of 6f.s(/3-chloroethyl) pentasulfide, results. A more complete characterization of these poly- sulfides was made possible by their synthesis. Oils having the characteristics of higher polysulfides were prepared by heating the known HS3, with excess sul- fur under moderate conditions, by allowing it to stand in the presence of sulfur monochloride, or by SECRET 36 Ml STAR!) <;VS \ M) OTIIKR SI I.Fl R MI STAKES heating it with methyl tetra- or pentasulfides. When these oils are stripped of their excess sulfur by the Cellosolve treatment or by moist ammonia, (hey re- semble the Cellusolve-st ripped Levinstein residues of composition corresponding to IIS, in appearance, odor, refractive index, density, polarographic be- havior. and analysis. HS., was also prepared by heat- ing MS* with excess sulfur under more stringent conditions, although sulfur monochloride and methyl polysulfides had no sulfuri/ing action on this disul- fide. All of the synthetic jjolysnltides, if allowed to stand without being stripped with Cellosolve, de- posit sulfur gradually until the composition of the residual oil approaches that of the pentasulfide. The solubility of sulfur in IIS, proved to lx* about 7 |x*r cent or 0.6 gram-atom so it is to be expected that sulfur would no longer be precipitated when the sul- fur content of the polysulfide had decreasedjo 5.6 atomsT unless some polysulfide solvent which does not dissolve* sulfur appreciably, e.g., Cellosolve, were added. The pentasulfide exhibits relatively high stability compared with higher |x>lysu Hides and was obtained in a state of high purity after methods had been worked out to remove various impurities always present in the nonhydrolyzable Levinstein residues. Attempts to distill HS, or HS,-mustard mixtures by ordinary vacuum distillation result in degradation of HS, to HS* and ITS., Ix»th of which are readily dis- tillable. When HS, is subjected to steam-distillation, HS* is obtained in the distillate, and the nondistillable residue consists of polysulfides higher than HS,. Autosulfurization of HS, appears to take place under these conditions, the removal of the volatile HS* forcing the reaction to proceed. Higher poly sulfides prepared in this way are similar to the |x»lysulfidcs isolated from fresh Levinstein mustard in that they slowly deposit sulfur over a |x*riod of weeks. This deposition of sulfur can be accelerated by the usual stripping methods to reproduce HS,. By steam-dis- tilling the impure polysulfide from Levinstein mus- tard for a short time to remove volatile impurities such as the disulfide and subsequently stripping the higher polysulfide residues with Cellosolve, pure HS, can be obtained as a light amber, slightly vis- cous liquid with a much less pronounced odor than that of the unpurified material "The molecular formula, CiH»Cl*S,. has lx-cn verified by ultimate analysis. Although it is stable to moist ammonia in the ah- sence of a solvent. IIS;, is stripped to IIS* by moist ammonia in the presence of a solvent such as ether or Cellosolve. .Metallic mercury also strips sulfur from US,, producing MS* and US., although the rate at which the stripping proceeds is much less in going from US* to MS,- than in converting MS, to HS*.35 The stability of MS, and IIS* and the degradation of IIS,, to yield MS* indicate that these compounds may be structurally related. HS; is known to be a linear disulfide. One sulfur atom may be added with difficulty to 11S2 but following this two additional atoms enter with comparative ease to give IIS,. Degradation of this MS,gives IIS*. These phenomena can be accounted for most easily by assuming that a sulfur atom enters the 11S2 molecule to produce a linear trisulfide of the structure t’K’Ild'IDSSSt Hj- CHT'l and that this molecule is then sulfurized to produce IIS, of the structure S t nci!fcHasssc,iw,n,n I . _ s The higher polysulfides could be accounted for by structures of the type S t S t cich..ch,sssch,cilci - \ s I s I etc. which would be in accord with the natural chain- forming tendency of sulfur atoms, and the limited stability of the higher polysulfides.35 During a study of the composition of British Levinstein H. British investigators arrived at similar conclusions.11" When treated with cold acetone their sample, prepared at Sutton Oak. produced a milky suspension which gradually deposited crystalline sulfur. Only a small amount of sulfur was produced, apparently that in solution in the Levinstein mus- tard. Attempts to distill the higher boiling fractions of Levinstein failed because of the decomposition of a “labile polysulfide” which could lx* removed by SECRET PRODUCTION I'UOCKSSES FOR H, IIQ, AM) IIT 37 preliminary refluxing of the higher boiling fractions with acetone. After this period of refluxing, the pre- cipitated sulfur was removed by filtration. The re- sulting material was found to l>e distillable under reduced pressure without decomposition to give I1S2 and HSj, which were compared with synthetic samples. Examination of the labile polysulfide obtained as a residue from the preliminary low-temperature dis- tillation of Levinstein mustard revealed it to be a yellow, viscous oil. nearly insoluble in alcohol, pe- troleum ether, and acetone, but soluble in benzene, chloroform, and carbon disulfide. Similar material could be obtained as a residue by extracting Levin- stein mustard with methanol. The polysulfide could lx> broken down partially to “2.2'-dichlorodiethyl trisulfide and sulfur . . . by heating at about 100 C for a few hours. . . This change could also be brought about slowly but completely by heating the labile polysulfide under reflux in the presence of acetone, since one of the products of this breakdown is soluble in acetone and the other, sulfur, is not. Synthesis of the labile polysulfide was accom- plished by heating the trisulfide with 3 gram-atoms of sulfur at 110 C for several hours. After extraction of the product with methanol to remove unchanged trisulfide and separation by filtration from a small amount of unreacted sulfur, an oil resembling the original labile polysulfide remained. The trisulfide could lx* obtained by distillation of the methyl alcohol extracts of Levinstein mustard without the formation of much sulfur, indicating that some trisulfide exists uncombined in the original mixture. The British investigators suggested that the labile polysulfide probably contains compounds of the type riCILCILS etc. s->s I nCHsCHjS -HX s or perhaps, since mono- and disulfides do not form polysulfides when treated in the same manner, struc- tures with the additional sulfur atoms branching from the central S of HS3. also appears to be a true level of stability, since 1LS., from different sources is stripped to this level on refluxing in acetone, and USa on heating with sulfur in boiling acetone for several days undergoes sulfurization to a product whoso composition ap- proaclics that of HS*.** A numhor of loss direct linos of evidence point to higher poly sulfides as tho principal impurities in Levinstein H. Thus, in a series of studies on fractional melting of Levinstein M samples, the densities and refractive indices of a number of fractions as functions of 11 content were recorded.21 If the densities and refrac- tive indices of various Levinstein H fractions are plotted against their H contents, straight lines are obtained, which can be extrapolated to give the cor- responding values for the impurity treated as a single component. From the equations of these two lines it is possible to eliminate the H content, giving an ex- pression relating density and refractive index. Kx- perimentally determined--values for the refractive indices and densities of synthetic_samples of 11S2, HS3, and HSj, all fell on or very close to this curve, but not so far out as the extrapolated values Tor the impurity, which were also on the curve. The indica- tions were that the impurity was a fn«(/3-ehloroethyI) polysuTfide containing six or seven sulfur atoms. Two independent investigations upon the compo- sition of solvent extracts and steam distillates of f evinstein II using cryoscopic methods have been reported.88247 In each, measurements of melting point and of average molecular weight, determined cryoscopically, of the samples have allowed calcu- lation of the mole fraction of the impurity and thus its average molecular weight. In one investigation 267 the average molecular weights of (he impurities in several ext racts were 311, 291, and 250. correspond- ing roughly to HS«, IISo, and 1LS4. In the other,84 the impurities in crude Levinstein were found to have an average molecular weight corresponding approxi- mately to HSj, whereas (he average molecular weight of the impurities in Levinstein stripped with moist ammonia or completely extracted with pentane corresponded approximately to HP,,. The results with steam-distilled material are divergent, indicating in one case 247 that low molecular weight impurities are present in sufficient quantities to bring the average molecular weight of the mixture below that of pure H. whereas in the other case 84 significant amounts of HSj are indicated. Molecular distillations carried out on three samples of Levinstein H at temperatures not exceeding 30 (' also indicate that the residues remaining after such distillations, during which the sample is not likely to have altered, are composed of polysulfides of coin- SECRET 38 MUSTARD DAS AND OTHER SULFUR MUSTARDS Table 1 .■'* Mixtures of II and IIS*. Mix.arcs i uf 11 and IIS x which have the composition of the mixture Mixt ures of II and I IS., one molecule of II plus one atom of sulfur produced by stripping Weight (per cent) Mole Mole Weight Caled. strippahlc Weight Chlorine Sulfur Mixture* ratio (per cent) (per cent) fpt in C sulfur Mixture* (per cent) (per cent) (per cent) I. II 3 75 62.5 5.6 0 11 62.5 37.2 33.5 US, 1 25 37.5 IIS, 37.5 2. II 4 SO 66.8 7.2 3.4 II 69.0 38,5 31 2 US, 1 20 33,2 MS, 31.0 3 11 5 S3 3 611.5 8.5 5.5 11 73.6 39.4 29.6 1IS7 1 16.7 30.5 US, 26.4 4. 11 6 ?*(/8-chloroethyl) sulfide and polysul- fides of varying composition and stability and of the general formula, (C1CH2CH*)jSx. Part of the excess sulfur is used in forming the stable linear bisulfide skeleton and this sulfur cannot be removed by ordi- nary methods of stripping. The pentasulfide repre- sents another stable level. Aged Levinstein mustard or Levinstein mustard stripped by the usual methods involving moist ammonia, consists essentially of a mixture of 6is(/J-chloroethyl) sulfide and HS5. Con- sideration of the amount of sulfur formed by the stripping process, the amount of pentasulfide which can be isolated from Levinstein, and the freezing point of fresh Levinstein leads to the conclusion that the polysulfide in freshly prepared Levinstein mus- tard may have an average composition varying from that of the hexasulfide to that of the decasulfide. Usually the polysulfide has a composition corre- sponding approximately to a heptasulfide. The com- position of the polysulfide as well as its concentration in fresh Levinstein mustard depends upon the con- ditions under which the reaction was carried out, especially upon the temperature and upon the rate of agitation, amount of seed charge, and rate of addi- tion of ethylene. Very likely many other factors, such as previous history of the sulfur monochloride, also have important effects. If there is no sulfur precipitated during the re- action, there must he a definite relationship between the composition of the polysulfide and its concen- tration, since one sulfur atom is produced each time a molecule of f»i«(/?-chloroethyl) sulfide is formed and this sulfur must nearly all he present as poly- sulfides. Thus the higher the sulfur content of the polysulfide, the lower need be its mole concentration. Table 1 illustrates this relationship. The sulfur and chlorine contents of all the mixtures before stripping are 113.5 per cent and 37.1 per cent , respectively, as required to correspond to one atom of sulfur per molecule of 6is(0-chloroethyl) sulfide with one atom of sulfur. The table lists other properties, based on theory, which the original mixtures should have. By making the assumption that Il-HSj mixtures are produced by stripping it is possible to tabulate the final content by weight of each constituent and the sulfur and chlorine content of the resulting mixtures. The theoretical values which appear in the table are in excellent agreement with the vast quantity of experimental data on Levinstein mustard compiled at Kdgewood Arsenal and other Chemical Warfare Service laboratories. Many of these data are sum- marize! 1 elsewhere.*6111 SECRET PRODUCTION PROCESSES FOR II, HQ, AND HT 39 6is(3-Chloroethyl) disulfide was formerly thought to be present in Levinstein 11 in amounts as high as 6 per cent 100 but methods which do not result in the degradation of higher polysulfides indicate a much smaller percentage for this component.34 82 Analysis of the stripped nonhydrolyzable residue from Levin- stein H (composed of 11S2 and HS5) and of the steam distillate from this (composed of I IS. and IIS*) by comparison of their refractive indices with curves prepared from known mixtures indicate 1 to 1,8 per cent of 118. in crude Levinstein H,*5 which is in good agreement with a value obtained by analysis of a fraction obtained by low-temperature molecular distillation of Levinstein II.®2 In addition to the higher polysulfides, several other impurities are usually present in significant amounts in Levinstein H. Of these, one of the most important is 1,2,2'-trichlorodiethyl sulfide (“trichloro II”). This material is formed by the chlorinating action of sulfur chlorides, particularly in processes where higher temperatures are encountered. The pressure instability of both Levinstein H and British sulfur dichloride mustards has been ascribed to the ready loss of hydrogen chloride from this and similar substances.21-2*5 The product, of dehydrohalogena- tion, 3-chloroethyl y3'-chlorovinyl sulfide (CKCVS), is thus also an impurity. Evidence for the presence of trichloro H in crude Levinstein H is mostly indirect but can l»e summa- rized as follows:21 1. Levinstein H contains an acidic impurity or an acid-forming impurity which is usually expressed as HCI, but which cannot be removed by air blowing as can HCU. 2. Vacuum distillation of Levinstein H is accom- panied by a weight loss, at least a part of which is Ht’l. The main impurity in the distillate appears to be /3-chloroethyl /3'-chlorovinyl sulfide. 8. Experiments on fractional melting of crude Levinstein H indicate that an impurity appearing in the first (lowest melting) fractions also appears in the distillate of such fractions. The does not indicate what the impurity is, but the inference is that 3-chloroethyl 3'-chlorovinyl sulfide is present in the distillates, whereas its precursor, trichloro H, is pres- ent in the original material. Semiquantitative esti- mates indicate that it may make up as much as 6 per cent of the crude H.21 Both trichloro II and /J-chloroethyl 3'-chlorovinyl sulfide have been synthesized, the latter by an un- ambiguous method making use of the addition of 3-chloroethyIsulfenyl chloride to acetylene as well as by dehydrochlorination of (richloro II.35 In addition to the above impurities, Levinstein H contains varying small amounts of a number of other substances. Among these may lie mentioned small amounts of volatile materials, including methane.57 hydrogen chloride,57 diethyl ether,57 chlo- rinated hydrocarbons, possibly 2-chlorobutane or ethylene chloride,2135 and sulfur. Levinstein H which lias been prepared or stored in iron always contains dissolved iron in greatly varying amounts, depending upon the length and conditions of storage and the conditions of the original reaction. One of the actions of hexamine as a stabilizer is to remove this iron.*2 The storage of Levinstein mustard in the presence of iron at 65 0 leads to the formation of an iron- containing polymer. This polymer has lieen shown to be a sulfonium salt of bis(£-ch 1 oroethy 1) sulfide, dithiane. and ferrous chloride,10 arising by the fol- lowing mechanism: Ferrous sulfide is formed by the action of poly- sulfide on iron, Fe + HS, —►FeS + HSlx. „ and the ferrous sulfide transforms mustard into di- thiane. FeS -f riCH2( ’HjSCH.CH2C1 —► CH2C'11j / \ S S + FeClj \ / CHjC’IIj This combines with mustard and the ferrous chloride to form the polymer 35 (’ll ATI. / \ j-S S -f j-fCKTlATIjIiS -(- jf/2FeCl*->- V / _ ■ \ / C’H2CHj " cr + ch2ch2 \ S S—(’H2CH?SCH2CH2 \ / i CH*CHj FeCb jT Levinstein H which has been heated or which has undergone storage at elevated temperatures also contains small amounts of Q, dithiane, and ethylene chloride, formed by the following series of reaction:31 SECRET ML START) (TVS AM) OTHER SI I.HR Ml’ST ARDS 2Cl('II..rH2—S- CHiCHsTl s+~(rn2cH2ci)2 + rr —►ric,H.riis-n ! C'H« +(CK,HtC,IIi S—CH*)* ■ 1 C’1I2 I S—(.’IIzCHjC'l ((ifHjCiijS-rH,),—> cii. -nu / _ \ nriitC’H*—sf s + ci —> \. / CH*—CHj C’Hj—<’HS / \ CJCHit’lWl + S S niH'H; The presence of (2 in stored I>evin.stein was inde- jiendently indicated by a series of toxicological ob- servations on the increased vesicanev of samples of H stored at fio C and above. 60h 1 1 3,2.r. The Mechanism of Formation of Levinstein H Any mechanism which is proposed to account for The fdrinat)<»n of Levinstein mustard must explain 35 1. The formation of />/.s(/3-chloroethyl) sulfide. 2. The formation of polysulfides derived from the linear US* skeleton. 3. The absence of more than traces of the disul- fide. 4. The absence of more than traces of free sulfur. A mechanism which fulfills these conditions has been developed. There is chemical and physical evidence 35 to sup- port the assumption that sulfur monochloride dis- proportion tes. to a slight extent at least, to give sulfur dichloride and sulfur tritadichloride according to equation (3). 2S2CT 2 + S,C'l2 (3) It is beyond the scope of this review to discuss this evidence, but, if this equilibrium exists, then it is possible for all three materials to react with ethylene according to equations (4), (5), and (6). + S( t2 —> rirHT'HsSriLrii.n (i) 2CjH 4 + SjC 1, —> C *K Tlyt' H*SSC ’IU' H,( ’I (5) 2('2hi + s.n, -> nrii2rii2ssscii2(’ii2n (6) Equation (<») would apply equally well if higher polythio sulfur chlorides such as S4C'l>, etc., were formed. The disulfide (HS2) produced according to equa- tion (5) has been shown to react readily with sulfur monochloride to produce chlo- ride and sulfur tritadichloride according to equa- tion (7).* ('!( I l,t 'HjSHt II,CH2('I + 3S..fb —> 2(,ICH2('ll2S('| d- 2Ss('!» (7) These products can react with ethylene rapidly to yield fn«(/3-chloroethyl) sulfide, and I IS,, or, in the reaction used to demonstrate the presence of the sulfenyl chloride, with cyclohexene to yield /J-ehloro- ethyl /L-cldorocycIohexyl sulfide. ( l(Tl -t HoSCI + C’sH4 —► ('K 11.( ‘H-StTI-l 1L( 'I («) The series of reactions corresponding to equations (5), (7), and (8) air reminiscent of the mechanism originally proposed by Conan I in 1920. According to Conant, the reaction passed through the following phases: S2( '12 S + SCI, C3H, + SC12—► ClCHoClUSCl C,H , + CICTI-CHaSCI —> CKTIjCHjSCH.C’UgCl These reactions were accompanied by a secondary reaction 2C1CH2C1I,SC1 + «S —> {(ICHsCIWA + S2Cb Convincing evidence of either the existence of /3-chloroethylsulfenyl chloride or its participation in the reaction was not advanced until 1943 4J when the pure material was prepared by chlorinolysis of US, and HSs. It reacts very readily with ethylene and with cyclohexene to produce H and /3-chloroethyI jS'-chlorocyclohexyl sulfide, respectively. /8-Chloro- ethylsulfenyl chloride also appears to be formed by the action of sulfur monochloride on H»S, and by the action of sulfur dichloride on I1S» and HS* since 0-ehIoroethyl /3'-chlorocyclohexv I sulfide can be ob- tained by the addition of cyclohexene to these re- action mixtures.34 As additional evidence that 0-chloroethyIsulfenyl chloride is an intermediate in the Levinstein reaction, it has been shown 55 that an equimolecular mixture of ethylene and cyclohexene vapor used in the Levin- stein process instead of pure ethylene produces, in addition to f>j«(0-chloroethyl) sulfide and bis-2- chlorocyclohexv 1 sulfide, the mixed sulfide, /3-chloro- SECRET PRODUCTION PROCESSES FOR If, HQ, AND HT 41 ethyl 2-chIorocyclohexyl sulfide in the expected mole ratio of approximately 1 1 2 respectively. Likewise, an equimolar mixture of ethylene and cyclohexene vapor passed through a solution of sulfur dichloride in mustard yields the same three products, indicat- ing the two-step nature of the reaction of ethylene with sulfur dichloride. The HSj formed according to equation (6) has been shown to be sulfur!zed readily at. 30- 3i> (’ by sulfur monoehloride to higher polysulfides (0). (10), and (11)] and again sulfur dichloride is the other product of the reaction. It immediately reacts with ethylene to produce more fcis-jS-chloroethyl sulfide. hs, + s,cuhs, + scu (o> ns4 + s.n. —> ns, + st % (io) HSi + S-Cl, —♦ IIS. + SCI., etc. (11) Because sulfur dichloride is introduced into the re- action mixture as a product of other reactions or as a result of the initial equilibrium, and since it reacts rapidly with ethylene, its concentration is low as long as excess ethylene is present. For this reason, the problem of overchlorination with resulting pressure instability of the product is not so significant in the Levinstein process as in the processes involving the use of sulfur diehloride directly. The net result of this series of reactions is to pro- duce* a mixture of (us-/3-chloroethyl sulfide and poly- sulfides corresponding to the monosulfide. The overall equation for the Levinstein reaction, (12), Is thus: 2j-C.il, + {x - 1)C1CH.CH.hSCHsCH2C1 + (C1CH.CH.)jS,4 , (12) The amount of impurity (as polysulfides) in the re- sulting Ijwinstein mustard depends upon the rate of formation of 1IS3 and on the rate of sulfurization of IIS. by sulfur monoehloride.*4 If it is assumed that the effect of temperature on this series of reactions is to increase the rate of all the reactions but that this is partially offset by the decreased solubility of ethylene at higher tempera- tures, tln’ii the net effect is to increase the rate of the sulfurization reactions relative to those involving ethylene. As fast as IIS* molecules are formed, they are sulfurized to higher polysulfides, and. since the addition of each sulfur atom results indirectly in the formation of one molecule of H (through SCI-), the product formed at higher temperatures should lie high in H content and in very high unstable poly- sulfides wliieh readily lose sulfur. At low tempera- tures sulfurization is slower, more 11S., is formed, it is sulfurized to a lower degree, and less II results. This interpretation is in excellent agreement with the known eharaeteristies of high-temperature. (60 (' process) and low-temperature (Levinstein proc- ess) II. The reaction of sulfur monoehloride with propylene to give “propyl mustard,” long believed 2,e and now known 11 to have the “normal” CHj,CH(Cl)CH- - S — (’H.CIlfCDCHj rather than the “iso" struc- ture. can also be accommodated to the above reaction scheme, since it has l>een shown that ft- chloroethylsulfenyl chloride, and by inference other sulfenyl chlorides, add to propylene in accordance with Markownikoff’s rule to give the “normal” struc- ture.41 It should he noted that even if the initial assump- tion of the scheme, i.e.. the disproportionation of sulfur monoehloride to sulfur dichloride and sulfur tritadichjoride is not valid, the following series of reactions provide a satisfactory mechanism: 2r.11 * -f s8ci3 —► rirn.rH. -s -s-cii3ch2ci (13) cich.ch, -s -s -ch3ch-ci + 3S,nt—► 2C1CH.CH8SC1 + 2S,C1, (or 2S -f 2S.C1.) (14) rirH.rn.sri + c.h4—> cich-cii8—s-cii-ch.ci (is) SsCl* (or S + S-C18) + 2r.n4 —► rk H.rHcSssc iu h.ci (16) 2nrH.rH.sr1 + 2S,ri.—> nchailssscha h.ci + sri. + 2S,n, a?) 2C.h4 + sri. —► cich-ch-schaha’i (is) Sulfurization of HS* to higher polysul tides by sul- fur monoehloride with the simultaneous production of H would proceed as already descrilied,*4 The assumption that sulfur dichloride can react in both linear and angular forms to give linear disul- fides and thiosulfoxides is not considered likely, since it has not lieen possible to isolate a thiosulfoxide or to obtain convincing evidence of the existence of this type of compound. The possibility cannot lie ex- cluded, however, since in one case it has been possible to isolate material of composition correspond- ing to a monosulfide plus varying amounts of sulfur which appeared to lie present in a very labile form.3*47 Evidence that trisulfides also exist in the linear form has l»een summarized.47 SECRET 42 MUSTARD GAS AND OTHER SULFUR MUSTARDS 5.2.6 The Purification and Stabilization of Levinstein II The storage and corrosion stability of Levinstein II as ordinarily produced is not entirely satisfactory and it cannot bo thickened for use as a spray. A re- view of the immense amount of effort which has l>een devoted to the study of its stabilization is beyond the scope of this report, and it is possible here only to describe the present position. A number of methods have l>een examined for the purification of Levinstein II, including treatment with ammonia, heat treatment, treatment with silica gel or charcoal, distillation under xarious pressures, flash distillation, carrier distillation using both steam and organic liquids, solvent extraction,1" and crystal fractionation.22 Of these, only vacuum distillation, steam distillation, and solvent extraction appear feasible for use on a plant scale. Pilot plant studies of all three have !>een carried out.24•'* 160 234 With- out a detailed discussion of the advantages and dis- advantages of each method, it can be said that solvent extraction using commercial pentane gives ex- cellent recovery (up to 95 per cent of the available H) of a product which contains 92.5 per cent 11 but whose pressure and corrosion stability is inferior to that of steam- or vacuum-distilled material; super- heated steam distillation which requires acid-proof equipment produces more stable material of 95 per cent or Ixdter purity in recoveries of SO per cent; whereas vacuum distillation of water-washed Levin- stein H produces material of exceptionally good sta- bility and 95-96 per cent purity with 86 i>er cent recovery. The vacuum distillation has the advantage of using existing plant facilities and appears to be the method of choice. Washing with water removes iron salts and acid and is an essential step; vacuum distillation of unwashed Levinstein H is unsatis- factory as a purification measure. A detailed description of the efforts to improve the pressure and corrosion stability of Levinstein II and to retard its decomposition without resorting to ex- tensive purification procedures cannot be given here. As a result of these efforts, which have included stud- ies on a variety of stabilizers, the addition of 1 per cent of hexamethylenetetramine (hexamine), whose efficacy as a stabilizer for Levinstein H was first recognized by the CWS-M1T Development labora- tory,44 to all munitions charged with Levinstein II and to all storage containers is now standard. This treatment confers satisfactory storage stability, as regards decomposition, corrosion, and pressure de- velopment, on Levinstein II, but is not sufficient to render this mustard satisfactory for thickening with polymethylmethacrylate. For this purpose it is necessary to use Levinstein H whose iron content is below 1 per cent, to add 1 per cent hexamine and 2 per cent nitrogen bases (either coal tar or petro- leum), and to store the thickened material in lac- quered containers.44 The stability of Levinstein mustard which has l>een purified by various methods is also improved by the addition of 1 per cent hexamine. The precipitate formed when hexamine is added to Levinstein H contains hydrogen chloride, iron chlo- rides, and sulfur. The action of hexamine as a stabi- lizer appears to be due to its ability to reduce iron content and acid, and has 1»een ascribed to these factors and to its high stability and low basicity, which was believed to be responsible for its lack of compound formation with H r- There is, however, disagreement as to the solubility of hexamine in 11 and as to the extent of compound formation between hexamine and H.33 132 and the mechanism of stabi- lization of Levinstein H by hexamine cannot lx- con- sidered completely clear. No attempt will be made here to describe the ex- tensive work on the thickening of H for use as an airplane spray or in airburst munitions. This subject is treated in detail elsewhere.1' 5.2.7 Pholosynlhelie Methods for the Preparation of II and Analogs As a result of work initiated in 1912 under Divi- sion 9 of NDRC, a novel method for the synt hesis of II and particularly of more complex relatives of II, such as Q, has been developed. The method gives excellent yields, is capable of great flexibility, and lias been applied successfully in a number of cases (see Table 2).b'J -77 The synthesis consists of the photochemical addition of hydrogen sulfide or mer- captans to vinyl chloride and related olefins in the- presence of photoactivators. The addition of hydrogen sulfide to vinyl chloride brought about by irradiation with ultraviolet light in the presence of peroxide catalysts was accom- plished in 1942 by NDRC groups59 but a publication appearing about this time indicates that the process had been studied earlier by a private group.277 Yields of 75-80 per cent accompanied by some /3-chloro- ethylmercaptan and polymeric material can be ob- r See NDRC Division II Summary Technical Report. SECRET PRODUCTION PROCESSES FOR 11, 11Q, AND HT 43 Tabi.e 2. Compounds related to II prepared by photo- synthetic methods. Compound Reference 1. (3-Chloroethyl ethyl sulfide 59 2. 6/s(d-Chlorocthvl) sulfide (II) 50 8. (J-Chloroethyl 3', (J'-dichloroethyl sulfide (2-chloro H) 42 4. d-Chlorocthvl pi'-hydroxyethyl sulfide (CH) 13, 142 5. J-( d-( 'hloroet hylt hio)et hvlrncrcapl an 125 6. 1,2-6i.t(3-Kluorfsithylthio)ethane 59 7. l,2-/)js(/8-Chioroelhyllhio)cthane (Q) 5 9, 122 el seq. S. 1,2-6(.v(^-Hroinijethylthio)etha.ie 59 9. ! ,3-6/s( (t-Ch!oroelhyl)-2-cliloropropane 59 10. 1,2-bi*(0-Chloroef hyl)-3-hydroxy propane 59 11. 2,3-6(s(j3-Chloroethvl)butanc 59 12. bix(#-Chloroet hvlthioethvl) ether (T) 59, 131 13. h(s((3-(^i-Chloroelhvllhio)ethyl)sulfide(di II) 129 11, a, -hloroelhyllhio)-p-xylene 59 15. 3-Fluorocthyl thiolaeetate 59 Methanol can also be used hut the product separates from this solvent as a second phase containing both methanol and ethanedithiol, and must he stripped, '['he possibility of using methanol in a continuous system has been recognized,147 but no development work along this line has been reported. The Q produced is of excellent quality and melts at. 53.6 C,1" but has a pronounced odor of ethane- dithiol, which may be removed by treatment with arsenic trichloride or I,.134 The heat of the photochemical reaction, which is exothermic, has not been measured but calculations from bond energies indicate a value of about IS kcal mole.12* In contrast to the addition of hydrogen sulfide to vinyl chloride, the addition of ethanedithiol proceeds without promoters or photosensitizers.59 However, the presence of these is very desirable and a number have been examined, particularly with respect to the redaction_of the light requirements of the reaction.147 'Hie most effective are peroxides of various types and disulfides.591:14147 Diphenyl disulfide is far superior to any of 18 other disulfide catalysis tried when the reaction is carried out in sealed tubes, but is no better than diamyl disulfide when used at atmospheric, pressure.147 Oxygen is ineffective.1311 The reaction appears to proceed by a chain mech- anism initiated, in the uncatalyzed case, by the photodissociation of ethanedithiol into radicals which attack the vinyl chloride molecule. Quantum efficiencies of about 1,000 are observed.147 Chain propagation occurs through the alkyl sulfide rad- ical : H S—C’-HjC H SI I HS—( ’H2C'Hs —S* + H* 11S—CHiC H 2—S' + C1U-CH- Cl —► HS -C II2CH*—S -Cl W;H—Cl HS —CIDCHs—S —CHjQH—Cl )HS—CH.CH.—SH | + lor 1 IS—CHjCH*—S—CHsCH/’l I HS—CH/ 'Hs—S—CH2CH3( 1 ) HS—C'H/Tl*—S* | + (or *S—-CHaCflo—S—CHjCHj- -('1 ( •s— S— CH/.’HjCI + CH; =CHCI —► Cl—('H2CH3—S— CH2C'Hj—S —CH.CHC1 ('KT DCHj—S—( H2CI1s— S~CH2CH—Cl + HS—CH2CH*—SH —> Q + HS—CIljCHg—S, etc. In the case of catalysis by photosensitizers, the initial photodissociation is of the catalyst molecule, and the tained. Illumination with ultraviolet light in the region 2,800 3,200 A is effective; peroxides and ben- zoin, a known photoactivator in the ultraviolet, pro- mote the reaction. Neither thermal nor catalytic activation will induce addition.5* It Inis been shown 277 that the photochemical addi- tion of hydrogen sulfide to olefins proceeds according to Markownikoff’s rule. Ultraviolet light alone was found to lie sufficient for the reaction to take place, but photosensitizers extended the range of effective frequencies. In the case of the synthesis of II from hydrogen sulfide and vinyl chloride, both irradiation with ultraviolet light and the presence of a catalyst apjtear to be necessary.59 The feasibility of synthesizing pure Q in high yields by the photochemical addition of ethane- dithiol to vinyl chloride has been demonstrated 59 and the reaction has been exhaustively studied at Edgewood Arsenal. The reaction is carrier! out as a batch process, and on a laboratory scale can l>e done either under at- mospheric pressure at the boiling point of vinyl chloride (—13.6 C) or at room temperature under approximately 4 atmospheres. Yields are 90 per cent in the first case and quantitative in the second when the irradiation is supplier! by an S-4 General Electric mercury vapor lamp and either benzoyl peroxide or sodium pcrcarbonate is present. The reaction can also be carried out at room temperature by bubbling vinyl chloride through the reaction mixture.125 Re- sults at 75 C have been definitely inferior to those obtained at lower temperatures.147 The presence of a solvent such as benzene is advantageous since vinyl chloride is not very soluble in ethanedithiol.155147 SECRET 44 MUSTARD CAS AND OTHER SULFUR MUSTARDS radical formed initiates a radical chain similar to the one above. Thus, the problem of reducing the light requirements of the reaction is resolved into one of finding photosensitizes which will yield radicals at longer wavelengths than that required for the photodissoeiation of cthanedithiol.'1 Disulfides absorb light even more strongly than cthanedithiol in the near ultraviolet, and provide alkyl sulfide radicals at longer wavelengths. Thus the 3,650 A mercury line is effective1 if diamyl disulfide is user! as a photo- sensitizer in the Q reaction,147 The diaryl disulfides, which are known to be easily dissociable,*75 absorb at even longer wavelengths (up to 4.200 A for di- phenyl disulfide) and are the most effective photo- sensitizers. Thus with diphenyl disulfide, which has been thoroughly investigated, excellent conversions are obtained w ith 300-watt Mazda lamps, and fairly good conversion is possible even with a 60-watt Mazda lamp. Among other easily dissociable com- pounds examined, the triaryl free radicals did not prove to be effective promoters. A novel “ionic chain” mechanism for this reaction received serious early consideration by CAN S work- ers 135 but was later abandoned.147 An early view that vinyl chloride was activated by light 122 was similarly abandoned, since vinyl chloride does no) absorb in the ultraviolet.147 Substances normally effective in acid-base cata- lyzed reactions such as boron trifluoride, phosphoric acid, hydrogen ion, triethanolamine, and water are ineffective as promoters in the photosynthesis of Q.I35H7 — A number of materials, e.g., copper, iron, tin. carbon, sulfur, and polysulfides, exert an inhibiting effect on the reaction, in some cases enough to com- pletely prevent reaction,5*,*4 and considerable diffi- culty has been experienced in scaling up the reaction because of this sensitivity to inhibition. Laboratory results indicate that stainless steel, aluminum, lead, zinc, and silver are no) injurious.135 The ethanedithiol required in the photosynthesis of Q can be prepared in 76 per cent yield by thiona- tion of ethylene chloride with aqueous sodium sulf- hydrate under hydrogen sulfide pressure.51 On a semi- plant scale, the expensive hydrogen sulfide has been replaced by carbon dioxide, w hich produces hydrogen sulfide from the sodium sulfhydrate.29 The efficiency of this process for production of cthanedithiol on a small-scale manufacturing basis has been demon- strated by the production of about 1,600 lb. The con- ditions were not studied exhaustively, but a cheap, practical process easily adaptable to large-scale man- ufacture was developed.29 S.3 TABULATION OF ORGANIC SULFUR COMPOUNDS EXAMINED VS CANDIDATE CHEMICAL \\ VRFARE AGENTS The many analogs of It which have been studied by investigators in the United States, Great Britain, and Canada are listed in Table 6. with references to the reports on their preparation and toxicological action. The closely related nitrogen mustard series is tabulated in Chapter 6. It can l>e stated that the study of analogs in the sulfur mustard series has not disclosed compounds superior to IT. Q, and T in po- tential general usefulness as chemical warfare agents. In the study of (lie mechanism of action of agents in this category, however, as will be noted later, ex- tensive use has been made of the data on the chemical and toxicological properties of the compounds re- lated to H. 5.1 PROPERTIES OF II. (,). AND T 5.1.1 Physical Properties The physical properties of II, Q, and T which have the most direct bearing on the effectiveness of these agents as war gases are the following: II Q T Density (liquid) g/ini at 25 (’ 1.27 1.24 Boiling point C 217 353 (calc.)5‘ 120/0.02 mm*" Freezing point C 14 56-57ls 102‘* Volatility mg I 25 C O-tMi"**' 0.0004"* 0.0028s"" Whereas II has sufficient volatility to yield injuri- ous vapor dosages, neither Q nor T presents vapor hazards. The vesicant action of Q and T depends upon contact 1 ret ween the skin and (he liquid or particulate agent. The low volatility of these two compounds gives them a much longer persistence than II as potential hazards on contaminated terrain.17,1 3 The uncatalyzed addition of ethanedithiol to vinyl chlo- ride occurs at wavelengths of 3,125 A and at all wavelengths helow this, in fair agreement with a probable threshhold wave- length (calculated from bond strengths) of 3,254 A for the photodissoeiation of ethanedithiol.u; SECRET TAHI I. VTION OF ORGANIC SL LFl R COMCOLNDS 45 Table 3. Organic sulfur compounds examined as candidate chemical warfare agents.t The general arrangement of the table is as follows: (!) mercaptans and derivatives; (2) sulfides and sulfide ethers; (3) polysulfides; (4) sulfoxides; (a) sulfoncs; (6) derivatives of thioacids; (7) sulfonium salts; (8) derivatives of sulfinie and sulfonic acids; (0) sulfur compounds of unknown constitution. The following abbreviations are used: nn', refractive index at 1 C; specific gravity at 1, C in reference to water at U (.’; inp, melting point in C'; bps, boiling point in (' at p mm Ilg; vp1, vapor pressure in mm Hg at 1 C; and vol', satu- ration concentration (volatility) in mg 1 at t V. Centigrade scale is used throughout the table. Compound Reference . . Physical properl ics to ’ synthesis Pr«iHTty Reference Reference to toxicity data 1. 0- Kluoroethvlmercaptan 59 bp«* 38.5° 59 34 nDi5 I .4290 59 2. £-{’hloroethvlmereaptan Mi, 53 bp:,u 114-120° 16 34, 54 3. d-1 lydroxyethylmercaptan 16 bp’ 47-50° Mr- 4. 1,3-Dietiloro-2-mercaptopropanc Old 34, 54 5. 3-Mercapto-l,2-propylene sulfiile 14 34 ti. Ktlianedithiol 29, 58 bp60 65 68° 58 34 ... n u?i — 1.5570 58 d4-s 1.1182 58 7 l-Chloro-2,3-flimercapto|>ropane ... .... -■ 34, 54 8. 1-H vdroxv-2,3-dimcrcaptopropane* (DT11) (HAL) 14,15,237 bp"1 115°±1 14 - 242b «i,3 1.570 1.573 14 - ... ■_ d,* 1.238-1,240 14 : t>. 2,3-Dime leapt opropionic acid _ 34_ 10- b/«(d-Mervaptoethyl) sulfide 8 bp’ — 101-106° 8 11. 1,0-1 lexanedi thiol 34. 54 12. o-Aminothiophenol* ...... . ~ 13. 2-{u>-(’lilon:iacet)iiniiio)-5-methvl-thiophenol* 14. Trichloromethvl sulfenvl chloride* (perchloromethyl mercaptan) 271 bp1** 148 149° 278 247 bp5" 73° 278 ,f> 1.722 278 - . . . _ , vol10' " 18 278 15. rt-Chloroethvlsulfenyl chloride 35 bp15 47-47.5° 35 34, 54 1 6. o-Xil rolicnzenesulfenyld«.<{/#-chloroel hyl )amine 39 mp 104-105° 39 34 17. hi*(0-Chloroefhylmercapto) chloramine 61c .... ... r 34 IS. Hulfiliniine of chlommine-T and 11 65a mp 144.6* 278 34 10. lelrakix-0-i ’hloroet hylmereaptosilicon 61 h bp‘» 98° 61h 34 — dd" 1.390 6 i h 20. Tributvlthiocthoxy tin 5 bp15 126° a d“ 1.132 5 21, Triethvl 0-chlorothioelhoxy lead Old 34 22. Thallous 0-cbloroethylmercaptide 61 f mp >300° Gif 23. fej’s(a-Chloromelhyl) sulfide* ... 24. Methyl 0-chloroethyl sulfide 11,23 bp” 52-53° 11 34, 54, 247 vol** 32.7 38 25. fl-llvdroxvelhyl methyl sulfide 23 bp15 63-66° 23 2ti. sulfide bp1* 105° 247 247 27. 3-Chloro-l,2-propylene sulfide* 8 bp5-' 38-40° 8 34, 54 »ua 1.5127 8 _ d16u 1.250 8 28. S-ThiiM’vano-1,2-propylene sulfide 53 .... .... 34 20. Methyl 2,2'-dichloroisopropyl sulfide* ... f 3(1. Mcthvl 2,2'-di hydroxy isopropyl sulfide* ... .... .... — . . 31. Divinvl sulfide* 32, fi/s-llcxachlorodivinvl sulfide 33 tf-C'hloroet by 1 vinyl sulfide*” 34. /3-('hloroethyl o-<-iilorovinyl sulfide* 189 * Not all the British re|>orta eoneerning compounds marki-d wit! an asterisk arc available in this country. References are contained in refer- f The table itidiuh** sulfur coin|Mmnds in which sulfur is linked to carbon with the exception of thiocyanates, which arc included In Table I, Chapter 14. Sulfur coni|H>unds containing cither arsenic or phosphorus are included in Tabic 1. Chapter 7. and Table* lr Chapter !» and have not been rc|n*atcd here. SECRET MUSTARD CAS VXD OTHER SULFUR MUSTARDS Table 3 (Continued). Reference Reference t° to r liysiClll pro|KTtlf*S toxicity ('(impound synthesis Property Reference data 35. d-Chloroethyl d'-chlorovinyl sulfide* 35 bp« 40° 35 34 ... bp" 15 30° 35 mp -24° 35 HU* 1.5480 35 . . 36. Equimolar-mixture of triethylpbosphorus and diethyl sulfide 611) 37. Ethvl d-ebloroethvl sulfide* 53, 59 bp*31 73-76" 59 34, 54 n i,“ 1.4858 59 vol20 16.57 38 38. d-Thiocyanodiethyl sulfide* 39. d-Chloroethvl d'-fluoroethvl sulfide 64a bp3" 91.5-92.5° 04a 34, 54 mp -44° 04a «ir" 1.4872 64a 1.228 04a — . . . 10. M«-Chh iroet liyl) sulfide* 8 bp'4 58.5 -59.5“ 8 41. a-Chloroethvl d-ehloroethvl sulfide* 42. Mustard gas See text See text See text 43. d-Chloroethyl d*,d'-diehlorocthvl sulfide (2-ehloro 11) 42 bpn W 08 69 42 34 'll.'" 1.5380 42 44. fiisfd.d'-Diehloniethyl) sulfide* .... 45. biX a,d,d'-T riehloroot hyl) sulfide* v . . 46. biM,d-1 tromocthy 1) sulfide 53 bp" 1 91 93° 53 31, 54, 247 mp 31-33° 53 47. fi/Md-Iudoethyl) sulfide mp 08 70° 247 247 48. d-Chloroethyl d'-eyanocthvl sulfide* 49. 6i*(d-( 'Vanoet hyl) sulfide* 50. d-Chloroethvl d'-hvdroxvcthvl sulfide* (CH) S, 43, 142 bp" 6 100° 43 n ii*" 1.5188 43 51. 6/s(d-Hydroxyethyl) sulfide Commercial 34 52. biti(d-( ’liloroformylet hy 1) sulfide 04a 34 53. 5i*(d-Cldoi'oaceioxyethyl) sulfide* .... . n 54. bidd-Triehloroacetoxyet hyl) sulfide* 55. his( d-Bn imoneetiixyet hy 1) sulfide* . . , 56. d-Hvdroxvethvl d'-evanoelhvl sulfide Ole bp15 186-188° Ole n,i!#—■ 1.5101 Ole dt-« 1.143 file 57. d-Chloroethvlthioacet vl chloride* ... — 58. Ethvl d-ehloroethylthioacetate* ... 59. Thiodiglyeolic acid 270 mp 129° 276 34 60. Methvl thiodiglveolate 11 bp* 102-104° 11 . . mi" 1.4748 II '-L-:1 ;■ • -- ■ .‘ • : — 1.230 11 61. All vl d-ehloroethyl sulfide* 53 bp" 64.5-65° 53 34. 243c vol!" 0.63 38 02. Ethyl d-chloropropyl sulfide* 03. Ethyl d, y-dicliloropropyl sulfide* 64. d-Chloroethvlpropvl sulfide* .... 243c 65. d-Chloroethvl d'-fhloropropvl sulfide* ■ — 41 54, 189 00. d-Chloroethyl d'.y'-dichloropropyl sulfide* — 67. p-Aminophenyl-(7-cliloropropylthio)ethylamiiie hydrochloride* 68. Aeelonyl d-chloroethyl sulfide 53 bp" :i 76-85° 53 mp 145-146° 53 09. d-Chloroethvlbutyl sulfide* .... 243c 70. d-Chloroethvl o'-methyl-d'-chloropropyl sulfide* . .T 71. /»*((’liloroallvl) sulfide* 72. biM. 7-Chloroaliyl) sulfide* — * Not ull the British reports coneerninn eomiMjunds marked with an asterisk arc available in this country. inferences are contained in refer- ence 176 TABULATION OF ORGANIC SULFUR COMPOUNDS 47 Tabi.e 3 (Continued). Compound Reference to synthesis Physical properties Property Reference Reference to toxicity data 73. 6i.s-()j-Ilromoallyl) sulfide* 74. 5js(rf-Cliloropr( ipyl) sulfide* 41 hp" ■“ 107-108° 41 34, 54 (propyl mustard) «ir° 1,4903-1.4917 75. ?))*( >-Cliloropropyl) sulfide* 8 hp- 111-112° 76. bis(f), y-Diehloropropyl) sulfide* hp* 34 77. Propyl d-hydroxy-y-fluoropropyl sulfide file 10.5-107 file 78. his{ x-Dicldoro isopropyl) sulfide 34, 54 79. bis{«-('hloru-o-acetoxy isopropyl) sulfide* SO. bis(d-Clilorobut vl )-3-sulfide Commercial 34, 54 81. ,i-( 'hlnr; .ethyl 2-ehh»n>eyclopcnty 1 sulfide 53 hp8“ 73° 53 34 «n!" 1 5336 53 82. Kthvl 2,4,6-triclilorophenyI sulfide* . . . .. 83. p-Lt hyltbiopbenyldiehlorostibine* 84. £-Chlorocthvlphenyl sulfide* 85. d-CiilorP! 115“ 53 34 «d" 1.5728 53 96. d-(‘hloroethyl lienzyl sulfide 53 bp* "* 95 97° 53 34, 243c vol18 0.115 38 97. d-Chloroethyl nonyl sulfide* 243c 98. Diphenyl sulfide* 34 99. 6/.s(/3-Chlorocyclohexyl) sulfide* 53 mp 73.5° 53 100. hyi uudecyl sulfide* . . . 243c 101. /C( hloroethyl chloromethoxymethyl sulfide* 102. fl-Ilvdroxvelhyl chloromethoxymethyl sulfide* 103. l-Melhoxv-2-(/3-chloroethvlthio)ethane* 243c 104. 1 -Met hy 11 h i o-2-( /J-chloroe t hylt hio)ethane* 243c 105. 6i«(/J-Chloroet hylthio) methane* 53 nip 31-32° 53 34. 54 100. 2-\lel hvl-4-chIoromet hyl-1,3-dit hioeyclopent line* 107. l-(^-Chloroothylthio)-2-ethoxyethane* 243c, 189 108. l-(#-ChIoroethoxv)-2-($-ehlor(>ethylthio)ethane* bp* 120* 179 179 109. l-(d-Chloroethyllhio)-2-cthylthi(H-thane* 243c 110. 1,2-/«s(d-FhKin*‘thvlthio)ethane 59 bp"’ 85° 59 111. 1,2-6/s(0-Chlorocthylthio)ethanc* (Q) See text mp 54“ . 59 Sec text 112. l,2-6/»(/3-Bromoelhylthio)ethane 53, 59 nip 78-79° 53 34, 54 113. 1,2-fc/s()3-Cyano«‘thyllhio)elhaiie* 114. 1,2-f»/s(d-Ilvdroxvethvllhio)ethane* 115. l,2-fe/s(/3-Ciiloroethylthio) acetaldehyde* 116. 2,2-Dimelhyl-4-chloromot hyl-l,3-dithiocyclo- — jientaue* 243c 117. l-(d-Chloroethylthio)-2-propoxyethaiic* 243c 118. Id fi-< ’hloroef hvlt hio j-2-propylt hioet hane* __ .... 243c 119. 1,2-fci>(0-ChloniethvIlhio)pn»paiie 8 bp"‘ 139-142° 8 34, 54 d,* 1.23:1 8 120. 2.3-6('s(d-Chloroethylmereaplo)-I-chIoropropane 5!) 34 121. 1,3-f>is( d-Chloroe t hyl 1 hiolpropane* 53 bp‘"e 101° 53 34, 54 * Not nil the British rcjw>rtjs concerning coiii|>ou»k1s marked eiHt* 176. with an asterisk arc available in this country. References are contained in refer- SECRET MUSTARD «.AS AM) OTHKR SULFUR MUSTARDS T.\BLB3 (Continued). Reference to Physical properties Reference to toxicity Compound synthesis I’mperty Reference dati. 122. 2,2-f»is(d-Chloriiet hylthio )pr»pane* 53 ••I**’"* 05s 53 34, 54 mp -15° 53 —: W-- 123. 2-Chloro-1,3-6ethyIthio)butane* 130. 2,3-6i>('hloroethylthio )bulane* 59 34, 54 131. 1,5-fci«(0-Chloroethylt hio)pentane* 132. 1 .fi-fifuf/S-Chloroct hylthio Jhexane* ... 133. l,5-6isO-(’hloropropyl(hio)pertlane* 134. n, a-bix(d-t1 hlorocl hv 11 hio)tohieno* 135. 1 ,S-6ix( /M.'hloroet hyl thiojfietane* 130. a,a'-bis(ti-( 'hloroet hylmeredpto)-p-xylene 59 mp 75,5-77 59 50 137. 1,9-hVs(fM.'hhiroet hylt hio)nonane* . . . 138. 1 ,l(hfci.s(0-C'hloroet hylt hio )deeane- -a., . . 139, 1,2-Diphcnyl-l ,‘2-hiM d-ehlorocl hylt hio lot hane* 110. 9,10-6»s(/Whloruethvlthio)Bt«iric acid* — - 141. b/jtfd-Kluoniet hylt hiomet hyl) sulfide* . . . hp1' 139.5° 238b 238b 142. fcjs()S-(’hloroet hylt hiomet hyl) sulfide* hp- . 125° 247 247 143. 0(s(d-C hloroet hvlt hio Inicthane 53 hp"™ 35—10° 53 34, 54 144. d-( /3'Chloroet hvlt hio)elhvl-d’-(d-ehloroothoxy )- — ethvl sulfide* — 145. h(,s[d-(ft-ChIonM'thyIthio)ethyl3 ether (T) See text bp! 174° 247 See text 1.2445 247 140. 6i>f0-O-C.*vanoefhvlthio)ethyl]ether* 147. W«|j8-(0-Chloroet hylt hio)et hyl] sulfide* (di 11) 53 mp 73-75° 53 f 48. 5(’xCd-(d-Hroiuoothylthiojelhvl] sulfide* 149- 6[d-( p-T h j oey a n oethylthioJethyl] ether* _ 150. his(jS-( 0-Phenoxyeth v11 h io )et hyl ] sulfide* 151. 6(«[/H/3-{2,4,6-Tiihromophenoxy)el hylthio)- ethyl] sulfide* 152. l,2,3-his(ff-l‘hloroet hylt hio) propane* . . , * — 153. fc/«[d-(d-Chloropropylthio>*thyl] ether* 154. hix[a-Methvl-tM0-chloroethvlthio)ethyl] ether .... 34, 54 155. 'hloropropyllhiojel hyl] sulfide* 150. 'hloroet hvlt hio )propvl] sulfide* — 157. 1,1, l-trisf/S-Chloroethvlthiomethyl) ethane 53 Op-'otm-.-.xliri 41 42° 53 34 158. fux[o-Methvl-f)-(d-ehlorethylthioJethi>xy)el hyl- -— ■ 1 hio Jethoxy let hyl] sulfide* 107, 6/..[d-[3-13-(»4-( M 3-( 'hlon «■! hylt hio )et hoxyV- el hylthio Jethoxy Set hylt hio]et hyl] ether* * Not all I lie British report* concerning enm|»ounil* marked cnee 17tt. with an n*t#*rl«k are available* in this country. Hefeicnee* arc contained in refer* SECRET TAIUI.VTION OF ORGANIC Sl LFl It COMPOl ADS Table 3 (Continued). • % e , to 1 hysteal projrerties to.xicitv Compound synthesis Property Reference data 168. #-()ximinoethvl i t hvl sulfide* 169, hloroct hvlt hio)ethvh rimel hylammonium chloride 2 170. /(-.\miuuphcnvl-d-(iJ-ch!oroctiiylthio)ethvIamine hydrochloride* 171. X-fJ-C'hlorocI hyllhiomorpholinc* 16 hp"4 77-80° 16 34 172, X-ff-t‘hloroct hvllhiomorpholine hydrochloride 10 mp 208 209 Hi 173. X-d-Hydroxyel hyllhiomorpholinc hydrochloride 16 nip 160-163 16 171. sulfide* 175. h(.xQj ( l»i«((3-l'hloriH‘lhyl )aminu)ethyl] sulfide* 26 mp 24.2 21.7° 26 34 «o-4 IP7 26 170. Methyl-fci.<((S-elhvlthioethvl famine f 241 f 177. Melhyl-fc«(/lK/3-ehloroel hvlthio)cthvl famine hydrochloride 36 mp 70 72' 36 34 178. hv- drochloride 39 mp 62 64’ 39 34 179. \,X-fux((5-((J-('hlororlh\lthio)ethvl)aniline hv- drochloride 36 mp 69 71° 36 34 ISO. InMii-i 3-C'hluri>ethylt hiojc'lhvl famine 181 nip 52° 181 181 181. «-Chloroniethyllhiophcne 8 hp'3 71-76° 8 182. w-Chlorimeet vlt hiophene* 183. to-Bromoacet vlt hiophene* ... — —77 184. 2-( "hloroacet vl-.j-nit rot hiophene* 185. Diphenvl-a-lhienvlstibilie* 186. Plienvldithicnvlstihine* 187. 2-Chloromercuril hiophene* 44 mp 183 184° 44 34 188. 2,5-h;s(Chloromerc4iri )lhiophene* 189. 2-(p-.\minophenyl)6-met hylbenzthiazolc* ... —— 190. X-d-( hloroethylphenothiazine 61 ii 191. Dimethyl trisulfide 53 i.p5" 64° 53 34 192. Dimethyl letrasulfidc 53 i»p* 56-69° 53 34 nii1" 1.6621 53 — ,r* 1.3008 53 193. 6is(/}-Cliloroethvl) ih.-ulfide* 8 hp1 80° 8 194. his(o-Chloroethvl) trisulfide* “ 195. 6/«(t)-C'hloroelhvl) trisulfide* 35 mp 30.5-31.5° 35 34, 54 196. 6/A-(^-Chloroethyl) jrentasulfide 35 1.6753 35 34, 54 197. his( 1,3-Dichldroisopropyl) disulfide ... 54 19.8. 6i*(2-Aminophenvl) disulfide , 199. 6(x(f»-C'hloromcthvl) sulfoxide* 200. Divinyl sulfoxide* 34, 54 201. d-Chloroethyl vinyl sulfoxide 34, 54 2(42. 6/x(j8-ChIoroethyl) sulfoxide* 8 mp 108-110° 8 34 203. Thiodiglyeol sulfoxide 05a mp 110° 65a 54 204. 2,5 (or 1,3) Dihvdrothiophenc sulfoxide 205. fe/s-Sulfoxide of l,2-6i*(f3-clilorocthyhhio)ctliane (2 isomers) 53 mp 148° 53 34, 54 206. fux-Sulfoxide of 1,2-5/x(d-h vdroxyet hyll liio)- ethane (mixture of isomers) 65c mp 90-101° 65c 34 207. bis(Klhoxvethvl) sulfoxide* 208. 6ix-Snlfoxide of bi«(0-((3-chloroethvlthio)e(hyl) ether (2 isomers) 51 mp 100-101° 51 56 mp 106107° 51 209. mono-Sulfoxide of h;x(d-(/3-i'hloroelhylthio)ctliyl) sulfide* - 210. Diphenvl sulfoxide* — 211. h)«{o-( ’Idorome 1 hv 1) sulfone* 212. Methvl vinyl sulfone* 213. Methyl d-efdoroethyl sulfone* %* ' ’ * Nut all IlieBnnsh rri*.rta eoneeminK eompounds marked with an asterisk are available in this countr\. Kefereneea are contained in refer- ence 170. — SECRET 50 MUSTARD G US VXD OTHER SULFUR MUSTARDS Table 3 (Couth ued). — Compound Reference to synthesis Physical properties Proj>erly Reference Reference to toxicity data' 214. Divinyl sulfonc* 53 34, 54 215. d-Rromovinyl vinyl sulfonc* bp" 137" 197 197, 241.1 216, his(d-Hromovinyl) sulfonc* nip 58 59 24 Id 197, 24 Id 217. Kthyl vinyl sulfonc* 2 IS. d-Chloroethvl vinyl sulfonc 53 lip'7 152-154° 53 31. 54 21!>. 0-('hloroet hyl ethyl sulfonc* — . . . 220. botfiJ-f'hloroet hyl) sulfonc* (H sulfonc) 53 1>P3 mp 155° 51.5-52.5° 53 53 34, 54 221. fcisfd-Bmmoclhyl) sulfonc* 222. d-C’hloroclhvl-ti', /S'-dibromocthvl sulfonc* mp 63 64 197 197, 24Id 223, his(Dihromocthyl) sulfonc (2 isomers) mp nip 138° 72-73° 24 Id 24 Id 197, 24Id 224* Thiodiglycol sulfonc 65a mp 56° 65a 34, 54 225. 2,5 (or 1,3) Dihydrotliiophcnc sulfonc* 226. 2-Chlorotctrahydrolhiophcne sulfonc* 227. X-d-Chloroethvlthiazan sulfonc* 228. N-d-Chloroctlivlthia/an sulfonc hydrochloride* 229. X-fl-Hydros vet hvl thiazan sulfonc hydrochloride* fTT- 230. Diallvl sulfonc* 231. 5i.«(’V-(,hloropropyl) sulfonc* mp 64 -65° 197 197, 24Id 232. Phenyl ehloromethyl sulfonc* . * ' 233. Phenyl vinyl sulfonc* _ 234. (i-i 'hloroel hvl plienvl sulfonc* 235. d-Chloroet hyl p-nit ropheny 1 sulfonc 34, 54 236. /J-C’hhrroethyl 2,4-dinit rophcnvl sulfonc 237. ti~( 'liloroclhyl p-tolyl sulfonc* 238. Diphenyl sulfonc* 239. f>i.s(2-Ch!orocyclobcxyl) sulfonc* 240. p-Thioxanc sulfonc* 65b 34 241. 6/ft-Sulfonc of 6/x(/5-chlonxUhvlthio)nicthanc* 242. 5i>-Sulfone of 1,2-5;*( vinylthio)ethane* 243. 5)s-8u!fonc of 1,2-fe/»(/J-chloroe(hylthio)e(hane* 53 mp 202-204° 53 34 244. 5/s-Sulfonc of l,2-6is(/S-hy«lroxyethylthio)cthanc 65c mp 113 115° 65c 34 245. 5is-Snlfoneof l,4-5/s(0-chlorocthvlthio)butanc* 246. his-( Met hoxyct hyl) sulfonc* . .7 247. hi.s( El hoxyct hyl) sulfonc* 248. 6/s-Sulfonc of 6;s(d-(d-chlorocthvlthio)cthvI) ether 51 mp 70 71° 51 56 219. 5»s(/3-(d-Chlorocthylthio)cthyl) sulfonc* 250. 6»*(d-lsoamyloxycthyl) sulfonc* 251. d-Kluorocthyl thiolucctatc 59 bp-'" 85 87° 59 34 — vop" 214 38 252. d-( 'hloroet hvl thiolacctatc 34, 54 253. Diet hylt hallium t hioacet ate 19 mp 181 183° 19 254. Triethyllcad thioacctalc* 241 mp 44“ 241 255. S-/9-ChIorocthyl fluorothiolacetalc 55 bp"' 80-81° 55 34, 238b 256. Phenyl fluorothiolacetalc 24 Ig mp bp18 36.5-37.5° 132° 24 Ig 24 Ig 238a 257. #-( ’hloroet hvl chlorothiolacctate* 7T, 258. Chloroacetyl thiophcnol* 259. 6i.«t(('hloroacctyl) sulfide* 260. (i*('hloroethvl brornothiolacetatc* 261. Methyl 7-fluorothiolbutyrate .33, 55 bps 54° 33 34 - ' no10 ,r-n vol?s 1.4587 1.1135 8.44 33 33 33 262. Methyl 7-fluoro-^-hydroxythiolbutyTatc 55 bp0-* 68-71° 55 31 nu*0 1.4872 55 cure * Not all the Hritish report* concern in* coni|>ounamate* 283. Thallous \-hutyldithioearhainate 19 .... 284. Thallous X,X-diisopropyldithiocarhamate 285. Dimethylthallium X ,X-diisopropyldithiocarha- 19 mate 19 hp' 130° 19 34, 54 hp4 5 145° 19 mp 150° 19 286. Thallous X-cyclohexyldit hiocarhamate 19 287, Thallous X,X-dibutyldith iocarha male 19 bp0,01 230-235° 19 nip 75 77° 19 288. Dimethylthallium X.X'-dihulyldithiocarhamate 19 bp" 4 147 118° 19 289. Thallous X,X-diisohut vldithiooarbamale 19 mp 165—165.5° 19 290. Dimethylthallium X,X-diisohutyldithiocarhamate 19 hp"4 104-105° 19 34 nip 73 74° 19 291. 6/«(/}-Chloroelhyl) trithiocarbonate 53 bp’ 85° 53 34 no'0 1.5505 53 292. 0-Chloroelhvldime(hvlsulfonium chloride 23 mp 147 118° 23 34 293. d-Hvdroxyethyldimethvlsidfonium chloride 23 294. Mel hv 1 -6/»(d-hydroxvet hyl) sulfonium chloride 23 295. Met hyl-h/ff-2-hydroxvethvl sulfonium iodide 23 296. Dithiane monomethiodide 61r mp 168° 61k 34 297. (m(d-Chloroethvl) sulfonium chloride 46 34 298. S,S-cnrfo-Klhv!enedrfhianc sulfonium dichloride 16 299. S-Vinyldilhiane sulfonium chloride 46 . 7 * Not all the British reports concerning compounds marked ence 176, with an asterisk are available in this roun t r y. R eferenees arc contained in refer- SECRET 52 Ml STAKD «; \8 \M) OTHER Sl'LKCR MLSTARDS Tablk 3 (Continued). Reference to Physical properties Heferenee to 1 toxicitv Compound synthesis Projx'rty Reference data 300. S-d-Chlomethvldiihianc sulfonium chloride 4f> mp 144° 40 34 301. S-£-I I vdruxycl hyldit hiane sulfonium chloride 302. Sulfonium salt of thiodiglvcol and hiM.d-cldoro- nip 170“ 197, 24Id 197, 24 Id ethyl) sulfoxide 303. .Sulfonium salt of thiodiglveol fus(/3-cliloroethvl) 34 sulfone 304. Sulfonium compound of f».f(2-chlorocthyl) sulfide .... 34, 54 and 2 moles of thiodiglvcol 8 mp 102 103° 8 34 305. Triethvllead cveloliexylsulfiimte 20 mp 132-134° 20 300. Triethvllead P'loluenesuifinate 20 mp 80 88 20 307. Melhanesul|ihonyl fluoride* 308. Methanesulphonyl chloride* 300. Chloromethanesulphonyl ehloriile 310. Triehhiromel hanesulfonvl cliloride* II • *>.• 241c 311. Kthanesulfonvl chloride 11 bp2" 73.5 75° II 34 1.370 11 - ... 312. 2-Fluoroethaiu‘sulphonyI chloride* 313. d-t’hloroethylsulphonyl chloride* 3)1. d-Hromoethanesulfonvl fluoride 04d bp-" 00 Old 315. t'hloropropanesulfonvl chloride Commercial 34 316. Hutanesulfonyl fluoride 04c bp" 54 50° 64c 34 317. Hut anesulfonvl chloride 64c b|»T 70 78 04c 34 318. Animonium 2-ehloroethanesulfonale ... 34 310, Sodium 7-fluoro-d-hydroxy propanesulfonatc 55 56 320. Cadmium m-nit robenzenesulfonate 0 7". . . 321. Lead w-nil robenzenesulfonate 3 322. Cadmium 2,4-dimtroben*encsulf«nate 0 323. Ixaid 2.1-dinilrohenzetu*sulfona!e 3 324. Triethvllead o-toluenesulfonate* 325. Tripropvllead o-loluenesulfonate 241 mp 87" - 241 320. Trimelhvllead y<-to!uenesulfonale* — — 327. Triethvllead p-toluenesulfonatc* ... — 328. Tripropyllead p-toluenesulfonate 241 mp 73 74.5° 241 320. Tribulyllcad />-lohienesulfonate 241 . mp 81-82° 241 330. Triel hvllead 2-ainino-5-toluenesulfonate 20 mp 210° 20 331. Triethvllead naphlhalenc-2-sulfonate* 332. Trihut vllead naphthalene-2-sulfonate* 241 mp 08° 241 333. Triethvllead l-amino-4-naphthalenesulfonale 20 mp 238 240“ 20 334. Dipropvllhallium d-eamphor-lO-sulfonate 10 335. Triethvllead d-camphor-lO-sulfonatc 20 mp 172“ 20 330. Triethvllead p-tolylthiosulfonate 20 mp 109° 20 337. 6(.v-Triethvllead methanedisulfonale • 2411) 2411) 338. Triethvllead methancsulfonamide* 241b mp 97° 241b 241b 330. Tripropvllead met hanesulfonamide* 241 h mp 07“ 241b 241b 340. Triethvllead methanesulfonanilide* 2411) mp 115.5° 241b 241b 341. 6i'jt'Trielhvllead methanedisidfonanilidc 2411) , 241b 342. Triethvllead elhenesulfoiianilide 241b mp 110“ 241b 241b 343. Triphenvltin lienzenesulfonamkle 241b mp 119° 241b 241b 344. Triethvllead l>enzenesulfonainidc 241c 345. Tripropvllead benzencsulfonamide . . , . . 241c 340. Triethvllead p-aminohenzenesulfonamide* 20,241b mp 173-174° 20 241b 347. Tri|)ropvIlead /j-aminolx'irzenesidfonatnide 241b mp 101° 241b 241b 348. Triethvllead o-toluenesidfonamide* 2411) mp 133° 241b 340. Triethvllead p-loluenesidfoiiamide* 241c 350. Triethvllead p-tohienesulfonanilide* 241b mp 134° 241b 351. Tripropvllead p-tohienesulfonaiiilide 2411) mp 101° 241b 241c 352. Triethvllead p-toluenesulfon-p-ehloranilide 2411) mp 111.5° 241b 241b 353. Triel hvllead p-toluenesulfon-p-hromanilide 24 Ih mp 117“ 241 b 241b 354. Tripropvllead p-toluenesulfon-p-chloranilidc* 241b mp 123° 241b 241b 355. Triel hvllead o-carboxvl>enzenesulfonimide 2411) mp 135° 241b 350. Tripropyllead o-c.arhosy1x*nzeneRulfonimide 241h mp 130“ 241b 241b * Not all (he Hritish reports concert!iiip rom|H>unda market! eiicr 170. with an asterisk arc available lit this country. Hr fern m s arr contained in refer- SECRET TOXICOLOGY 53 The high freezing point of II is a disadvantage in some instances. For aircraft spray tanks, in particu- lar, there was interest in a vesicant mixture which would withstand high-altitude temperatures of — to — -IOC for several hours without freezing. This degree of lowering of the freezing point could lie attained only by the use of a relatively large |>er- eentage of an inert diluent such a* acetone or ben- zene. Such mixtures suffered from the relatively low pay load of active vesicant agent. The freezing point could be lowered a small amount by mixture with Q or T. The freezing points of eutectic mixtures of II with pure Q or T are as follows: HQ (68 32) 4.5 and IF!' (35 65) approximately — 8C.2U The principal U. S. standard charging was H which with its 30 jaw cent nonvolatile impurities melted at about 8 (’. The standard British chargings included IIT ((50 10) melting at about 0 C and solutions of H containing 10-20 per cent lienzene. The v iscosity of H is an important property not included in the above tabulation. Thr size of drops of 11 from spray munitions is a function of the re- sistance of the charging to shattering forces upon emission and (his property is related to the viscosity of the material. The addition of “thickening” agents, such e adequately assessed only against hu- man skin, the availability of volunteers has been of major importance in this program. The performance of skin tests under conditions which permit quantitative interpretation of the re- sults requires that the testing procedure lie carefully controlled. The introduction of specially designed micropipets (see Chapter 16) has facilitated the routine application of very small volumes (0.01 mm* or less).717 54 Evaporation is a variable entering into tests with volatile agents. The term “absolute” vesicaney has been introduced by British investi- gators to indicate the activity of the compound when it is applied to the skin anti then covered to prevent evaporation. For example, in the usual “ojjen” vesi- cation test, Q is many times more potent than H. Under conditions of “absolute” or “closed” vesica- tion, which is important in the study of the intrinsic activity of different molecular structures, Ore two agents are much nearer to each other in activity. For the study of the vesicaney of the vapor of an agent, rather than that of the liquid in contact with SECRET MUSTARD GAS AND OTHER SUITER MUSTARDS T \Bi-K 4. Relative vesiraneies of II, Q, tnd T. Compound Uncovered Reference Vesica ney Covered relative to H Reference Vapor Reference li 1 Q 5* 199 T 3* 109 Methyl-0-chlomelhyl sulfide .. ... Kthyl-tf-chloroet hyl sulfide 1 2 <1 0.06 0,00 100 108 100 20*2 1 <0.1 <0.1 30 30 * More recently i let er mined ami higher relative iioteoeif* bas'd mi median in dioxane) are given elsewhere.5* (8ee Chapter 23.) vesicating doses of 32 mg for H, 1 f .«■ T. and 0.3 for Q (dissolved the skin, several techniques have Iwen developed. The effect of the vapor on animals in body exposure experiments, as outlined in the section on /.(C/Ws. gives evidence that liears on the problem. The most direct and important of the experimental procedures is the use of chambers for the exposure of volunteers under conditions of temjjerature and humidity ap- proximating as closely as possible field conditions of exposure 173 (see also Chapter 23). For laboratory screening of agents on human skin, however, vapor cups or other mechanisms for producing bums on small areas of the skin of the forearm have been em- ployed. In dynamic tests, in contrast to static tests, a stream of a known concentration of (he gas in air may be directed at a small area of skin for a given time.30 M The relative vesieaneies of IT, Q, and T are list eel in Table d. The references to the, vesicancy tests on (lie wide variety of sulfur mustard compounds are included in Table 3. British surveys of the relation- ship of structure and vesicancy are included in the Bibliography.176 ,*s m 202 ** Vesicancy data are sum- marized and tabulated by NDRC (see also Chapter 23). The data on two of the more volatile analogs of H are included in Table 1 for comparison. There would he interest in a compound as vesicant as H but more volatile. The vesicant potency of II is not ap- proached, however, by any of the more volatile jS-chloroethylthio compounds. Only among the com- pounds with relative low vapor pressure have more potent analogs l>een found. These have the advan- tage of greater effectiveness in contact with the bare skin but they have poorer penetration through cloth- ing and negligible vapor action. The laboratory tests give indications of the relative activities of the different compounds. The actual Cl's or liquid contaminations required under field conditions to incapacitate troops arc determinable only through chandler and field trials (see Sec- tion 5.6). 3.5.2 s of IT, Q, and T Tlie L{Ct);.n values for total exposure of different species are summarized in Table 5. For agents in this class the L(C()u for the mouse or the rat servos as a useful index of the potency of agent even though final assessment may rest on vesi- cant action. H has an L{Ct)bo for the mouse or the rat of aland 1,000 mg min/m* and this figure became an informal base line in the screening of new agents. Potential persistent agents showing an L(Ct)m near to or less than 1.000 were usually selected for more detailed study. In the case of H, it will be noted that the species variation is small. The rate of detoxifica- tion of I! is known to be low in tests on human skin ,Mand eyes and in most of the L(Cl)m experiments, although the data in the case of the mouse show an increase in L{Cl)50 with time which is not in line with this generalization. From the data on rats the calculated detoxification constant is 0.002 mg/l.*1 The high toxicity of Q under laboratory conditions is a value which could be attained in the field only if equally efficient means for production of the aerosol could he achieved. The laboratory results van’ with the particle size obtained in the aerosol (see Chapters 12 and 15). Among the volatile analogs of IT, none of them exceed II in toxicity, bis(/3-Bromoethy 1) sulfide has about the same L{Cl)„0 for the mouse as does II.60' The methyl, ethyl,*nf ami benzyl-0-ehloroethyl sul- fides, for example, are much less active. A summary of the relative toxicities of the analogs of 11 is given elsewhere.60’1 The L{Cl)io’s tabulated in Table 5 are a function of the action of the vesicant agent on and through SECRET TOXICOLOGY Table 5, L(Cf)5„’s of II, Q, and 1’ for different species. (Exposure of the entire animal* — 15-day observation |ieriod.) UCt)M (mg min/in1) ur suggested value XuiuIkt Explore where manlier nf ,4 = anal. cone. of t hues animals is animals Agent Species (min) small X = nom. cunc. list'll Reference 11 Mouse 2 860 A 160 80 10 1,200 A 230 50 10 1,200 A ISO 123 60 1,380 A 140 SO 360 4,110 t A 277 80 Hat 2 840 / A 50 80 10 800 / X 48 50 10 850 A so 80 60 900 A 00 SO — 360 1,512 A 40 80 Guinea pig 10 1,700 X 19 50 Rabbit 10 900 X 8 50 ea. 30 1,025 V SO 83b Cat 10 — 700 X 10 50 I),.g 10 600 X 8 50 —- Goat 10 1,900 A 60 166 Monkey 10 800 X 3 50 Q Mouse —— 10 170 A KM) 50 10 350 A 120 10 270 A 200 82 Hat 10 400 X 34 .50 Guinea pig 10 >1,600 A 26 .50 Rabbit 10 2,000 X 12 50 — Cat 10 ‘MX) X II 50 __ Dog 10 1,400 X 6 50 T Mouse 10 1,050t A 180 18 HQ 90/10 Mouse 10 820 A 200 120 1 IQ 75/25 Mouse 10 770 A 240 120 NT 60'40 Mouse 10 820 A 260 123 * Tin- toxieities in this table arc for total cxjMKsurc of the animals at low rates of aii 1 flow. (Sec TaWo «.) t The aerosol w as gpiicnitwl und it conditions different from those prevailing in the preceding tests. Under the conditions nf this experiment. Q cave an L(Ct)iu of 700 ■ -— the skin of the animal as well as on the lungs, toxi- cological techniques have been worked out in de- tail 45 for differentiating the relative contributions of the actions of the agent on the lungs and on the body surface. In the case of agents such as T or Q, which were dispersed by atomization into the exposure chambers, the rale of air flow was an additional vari- able which affected the L{Ct)i0 by influencing the impingement of the aerosol particles on the skin. The most extensive studies have been made with the mouse. The results with HNl, UNH, and L are in- cluded for comparison in Table 6. The laxly exposure exiierimenls were carried out in an apparatus which exposed the body of the ani- mal to the toxic agent while the head was in uncon- taminated air. In the inhalation experiments only the head was exposed to the toxic atmosphere. The linear velocity of air flow through the chamber was Table 6. K fleets of flow rale ■ and type of exposure on !,(('!)-M’s of vesicant agents for mice.* i0 Type of Degree of explore airflow H Q I INI HX3 t. Total Low 1,200 170 000 590 1,400 High 1,400 100 900 300 900 liodv D.w 3, 500 1,500 4,800 1,000 1,1)00 High 3,400 310 3,100 370 1,200 Inhalation Low 1,600 High 1,600 280 1,300 230 1,300 1,100 1,200 1,400 1,500 * Kxpueure urr 10 mimitrs ( tit II Nl ami ranging from 5 to 15 •xorpt for some body ex injures minutes. 116 rnpli at the low flow rate and 3 mph at flu* liigh flow rate. With 11, which is present entirely in the vapor form, the L(Ct)*0 for the three types of exposure is SECRET 56 MUSTARD GAS AND OTHER SULFUR .MUSTARDS not significantly altered by change in flow rate. It might be anticipated that an increase in wind speed would tend to disturb the cushion of air held in the fur of an animal and increase the concentration of vapor at the surface of the’ skin. The experiments with HXl give an indication of such an effect fait those with II give none. In no case is the inhalation toxicity significantly affected by flow rate. With Q. which is present as an aerosol, the body toxicity increased markedly with increase in air How. This is also true of HX3, which is present in part as an aerosol, and of L (see Chapter 7). (The authors of Table 2 point out that the same toxicity for I. by total exposure and by inhalation at low flow is an anomaly not in line with the rest of the data.) For II, HXl. and 11X3. the reciprocal of (he /,(Ct),vi by total exposure corresponds fairly closely to the sum of the reciprocals of the L{Ct);,i,’s by body exposure and by inhalation, respectively.5" The body toxicities become less significant relative to the inhalation toxicities as the size of the animal increases. For the dog and the monkey the body L(Ct):,us are about 11,000 and 14,000 respectively.50 It is reasonable to assume that the />(C/)i0 of H for man by exposure which includes inhalation falls within the range of values in Table 5, namely, 1,000 to 2,000 mg min m3. In calculations on the effective- ness of II against troops, however, in view of the ade- quacy of the modern gas mask, this value has logi- cally received less consideration than those based on the production of casualties by body exposure. The power of H lies primarily in its ability to produce casualties despite the mask. In the case of man, the evidence indicates that the actual production of death by body exposure requires Cl's of more than 10,000 in temperate weather. There are no data to establish with certainty whether the L(Ct)M's of II for man by body exposure fall within the range that can be attained by feasible munition expenditures. But death is not the objective which requires primary consideration in the assessment of II. Sublethal skin injuries from IT are capable of totally disabling troops for periods of weeks and. as outlined in Sec- tion 5.6, sufficient data are available from tests on man to indicate the dosages required to yield differ- ent degrees of disability. Since man is a sweating animal and the effectiveness of H on skin is markedly affected by the degree of surface moisture (see Chap- ter 23), the results on body exposure of animals to H have no direct bearing on the estimation of casu- alty-producing dosages for man. The experiments on body exposure have had a direct use in supplying animals for study of the systemic effects from ab- sorbed H under conditions where the lungs are protected.'3* 5.5 .1 \ction of II on the Kyos The toxicity of H vapor to the eyes is a subject of special importance. Serious injury may be produced by low dosages, and loss or impairment of vision is a casualty effect of primary significance. In addition to the L(Ct)w tests, the principal vesicant gases were screened for effectiveness against animal eyes.60,1 K in the dog corneal ulceration is produced by II at C/’s of about 400 mg !.6M In the rabbit severe corneal opacity is produced at Ct 800 with clearly char- acterized ocular lesions of graded severity over the range from Ct 200 to Ct 1,200.94 ,fi7 This gradation was utilized by the Bushnell, Florida, installation as a practical bioassay for determining Ct values of II in field trials.167 The animal tests on eyes have proved useful for preliminary tests on toxicity but tests on man have been essential for the establishment of casualty- producing dosages. British investigator's have shown by chamber tests that the human eye is about four times as sensitive to H as the rabbit eye.'*7 A Cl of 100 will eause serious impairment of vision for 24 IS hours anti it is estimated that a Cl of 200 will pro- duce temporary blindness for a week or more. Liquid H and T and particles of the solid vesicants in the eye all produce extremely severe ocular lesions. The subject of the action of vesicants on the eye has been reviewed in detail by (he Committee on Medi- cal Research [CMR].66 A widevarietyof organic com- pounds was supplied by NDRC Division 9 for the studies of possible therapeutic agents for liquid vesicants in the eye. The' pathology and the physiological mechanism of action of vesicants are treated in Bait III of this report. The data reviewed there together with the sections on these subjects in the reference just cited give the basis for (he conclusion that prompt decon- tamination is the only treatment of special value in mustard burns of the skin or of the eye. 5.5.4 Toxicity in Drinking Water In connection with the program on decontamina- tion of water supplies (Chapter 39) it has been neces- sary to determine the toxicities of compounds which might lie produced by hydrolysis or in the course of chemical treatment. Methods based on oxidizing SECRET EVALUATION VS WAR CASES 57 Tabi.e 7. Toxicity in drinking water. Cone, in water (ppm) Duration of administration (days) Results Deaths on mice Weight gain Reference II-sulfoxide lOt) 30 0 30 Normal tan. H-SulfolK* 100 30 0/30 Normal 601) Sulfonium salt of II and 2 moles of thiodiglvcol 100 4 13 30 60b (in 30 days) ■ --- - - ----M.-: - -- - _-=r - 100 1 6/30 . . . , 60d 100 30 6 30 60d 50 30 0/30 Normal 60c 25 30 0/30 Normal 60c Sulfoxide of the alwive sulfonium salt 1,000 30 15/30 60h too 30 0 30 Normal 60b Sulfilimine of 11 and chlorarnine-T Silt. sol. _-<100 30 2/30 Normal tan. Thiodiglyec >1 sulfoxide 1,000 28 f 30 60a Thimliglycol sulfonc 1,000 28 0 30 60a (J-sulfoxide 100 7 0 20 60e 1/20 60e agents would yield sulfoxides and sulfones. Repre- sentative data on the toxieities in drinking water of derivatives of li are given in Table 7. For 11 and related 0-chloroethvl vesicants the maximum safe concentration is considered to be 2 ppm, as indicated by the DH-3 test, for water to be used for a period not greater than 1 week.28 In the tests on mice the sulfoxides and sulfones in this series are shown to be relatively nontoxic. The sulfoniurn salt of II and thiodiglyool, however, retains consider- able toxicity. It reacts with DB-3 and this color test thus serves to indicate active sulfoniurn salts present as well as unchanged II. b/T„ doses of sulfur mustards by intravenous, sub- cutaneous, or percutaneous routes of administration are tabulated in Chapter 22. 3.6 EVALUATION AS WAR OASES Among the more volatile vesicant agents II retains its position as the most effective war gas in this class. For special purposes the nitrogen mustards would have some uses (see Chapter 6) but, among the hun- dreds of analogous compounds that have been stud- ied since II was first used in 1917, no agent has been found to have a more advantageous combination of toxicological, chemical, and physical properties. There would have been interest in an agent as vesicant as 11 vapor but much more volatile. In the absence of an agent meeting these specifications, two approaches were, made to (he problem of increasing the rate of evaporation of H in the field. At the close of World War II, these two approaches had not reached a stage of completion permitting final assess- ment of their potential usefulness. The first was the thermal generator bomb under development-by Di- vision 10 NDRC. The second approach was the addi- tion of several per cent of a pyrogenic material, such as white phosphorus, to the H charging, an inter- esting modification developed By Division 11 NDRC/" The British, Canadian, Australian, and United States field test installations greatly extended the technical knowledge of the field behavior of H over the status of the information at the close of World War I. Division 9 NDRC' was a participating agency throughout the studies on H munitions at the Chem- ical Warfare Service Mobile Field Unit operating at Hushnell, Florida, during 1944 and 1945. The results of this extensive program, which was one of the most important phases of chemical warfare research, have been thoroughly summarized in the formal reports from Dugway Proving Ground and elsewhere.173 The chamber trials on H carried out on human volunteers in conjunction with the research pro- grams of the field installations have provided docu- mentation for the estimation of the casualty-produc- ing power of H vapor on man. Table 8 173 summarizes the quantitative information on the amounts of H vapor required to produce physiological effects of military significance. The nonvolatile vesicants Q and T, in mixture with H, possess the advantage of providing a contamina- tion of ground and materiel which would remain a potential contact hazard (but not a vapor hazard) for days under meteorological conditions where TI SECRET 58 MlSl’ARD GAS AND OTHER Sri.FUR MISTAUDS Taiile S. Dosages of H vapor for production of injuries in man.17* . _ II dosage (mg min mr) Protection F.ffect Hot and humid weather, temp, above 80 F, sweating skin Warm weather, temp. 60 80 F, skin not wet with sweat Cool weather, temp. 10 00 F, cool, dry skin Disability Time of onset Duration (Xo mask or protective Xo significant injury; maximum safe dosage 50 50 50 - clothing) Eye damage of threshold military significance _J 100 100 100 Partial 0-24 hr 1 3 days Temporary blindness 200 200 200 Total 3-12 hr 2 7 days Mask (Xo protective clothing) Xo significant injury; maximum safe dosage 100 150 too Skin burns of threshold military significance 200 ;too 1,000 Partial 2 12 days 1 2 weeks Severe genital burns 500 1,000 2,000-4,000 Partial 2 7 days 1 4 weeks Severe generalized burns 750 2,000 4,000 4.000 10,000 Total About 24 hr 1-2 weeks (Partial) (4-12 hr) (3-6 weeks) would persist for a number of hours. In contact with the bare skin, Q is the most powerful vesicant known. The disadvantages of mixing Q with H relate chiefly to the use against a target which it is desired to oc- cupy quickly, since the presence of Q would present a persistent hazard to the occupying troops.6' SECRET Chapter 6 NITROGEN MUST A R DS * Arthur C. Cope, Marshall dates, ami Hinlsey lienshaw 6.1 INTRODUCTION Dint no the 1030’s (he synthesis of various ter- tiary ftfs(/3-chloroethyl)amines, now calk'd nitro- gen mustards, was descrilied in the open literature and references made to their vesicant actions. As a consequence, these substances were investigated by the chemical warfare services of most or all na- tions before and during World War Tl. The nitrogen mustards that were found to merit the most seri- ous consideration were ethyl-b/,s(/3-ehloroethvl)amine (HX1), methyl-b/s(0-ehloroethyl)amine (HX2), /;;.v(/3-chloroethyl)a mine (HX3), a nd ~ Lsopropyl- t>?.s(d-chl( >roet hyl)amine. The toxicity, vesicancy, eye-injurant action, potential effectiveness as water poisons, relative tack of odor, order of volatility, and low freezing point of these compounds made them potential competitors of the standard persistent agent, mustard gas, bin{0-chloroethyI) sulfide (II). HN2 is not now seriously considered for use as a war gas because of the degree of instability it has l>een found to possess, and i sop ropy I -b is (fi-e h I oro- ethyl)amine is disqualified localise its toxicological potencies are somewhat inferior to those of HX1 and HX3. TTNl and HX3 remain as potential substitute agents for II. Although they are not believed to pos- sess the general utility of H, they may be of value under special circumstances. In particular, 11X3 would seem to be admirably suited for use in high explosive-chemical shell, and it was the intention of the German Army to use it in this way in the event of chemical warfare. In so far as classical chemical warfare continues to lie of military importance, the present reviewers believe that this method of em- ploying HN3 merits careful consideration from both the offensive and defensive jioints of view. This chapter is not in itself a complete review of all available information relating to the value of the nitrogen mustards as chemical warfare agents. It should lie read as a supplement to the several previous assessments that are already avail- able.11*im.im Xo attempt is made to dupli- cate the chemical and physiological phases of the subject that are covered in detail in Chapters 19 to 23. 6.2 SYNT1IKSIS VND PKOPKRTIKS 6.2.1 Synthesis Many nitrogen mustards and related compounds were prepared during World War II for evaluation as possible chemical warfare agents (see Table'I), The following four tertiary 6/.s(/3-chIoroethvl)ainines proved to have (he greatest practical importance and were studied most intensively. CHiCIIgCl CH..CH..C1 / / CH,-N CHjCH*—N \ \ CHiC'IIjCl (ll,<'ll,rocthyl)- cthyl)umine (HX2) amine (HX1) CH2(TI2CI CH*CIIzC1 / ‘ / N—CII2CH2( ’1 (( ’H3)jCII- -N \ \ CH*CH*CI (TU’1I,CI Zris(0-Chloroethyl)- Isopropyl-i>/.s(#-ehlor(>- amine (IIX3) elhyl)amine The most practical method for preparing all com- pounds of this class is from the corresponding hydroxy compounds (ethanolamines) and thionyl chloride, e.g., n(ch2(II2oh), + asocij N(CH2CH2CI),HC1 + 3S()s_+ 2HC.JI The free amines or their hydrochlorides may be used in the reaction, which is conducted either in a solvent such as ethylene chloride or without a solvent. The resulting hydrochloride salts are converted to the free bases with aqueous sodium hydroxide. This method for preparing HX3 is described in the open literature.-012#2-2M-l,s Similar preparations of HN2 are also described. The application of the method to the preparation of the large series of homologs listed in Table 1 is dis- cussed elsewhere.s-4-i-7-*-,et h ylfluoroacel amide 00c, 178 - bp** 77° 178 29 inp 65° 178 7. X-d-4 '11tons’thvlehloroacetamide* 7 mp --54° 7 8. X-d-Chloroethvll richloroacetamide 7 nip 75° 7 (t. Kthvl X-d-chloroethvloxamale 7 inp 08° 7 10. Methyl X-d-chloroethylcarbamate 7 1.4575 61 29, 134 bpM 100° 7 11. Methyl X-d-chloroel hvl-X-nit rosoca rlaimate 7, 110. 121, Hu1’ 1.4600 no 29,41. no, 102, 55 xy-p- 42 mp 106.5° 42 29, 41 phenvlenediamine 42 mp 102-103 42 29, 41 87, 2-(ft-Chloroethylamino)qninoline hydrochloride 88. «-Benzylamino-0-chlort»-0-pheiiylprop1ophenone »8k mp 140 152 48k 29,41 hydrochloride 89. 3-Bcnzylamino-a-bromo-d-phenylpr«piophenone 47 mp 152-156° 58 29 hydrobromide 90. o-Henzylamino-d-bromo-^-phcnylpropiophenone 47 mp 147-149° 58 29 hydrohromidc C. Dermilives of tertiary amines • 7 mp 144 147° 58 91. X-Mcthvlcthvleneimine -.. 29 92. d-Hthyleneiminopropionilrile 53j bp,! 67 69 53j 29 93. Methyl 3-ethylenciminopropionate 53j bp"’ 61 -64° 53j 29 94. j3-Chloroethyldimelhylamine* 134 95. Dimethyl 0-chloropropylamine If. no*1 1.4214 16 29, 41 p 123 124° 177e 177c 98. Methyl bis(0-cl11 oroe 1 hy l)a m i nc * 2, 5 Hll18 1.4679 2 29, 41, 121, 134 . ‘V ■ .. - ■ ' • • #11* 1.4682 115 1.11S 82 — ,r5 1.1203 115 — bp!u 50.0-50.5° 2 vol*° 2.487 15 99. Methyl 6(’*(/}-ehloroethyl)aminc hydrochloride* 2 mp 107-108° 2 29, 41, 129, 177a 100. Methyl b/s(d-chloroethvl)jimine formate 51a ... 101. Methyl fe/«(d-ehloroetliyliamine picrate 102. Horon fluoride complex of methvl-h(s(d-ehloro- 51a ethyl)amine 103. Reaction product of methyl 6/x(fPchloroelhyl)- .50 amine and titanium tetrachloride 104. Methyl 6/s(/3-chloroelhyl)aniinc oxide hydro- 51 e chloride 29 105. Methyl 6is(d*cyanoethyl)amine 53e bp1" 177-187° 53c 29 106. Methyl 6/s(tJ-thiocyanoefhyl)ainine 23 Decomposition on distilla- tion at 1.5 mm 23 29 107. Methyl hydrochloride 23 mp 117-118° 23 29, 41 108, Methyl 109. Methyl 0-chloroethyl-/J-hydro.vycthylaminc hy- 12, 126 ... 29, 41 drochloride 12 29 110. Methyl fl-chh.rocthyl-/3-hvdroxyethylainine picrate 12 mp 72 73° 12 ... — 111, Methyl /3-acetoxyethyl-d-ehloroethylamine* 32, 126 n ir‘ 1.4474 32 43, 106b bp"14 540 32 bp" 111 46° 32 bp" w 41° 32 SECRET SYNTHESIS VM) PROPERTIES Table 1 (Conti n ued). Compound Reference to synt hesis Physical properties Property Reft ‘nmee Reference to toxicity ami vesica ncy data 112. |)imeiliyl ,t, d'-dichlortr-fcri-butvlamine* 113. Methyl /}-chloroethyI-/3-chloropropylamine* 134 114. Methyl d-chloroet hyl-d-chloropropylamine* ... 134 115. Diethvl (4-chlor(x‘l h vlaminc* 116. Diethyl 3-chloroethvlamine hydrochloride 16 mp 210-211.5° 16 117. Kthvl bix(#-chloroethyl)Hii)iHe* 2, 10, 120 «n5* 1.4639 2 29, 41, 134, _■ 143 d 1.083 2 hp» ‘ 49.0-49.5° 2 TT vol" 1.59 31 118. Kthvl bix{ /S-chlomethyl (amine hydrochloride* 2, 10 nip 139-140° 2 137 119. Kthyl /S-ehloroct hyM-hydroxycthylamine picryl- sulfonate 17 mp 110 111 17 _ 29 120. 0-Methoxvethvl 6f's(j8-chloniethyl)amine 23 «n** 1.4671 23 29, 41 y 1 hix( fi-chloroet hyl)aminc* 16, 120 « n27 1.4029 16 29,41, 134, — 1 13 ,r-« 1.092 16 148 bp* 62-63° 16 vol2" 0.783 31 SECRET N ITROEEX MUSTARDS Table 1 {Continued). Reference to Physical projicrtics Heference to toxicity and Compound synthesis Property Heference vesicancy data 144. Propyl bisl 3-chloroethyl)amine hydrochloride* 16, 120 mp 118-120° 16 137 145. 146. lsopn>pyl-6iJi(d-cMoroetliyl)anune* 2,9 n hM 1.4641 2 29, 41, 143 d 1.053 2 bp2 5 67.0 68.0T 2 ' “ mp 13.7 S3 - V«>l“ 0.869 15 147. Isopropyl-5ts(/J-chloroelhyl)amine hydrochloride* 2, 9 mp 210 213 (dec.) 2 It, 137 148. X-/J-Chloruethylpij)cridine 11 bp* 40 11° 11 29. 41 140. X-0-Chloroethylpiperidine hydrochloride 11 mp 230 11 150. Hutyl-6/x(d-chloroethyl)amine 16 no5* 1.4637 16 29, 41 d26 i ,027 16 __ , ... bp2-* 89.(V 89.5° 16 ‘ vol-® 0.321 31 151. Hut vl-5is(d-chloroethyl)aniine hydrochloride 16 nip — 96-97* 16 152. 7 -Chlorobut vl-6ix(d-chloroethyl)ainine 29, II 153. y-Oxobutyl 5»«(0-chloroethyl)amine hydrobromide . . . 29 154. xrC'Hutyl-6i.sO-chlori ir‘ 1.4655 16 29, 41 d21 1.028 16 bp* 84-84.5° 16 rr. v«l,# 0.394 31 155. «er-Rutyl-fc(s(/3-chloroethvl)amine hydrochloride 16 mp 132-138° 16 156, Isob«ilyl-/)ix(/J-chlorocthyllamine 16 «,.2? 1.4597 16 29, 41 - _ .r< 1.0078 16 bp1 81-81.3° 16 - . . vol20 0.508 31 157, Isobutvl-6/x(d-chloroethvl)ainine hydrochlorides- 16 mp 107-108° J6 158. 0T/-Hutyl-b/8(#-chloroclhyl)amlne 16 n tf° 1.4710 16 29, 41 • — if-' 1.032 16 • , bp1* 68-69° 16 • vol2" 0.581 31 159. lcr/-butyl fcr.s-(0-chloroethyl)- amine hydrochloride 23 mp 112.2-114.2° 23 41 162. /3-Chloroethy!-6i*(d-chluropropyl )aminc* 163. 0-Chloroethyl-fws(0-chloropropyl)amine hydro- .... . chloride* .... 164. 1 urfuryl 6/»((?-chloroelhyl)aminc 23 nr.a 1.5033 23 41 1.171 23 bp11-"1 106-107° 23 165. Furfurvl-6is(/3-chloroet hyl )amine hydrochloride 23 mp 88.5-89.5° 23 29, 41, 43 166. Tetrahydrofurfuryl-hisO-chloroethyl )aminc 23 nr.24 1.4877 23 29, 43 d” 1.129 23 “ ‘ . bp«* 82-84° 23 167. Tetrahydrofurfuryl-6/jt(0-chloroethyl)amiiie hy- drochloride 168. d/-X-(/J-Chloroet hyl)-2-chloromelliylpiperidinc 23 mp 117 118° 23 hydrochloride* 169. 6(or 7)-Chloroetronccane 30 nn” 1.4913 30 29 170. 6(or 7)-Chloro-l-ehloromothyl-l ,2-dehydropyr- bp” 111 112° 30 roiizidine hydrochloride 30 mp 122-123° 30 29 171. triaf/J-Chloropropyl)amine II bp2 99-103° 11 29 172. X-Kthyl-X-C^-chloroethyDaniline 11 bp°* 102 109 It 29, 41 173, X,X-hid d-( 'hloroet hyl laniline* 11 bp0-7 123° 11 29, 44 mp 43-44° II SECRET 65 SYNTHESIS \M) PROPERTIES Table 1 (Continued). Reference to Physical properties Reference to toxicity and Compound synthesis Pro|>erly Reference vesica ncy data * 174. \, N -hig{ (3-C hh in >et hyl t-p-nitrosoaniline 53c nip 72 53c 20. 41 I7.i, Cychihexyl-fH*(d-chhinx‘t hyl )a mine 16 n n5i 1.1040 16 20, 41 d-' 1.077 16 bp11 105-105.6° 16 vol*° 0.0383 31 17(1. Cvclohc\vl-fci»(itJK,liloroethvl)ainino hydrochloride 16 mp 174 175’ 16 177. Benzyl-hisfd-rhlonxOhynnmine 16 Hi)55 1.5334 16 20. 41 (f3 1.112 16 » b|)- 138-130’ 16 178. Itenzvl-b/x(jJ-chlori <1.0 2 20 1 SO. 1 lop) vl-bix{d-chloroethyl famine hydrochloride 2 41 181. Phenethyl-h»s(d-chloroethyl)ainine 182. X, X. \'-Mrfi/r(«(d-Chliiroelhylfelhylene- 53r bp* 06° 53k 20 .1. diamine di hydrochloride 183, l,3-h(»(fei#(d-Ch!orocthyl)amii»o) propane dihydro 40 20, 41 chloride. 184. 1, 3-fu«[ bis( ii-( hloroelhyl )amino]-2-ch!oropropane 32 nip 138 130’ 32 20 di hydrochloride 32 nip 141-141 2U 32 20 1 So. hix{ff-(his(d-Chlonx*f hylfaminof-ethyl] sufide* 23 'll.'* 1.5287 23 20 186. 6. Derivatives of quaternary ammonium mils 106. Trinielhyl-d-fluoroct liylaminonium bromide* mp 244° (dec.) I77d 177d 107. 1 )imethyl-bi«(d-chlorocth vl)ammoninm chloride 108. d-.\rcloxvcthvl-d-chlon«‘lhvldimclhylammonium 29, 41 iodiile* 126 mp 150° (dec.) 126 100. Mcthvl-/r/.s(d-chloroelhvl)ammonium chloride* . . . .... 200. MethyHri«(d-chloroethyl)amn!oni»im sulfate* 201. Elhylvinyl-5is(d-chloroelhyl)ammonhim chloride 32 mp 102-106° (dec.) 32 20 202. Triethyl-d-fhionx'thylammonium bromide* 203. d-Carbaimixyel hylet h y 1 -6/*(d-chi oroe t hylfammo- mp 237° (dec.) 177c 177c nium chloride 23 20. 41 204. d-KIuoroethylpyriiliniuni bromide* 205. X,X-f»is(d-Ch!orocthvl)pjix'ridinium chloride, mono- mp 180’ 177c 177c hvdrale . . . 206. Polymer of mefhyl-fei.’Kd-chloroethyDamine (« = 21 2 20 207. Polymer of met hvl-hi>(d-ehIorcx't hyl (amine 2 20 208. l,4-f»s(d-f ’hlonxdhylf-l ,4-diethylpi|>eraziniuiii di- chloride 200. Ammonium comiKHind from 1 mole of methvl-fc/«(d- ... 20 chlornethvl)amine ami 2 moles of methyhli- ethanolamine 20 Detailed studies of the preparation of the four most important nitrogen mustards on a laboratory, pilot plant, or manufacturing scale are reported in the following references. SECRET 66 NITROGEN MUSTARDS Agent References HNl 2, 4, 10, 71. S7, 05, 07, OS, 90, 136. 182d HN2 2, 4, 5, 82, 120, 158, ICO, 182a, 185a, 185b, e, <1. 102, 104, 196 11X3 2,4,5, 68, 80, 02, i)6, 157, 170. 171, 172 Isopropyl-6is(0- chlorocthyl)amine 2, 4,83,136 Work has been done on alternate syntheses which do not employ thionyl chloride. Results have been discouraging.*4 ** The best alternate method for HNl uses phosphorous trichloride in place of thionyl chloride, and gives yields approaching 75 per cent.16 Other reagents used with less success are phosphorous pentaehlondc,206 phosgene,1'41’-'1 193 and hydrochloric acid.1'4*’ The alkyl-6is(d-hydroxyethyl)amines and hydroxyet hy 1) a mine required for synthesis of the nitrogen mustards have liecn prepared commer- cially by reaction of primary amines or ammonia with ethylene oxide. Some work has been reported in the classified literature on such reactions and on the purification of technical ethanolamiues for use as nit rogen mustard intermediates.'s«.iei.i«.i*7,iB». 16»aT2,lS5a,r.l92,194,196 Because of the possibility of a short supply of ethylene oxide in the event of large-scale nitrogen mustard manufacture, methods which did not em- ploy ethylene oxide were investigated for preparing alkanolamines, particularly RN(riI,(TI2()H)2 (where R is ethyl, methyl, or isopropyl). The most success- ful method developed utilizes formaldehyde, hydro- gen cyanide, and ethyl alcohol as the basic raw materials and follows these steps: 1. HCN + CHjO CHjOHC’N 2. rn,oiR’N + (C.H:.0)2chs —► C*Hi(X'H20( 'Il*(’N T CjlUOH 3. 2(',IIiOCTljOCH2CN + 1H2 (f,2H5OCH2OCH2CH,)2NH + Nil, 4. 2(C2H;,0(TI2OCI 12('H2)2NH -(- (C2H;,)2S()4 + Xa.ro, —> 2(C,HiOCn2OCH2CHI)2Nr,Hi + Xa2S04 + h,o + ro2 5. ((‘2H5orH2oriU'ii2)2xr2H, + hci + (,11,011 —► (CH2OHCH.)2NC2Hi-H(,l T- 2rH,(0( ,11.5)2 This “formal” route, in which formaldehyde cyanohydrin is converted to a less sensitive formal derivative before hydrogenation, is estimated to Ik* capable of producing N-ethyl diethanolamine hydro- chloride at a cost of 25 to 30 cents per pound, at an annual rate of 10.000,000 pounds.2" A more direct route, in which formaldehyde cyanohydrin is hydro- genated directly to diethanolamine (subsequently alkylated), gave poorer yields and appears to In* a more expensive process.2" Methyl-6>«(j8-liydroxy- ethyl)amine has been prepared successfully by hy- drogenation of diethanolamine in the presence of formaldehyde.19 Other less advantageous routes to the alkanolamine intermediates for HXl, HN2, and HN3 have been explored.1" In addition to the stand- ard method of preparation from isopropyl amine and et hylene oxide. isopropyl-Iu’s(/3-hydroxyethyl)amine has been prepared by hydrogenation of a mixture of acetone and diethanolamine,19 or by the react ion of ethylene oxide with isopropyl-d-hydroxycthylamine. The latter compound is prepared by hydrogenating a mixture of acetone and-cthanolamine.199 _ 6.2.2 Physical Properties The nitrogen mustards ai*e oils of limited water solubility. They are miscible with ordinary organic solvents. Their physical properties have been ex- tensively studied. i«7.i«s.i89.jwb.c gome 0f t tie constants having most- liearing on chemical warfare are presented in Table 2. 6.2.3 Chemical Properties The nitrogen mustards are basic amines which form stable salts with strong acids such as hydro- chloric acid. They are active alkylating agents, and the physiological reactions responsible for their toxicity are primarily alkylations. Their reactions from the biochemical, physicochemical, and physi- ological mechanism standpoints have been studied in great detail and are summarized in Chapters 19, 20, and 21. A primary intermediate in their reactions is a l-(/3-chIoroethyl)ethylenimonium ion, formulated 1 >elow for HN2, which is analogous with (he ethylene- sulfonium compound1* intermediate in the reactions of H (see Chapters 19 and 20).33 125.127,130.135,151 .ISltt.b Self-alkylation is responsible for the dimerization which occurs slowly when the lower molecular weight alkyl-6f«(/J-chloroethyl)amines are allowed to stand. The reaction is rapid in the presence of water. It re- sults in the deposition of crystalline solids such as the “dichloroeyclie dimer” which is formed from HX2; '■ Ktliyloni'-Siilphoninrn Compound ClCH;CIIj + SCHjCH.CC SECRET SYNTHESIS \ND PROPERTIES 67 Tabi.r 2. Physical [»reen exploded without evidence of gross decomposition except in 75-mm shell under condi- tions more severe than are encountered in the field.M 66 89 However, in the most quantitative in- vestigation that has been made, one of six M47A2 bombs charged HXl flashed on static detonation.66 Although considerable lexicologically effective vapor was subsequently evolved from the terrain contam- inated by the explosions in these tests, the dosages were less by an amount approaching 30 per cent than would have been predicted from similar tests with H had no decomposition of HXl occurred during the explosion or, subsequently, on the contaminated terrain.66 -— The available data for I1N2 are of a rather quali- tative nature. It appears that this agent can be dis- persed without complete destruction by explosion of various chemical munitions but that the toxicological effects of the initial clouds so produced are inferior to those produced by H or HT.7* 7"7114,47 IH!M*0 Stability on Terrain Two imjiortant characteristics determining rate of inactivation on soil and vegetation are solubility in water and rate of reaction with water. The approxi- mate data given in Table 3 lead to the prediction of HXl and IIN2 vapor wore greater than those of If, as predicted from the relative volatilities, the total dosage (C7) of evolved vapor was in the eases of HNI and HN2 only one-half of that anticipated from similar trials with H. In annulus trials with HXl and If in Florida, large drops were used on relatively dry terrain and the 1-hour vapor dosages failed to reveal significantly greater ground losses for HX3 than for If.6* As stated almve, however, in trials with single, statically fired homhs the vapor evolution of HXl was somewhat less than would have been pre- dicted on the basis of tests with If. Fart of the loss may have occurred during the explosion of the bombs, and part subsequently on the terrain. Ter- rain losses would have been facilitated by the dis- pei-sion of the agent into small droplets during the explosion. 6,2.3 Detection and Analysis Excellent methods for the detection and analysis of the nitrogen mustards arc available (see Chap- ters 31 and 37). For purposes of detection the use of the DH-3 re- agent (see Chapter 34) is perhaps the most useful. As used in the United States MO Detector Kit, this test is approximately as sensitive for the nitrogen mustards as for H. Collection of 0.1 to 0.2 Mg of HN 1, HX3, or H from air containing 0.2 Mg 1 or more of these agents suffices to give a positive reaction.112 A supplementary test to differentiate nitrogen mustard from H is somewhat less sensitive. For purposes of analysis, among the most useful methods are those utilizing the DB-3 reagent and those dependent upon the mercurimetric titration of the chloride obtained by hydrolysis from the nitro- gen mustard (see Chapter 37). 6.2.6 Decontamination and Protection The gas mask canister offers complete protection against the nitrogen mustards. In general, standard methods of decontamination effective for H are useful for the nitrogen mustards, but destruction of the nit rogen mustards by chemical reaction with bleach or with currently used chlor- amides is notably less efficient and rapid than in tin* case of II 117 (see Chapters 21 and 32). 6 3 CHEMICAL STRUCTURE IN It ELA- TION TO TOXICOLOGICAL POTENCY Among the nitrogen mustards and related com- pounds that have been studied (see Table I), the Tabi.k 3. Characteristics influencing rate of inactivation of II, fIN' 1, 1I\2, and I1N3 on terrain. Approximate solubility Approximate half- in water at room tom- life in water at Agent peraturc (ppm) 25 C (minutes) 11 500 8 HXI 1,000 + 1.3 IIN2 13,000 + 4.0 II N3 SO _ 2.4 greater losses with HXl and HX2 than with HN3 and TL This prediction is confirmed by the available field data. There is no evidence in the semiquanti- tative data of bomb and annulus trials that losses on moist terrain are greater for HN3 than for II, in spite of the greater persistence of HN3.*S The results of British annulus trials with HXl and HN2 on alkaline sod (Porton downland) indicate that ground losses are significantly greater than in the ease of H.1,5 IS* Although the initial dosages of evolved SECRET TOXICOLOGY highest toxicological potency is found among the tertiary 6fs(/3-ehIoroethyl)amines, and, in particular, in HXl, HX2, HX3, and the propyl and isopropyl analogs of HXl. The toxicity, vcsicancy, and eye- injurant action of these compounds is reviewed in the following sections. 6 1 _ TOXICOLOGY 6.1.1 Detectability by Odor and Sensory Irritation HXl, HX2, and 11X3 arc markedly less detectable by odor or sensory irritation than is H. Testimony to the insidiousness of the vapors of all three nitrogen mustards comes from plant accidents in which men. informed of the potential hazard, were incapacitated without being aware of having been exposed until eye and respiratory symptoms developed after a lapse of several hours.*9174*175 Laboratory (osmoscopic) determinations of the median detectable concentrations of H, HXl, HX2, and HX3 are given in Table L Attention is directed odors. The pilot plant HX3 that was made in Eng- land has a faint geranium-like odor. Inasmuch as oral reports indicate that laboratory-prepared sam- ples do not possess this odor, it may be suspected that the geranium-like odor was due to an impurity, possibly associated with the preparation of the ma- terial in equipment previously used for lewisite. This pilot plant material was used in the osmoscopic de- termination cited in the preceding paragraph. Thus, it is possible that other samples of plant run ILX3 would be even more odorless, and therefore more insidious. 6. Toxicity Toxicity data for animals totally exposed to air- borne HXI, 11X2, and HX3 are set forth in Tables 6, 7, and 8. From the summary presented in Table 5 it Tabus 5. Summary of toxicides of H, 11X1, I1N2, and HX3 in the form of vapors. (See Tables 6, 7, and 8 of this chapter and Table 5 of Chapter 5 for more detailed data.) Agent (mg min nr1) Mouse* llange for (/ = 10 min) other species) Ms! i mated relative toxicity (H_» 100) H HX1 TIN2 HX3 1,100 IKK) 2,800 _ - 900 500-3,000 2,000 1,000 6,000 550 500-2,000 100 »100 50 5 100 ♦ Fiftocii-diay observation period, t Approximate. T vbi.k 4. Median detectable concentrations of 11, 11X1, HX2, and 11X3 as determined in the laboratory by the osmoscopic technique. A Rent Purity Median delectable 11 Plant run Ixtvinstein 0.6 00 Vnctiuni-disl ille- parent only at much higher concentrations.141 Presumably use of this technique with H and other nitrogen mustards would give values correspondingly low in relation to the median delectable concentrations as determined in the labora- tory. SECRET 70 MTROGRN ML STARDS Tabi.k 6. Toxicity of 11 XL The animals divcn in parentheses. were totally exposed, /-K’0,„’s that are estimated very approximately are i HCD* (mg min in3) Exposure time (min) Observat ion licriod (days) Analytical (A) or nominal (X) cone. Xiimler of animals Notes Reference Mouse 900 10 15 A 2S0 Low-flow chamber 37 900 10 15 A 89 High-flow ehamlier 37 1.300 10 10 X 1 10 Low-flow ehamlier 85 < 1.200 9ti0 30 20-100 15 15 A A 30 140 Static chamber Large ehamlier; 90 F; wind 143 1,100 20 100 10 _ A 140 sjieed without effect I O le Hat— (7.50) 10 30 X 10 Low-flow chamber 37, 14a <1,200 30 15 A 34 Static ehamlier 1 13 800 20-100 10 and 15 A 84 I.argeehamlier; OOF; wind s|*eed without effect 104e Guinea pijr (2,500) 10 30 X IS Low-flow chamber 37, 14a (1 ,.500-3,000) 30 15 A 36 Static ehamlier 143 Rabbit (1,000 3,000) 10 30 X 5 Low-flow ehamlier 37, 14a ‘MX) 30 15 A ot> Low-flow chamber; 90 F 71 (1,000) 30 15 A IS 1/ow-flow ehamlier; 73+ F 71 ( > 4.000) ;io 15 A - 15 Static ehamlier 143 900 1,100 2a loo 20-100 15 10 A A 84 84 Ijarge ehamlier; 90 F; wind speed without effect 104e 910 300 15 A 54 Ijow-flow ehamlier; 90 F 71 Cat (400) 10 10 30 X 12 Low-flow ehamlier 37, 44a lh>K (800) 10 10 30 X 14 Low-flow ehamlier 37, 44a Goat (1,500 3,000) 30 15 - A 9 Static ehamlier 143 Monkey (1,500) 10 15 X G Low-flow ehamlier 37 flow rate, temperature, use of an anesthetic, and variable delayed deaths due to secondary infections. Wind speed or flow rate is only of marked im- portance for toxicity when aerosol is present.37 "*4'* HNS when present in part as fine drops is much more toxic at high flow rates than at low flow rates.37 '40 At high flows a greater liquid dose is deposited on the skin, from which it may be absorbed after exposure both directly and indirectly as a result of licking and inhalat ion of vapor.37140 A number of data are available on the toxieities of the nitrogen mustards to animals exposed totally, by inhalation only, and by laxly only.37 131 144 In the ease of mice totally exposed to HNS, the absorption of the agent from the body surface compares in im- portance with that directly inhaled.37 It is doubtful, however, that casualties among troops in the field would be produced by the systemic effects of nitro- gen mustard absorbed through the skin except when the exposures are already more than sufficient to produce vesicant effects of incapacitating sever- ity iM.m Relatively high concentrations of the nitrogen mustards produce symptoms of irritation during ex- posure •4.o.7i.7»,*,,>t»..12'.o' hut lower concentrations arc without immediate effect.7'131 Symptoms then develop only after a latency of one to several hours and the most conspicuous pathological changes ap- pear in the eyes and respiratory tract.84-7'-*3 m I)ea(h in lethally dosed animals is usually delayed for one day to two or more weeks, depending on dosage. Detailed pathological studies have been made rr.rs.si ,»,m is»,ho ui ,t« Data on the toxieities and pathological actions of nitrogen mustards administered percutancously, orally, and by injection are presented in Chapter 22. From the practical point of view it may be noted that production of casualties from (he drinking of con- taminated water could easily occur. The available chemical tests are, however, sufficiently sensitive to reveal potentially dangerous concentrations of the nitrogen mustards and their toxic products of partial hydrolysis11 ,1,200) 2 ? A 24 Static chamlier 121 (3,000) 5 10 A 24 Static chamlier 121 (5,500) 10 15 X 12 Low-flow chamlier 44 a (3,500-7,000) 10 MKT) X 16 Static chamlier 177b (3,000- 6,000 r 10 5 A 20 Static chandler 121 (4,000-8,000) 20 10 A 30 Static chamlier 121 (3,000-6,000) 30 7 A 30 Static chandler 121 (>3,800) 60 120 9 A 8 Low-flow chamlier 13! (2,.500 5,000) 240-450 9 A 14 Low-flow chandler 131 Rabbit (> 1,200) 2 25 A 24 Static chamlier 121 (1,000-3,500) 5 26 A 24 Static chamlier 121 (4,400) 10 15 X 4 Low-flow chamber 44a (7,000 14,000) 10 io< ?) X 12 Static chamlier 177b (3,000) 10 15 A 10 Stat ic chamlier 121 (2,000-8,000) 20 28 A 30 Static chamber 121 (3,(XX) 6,000) 30 26 A 20 Static chamlier 121 Cat <1,400 10 10-30 X 8 Low-flow chamlier 4 la- Don (2,000) 10 10 30 X 4 Low-flow chamber 44a Goat (1,000) 2 13 A 8 Static chamber 121 ( < SOO) 5 22 _ A 8 Static chamber 121 (< 1,700) 10 9 A 6 Static chamber 121 (<2,000) 20 9 A 8 Static chamlier 121 (1,000 2,000) 30 9 A 10 Static chamber 121 through clothing is of more practical importance than action on bare skin because usually most of the laxly surface, including the areas that are at the same time the most sensitive and the most critical for incapacitation, is clothed. In addition to vesi- cancy through unimpregnated clothing, the degree of protection offered by chlorarnide-impregnated clothing and clothing containing activated carbon mjnires considc• ration. A further differentiation must l>e made lietween situat ions in which the skin is relatively cool and dry and situations in which it is hot and moist. As in the case of H. high temperatures and humidities and physical exercise augment the sensitivity of men to SECRET 72 NITROGEN MUSTARDS Table 8. Toxicity of 11X3. Riven in parentheses. The animals were totally exposed. /.(Cf)5„’s that are estimated very approximately are Analytical' . (A) Ol KxposiiPe Observation nominal Xumlier iACtho time period (X) of Species (mjj min/m1) (min) (days) cone. animals Xotes Reference Mouse (1,700) <2 18 A 132 Fine aerosol 144 500-000 10 14-15 A 58 Aerosol-free vapor 101a .500 10 15 A 230 Low-flow chamber 37 300 10 15 A 60 High-flow chamber; aero- sol present 37 (10.-,) 10 15 A 20 Vapor; wind tunnel; 05 K 37 1,700 10 10 N 160 lane-flow chamber 78 .-,70 10-100 15 A 130 Vapor, 00 F, 85% humiditv 1 Ole Hal (800) 0.25-2 20 A 104 Fine aerosol 144 1,700 10 15 N 28 Low-flow chamber .54 (SOO 1,500) 10 15 A IS Low-flow chamlser 37 (? 1,000) 30 ? A 50 Static chamber; 85 F 139 670 10-100 15 A 60 Vapor, 00 F, 85% humiditv 104 c Guinea pig >2,300 10 ? \ 10 Low-flow chamlicr 37 >1,000 30 ? A 45 Static chamber; 85 F 130 Rabbit (585) 3-15 15 A 12 Vapor; wind tunnel; 5.5 mph 05 F —- 37 (1,000-3,000) 10 10 X 11 Low-flow chamber 37 (500) 10-18 15 A 8 Low-flow clmmlicr; vapor only; 100 F 37 (830) 18 50 15 A 30 Low-flow chamber; vapor only; 72 F 37 (>1,000) 30 ? A 31 Static chamber; 85 F 130 635 10 100 15 A 70 Vapor; 00 F, 85% humidity 104c 550 ± Ioiir 15 A 60 ± Field tests 6.5 Cat (100 1,000) 10 ? A 32 Low-flow chamber 37 Dor (400-1,500) 10 ? A 36 Low-flow chamber 37 <1,350 30 ? ? ? I/Ow-flow chandler 187 Goat (500 1.000) 30 ? A 18 Static chamber; 85 F 130 the vesicant effects of the nitrogen mustards (see Chapter 23). So far as is known the time course of development of injury and incapacitation due to skin injury ap- pears to be comparable for H and the nitrogen mus- tards (see Chapters 5 and 23). Some evidence exists that nitrogen mustard bums are shallower than II burns and heal more quickly.*9108 n715* On the other hand nitrogen mustard burns have been referred to as more tender and painful than H burns.*9191 How- ever, a sufficiently complete and realistic determina- tion by means of performance tests of the relative extent and duration of incapacitation produced by lesions due to H, HNl, IIN2. and HNS remains to be made. Thusat present evaluations must l>ebased prin- cipally on lesion-producing effectiveness rather than on the more pertinent criterion of casualty-produc- ing effectiveness. Furthermore there is no informa- tion as to the effects of large dosages of nitrogen mustard vapors upon masked troops. In the case of H it is known that severe exposures under tropical conditions produce incapacitation within one hour of exposure tine to temporarily incapacitating nausea and vomiting followed rapidly by the development of very severe cutaneous injury."3 No evidences of systemic injury have been apparent in any of the man-chamber trials with HNl and HNS at the rela- tively low dosages that have been utilized.1041’-*-'111 Vesicancy of the LiQrms 1. Cool and temperate conditions. For the produc- tion of lesions on bare skin when free evaporation is permitted and decontamination is not practiced, tin* order of vesicant potency is fl > HNS > HN2 > HNl. The relative weights of the small liquid drops required to produce blisters at 50 per cent of the sites of application are;39 44'•e f*h i "91108 II 1 HNS 2-4 HN2 - 4-8 HNl >8 SECRET TOXICOLOGY 73 The agents fall in the same relative order when evaluated by more realistic tests in which the sizes of the lesions produced by large drops are com- parcd.,4VIM-,i“ None of the other nitrogen mustards and related compounds are as vesicant as HN2 or, probably, as vesicant as HNl (see Table 1).o.h*u»i When effective decontamination is practiced I to 5 minutes after contamination, II produces markedly greater lesions than HN3.1*7 The positions of HNl and HN2 are not known with certainty; HN2 may lo only slightly inferior to H m and IlNl somewhat inferior to HN2.*49 Antivesicant ointments (i.e., United States Mo and British A.G. No. 6) available to Allied troops during World War II do not destroy the nitrogen mustards as they do H. but. their bases are good solvents for the nitrogen mustards. The latter are effectively decontaminated by solvent and mechanical action when large amounts of ointment are applied and then wiped off.,w4*n*197 If the oint- ment is left in place on the skin, the dissolved but undestroyed nitrogen mustard may slowly exert its vesicant action and the lesions produced by small doses of H and 11N3 bi*come comparable in sever- itv.M.m.e Dilute acids also exert a solvent, action on the nitrogen mustards, and such oxidizing agents as permanganate in aqueous solution may l>e utilized as decontaminants. In addition, some chloramides not in general use by the Allies during World War II do destroy nitrogen mustard readily. Notable among these are 8-43(5, NCI, NCI, I I C.HS—C-=N—C =N—C=N 1 j and the German Decontaminant 40, O ('1 O Cl O Cl 111 II I II I C—N—C—N-C-N L_ ~~ 1 (see Chapter 24). Through one or two layers of unimpregnated cloth (he order of lesion-producing potency is II > HN2 > HNl ~ HNS.5"14*14* The order found for bare skin is modified because of the importance of vapor pressure for the transport of the agent through the cloth and to the underlying skin. Through cloth impregnated with CC-2 the nitro- gen mustards gain in effectiveness relative to H lo- calise of the comparative ineffectiveness of CC-2 as a decontaminant for nitrogen mustard. Laboratory data insufficient to permit a conclusive estimate sug- gest the following order of potency; HN2 > H > HNl > IIN3.9' Realistic trials under field con- ditions are lacking. 2. Hot and humid conditions. The scanty avail- able data do not permit an evaluation of the relative potencies of the three liquids under tropical condi- tions. The lesions produced by small doses of the liquids on resting men and on men exercised to the |K»inf of sweating under temperate conditions suggest that the differences which would he observed among the agents if they were tested under severe tropical conditions might he less pronounced than those which have been obtained on relatively cool, dry skin.44' Vksu anov of thk Vacuus 1. Laboratory evaluation of potency. Laboratory data which relate to the production of lesions on limited areas of the skin of the forearms of men not acclimated to hot summer weather and exposed un- der relatively moderate ambient conditions of tem- perature and humidity demonstrate that on a dosage basis HNS is equal to or slightly more effective than II. that IIN2 is definitely inferior to both Tl and HNS, and that HN1 is greatly inferior to each of the three other agents (Tables 9 and 10).58M Table 9. Vapor train tests of the vesicant potencies of the vapors of H, 11X1, HX2, and 11X3.“ The subjects were at rest. T = 80 F. Analytical Ct (mg min/m3) for 50 per cent resfMinses Relative Agent Krytheinas Hlistcrs dosage* H <430 2,300 I 11X1 2,700 + >21,000 >8 11X2 1,200 + 3,800 2 + HX3 400 ± 1,800 0 .7 t- * Reciprocal of vesicant potency. Tabi.e 10, Vapor cup tests of the vesicant potencies of the vapors t .f 11, UN 1, and HN3.« The subjects were at rest. T = 72- 73 F. Kstimated median vesicating dosage in mg min/in3 Relative Agent (/ = 5-60 min t dosage* 11 3,500 1 11X1 IS,000 5 + HX3 3,700 1.1 * Rwiprofftl of vifUfant latency. These relationships for II, HNl, and HN3 are con- firmed by arm-chamber studies at high temperatures SECRET 74 NITROGEN ML'ST ARDS Tahi.k 11. Basie man-chamber tests with II and nitrogen mustard vapors: Cubed States Army data.,""M‘-,l-r T — 90 F. Relative humidity = 85 per rent. All subjects wore gas masks, shoes, and socks. Additional Vapor exposure Genital clothing Number dosage time Season protection and protection of men (mg min in1) (min) Kffeets // Summer ('C-2 impregnated shorts None 3 106 10 Moderate erythema of neek. back, and legs. Summer CG-2 impregnated shorts None 6 200 20 ± Severe erythema of neck. — thorax, abdomen, and legs; — some delayed superficial vesication. - ■ uni SnmmiT (’('-2 impregnated — shorts None 3 107 11 No effects. Summer CC-2 impregnated shorts None 3 211 *22 No effects. Summer CC-2 impregnated shorts None 3 285 30 Questionable erythema of neck. Summer CC-2 impregnated - shorts None 3 520 34 Mild erythema of neck; 1, mild erythema of back. Summer CC-2 impregnated shorts None 3 689 41 Mild erythema of nock and — body. Summer CC-2 impregnated — ‘ - . ■' shorts None* 3 940 44 i moderate and \ moderate erythema of up|MT trunk. Summer CC-2 impregnated shorts None 3 1,030 29 | moderate erythema of axil- — ■ larv folds; J mild cry- — thema of upper back and neck. ■ — - HN.i* Winter or I’n impregnated early spring shorts None 2 90 15 No genital injuries: minimal Winter or Carbon-eontain- erythema over exposed early spring ing shorts None 2 90 15 skin, marked over neck, - - back, and anterior axillary _ folds. Winter or Carbon-eontain- - “ early spring ing shorts None 3 150 25 Generalized moderate cry- thema at 20 hours which had reached its maximum and begun to decrease by 96 hours. Krythema most — pronounced on neck, hack, — and anterior axillary folds. Winter or Carhon-eont ain- early spring ing shorts None 4 200 ? 3 slight and 5 moderate erv- thema of trunk and neek; \ minimal erythema on legs. Winter or Carlxin-rontain- early spring ing shorts None 3 250 ? Slight erythema of trunk, ... moderate erythema of neck; § minimal erythema of legs. * Wind apecd in the chamber seemed to lx* without effect and has been dforcgi ided in compiling this table. SECRET TOXICOLOGY Table 11 (Continued). Season Genital protection Additional clothing and protection Vapor Exposure Number dosage time of men (mg min in') (min) Effects Winter or Carlton-conlain- U\J* — early spring ing shorts None 8 300 ? * slight, ( moderate, and I marked erythema of t runk; more pronounced cry- Winter or ('arbon-contain- thema of neck; minimal erythema of legs. early spring ing shorts None 1 390 ? Areas of vesication on trunk and neck; marked ery- thema with edema and moist desquamation of ears and preauricula r — areas; slight erythema of scalp; minimal erythema of legs. Winter or Ca (boils contain* Xonimpregnated t 3n0 ? ( slight erythema of neck; early spring ing shorts 2-piece herring- — J vesication of neck. twine (will suit, M5 ointment on neck. * Wind speed in the chamber seemed to be without HTcct and ha** been d»regardf*d in eoinpiliiiK this table. and humidities with the exception that, when tests were made with sweating observers acclimated to hot summer weather, UN I assumed a much more favorable relative position, requiring only 1.2 to 1.6 times the dosage of H to produce equivalent lesions.109 The effectiveness of II. HNI, and HNS vapors in these tests was little affected by the inter- position of a layer of unimpregnated cloth.109 2, Man-chamber evaluation of potency. The only available man-chamber tests of the effects of nitro- gen mustard vapors on observers wearing no cloth- ing or unimpregnated clothing are summarized to- gether with representative data for IT in Tables 11 and I2.,tab e ,, r 110 1,1 These data relate to a chamber temperature of 90 F and relative humidities of 65 and 85 |x*r cent, and to the production of injuries corresponding only to relatively mild partial dis- ability.11* Although the two groups of data show some dis- crepancies. it seems reasonable to conclude that, in warm or hot weather and against troops provided with gas masks but not with protective clothing, HN3 vapor may approach H vapor in potency as a casualty-producing agent, particularly when the genital region is unprotected. HN1 vapor, except possibly on freely sweating men acclimated to hot weather, appears to lx- definitely inferior to II and HN3. The laboratory findings (see the preceding section) suggest that HNI vapor would be markedly inferior under cool or temperate conditions. 3. Evaluation of protective. clothing. The merits and limitations of the ('C-2 impregnated and the earbon-containing type's of protective clothing are reviewed in Chapters 26 to 30. In brief, the data 72 73- ■09.111.ns reveal that CC-2 impregnated clothing offers excellent protection against II, considerable pro- tection against HN3, and relatively little protection against TIN I. Thus, in the tropics against troops protected by this clothing, HNI vapor may be a more potent casualty-producing agent than H. The relative positions of ti and HN3 are not known but may not lx* important because of the high degree of the protection afforded against both agents. The best experimental types of carbon clothing now available offer protection against such large dosages of II. HNI, and IIN3 (presumably also HN2) that differences between the dosages of the agents required to "break” this clothing become of minor consequence. 1. Protection afforded by ointment. Prophylactic use of S-330 ointment, and presumably other oint- ments containing the chloramides available to Allied troops during World War II, offer little protection to skin exposed to nitrogen mustard vapor.109 6.1.1 Eye-Injurant Action Numerous observations on the effect of the nitro- gen mustards on human and animal eyes demonstrate that HNI, HN2. and IIN3 are eye-injurants more insidious than IT and more or less comparable with it SECRET 76 NITROGEN MUSTARDS Table 12. Basic man-ehaniber tests with 11 and nitrogen mustard vapors; I'nited States Naval Research Laboratory data.'1"111 T = 90 F. Relative humidity = 65 per cent. Fxposure time = 60 minutes. All sub- jects wore gas masks, uniinpregnatcd outer and under clothing, caps, shoes, and socks. The maximum lesions sustained on various parts of the body over a period of approximately a week were graded according to the following numerical scale: 0 = No reaction. 1 = Mild erythema. 2 = Moderate erythema. 3 = Intense erythema. -1 = a. Krylhema with edema. b. Maceration of axillary skin. c. Dry sealing of scrotum. 5 = a. Vesicle. b. Numerous pinpoint vesicles. e. Crusting or ulceration of scrotum or axilla. No. of crusted or Va|M»r Severity of injury ulcerated Numl>er dosage Rest of scrotal Season of men (mg min m3) Neck Scrot um body lesions — II March 6 . 50 0.3 0.0 0.1 Julv 5 50 1.2 0 2 0.5 - March 6 100 1.2 1.2 0,7 July 5 100 1 .!> 0.8 0.9 April 10 150 2.0 0.3 0.7 July 6 150 3.0 2.2 2.1 April 10 200 2.2 2.1 1.2 •JufjT 0 200 4.0 3.2 2.4 April __ 15 250 2 4 3.2 1.0 Julv 6 250 4.2 3.7 2.8 November 0 300 3.3 0,0* 2.4 ... - — — March 5 300 3.4 0.0* 2.9 — — JIM August 10 100 1.3 1.2 0.3 0/10 August 10 200 3.4 1.4 1.2 0/10 August 10 300 3.3 4.6 1.7 7/10 January 8 300 1.0 0.6 0.3 0/10 January 4 450 1.8 1.5 0.6 0/10 January 6 700 2.2 4.0 0.6 4/6 . n . - — uns -— . September 8 50 1.8 0.5 0.2 0/8 Septemlier S 100 3.0 1.9 0.8 1/8 September 8 150 4.0 4.0 I.S 6/8 August 8 150 2.0 0.0* 0.2 OS* February 6 150 1.5 0.3 0.7 0/6 February 6 250 . 2.5 1.5 0.8 0/6 February 8 350 4.9 3.6 1.7 6/8 * Subjects wore f’C-2 impregnated shorts. - in potency .sh.jo.jmi.nub. c.l86a,112,US,ll!t.l2S.I32,lJS,lM.l3s.M2,141»,U2,lS«.t74.176.1SO.I84 pyl-6fs(d-chloroethyl)amine and isopropyl-6is(0-chlo- roethypaminc appear to lie somewhat less potent.44'- 81 .134 Effects of the Vapour in* Small Dosages ox Ik man Eves The results of observer tests with H, HNl, IIN2, and HNS as vapors demonstrate that all four agents are roughly comparable on a potency (dosage) basis in eye-injurant action. Provisionally it would appear that HN2 and HNS may be somewhat more potent than H, and HXl somewhat less potent, but the dif- ferences cannot be considered to have been estab- lished with significance (see the following section for animal data). The importance of the human eye data merits their more detailed review as follows. 1. II. Critical summary and review 112 m of the four available sets of data n>r» n».H7«.i9«. suggest that SECRET TO \ ICO LOG \ 77 50 mg min raJ (/ < 8 hours) is the maximum dosage to which unmasked personnel may lx* exposed with- out danger of significant eye damage, and that 100 mg min m3 (t = 0 minutes to 7 hours) is the threshold dosage for production of partial disability. Extrapolation from the data leads to the estimate that for offensive purpose's 200 mg min m* (I = 6 minutes to 7 hours) would suffice to produce in- capacitating conjunctivitis and blepharospasm, with lacrimation, photophobia, and soreness, and perhaps with some corneal damage, in the majority of men for a period of 2 to 7 days, beginning 3 to 12 hours after exposure. II vapor is somewhat less effective at very short (i.e., 1 to 2 minute) and very long ex- posure times. 2. 1IN1.14- A dosage of 90 mg min m* is believed to represent the beginning of the human casualty zone, on the basis of tests in which one eye of each of 21 observers was exposed in a respirator facepiece to 5 I min of 11N1 vapor (Cl = 37 to 90. t = 5 to 67 minutes). There was no serious change of vision ex- cept for three men, exposed to dosages of 41.56, and 90 respectively, who did not think they could shoot a rifle for 48 hours. Only one of three men exposed to a dosage of 90 was a “casualty” in this sense. There was an average delay of 12 hours in (he development of symptoms, which included gritty feeling, lacrima- tion, photophobia, blepharospasm, headache, blurred vision, conjunctival hyperemia, corneal flecks, epi- thelial* I >edewing, and punctate staining with flu- orescein. Minor symptoms persisted in one case for as long as 21 days. The conclusions of (he re[>ort are transcrilted verbatim as follows: a. A dosage of I1N1 of 90 mg min ra3 Is prob- ably the beginning of the human casualty zone, but with ocular idiosyncrasy casual- lies can occur at lesser dosages. b. The average interval between exposure and onset of symptoms was 13 hours. c The most common complaint was “gritty” foreign body sensation in the eye. d. The most common lesion was flecks of the corneal epithelial surface which disappeared spontaneously in 1 to 15 days. Conjunctival hyperemia occurred almost as frequently. e. The most annoying symptom was pain in and behind the eyeball. f. Other complaints were lacrimation, photo- phobia, and blurred vision, although there was never any reduction in visual acuity or accommodation. g. Blepharospasm occurred in only two ob- server and myosis in only one. h. So far as can be judged from the results ob- tained, the dosage (Cl) of HNl vapor is a sufficient index to the degree of damage anticipated, even though the exposure time be varied from 5 to 60 minutes (but see below). 3. HN2.15* Dosages of 40 to 55 mg min m* (t = (5.5 and It) minutes, respectively) are believed-to represent the lowest limits of exposure necessary to produce “disablement” — i.e., certain cases would call for medical aid and. to an extent depending on transport and medical facilities, would lx; unable to take part in operations for a minimal period of 1 to 2 weeks. This conclusion was based on experiments in which an unstated number of men wearing oro- nasal masks were exposed in a man-chamber to dosages of 10 to 55 mg min ra*. The performance of additional human experiments at higher dosages was considered to involve an unreasonable risk. There were no subjective symptoms during exposure. From 8 to 15 minutes after exposure lacrimation and a feel- ing of grittiness under the lids developed. After 6 to 10 hours the following symptoms had set in: lacri- mation, photophobia, blepharospasm, and pain in the eyeball severe enough to prevent sleep. At 24 hours the symptoms were similar but the pain had become less seven*. There was pupillary constriction, conjunctival congestion, deep ciliary congestion, and threshold edema of the corneal epithelium, but no staining with fluorescein. The condition was resistant to mydriasis with I percent homatropine but pupil- lary dilatation and relief of blepharospasm was achieved by two applications of 1 per cent atropine. The observers gave their opinion that their efficiency as soldiers would have been seriously impaired from 6 to 10 hours onward. The duration of the symptoms was not stated. The report recommends that a dosage of 70 mg min m3 be aimed at as a minimum for of- fensive purposes. 4. HN3.""1’'' Of four observers exposed to a dosage of 20 mg min m3 (I = ?), none experienced any sub- jective symptoms but all showed moderate conjunc- tival injection. Their corneas were grossly normal but examination with the slit lamp revealed moderate to marked epithelial edema. Of three observers ex- posed to a dosage of 42 mg min/m* (1 = 7 minutes), SECRET 78 MTKOGEN Ml STAKUS Table 13. Eye damage produced in rabbits by the vapors of H, HXt, and Eight animals were exposed to each agent, at each dosage. The eye damage was graded according to an arbitrary numerical system1"** which took account of changes in the iris, cornea, conjunctivas, ami lids. The analytical dosages of the agents were determined by methods adequate to integrate low concentrations over long times. K\|>osure time (min) I )osagc (mg min/ni 11 11X3 Eye Dosage i3) damage (mg min nr1) Kye (tamale MX I Dosage Eye (mg min/in*) damage 2 440 20 353 21 485 30 384 19 439 29 10 3:40 29 410— 23 650 15 370 24 389 12 CO 420 23 4 IS 25 4:4,5 12 434 24 400 10 . 200 420 I7 240 347 21 4lt 16 530 11 406 10 360 330 13 all developed lacrirnation, photophobia, and a feeling of grittiness in the eye. They exhibited marked con- junctival injection. Their corneas were grossly nor- mal and did not stain with fluorescein but examina- tion with the slit lamp revealed epithelial edema and slight infiltration of the anterior stroma. One de- veloped moderate edema of the lids. All three were improving both subjectively and objectively on the fourth day after exposure. On the basis of the brief available description it would appear that HN3 pro- duced effects comparable to those found for 11 in one investigation l9a b and more severe than those found for 11 in two other investigations.M9,47“ The clinical reports of plant accidents indicate t hat the development of eye symptoms due to the vapors of IINl and HN3 were delayed for several hours.*9174 The same delay was experienced by some workers ex- posed to HN2 vapor, but others developed eye irri- tation, lacrirnation, and photophobia immediately after exposure.17* Effects of Vapors on Animal Eyes Although the animal (i.e., rabbit) eye is consider- ably more resistant to H and nitrogen mustard va- pors than is the human eye,7S11*,14! it may be assumed that the relative potencies of the different agents can lie determined in animal tests. The most satisfactory available set of comparative data is summarized in Table 13.,4h l0** The results suggest that the rabbit is approximately as suscep- tible to HN3 as to H. IINl is probably more potent than H and HN3 at very short exposures (i.e., 2 min- utes) but significantly less potent for exposure times of 10 to 240 minutes. The results of less rigorously controlled earlier work witli dogs exposed for 10 min- utes suggest that HN1, HN2, and HN3 produce threshold eo meal ilam age at somewhat lower dosages than H; that at low dosages HNS is the most potent eye-damaging agent, followed by FIN1, HN2, and H; and that at Itigher (buTstill moderate) dosages the differences among the hair eompounds are less con- spicuous;Mc In tests with relatively large vapor dosages which produced severe ocular injury, it was found that the dosages required to produce equally severe super- ficial corneal and conjunctival injury were about the same for each of the t hree nitrogen mustards.67b With equally severe injury to the superficial corneal tis- sues, however, the damage to t he deeper tissues (i.e., iris and ciliary body) was much the greatest with 1IN2, intermediate with IIN3, and least with I IN 1 and H.s7b The severity of the deep ocular effects pro- duced by HN2 make it a particularly dangerous agent from the standpoint of severe and permanent eye injury. The results of additional studies on the effects of nitrogen mustard vapors on animal eyes are to be found in the following references, some of which con- tain more or less complete histopathological analy- S(.S 44(1,43,S7»,.-,S8,70,71.84. H5,123,138.142 The clinical and pathological studies with I1N2 have been reviewed in detail.“ Liquid Contamination of the Eve Tests on animal eyes with small liquid drops (i.e., 0.5i mg) of H, IINl, HX2, and HNS demonstrate that all the agents produce such severe burns, fre- quently with permanent loss of sight, that any differ- SECRET KESLLTS OF FIELD TRIALS 79 euees in potency which may exist are relatively un- important to an evaluat ion of their relative merits as offensive agents.4**’<'1"1 ,23 , 52 1“ ’4# The observations tend to emphasize the similarity of the lesions pro- duced by II and HX3 and the more severe character of the injury that HXT2 produces in the deeper struc- tures of the eye. The effects of small droplets, and in the wind tun- nel of sprays consisting of fine droplets and vapor, have also been studied in animal experiments in order to assess (he relative effectiveness of the agents in the initial clouds produced by bursting muni- tions.1-’3 ,SUIK The results indicate that 11X3 may be slightly less damaging, and HX2 slightly more dam- aging, than IT. In any event the differences are not marked. Decontamination and Treatment Decontamination can be effected practically only by prompt lavage of eyes contaminated with (he agents in the liquid form. There is some evidence that lavage is of less value with 11X3 than with II.140 Prompt use of dithiocarbamates or of BAL ointment may be of limited value.46" 57c "» The subject of treatment has been authoritatively reviewed.62 The susceptibility to infection of eyes in- jured by nitrogen mustard and the value of various types of chemotherapy have recently been investi- gated, IMo‘l 6.5 RESULTS OF FIELD TRIALS Field trials with the nitrogen mustards have in- cluded tests of the vapor return from contaminated terrain and study of casualties in animals exposed to clouds of liquid drops and vapor produced by burst- ing munitions. Xo observer tests have been made to determine the vesicant effects of evolved vapor in the field or the hazard to trav ersal and occupation which is presented by the liquids on soil and vegetation. The results of (he tests reviewed in Section 6.2.4 attest to the excellent stability of HX3. the probably adequate but marginal stability of HX1, and the questionable stability of HX2. HNl, 11XT2, and 11X3, as well as H. dispersed from explosive munitions as clouds of liquid drops and vapor can produce profound eye damage and serious, often fatal, respiratory injury in unprotected ani- mals exposed on open terrain (see references cited in Section 6.2.4). However, such trials may have only limited bearing on the general utility of the agents in warfare. Kvoution of Vapor from Contaminated Terrain Results of field trials (see Table 14) conducted dur- ing warm weather at Hushnell, Florida, are avail- able.64** The tests included both annulus trials and trials with single, statically exploded M47A2 bombs. They lead to the following tentative conclusions."2 1. When terrain is similarly contaminated with HX3 and Levinstein H, the vapor dosage of 11 evolved during the first few minutes is five to eight times us great as that of HX3, as would be predicted from the relative volatilities of the agents. With the passage of time the relative dosage of evobed HNS vapor becomes progressively greater until, after the lapse of sufficient time for the completion of the evaporation process, the total dosages of the two agents become approximately equivalent. The time interval after which the evohed dosage of HX3 at- tainsany specified fraction of the U dosage depends on the meteorological conditions and the size of the liquid drops with which the terrain is contaminated. 2. In trials under semitropical meteorological con- ditions with single, statically fired M47A2 bombs charged HX3 or Levinstein II, the areas over which toxieologically significant dosages of HX3 vapor were obtained within 4 hours amounted to substan- tial fractions of the areas over which equivalent dosages of H vapor were obtained (see Table 14). 3. It is estimated 64 that in large-scale attacks un- der the semitropical conditions prevailing during the Florida trials the 4-hour vapor dosages obtained from equal expenditures of M47A2 bombs charged HX3 or Levinstein H would be: 4-hour vapor dosages, Meteorological conditions HX3 as per cent of 11 Woods, clear day 45 Woods, clear night 20 Open, clear day 65 4, At the lower surface temperatures character- istic of cool or temperate weather, the times after contamination at which the evolved HX3 vapor dosages would attain the above percentages of the H dosages would he greatly prolonged. 5. Under semitropical meteorological conditions the persistencies of vapor evolution by 11 Nit and Levinstein H arc not markedly different. Both are, of course, much less than that of HT (see Chapter 5). 0. 11X3 vapor evolved from contaminated terrain in the annulus and bomb trials was proved by bio- assay tests to be toxicologically effective. On the basis of the respiratory and ocular lesions produced in rabbits exposed at intervals up to more than 24 SECRET 80 N IT ROC EX MUST A RDS Table 11. Results of field trials with IIX1, 11X3, and II; single bomb tests,“MMir Avg Avg temp Area (artillery squares) within the contours for the wind Average gradient stated dosages (Cl's in mg min/nv’) for 0 to 0 + Agent and sjx-ed at ground r,m - t 4 hour sa mpling at a height of 12 inches. test Bomb 2m (mph) temp {(') in the o|X‘ti 50 100 250 500 1,000 2,500 Meadow, lapse < •otulilions 11M, lost 4 M47A2 4.43 23.SI -1.11 0.98 0.01 0.32 0.17 0.09 II, predicted M47A2 4.43 23.81 -1.11 1.01 0.59 0.29 0.17 0.10 11, observed M47A2 4,3 17.0 -1.2 0,81 0.51 0.22 0.13 0.07 11X1, tost t> M47A2 4.0 23.0 +0.7 1.72 1,(3 0.48 0.27 0.15 0.06 II, predicted M 17A2 4.0 23.0 +0.7 1.77 1.03 0.51 0.29 0.17 0.08 II, observed M47A2 4.41 21.09 +0.52 2.18 1.22 0.55 0.31 0.17 11X3, test ti M47A2 3.3 35 2 -1.1 0.76 0.50 0.30 0.18 0.11 0.00 11, predicted M47A2 33 35,2 1.1 1.34 0.79 0.40 0.23 0.14 0.07 11. observed M47A2 4.S 32.8 -1.32 0.77 0.48 0.25 0.16 0:10 0.06 II, observed M70 4.2 36.2 -2.24 0.71 0.45 0.23 0.14 0.09 0,05 Forest, lapse conditions HX3, test 1 M47A2 1.06 29.3 -1,05 0.57 0.35 0.20 0.13 0.09 0.06 11, predicted M 47A2 1.06 29.3 - 1.05 0.92 0.59 0.35 0.24 0.10 0.08 11. observed M47A2 0.1) 27.5 -1.10 1.10 0.72 0.41 0.25 0.10 0.09 11, observed M70 0.5 26.5 -1.03 0.90 0.67 0.45 0.32 0.22 0.12 11, observed M70 0.9 28.S -1.08 0.45 0.30 0.20 0,14 0.10 — 0.07 Forest, inversion conditions 11X3, test 5 M47A2 0.5 24.5 +0.3 1.18 0.04 0.24 0.12 0.08 0.05 11. predicted M47A2 0.5 24.5 +0.3 3.83 2.38 1.21 0.71 0.38 0.19~ 11, oliserved M47A2 0.6 21.5 -1.45 1.16 0.00 0.36 0.23 0.13 0.06 II, observed M 47A2 0.5 19.5 + 1.70 4. IS 2.78 1.27 0.70 0,20 0.08 II, observed M70 0.6 25.5 +0.80 2.75 2.02 1.38 0.84 0.47 0.17 hours after exposure, HN3 vapor was significantly more potent than H vapor. 7. When terrain is similarly contaminated with HNl and Levinstein II in the form of large drops in annulus trials conducted in the open in warm weather, the initial rate of vapor evolution was greater for HNl than for H, as would be predicted from the relative volatilities, and the 4-hour dosages of HNl were nearly twice those of II. 8. In the available single-bomb trials in the open under semitropical meteorological conditions the 4-hour dosages of HNl vapor were approximately equal to those obtained with H in similar tests (see Table II). Approximately 90 per cent of the total evolved dosage of HNl vapor had been attained within this time. 9. Analysis of the data indicates (hat the destruc- tion of HNl during the explosion or, subsequently, by inactivation on soil and foliage may have been as much as 30 per cent greater than the loss of II. Tak- ing these results in connection with those of British annulus trials155 which indicated 50 per cent de- struction of HNl on soil, it seems probable that large variations in per cent destruction may be expected, depending on the munition utilized and the char- acter of the terrain upon which the agent is deposited. Even greater variations might he expected in the case of HN2. 10. HNl vapor evolved from contaminated ter- rain in (he annulus and bomb trials was proved by bioassay tests to be toxicologically effective. In terms of the respiratory and ocular injuries produced in exposed rabbits, it was somewhat less effective on a dosage basis than HNS vapor under similar con- ditions. 6 6 EVALUATION VS WAR GASES The instability of HN2 disqualifies it from serious consideration for use as a war gas. Isopropyl-fus(j8- chloroe(hyl)aminc is also disqualified because its somewhat, inferior toxicological potencies are not counterbalanced by other advantageous properties. Thus only HNl and HN3 remain as potential sub- stitute persistent agents for II. In Table 15 are sum- marized the properties of II, IINl, and HN3 which bear most directly on an evaluation of their relative merits and limitations. The judgment of the present reviewers is in accord with the principal conclusions of previous assess- ments:1,2 l,H (I) that IINI and HN3 do not possess the general utility of H as an offensive agent; and (2) that in so far as incapacitation of masked enemy SECRET EVALUATION VS WAR CASES Tabi.k 15. Properties of If, HN1, and Il\3 hearing on their potential effectiveness as war gasos. Pri>|srty 11 UN i 11X3 Storage stability GimmI Satisfactory Kxeellent Stability On explosion of Good Probably sufficient Good munitions Stability on terrain Good Good to poor, depending Good on the nature and Density (g ml, 25 (') 1.27 moistness of the ter- rain 1.09 1.23 Load carried bv M47A2 tilt (pure 11) 71 (Levin- til 67 Iwnnb ( lb) stein 11 = 53 lb of active agent) Freezing point ((') 11.2 (pure) ca. 8 (I feasible . ages layers of CC-2 impreg- _ naled clothing Injurv-prodneing effective- Ineffective in reasonably Ineffective in reasonably Ineffective in reasonably ness of vapor against attainable dosages attainable dosages attainable dosages masked troops equipped with clothing containing - activated carbon Relal ive injury-producing 1 l_l 3 1 <8 effectiveness of liquid on bare skin Relative injury-producing ? ? ? effectiveness of liquid - through (.'( -2 impreg- — nated clothing troops not equipped with chloramide-impregnated clothing is the primary objective in the use of a per- sistent- agent, HN1 and IIN3 do not possess the of- fensive potential of H. At the present time, however, it is pertinent to arid a discussion of two additional points. 1, The lack of reactivity of 11X1 and 11X3 with the chloramides used in the United States and Brit- ish impregnated clothing of World War II led to the inference that this ty|ie of clothing would afford little protection against the vapors of these agents, and that they would therefore lie more effective casualty-producing agents than H against troops so equipped."2118 Recent man-cham1»er tests at 90 F reveal, however, that subjects exposed in 2 layers of CC-2 impregnated clothing to 5,000 mg min/m*, and in 1'2 layers to 1,000 mg min/m3, of HN3 vapor failed to sustain injuries of incapacitating severity.7* 'Phus CC-2 impregnated clothing affords marked protection against IIX3 vapor, although not neces- sarily so much as against II vapor. The explanation of this unexpected finding is not at hand. On (he other hand it has been confirmed that CC-2 impreg- nated clothing affords little protection against UNI.77 However, this lack of protection is at least partially offset In the additional evidence that HX1 vapor is relatively ineffective as a vesicant, except possibly in very hot weather.72 2. It was the intention of the German Army to use HN3 in high explosive-chemical shells. In the SECRET 82 NITROGEN MISTAKDS opinion of the reviewers, tins means of exploiting HNS merits careful evaluation.When HNS is used in this way as a harassing and casualty-producing agent, no other known gases except the Trilons (see Chapter 9) would be expected to approach if in ef- fectiveness. It is believed that in high-explosive bombardments an occasional high explosive-chemical shell charged TINS and indistinguishable upon de- tonation from ordinary high-explosive shell would have l»een used. HNS possesses the stability to with- stand destruction during the explosion of the shell and the lack of odor to escape ready detection except by chemical methods. It is believed that the poten- tial harassing and casualty-producing effects of the vapor slowly evolved from the contaminated terrain might exceed those of the initial cloud. The duration of danger from the vapor, the time intervals required for the evolution of casualty-producing dosages, and the areas over which effects would be produced would depend on meteorological conditions. As an example of the order of magnitude of the hazard, however, reference may be made to the field trial data re- viewed in Section 0.5 and Table Id. It will be noted that in warm weather explosion of a single M47A2 bomb (containing 07 pounds of HN8) resulted within I hours in the attaining of a dosage of 100 mg min m* of vapor over approximately one-half of an artillery square, and of 250 mg min nrTover about one-fourth of an artillery square. A dosage of 250 mg min in* should more than suffice to produce total disability of several days’ duration due to eye injuries, and possibly seven' respiratory injury as well. SECRET Chapter 7 ARSENICALS Marshall (lairs, Jonathan IT. Williams, and John .1. Zapp 7.1 INTRODUCTION IN ji'lv 1917, the Germans not only introduced mustard gas into World War I, but also employed for the first time an arsenical chemical warfare agent, diphenylchlorarsine (DA). Other arsenical agents were employed by the Germans in rapid succession, phenyldichlorarsine (PD) in September 1917, ethyl- dichlorarsine in March 1918, diphenylcyanoarsine in May 1918, and ethyldibromoarsine in September 1918. Although lewisite ami adamsite were not actu- ally used in battle, the Allies were preparing at the end of World War I to use /3-chlorovinyldichIorarsine (lewisite) and diplienylaminechlorarsine (adamsite), and were seriously considering the use- of methyl- dichlorarsine and arsine itself. There was a distinct feeling on the part of the Allies that the Germans did not obtain (he maximum effectiveness from the arsenicals which they used lie- cause of technical difficulties in methods of disper- sion, and further that some of the agents which did not receive battle trial (e.g., lewisite and adamsite) might become the most effect ive agents of their class. In view of this, it was natural that attention again be turned to the arsenical agents at the beginning of World War II. Accordingly, both the British and the Americans carried out extensive investigations on (1) improved methods of preparation of the known arsenicals, (2) the preparation of small quantities of new arsenicals, and (3) the physiological action, toxi- cology, and assessment of military value of these agents. Although considerable progress was made in the first two categories, none of the arsenical agents proved to offer much promise of success in battle for reasons which are detailed below. 7.2 CHEMICAL SECTION 7.2.1 Lewisite Lewisite. develo|»ed during World War I, is un- doubtedly still the best arsenical for gas warfare. (For a summary of work to 1940, see the bibli- ography.)'" The preparation of the agent by the original procedure 104 was complicated and danger- ous; it involves (he reaction of acetylene with arsenic trichloride, uring aluminum chloride as a catalyst. The reaction yields three products: Asti, + II--C==C—. H —>■ C1CH -CTIAsCl/—► 1^1 (CK’H =CH),Ast'I I.-2 L3 When aluminnm chloride is used as the catalyst, the very vigorous reaction leads to a mixture in which 1.-2, 1.-3, tar, and an explosive material are present with the desired lewisite. The optimum yield of L-l in this scheme is about 20 per eent.M*"M< 11 was highly desirable, therefore, to search for other catalysts. The first work with a catalyst other than AK'U was carried out in Great Britain in 1938,28Sa where it was shown that acetylene can be made to react di- rectly with arsenic trichloride in hydrochloric acid solution using mercuric chloride as a catalyst. The yield of L-I was 80-85 per cent based on the arsenic trichloride and 75 per cent on the acetylene. The main drawback to this process was the very corrosive nature of the catalytic solution. A pilot plant oper- ated by the British at Sutton Oak was found capable of producing 10 tons per week of “stripped lewisite,” which analyzed: L-l, 83.7 per cent; L-2, 11.5 per cent; arsenic trichloride, 2.8 jjer cent; solvent (chlo- rinated hydrocarbon) 2.0 (>er cent.2**6' W ork in this country13*'4'14* showed that a batch process for L using a mercuric chloride catalyst is economically advantageous. Work on other catalyst systems proved cuprous chloride used in conjunction with ethanolamine hy- drochloride to be one of the best, both for batch and continuous operations.*-,tt-,*ll88’,®*-200-2',*f*!*0* Although the reaction rate is somewhat slower than with IIgCl», the product is cleaner and there is less of a corrosion problem. It was also shown 2011 that (he cuprous chloride process gives 50 per cent more production and 5 per cent greater acetylenation efficiency, and that only one-half the amount of thionyl chloride or phosgene-hydrochloric acid is needed in treatment for sludge removal. A plant, operated by this process at, Sutton (3ak produced 10 tons per week of “stripped lewisite.”28*' Many workers recognized the desirability of a con- SECRET 84 ARSENIC \LS tinuous vapor phase process for the preparation of L whereby a mixture of arsenic trichloride vapor and acetylene could be passed continuously over a cata- lyst. Some degree of success was attained by the use of mercuric oxide suspended on alumina in an all- glass reactor.*4 With antimony trichloride as an activator for the mercuric oxide catalyst, the con- version was from 30 10 per cent, with yields of 40- GO jxw cent during the first hour; however, the life of the catalyst was <|uite short.55 Early in World War II, it became apparent that there existed a shortage of pure arsenic trioxide used in the preparation of arsenic trichloride for lewisite production. Consequently two programs were in- augurated: (I) the conversion of crude arsenic tri- oxide to arsenic trichloride; and (2) the use of arsenic trichloride containing impurities in lewisite produc- tion by the mercuric chloride process. In a study of the latter problem it was shown that arsenic trichlo- ride from crude arsenic t rioxide can lx* used directly in a lewisite plant. Incidentally it was indicated that slightly higher absorption rates were obtained when either 2 per cent antimony trichloride or 1 per cent ferric chloride had been added to the arsenic tri- chloride,4-’ This demonstration led to the observation that, when antimony trichloride is included in the catalyst layer in the mercuric chloride process, the output of lewisite is materially increased.55 In pilot plant operations, it was found that, when the same volume of SbC'L-containing catalyst (26 per cent Sb(’L added to the standard HgCI* catalyst) is used in the standard Hgt’b batch process, the time re- quired for acetylenation is reduced by about 40 per cent, whereas the amount of Hg present is 72 per cent of normal.2"5 The problem of using crude white arsenic in the production of arsenic trichloride was investi- gated first on a laboratory scale and then in a pilot plant.50 57 1% 197 With the use of three different raw- materials, one of them containing only 51 per cent arsenic trioxide, for reaction with sulfur monochlo- ride, yields of 95 per cent baser! on both arsenic anti chlorine were obtained in pilot plant runs. \\ itli this experience as a background (he process was trans- ferred to the Pine Bluff Arsenal,*2 where about 80 tons of specification-grade arsenic trichloride was produced from two lots of crude arsenic trioxide re- covered from ore of the Gold Hill, Utah, deposit. A yield of 95 |H*r cent was obtained based on the arsenic content of the crude arsenic trioxide. Prac- tically all of the arsenic trichloride produced in the experimental runs was consumed in the lewisite plant with satisfactory results. It was also demonstrated that arsenic trichloride of high purity can be prepared from either refined white arsenic or from low-grade arsenic crudes and hydrogen chloride in yields of 97 to 99 per cent based on the arsenic content of the raw' materials.51 In connection with the use of lewisite as a chemical warfare agent it was necessary to study its corrosive effect on shell steel. It was shown that plant-grade lewisite produced by the mercuric chloride process is practically without action on shell steel (No. 1045) and may be stored in such steel for long periods of time at tropical temperatures with insignificant cor- rosion.-* Under these conditions no pressure is devel- oped and no deterioration of the lewisite results. Phosphorus pentoxide may be. used to decrease cor- rosion slightly, to eliminate the slight rust formation, and to prevent tin1 increasedn moisture content under damp storage conditions. Through other st udies it was found that a 1 1 mix- ture of lewisite and Levinstein mustard is far more corrosive than either constituent alone, and that a 1 1 mixture of lewisite and (hiodiglycol mustard is only one-tenth as corrosive as (he other mixture.29 31 The conclusion reached, therefore; is that pure mus- tard must be employed if mixtures of it with lew isite are to be used in chemical warfare. Several investigations were made in order to dis- cover agents other than BAL (2,3-dimcrcaptopro- panol-1) which might serve, to detoxify lewisite or act as antivesicants for it. In a study of the reaction products of lewisite and six different dithiols 10 it was found that the properties of the compounds are best explained by cyclic formulas of the types: S—CUR S—CHR z / \ CICH -ATI As CK’II—CHAs CHIU “ \ — \ -/ S-CH2 S—CHR In a study of the reaction of lewisite with thiols, al- cohols, and amines it was shown that the competitive rates of formation, or the stability at equilibrium, or both, of bonds involving arsenic are in the order As—S > As—O > As -N; hence o-ditIdols appear to be the most satisfactory reagents for detoxification of lewisite.10-33 It has been shown that urea peroxide reacts readily with lewisite to give a nonvesicant product.14 How- ever, a careful investigation failed to reveal a suitable SECRET CHEMICAL SECTION 85 method of stabilizing urea peroxide at 60 C for field use.-’4 Other peroxides were studied and it was found that 10 g of a 1 1 mixture of sodium perlwwate mono- hydrate and sodium dihydrogen phosphate mono- hydrate, either in the form of a tablet or as a powder dissolved in 50 ini of water, gives a solution equiva- lent in active oxygen content to a 3 per cent hydro- gen jK'roxide solution. The conclusion was reached that HAL if quickly applied is somewhat more ef- fective- as a preventive for lewisite bums than the IMuborate-phosphate mixture; however, the latter is non toxic.M _ T.2.2 Miphatic Arscnicals Aliphatic arscnicals in wide variety have been pre- pared for testing as candidate chemical warfare agents. Major emphasis was placed on alkyldichlor- arsincs, as it was thought for a while that memliers of this series might show toxicity equal to that of lewisite and at the same time exhibit greater chemical inertness, particularly in reactions with water. How- ever, it was finally established (hat n-amyl-, isoamyl-, and /t-hexyldichlorarsine, for example, undergo the same general reactions as lewisite and react at ap- proximately (he same rate.36 i here is an opjxirent difference in the behavior of the alkyldichlorarsines as compared with lewisite in that the former do not IiIterate a gas when treated with sodium hydroxide and the alkylarsine oxides remain in solution longer than does lewisite oxide. The alki/Mirhlomrsincs have usually been prepared by the use of one of the following three schemes; 1. The Meyer reaction. RX + XajAsO., —> RAsOjNa, + XaX RAsOjNa* + 211+ RAsOjII, + 2XV RAsOjH, + SO, + 2HC1 —► RAsCl, + II,S04+ H20 2. The Kharaseh lead alkyl process. PbR, + 3AsClj ->- 3RAsCl, + PbCl. + RC1 3. From arsenic trichloride and tertiary arsines. RjAs-F 2AsClj —3RAsCl, The Meyer scheme is the one most frequently used.16 -ij) Incomes less efficient with the higher alkyl halides, such as heptyl bromide. The Kharaseh process is particularly good for the prepa- ration of ethyldichlorarsine in view of the availa- bility of tetraethyllead.ls The suitability of (he prow- ess for large-scale production has I wen demonstrated by pilot plant operations in which the reaction went readily and smoothly giving a 90 per cent yield.6* For tho preparation of (Unlkylchhrarstnes, four principal routes have been followed. 1. The Meyer reaction. RAsCl, + INaOlJ —> RAs(ONa)* + 2NaCl + 2114) RAs(ONa), T R'Br - ■> RR1 As(),Na -)- NaBr RR'AsO,Na + SO, -f lit I —> RR'AsCl + XaHSOi 2. The Kharaseh lead alkyl process. SRAsClj + R.Fb—*. 3RR'AsCl + R'CI + PbCI* 3. The cacodyl process. 4RCOOH -f AsjOj —* 2H.O + ICO, -f (R,.\s)gO (H,As),0 + 211 Cl -> 2R,.\sCl + 11,0 I. From arsenic trichloride and tertiary arsines. 2R3As + AsClj —► 3R,AsCl Here, as in the ease of the alkyldichlorarsines, (he Meyer reaction scheme is the one most commonly followed.1 5.2X.W.3M.M However, work on the cacodyl process * o .m has resulted in a marked improvement in this classical reaction. The improvement is in the form of a continuous catalytic process wherein va- pors of the acid and arsenic trioxidc arc passed over an alkali salt on a pumice support. Although this process was previously identified only with the pro- duction of dimethylarsine derivatives, it has been demonstrated that higher homologs may be pre- pared in fair yield.17 In the preparation of tertiary arxinrs, three general reaction schemes have been used: 1. Reaction of Grignard reagents with AsClj, RAsCU, or RjAsCl. 3RMgCl + AsClj —> RjAs + 3MgCl* 21lMgCI + R'AsCl,—>R,R'As + 2MgCl* RMgCl -f —> RR,As + MgUl, 2. Reaction of alkylmercuric chloride with arsenic trichloride. SRIIgCl + AsClj —> RjAs + 3IIgCl2 3. Disproportionation of RAsCl, or R,AsCl. 2RAsCI2 R,AsCl -f AsClj 2R,.VsC| R3As + RAsClj RAsCl, + R,AsClRjAs + AsClj It should be noted that all these methods are labo- ratory procedures and (hat no large-scale prepara- tion of an aliphatic tertiary arsine has been at- tempted.1 6 M:,s SECRET 86 ARSENIC AI.S 7,2.:$ Aromatic Arscnicals The standard approach to an aromatic arsenical is the Bart reaction l>etween an aryldiazonium halide and sodium arsenite: ArNsCI + NajAsOj —► Ar.\sO,Na. + NaCl + N, ArAsO,Na. + SO. + 2HC1 —> ArAsCl, + Xa,SO, + H.O Many aromatic arscnicals desired in the toxico- logical testing program have Ix-en prepared in this manner.5 5*,m However, the only aromatic arscnicals produced on any sizable scale during World War II are diphenylchlorarsine (DA) and diphenylcyano- arsine (DC). Considerable attention was devoted by the British to process development studies of those compounds.280 -90 307 They carried out laboratory and large-scale tests on two processes for DA prepara- tion: 1. The Bope-Turner process. CJFAsCl. + H.0 —► C’ellaAsO + 2HC1 C’sHvVsCh + 3(‘eHiAsO —> 2(CcIU)1AsCl + As,!), 2. The double diazotizat ion process. CJT.X.C1 + XasAsO, —> r«H4AsO(ONa)j + N, -f XaC’l C6H,As()(OXa). + XaHSOj —> CsIl>As(ONa), + NaHSO, 16H;,As(OXa). T ( sHiX.t 1 (C6H &)j AsOOX a + X. + NaCl (CsHs).As()OXa + SO. + HC1 —► (CgllnhAsCl + XallSOi A considerable improvement in the Pope-Turner process was effected by the British workers,290 who worked out the proper conditions for partial hy- drolysis of phenyldichlorarsine to a stoichiometric mixture of phenylarsinc oxide and phenyldiehlor- arsine (3 1 mixed oil), which, when healed to 240- 250 C, was converted to DA in gooil yield. DA is readily transformed to DC by reaction with 30 per cent aqueous sodium cyanide at 35-40 C.290 7.2.1 Heterocyclic Arscnicals From the standpoint of large-scale preparation work, only one member of this group, adamsite (DM), was considered important during World War 11. However, representatives of several other heterocyclic types were prepared for toxicity testing. Adamsite is still prepared by the standard pro- cedure worked out during World War I and involv- ins tlie reaction of diphenylaminc with arsenic trichloride: 0 '0 ~(XbO"“ -As \ Cl It has been shown 3 that a considerable part of tin1 arsenic trichloride called for in this equation may l>e replaced by the less expensive arsenic trioxide with- out a sacrifice in yield. Furan arscnicals were studied both in this country and in Great Britain.25101* They were prepared by reacting a-ehloromerc; ifuran with arsenic trichlo- ride to give trifurylarsine. From this tertiary arsine the mono- and di-furyl-chlorarsines were made by reaction with arsenic trichloride. Similarly thiophene arscnicals were made from a-thienylmagnesium bromide and arsenic trichloride ’ and pyridine arsen- icals were obtained from 3-aminopyridinc by the Bart reaction.5* Other miscellaneous heterocyclic arscnicals pre- pared for toxicity testing include 5, lO-dichloro-5, 10- dihydroarsanthrene.4 307b i i k m dibenzarsinole chlo- ride,5’3071’ and 10-chloro-9,10-dihydroarsacridine.58, 307ij k.m.n 'The preferred methods of preparation are illustrated by the following equations. 1. 5,10-Dichloro-5,10-dihydroarsanthrene. 0 0-N^C1 /\ AsOA'a, + Naj r\sOj ► I I -xo2 _ V-N0* —AsOjHs -X..C1 O OH 0—AsOjlli A-AstONa), /\-A»-/\ — +U -*U U It.Or. Ah O OH H2OjAs cHb - o^b CljAs q SECRET PHYSIOLOGIC-\L SECTION 87 2. Dilienzarsinole chloride. — + .\:ij.\s(), > o / XajO,As IlsOjAs <0 or-A A IljOaAs V/\ V () Oil o Oil Cl 3. !()-(’hloro-9,10-dihydroarsacridine. /\ -CH2-/\ + NajAsOa—► v-x" V OrO-Or*t) AsOaXai AsO.H. 0—ch.-ZN ih*u i \J mr ~ AsOjHi COO — o oh .. a PHYSIOLOGICAL SECTION 7.3.1 Lewisite When the United States became actively involved in chemical warfare during World War I, high hopes were held for a new agent, /3-chlorovinyldichlor- arsine, which was prepared and suggested as a candi- date. agent by (’apt. W. Lee Lewis in 1917. On the basis of relatively meager laboratory data it was de- cided to produce this agent, Lewisite (L), in quantity and to use it in battle. A shipment was on its way to Europe when the war ended in November 1918. During 1918, and particularly during the latter half of the year, the toxicological properties of L were studied intensively in various laboratories of the Chemical Warfare Sendee. The .lata obtained dur- ing this period are well summarized 124 and will not lx* discussed in detail in this report. However, two reports'224 225 issued in 1919 are particularly interest- ing in that they not only summarize the toxicological data acquired during (he war period but also attempt to assess the military value of L as a chemical agent. Since the conclusions of 1919 offer a convenient start- ing point from which to consider the later develop- ments which took place in the interval between wars and during World War II, these conclusions will be briefly stated. The effects of liquid L on the skin were studied in detail on dogs and rabbits.224 It was felt that L was definitely more damaging to the skin than II and that the danger of systemic poisoning from I. was con- siderably greater than with H. It was concluded (hat, if man were as susceptible as dogs to systemic poison- ing from L, the minimum lethal dose for man would lx* 1.4 ml distributed over an area of 5 square inches for an individual of average size. No systematic study of the effect of liquid L cm human skin was carried out. However, it was stated that ;224 Laboratory workers who have been accidentally burned with liquid 1. have- given strong evidence for the greater ef- fectiveness of this substance in man than liquid II. The b lesions develop with extreme rapidity, are painful and associ- ated with definite constitutional symptoms. The lesion is not confined to the skin, fait extends to the deeper tissues. In heal- ing, dense scar tissue forms, the skin loses its flexibility and contractures may develop. With liquid II skin burns in man, pain is less or absent, there are no constitutional symptoms, the amount of skin destruction is less, and healing occurs with- out extensive scar formation, formation of contractures, or permanent disability. . lu view of the divergence of these views from those currently accepted, it is well to bear in mind that these were accidental burns and hence were probably treated, that the accepted treatment at the time was application of 5 per cent sodium hydroxide to the lesion for a period of 80 minutes, and that sodium hydroxide itself in that strength produces a very destructive skin effect. The effects of 1/ vapor on the skin were studied with dogs, rabbits, and man, and are summarized in Table 1 « 2. Dibenzursinole chloride. Table 1. Approximate concentration to produce skin lesions in 30-minnte exposure. Rabbit Dog Man 1-ewisite (L) 0.025 mg 1 0.050 mg/I 0.200 mg/I Mustard (11) 0.200 mg/I 0.050 mg/I 0.025 mg/I The comparison indicated a lower sensitivity of man toward L vapor than toward H. The degree of SECRET 88 arsenic vls protection afforded by ordinary wet and dry clothing against I, and H vapor was also studied. 11 was concluded:224 An approximate concentration of .200 mg 1 (of L) is neces- sary to produce skin lesions in man on exposure of one-half hour. To lx- effective on parts of the luidv covered with cloth- ing, il would lie necessary to raise this concentration from three (3) to one hundred (100) limes, or approximately to a concentration of .(UK) to 20.0 mg 1 . . . So far as the concentra- tion required under field conditions to produce cutaneous lesions in man, 11 should lie regarded as from eight (8) (on unprotected skin) to a thousand (1000) times (a single layer of wet wool) more effective than b. The eye effects of L vapor were studied on rabbits and dogs. As with skin effects, it was found that rab- bits were more susceptible to I. vapor than were dogs, and a comparison with H revealed that ibe relative susceptibility of the sjteeios toward I, and II vapor paralleled that Of the skin effects. The data are sum- marized in Table 2."5 — from tin* laboratory, tin- cxjieriinental field and the field of war, our knowledge of the latter is confined entirely to data front the lalwiratory, 1 he abrupt cessation of experimental work at the American I niversity following the signing of the Armistice in November 1018, prevented the carrying out of field tests with I., preparations for which were already under way. In summary of the situation in 1018 it was stated: 224 \\ e regard t he laboratory data as offering si rong support for the probability that 1. will prove to have great military value. Its actual value can only !«• definitely determined, however, by further ex])erimenl:d data, cs|n-cially those obtainable by field tests. It would furthermore seem clear that the usefulness of I, in war would differ quite widely from that of II. kittle effect should lie expected from the vapor when used against troops supplied with an efficient mask equipment, because of the low skin vajair toxicity and the resistance of clothing to penetra- tion of the vapor. This is the condition, on the other hand, m which II has liecn found most effective. The usefulness of |, would lie eon fined to the effect of the substance reaching troops in the liquid phase (splash or mist) by their coming in contact with contaminated material, the influence of the hydrolytic products in contaminating the ground and objects, and the respiratory effects ami possibly the eye effects of the vapor in the ease of troops unprotected by mask equipment. In those respeets L offers many advantages, so far as can be concluded from the data at hand, over H. Wc feel that L offers sufficient promise to warrant the most careful further consideration. Data which are not at present obtainable and which are most, desirable in ibis connection are as follows; 1. The keeping qualities in steel. 2. The ability of the substance to withstand detonation. 3. The vajsir concentration which it is possible to secure and maintain in field tests. 4. The vapor concentration necessary to produce eye le- sions in tests on man. o. The relative importance of burns by liquid II and vapor of H in actual warfare. In conclusion we wish to repeat: We believe that L w ill not replace II in warfare, and that in any plans for military oper- ations the production and utilization of II should remain one of the most important propositions. While very promising, the military value of L remains to lie established. Fit the interval between 1919 and 1910 relatively little research on the toxicology of 1, was carried out by (lie Chemical Warfare Service, with (he exception of a dt'tailed study which was published in 1923.1,4 'l itis report has been critically reviewed 124 and will not l>e discussed in detail, although a few of the re- sults will be mentioned later in the present report. It was concluded 1,4 that L is superior to II in that it gave deeper and more severe bums as well as sys- temic disturbances leading to death, but the diffi- culty of setting up effective vapor concentrations was recognized. Following (he publication in the open litera- T\bi.e 2. Approximate concentration' to pro- duce eye lesions in 30-mimile exposure, Hahliit Dog Man D'u-isiic (L) 0.001 me 1 0.020 mg 1 Mustard (II) 0.030jn*/l 0.020 nig/1 0.001 mg 1 The statement was made;224 “If we may lx- allowed to infer or judge of (he susceptibility iu man without having an actual determination, the conclusion would l>e that the eye of man is less susceptible to L than to II, but such a conclusion can never convex* the conviction as one based on actual determina- tion.” No experiments involving the effects of liquid L on the eye were reported. I lie respiratory effects of I, vapor were studied on dogs and compared with the effects of II vapor, it being found that the dog was approximately twice as susceptible to L as to H. It was pointed out '225 that the concentration necessary to produce death in man on respiratory exposure is not known in the ease of either II or L. hut that in the light of our present knowledge we can only conclude that on respiratory exposure, I. is to In- regarded as approximately twice as effective as II as determined by the concentration necessary to kill. I his conclusion, as applied to man, must be made with reservation due to deficiency of data.” With resjtecl to the relative military value of L and II. il was stated; In attempting a comparison of the relative military value of the substances II and b, we meet with the fact that while with the former sulislance we have a very large exjieriencp SKCRFT physiomm;i<:\l siir.no\ 89 ture 335 337 of information that L had been seriously considered by the Americans as a war gas, the agent was studied in the laboratories of other nations. The published German reaction was unfavorable. The compound was tested in Germany in 1910 338 and the conclusion reached that it was not reliable as a war gas because its toxic effects were less lasting than those of mustard and the irritant effects were so marked that men would lie warned in time of its presence. The opinion was offered 340 that the Ameri- cans were spared a great disappointment by living unable to use L in World War I. A series of experi- ments was carried out 339 in which the effects of rela- tively large doses (one to two drops from an ordinary eye dropper) of 11 and L on human skin were com- pared. These exjieriments,- published in 1932, led to the conclusion that L was inferior to II in producing skin injury and that its potentialities as a war gas have l>een greatly overrated. In reference to the cal- culation 284 331 that 1.4 ml of L applied to the skin of a man should lie the approximate minimum lethal dose, it was asserted that this amount was applied repeatedly to the skin of human livings without giv- ing evidence of systemic intoxication.339 The Japa- nese used a 1 1 mixture of II and L against the Chi- nese at Ichang in 1938, but subsequent information obtained by (he interrogation of Japanese officers revealed that the L was added mainly to lower the freezing point of the H. The value of L as a chemical warfare agent still re- mained to be established in 1941. The published Ger- man opinions were looked upon with distrust, and. as in World W ar I, the United States undertook the quantity production of L. The discrepancies in the literature as to the toxicological effects of L had to lie resolved and intensive research was carried out both in the United States and Great Britain. Properties of Lewisite Plant run L is usually dark brown in color and pos- sesses an odor reminiscent of geraniums. Both the color and odor are due to impurities, which can be removed if the extra effort involved is considered worth while. Vis- and leans- isomers exist which have almost identical toxicides.171 L freezes at —18.2 C to 0.1C, depending on the purity and isomers present. The density of liquid L is 1.886 at 20 C, whereas the density of the vapor is 7.1 com- pared to air. The volatility of L is greater than that of II and increases somewhat less rapidly than that of II with increasing temperature. The following data for I. are calculated from the vapor pressures;;o: comparative data for 11 are also given.9" Teinjicratiire Volatility (mg 1) I, II C I, II 0 1.06 10 2.23 15 3.2!) 0.41 8.0 20 4.48 0.65 6.0 25 6.14 0.06 6.4 30 8.62 1.30 5.1 35 11.32- 40 15.75 2.82 5.6 L is fairly stable on storage in glass or steel hut is degraded to a considerable extent on detonation.1,4 The reaction of 1. with UAL and certain related dithiols30,f 3m,r 3U to form nontoxic complexes has assumed great importance in the treatment of L lesions and of arsenical poisoning from L or other sources. — The chemical properties which most sharply limit the usefulness of L as a chemical warfare agent are the ease with which it reacts with (1) water and (2) alkalies. In contact with water or moist surfaces, lewisite is readily hydrolyzed to the oxide which, although mildly vesicant, is nonvolatile and insolu- ble in water. Since L “precipitates out” in contact with moist surfaces it is impossible to maintain high vapor concentrations in humid atmospheres. Alkalies decompose L rapidly at ordinary temperatures, and even alkaline soil315 rapidly destroys the liquid and imposes a further limitation on its use as a ground contaminant. 'The maximum efficiency of L is only attained, therefore, under conditions of low temper- ature or low humidity, both of which minimize hy- drolysis, and on dry nonalkalinc terrain. Physiological Action Lewisite Vapor. The qualitative effects of L on the eyes, skin, and respiratory tract have been described in the open literature21433" and have also been re- cently summarized.3'® They may be very briefly re- stated as follows: 1. Eyes. L vapor is extremely irritating to the eyes, causing pain, lacrunation, and blepharospasm. The lacrimation and blepharospasm protect in a large degree from further exposure to the vapor but if the Cl is sufficiently high the irritation and pain persist and after a few hours are followed by edema of the eyelids and conjunctivitis. Permanent damage is, however, apt to result only from very high con- centrations difficult to achieve in the field. Liquid L is capable of causing severe damage to the SECRET 90 eyes. Pain, lacrimation, and blepharospasm appear immediately, ami are followed by edema of tin* lids, iritis, and conjunctivitis. In severe contamination, ulceration, necrosis, and secondary infection may lead to blindness or to permanent impairment of vision. 2. Respiratory tract. L vapor is irritating to the nasal passages and produces a burning sensation followed by profuse nasal secretion and violent sneez- ing. On prolonged exposure coughing results and large quantities of frothy mucus may Ik* brought up. The effects of L vapor are so prompt and striking that men usually mask before enough of tin* com- pound is inhaled to produce serious injury. However, in cxjx*rimental animals exposed to vapor in a gas chamber, injury to the respiratory tract is essentially similar to that produced by mustard. Edema of the lung is often more marked and is frequently accom- panied by pleural fluid.-’18 3. Skin. L vapor usually produces no more than erythema of the skin, although if the skin is hot and dry and the vapor concent ration is high, small, shal- low. turbid blisters may develop and may coalesce to form large vesicles. Such conditions would seldom Ik* ivalized in the field. Eiqnid Lon the skin produces an immediate sting- ing sensation which fortunately warns of its presence. If L is allowed to remain on the skin for 5 minutes, the site of application assumes a cooked appearance, somewhat resembling that from an acid hum. Ery- thema develops in a short time around the site of contamination and is followed by vesication of the cntiie erythematous area. L can penetrate the skin, subcutaneous tissue, and muscle, causing extreme edema and neemsis. The fluid coinained in vesicles produced by I, tends to be more opaque than that found in mustard blis- ters, although it is frequently impossible to distin- guish L vesicles from mustard vesicles by their appearance. The fluid from an I, blister contains 0.8 to 1.3 y of arsenic per cubic centimeter, equivalent to 2.5 to 4.0 y of original L.'*7 4. Systemic effects. The absorption of a sufficient amount of L through the skin of dogs may lead to death within 24 hours and usually within 10 hours. Table 3. Toxicity of T. jierioti 10 days, except a ■ vapor. (All figures are lACl).,,, in rng miriTT, s noted.) exposure time = 10 min, observation Total Inhalation only Hotly only Species exposure exposure exposure Mouse 0.0-1.4 (nom.)71 1.4-1.5 (nom.)75 1.2-1.9 (nom.)77 Mouse 2.8 (nom.)*5 1.6 (nom.)85' 0,3 (nom.)*1' Mouse 1.5 (anal.)43 1.5 (anal.)85*’ 7.0 (nom.)14 Mouse 2.5 2.8 (nom.)17' Mouse 0.5 (anal.)*6*1* Rat 1.5 (anal. )**-f _ 20.0 (nom.)14 Rat 0.58 (anal.)***-J , Guinea piR 1.0 (nnal.)“*‘* 20.0 to 25.0 (nom.) 14-J Guinea pig 0.47 (anal.)*4’1 Rabbit 1.2 (anal. PHI 15.0 (nom.)14 Rabbit 1.5 (anal.)*4"’* Goal 1.25 (anal.)*48 ** Cat — 30.0 (nom.)14-ft Dor 1,4 (nom.),M §§ 30.0 (nom.p- n 40 0 (nom.)* ♦ 11- to 14-min exposure. 21-day observation )ieriud. 1 9- to 25-min exjxieurc. 21 -day observation jieriod. x tiO- to 180-miii.exposure, 21-day < observation period. § lO- to 10-hub ex|MK-ure. • 1! 7 5- to 13-tnin exposure. 21-day observation |*riod. r 60- to 310-min exposure. 21-day ohm* r vat ion period. ♦♦ 1 Ott- to 255-min ex|H>turc. 21-day observation period. ' n 30- to 45-min exposure. XX 30- to 00-min exposure. if (<'l = 1.32 for 7 J-mm exposure and I II for 15-nin 9 »-h >ur obiprvatiuii period. The report .states th it concentrations were determined both as nominal and analytical but only one set is given ami it » not charac terized.) ““ -Vufr. N’om. » nominal concent rut ion; i.e., concentration calculat'd from the amount of L volatilized. the flow rate, ami the duration «*f flow. Amount volatilized (mg) Nominal coiwentration — Flow rale (l/min) X lime (min) Anal. = analytical concentration: i.c., concentration determined by sampling and chemical analysis of the atmosphere. SECRET PJI\ SIOLOGICAL SECTION 91 A few hours after application, the dogs show evidence of severe intoxication and appear almost moribund. Death apparently occurs from an intoxication which interferes with certain vital processes without pro- ducing sufficient anatomical lesions for complete characterization of the immediate cause of death. A frequent accompaniment of systemic intoxication is a change in capillary permeability which permits loss of sufficient fluid from the blood to result in homo- concentration and profound shock. The blood vol- ume of dogs was observed 224 to fall as low as 3.9 per cent of laxly weight in burned animals (normal = 9.7 per cent ). Nonfatal cases may develop a hemolytic anemia, focal necrosis of the liver, and some injury to the in- testinal mucosa. — Toxicity. There is no disagreement over the fact that L is a highly toxic compound and that it can produce the physiological effects which have been described. In order to evaluate the usefulness of I. as a chemical warfare agent, however, several things must Lie known. These arc: 1. What dosages of I. are required to kill men or at least to make them casualties? 2. Can these dosages Lie attained in the field with a reasonable expenditure of munitions? 3. How easily can the soldier protect himself against the effects of b? 4. Are the results obtainable through the use of L in the field likely to be better or worse than those obtainable with the standard vesicant agent, II? Toxicity Data The answer to (1) can only l>e approached experi- mentally through studies on animals. The toxicity of I, vapor toward animals of different species is shown in Table 3. The L{Ct)„0 of L vapor for man is un- known, but may be estimated (from the data of Table 3) to lie of the order of 1.2-1.5 mg min/l (ana- lytical). The L{Ct)bo for body exposure only has been estimated to be of the order of 100,45 on the basis of animal experiments and with the assumption that the absorption of b through the skin is a function of the ratio of surface exposed to laxly volume. The toxicity of liquid b applied via the skin for animals of different species is shown in Table 4. On the assumption that man would lie as susceptible as the dog, it was calculated in 1919 224 that the LD-„o for a 70-kg man would be of the order of 1.4 ml of b applied over an area of 5 square inches of skin. It is stated, however, that doses of 1.4 ml can lx* applied Table 4. Toxicity of lewisite by skin application. Animal LI)infing kgt Reference Mouse 15 87 (Cited bv Smith) Hat 24 300f Hat 15 318 Rat 24 240 Rat 20 318 Ralihit 5 318 Rabbit ti 24 it Rabbit « 133 Guinea pig 12 24!t Dog 38 224 Dog C!». 70 295a Goat 24 241 Quit 10 217 repeatedly to men without eliciting any clear-cut symptoms of arsenical poisoning.”** The I. Dm for man is probably much greater2*™ than the 40 mg kg sometimes assumed, A case is reported in which a worker at Pine Bluff Arsenal suffered accidental lewisite burns over 20 per cent of his body surface (mostly on the legs). Tie showed an anemia 10 to 15 days after the burn, but no clear-cut signs of systemic arsenical poisoning. Tt appears, therefore, that man is not nearly so susceptible to systemic arsenical poison- ing from skin contamination with L as was originally believed. The toxic dose of L when administered pa rente rally is much lower than that required by skin absorption. For example, the LD„n for rabbits is stated in one British report 227 to be 2 mg kg by either intravenous or subcutaneous injection, and in another 249 to be 0.5 mg kg by intravenous injection. The intravenous LD:to for dogs was found to be 2 mg kg as compared to 38 mg kg by skin absorption.2-' Two mg kg, injected intraperitoneally, has been given as the minimum fatal dose for guinea pigs.295" It is difficult to see, however, how the enhanced toxicity by parenteral administration can be utilized in warfare. Casually production by L may result from the action of the vapor on the respiratory t ract, or of the vapor or liquid on the eyes and skin. Assuming that men will be masked, the probabilities of casualty production from the inhalation of vapor are small. Relative to (he eyes, it has been shown that for L to produce moderate corneal damage in dogs a vapor Cl of 2.8 mg min 1 (nominal) is required; whereas a destructive lesion is produced by a Ct of 5.5 (nom- inal).S2f Analytical concentrations in the above ex- periments were approximately 50 per cent of the nominal so that an analytical Ct of the same order as SKCRKT 92 \RSEMC\LS the fACt) by inhalation is required to produce mod- erate eye damage. Since the immediate response of the eye to b vapor is lacrimation and blepharo- spasm, both of which protect against further expo- sure, serious ey e casualties from 1, vapor are not to lx* expected in conscious men. biquid b in the eyes is capable of producing de- structive lesions. It has been estimated176 that a drop 170 n in diameter in the eye of a man would make him a casualty for over a week unless immedi- ately treated. A 0.1-nig drop in the rabbit ey’e caused IKM'foration of the cornea in approximately 75 per cent of the cases and permanent disability (as judged by the1 jx-rsistence of corneal haze) in nearly all cases.’-’14 In the rabbit eye a 0.1-mg dose of liquid b produces a maximal lesion. With doses greater than 0-1 mg the severity of the ocular reaction did not appreciably increase. It has been stated that a dose of 0.01 to 0.02 mg of liquid b will produce permanent ocular damage (in rabbits) approximately equal to that produced by 0.1 to 0,2 mg of liquid H. With 0.05 mg of b most of the eyes are completely de- stroyed. whereas even 1.4 mg of H does not produce an equally' severe lesion. Mild, self-limiting injuries of comparable severity are produced by 0.005 mg of I, and 0.02 mg of H. It is thus apparent (hat the severity of the b lesion increases steeply with in- creasing dosage and rapidly reaches a maximal lesion, whereas the curve relating severity of the lesion to dosage of H is much more flat and very large doses are required to destroy an ey e completely'. I he threshold Ct for vesication of bare human skin (forearm) has been estimated as 1,0 mg min 1 (analytical) for a temperature of 55 F and relative humidity = 70 per cent.24* A Ct of 1.8 at T = 90 F and relative humidity = 49 per cent caused vesica- tion of the bare hand in 50 per cent of the men ex- posed.30* A Ct of 1.5 (analytical) caused vesication of the neck of six men exposed in the field at T = 60 F and relative humidity = 41 per cent, but no effect was obtained on skin covered by ordinary battle dress.24* A Ct of 1.5 (analytical) at T - 90 F and relative humidity = 65 per cent caused vesication on the skin (forearm) of three men (3 3), whereas a Ct of 1.2 produced vesication in none of three men (0 3) under the same conditions of temperature and humidity.21* biquid b on the bare skin is a very potent vesicant, the median threshold blistering dose for man Ix'ing 14 ns as compared with 32 ng for II.7* Contrary' to the opinions held in this country prior to World War II, recent work has tended to establish the view*** that in relatively large amounts I, does not produce as severe skin damage in man as does H. Although with doses up to about 1 mg of liquid l„ produces skin lesions in men not perceptibly differ- ent from those resulting from the same amount of liquid H, the response to larger doses of the two agents is different. For 2-mg dosages of b and of H, the lesions produced by I, are less severe and heal in 2J4 to 4 weeks compared to the 5 to 9 weeks re- quired for healing of the mustard lesions. One investi- gation,*** using much larger doses, placed two large drops (from an ordinary eyedropper) of b on one forearm and of H on the opposite forearm of a man. He reported healing of the L lesions in 2(1 days and of the II lesions in 63 days and stated that these re- sults were typical of other experiments. It has l>een pointed out that in rabbits tin- damage produced by 2 mg of liquid L is more severe and slower to heal t han that produced by 2 mg of liquid 11. The reaction of rabbit skin toward L is, therefore, not character- istic of the reaction of human skin. In an investiga- tion conducted at Port on -,&s it was concluded that b burns heal more quickly than II burns, are less prone to infection, and cause less pain during healing. The question of the comparative severity of lesions pro- duced by H and b on human skin has recently l»een reinvestigated,”0 with the result that b Irsrnirs were- found to be less severe and to heal more quickly than those caused by the same amount of II (either by weight or by volume, the dose being 1.0 mg or 0.5 microliters). It may be noted parenthetically that in 1941 a statement appeared in United States official chemical warfare manuals to the effect that the fluid from lewisite bullae was itself vesicant. However, experi- ments have been reported *** leading to the conclu- sion that b blister fluid was neither vesicant nor irritating and an American investigation in 1943 137 confirmed this conclusion, with the result that state- ments regarding the vesicancy of b blister fluid have been withdrawn from recent editions of United States official manuals. The toxicity of I, for man is summarized in Table 5. T he dosages required for b to produce casualties in men or to kill them appear to have been as well established as would be possible through the use of experimental animals in lethal experiments and hu- man observers in marginal experiments. As was aptly stated in 1919 255 (he value of b as a military’ agent depends in large degree on whether SECRET PHYSIOLOGIC A I, S liCTION 93 Tablk 5. Toxicity of lewisite for man. VajKrr approx. L{Ct)ii, (analytical) mg min 1 Liquid close mti5" Death (bv inhalation) 1.2-1.5 (est.) Death e completely destroyed. The British23* attempted to assess the danger of systemic intoxica- tion from liquid I. released in bomb explosions. On the assumption that the lethal dose for man would be 1.9 g (a dose which is probably not fatal) it was concluded that the risk of receiving serious injury from a bomb charged with L would be no greater than from a bomb of the same size charged with high explosive. When unthickened b is released from an airplane spray tank, the droplets formed are less than 1 mm in diameter.'** Since it has been reported *"*-230 (hat L droplets of less than 1 mm in diameter evap- orate completely while falling through 2,000 feed, it is apparent that the employment of unthickened L from medium altitudes (>2,000 feet) as airplane spray would be useless. I. may be thickened with methyl methacrylate and similar materials. The use of thickened L as airplane spray results in larger drops (55 per cent of drops >0.5 mg as compared with 8 per-ccnt of drops >0.5 mg for unthickened L).1*3 However, when droplets of thickened L strike a surface, they tend to harden. This effect may be due to the formation of a skin of L-oxide on the sur- face of the drop.1*3 A comparison of the casualty-producing effect of thickened and unthickened L when used as an air- plane spray from low altitude (100 feet) revealed that thickened L was less effective in producing casualties in goats than unthickened L, and that the eye dam- age caused by the unthickened L was more severe than that caused by thickened L.163 The tactical value of producing L blisters on hu- man skin is thrown into very serious doubt by recent Canadian experiments 3,3 in which observers clad in battle dress and shirts over long-limbed underwear and wearing respirators and steel helmets were ex- posed to airplane spray of L to which had been added 0.55 per cent of thickener. The temperature was 75 F with relative humidity = 89 per cent, and the contamination density was 0.7 to 5.4 g in2. T he drops varied between 1.3 and 5.0 mm in diameter. Of 30 men hit by the spray, 20 developed lesions which in 7 cases were numerous and prominent but in other cases were trivial. It was noted that the in- dividual lesions produced were discrete and circum- scribed in contrast to the diffuseness of the typical lesion produced by H spray. After 9 days of compara- tively strenuous exercise, none of the observers was the neci -ary dosages can lie set up in the field. Suf- ficient field exjieriments have now been carried out to indicate that the requisite dosages are probably not attainable with any reasonable expenditure of munitions. Fiki.d Tkst Data The concentration of vapor obtained from pouring 50-75 g of I. |s'r square yard on the ground is low and Cl values obtained are usually not over 4.0 mg min I.'24 240 The vapor concentration obtained di- rectly over the contaminated area fell steeply during the first 30 minutes of the experiments and thereafter was not dangerous. In experiments conducted at Edgewood Arsenal four M70 bombs charged b (total 360 pounds) were fired statically. Twenty-five yards downwind from the burst the initial concentration was 0.060 mg 1 but fell to 0.013 mg 1 in 10 minutes. The Ct for 15 minutes was 0.395 mg min I.216 In a further test at Edgewood an airplane sprayed 610 pounds of un- thickened L from an altitude of 75 feet over an area of 76,250 square yards.1*3 Significant vapor concen- trations directly over the contaminated area were recorded only for the first 10 minutes and the total Cl recorded was of the order of 3. It is apparent from (he above examples that dan- gerous concentrations of L vapor are difficult to at- tain in the field, 'Hie reason for this is apparently the rapid hydrolysis of the vapor and liquid in con- tact with a moist environment, with possibly the destruction of some I, by alkaline soil, together with the fact that the agent may he partially destroyed by detonation when loaded in munitions. In ex- tremely hot and dry climates more effective vapor concentrations may be anticipated. The effects of liquid L on bare skin might lx* achieved through ground contamination, bursting munitions, or airplane spray. However, L is so un- stable on contact with moisture (hat under ordinary conditions of humidity it is rapidly hydrolyzed on the surface of soil or foliage, leaving behind a residue of L-oxide. The L-oxide. while weakly vesicant, is SECRET 94 VttSEMCALS in such condition that he could not carry out military duties, and in no case had secondary infection de- veloped. It was concluded that the casually-produc- ing propensities of II spray are definitely greater than those of L spray. Protection* against Lewisite Lewisite Vapor. The median detectable concen- tration of L vapor by odor is stated to be 0.014 to 0.023 mg 1. However, the irritating effect of the gas on the eyes and respiratoiy passages is noticeable at far lower concentrations, variously estimated as 0.008 mg 1339 and as 0,006 mg I.124 On the basis of these figures, a concentration of 0.006 mg 1 should certainly warn troops of the presence of gas and should lead to masking or to withdrawal from the toxic atmosphere. The service respirator gives en- tirely adequate protection to the eyes and respiratory tract against the effects of L va|K>r. Even in the ab- sence of the respirator, serious eye effects from L vapor are unlikely to occur in conscious men since the immediate response of the eye to L vapor is lacri- mal ion and blepharospasm, both of which protect against further exposure. Ordinary* clothing affords considerable protection against L vapor. It has been estimated 224 that a single layer of dry cloth would protect against ap- proximately three times the concentration of L that would produce a reaction on bare skin. The British 258 estimated that a Cl of 3.0 4.0 mg min I would be required to produce an effect under a single layer of dry serge. In another report, it was found 257 that the penetration of cloth by L vapor decreases with in- creasing humidity, and it was suggested that the reason lies in reaction of L with moisture on the fibers of the cloth. Complete protection against L vajKir was afforded by ordinary dungaree shirt ma- terial, S-330 ointment, and CC-2 impregnated cloth up to at least Cl 3.3 (analytical) under exposure con- ditions of 90 F and 65 per cent relative humidity with 4 hours wear of the clothing after exposure.219 Wet clothing is much more effective in protecting against L vapor than dry clothing. It has l>een esti- mated 224 that 100 times the concentration of L that would produce an effect on bare skin would be re- quired to | tenet rate a single layer of wet cloth. In fact the British state that 1. vapor will not burn through wet clothing. Liqud Lewisite Liquid lewisite in the eyes is capable of causing se- vere damage. However, complete protection against liquid I- is afforded to (ho eyes hy wearing (lie respi- rator or (ho eye shield or even by closing the eyes. Liquid L will penetrate ordinary dry clothing, a drop of 2.5 mg (1.5 min in diameter) generally caus- ing vesication through dry service clothing in temper- ate climates.2*1 Under tropical conditions a 0.4-mg drop (0.77 mm in diameter) may produce vesication through light dry clothing.334 Wet clothing protects against liquid L by forming (he insoluble and nonvolatile L-oxide before the agent can penetrate to the skin.314 CC-2 impregnated clothing offers more protection against liquid f. than does unimpregnated dry clothing, although 5.7 mg of L produced vesication through a single layer of CC-2 impregnated cloth,179 indicating that the pro- tection afforded against L is less than that against II. ( '<*\tI’AH 1SO.V WITH Ml-STAUU The toxicity of L vapor and II vapor by inhalation are of the same order of magnitude. However, to pro- duce systemic effects through the skin, eye damages, or skin vesica! ion, significantly higher Cl's are re- quired for I. than for H. Because of the rapid de- struction of I. liquid and vapor in contact with moisture or with an alkaline environment the requi- site Cl's for L would be extremely difficult to attain in the field. Further, L vapor, unlike II vapor, is not insidious but gives adequate warning of its presence by irritation of the eyes and respiratory passages. Liquid L is more vesicant, than liquid H but the burns from I. do not. incapacitate men to the same extent as do burns from H,313 and the L burns heal more rapidly and are less painful than those from If. Liquid L on the skin or in the eyes produces an im- mediate stinging sensation which warns of its pres- ence. whereas mustard is nonirritating at (he time of application. Mustard penetrates ordinary clothing much more readily than does L, and, since H is more stable than L, is a Ix-tter choice both for terrain contamination and vapor return. Mixtures of H and L have been suggested but have no advantage over II used alone except with respect to lower freezing point. Thkrapv In 1941, the discovery of a powerful therapeutic agent against L and other arsenicals was an- nounced.3"01 This substance. 2,3-dimercaptopropanol- 1, variously known by the code letters HAL and DTK, will not only destroy arsenicals on contact, but is capable of minimizing the damage from liquid SECRET PHYSIOLOGICAL SECTION arsenicals in the eyes if applied from I to 10 minutes after exposure, and from liquid arsenicals on the skin if applied up to 1 hour after contamination. A discussion of BAL is lieyond the scope of this report except to say that an ointment containing BAL was available for issue to United States soldiers. This ointment was suitable for application to the skin or eyes and placed in the hands of the soldier a method of self-help for minimizing the effects of con- tamination from liquid arsenical agents. Prepara- tions of BAL were available to physicians for paren- teral administration and are effective in combatting systemic intoxication from arsenicals. Summary By the end of the World War II, the toxicology of Lhad been worked out to the point where the dosages required to produce casualties or death in human liv- ings were known with a degree of approximation that is probably sufficient for military purposes. Field tests, however, showed little promise of at- taining the requisite dosages of L vapor with any reasonable expenditure of munitions. The use of liquid L for gross contamination of personnel seems feasible only when the agent is dispersed as low- altitude airplane spray, and the effects produced on contaminated personnel are so inferior to those pro- duced by mustard as to create strong prejudice against the use of L. Since the powerful antiarsenical agent, BAL. avail- able to Britain and the Uniter! States in World War II. will be available to all in the future, there seems to lie little likelihood that there will ever lie any incentive for the use of L as a chemical warfare agent. 7.3.2 Chlorarsine Derivatives Other Than Lewisite Lethal Agk.nts In the Spring of 1918, ethyldichlorarsine (ED) was us»>d by the Germans as a skin and lung irritant suit- able for gassing operations to be followed by infantry assaults.331 *28 There is no mention in Allied official records of casualties attributed directly to ED, but the Germans held the compound in high regard. 1 he United States Chemical Warfare Service investigated methyldichlorarsinc (MD) during the latter half of 1918 but the compound was not used in battle. In 1989, the results of a preliminary investigation by the Chemical Warfare Service 12‘ revealed a lack of sufficient data for making a definite decision as to tlu* value of ED as a military agent, hut stated that “the present available data indicate sufficient poten- tial value to warrant further study and develop- ment.” Accordingly, the National Defense Research Committee [NI >HC] was asked to screen the arseni- cals for toxicity and stability in order to determine whether any members of the group were sufficiently promising to warrant further study or development as chemical warfare agents. A number of chlorarsine derivatives were prepared and were studied for toxic- ity at the University of Chicago Toxicity Labora- tory [UCTL]. Physiological .1 rtion. The toxic chlorarsine deriva- tives produce effects which are qualitatively similar to those produced by I- (q.v.) hut which differ in degree. Thus, they are all irritant to the respiratory tract and produce lung injury on sufficient exposure. The vapors are irritating to the eyes and the liquids may produce serious eye lesions. The absorption of either vapor or liquid through the skin in adequate dosage may lead to systemic intoxication or death. Local skin damage leading to vesication in man is usually produced by sufficient exposure to the vapor or by contact with the liquid. Vapor Toxicity. The chlorarsines originally screened for vapor toxicity at the UCTL27 are listed in Table (>, which shows the results of tests against Tabik fi. Toxicity of vapor figures for L(Ct)-,» are in mg of clilornrsincs for mice. All min/1 (nominal). Compound L(CI) - (Mouse) lewisite, isomer I L(Cl)iu = 2.8 lewisite, isomer IT UCl),u = 2.8 Plant run lewisite, isomer I Phenyldichlorarsine lACtU = 3.7 HCI)90 ~ 13. d-Methoxvcth vldichlorarsine unstable 5-Ethoxyethyldichlorarsine unstable 0-C’hloromethoxypropyldichlor- arsine (No deaths at Cl = 8.7) Allyl phenylchlorarsine Phen y 1 ((3-chh troviny 1 Jclilor- (No deaths at Cl = 24.44) arsine UP On. ~ 1. Tsoamyldichlorarsine UCtU ~ 2. .w-Hul vldichlorarsine urt),u ~ 12. 6/s(CliloroinclliyI)ehlorarsine UCtU - 4.5 C 'liloromet hyldichlorarsine (No deaths at Cl = 43.5) 4-Fentenvldiehlorarsine L{Cl)u -3.7 A m vldichlorarsine L(Ct),„ = 2,5 Hut vldichlorarsine />(COio ~ 3.5 El hvldichlorarsinc UCtho - 3.5 0-Kurvldiehlorarsine (No deaths at Cl = 2,3) 1 leptyldiclilorarsine UCtU — 13.1 d-Mcthylhutyldichlorarsine unstable 1 lexyldichlorarsine UCtho - 3. Dimethylehlorarsine L{Cl)lu — 10. SECRET VKSEMCALS mice by total exposure for 10 minutes. On the basis of the information listed in Table fi and information from the Chemical Warfare Service on ED,129 butyl- dichlorarsine,1*0 amyldichlorarsine.157 and isoamyl- dichlorarsine,147 a more detailed investigation was made of the toxicities toward mice (by total expo- sure) of the vapors of the alkyldichlorarsinea from methyl- through hexyl-.49 In order to avoid errors known to result from different degrees of humidifica- tion of (he animal’s fur, the mice were exposed for 1 hour to a relative humidity of 20—HO per cent before exposure to the toxic arsenical. The dichlorarsines were vaporized with dry nitrogen at 25/30C and were passed through the 4-1 glass chamber at 11.2 1pm. The relative humidity of (he gases in the cham- ber did not exceed S |H*r cent. The vapor toxicities of the alkyldichlorarsines are given in Table 7, together with the toxicity of phenyl- dichlorarsine (I’D) and of L for purposes of com- parison. The toxicity of several diehlorarsines when applied to the skin (shaved) of mice is shown in Table 8. Table 8. Pcrcut aneons toxicity of arscnicals for mice.2* Compound Dost* mjt No. of mice Percent mortality 10-day period Comparison with lewisite I, (plant run) 0.1 10 0 0.3 10 50 0.5 10 UN) KI) 0.1 4 0 0.5 1 25 <5 L 1.0 4 25 X-Putvldichlor- 0.5 10 10 arsine 1.0 10 30 <5 1, 0-Methylbutvl- 0.5 4 25 dichlorarsine 1.0 4 100 - :■ i. X-Amyldichlor- 0.1 7 It arsine 0 3 10 80 = i. 0.5 10 100 Hexvldichlor- 0.1 4 0 arsine 0,5 4 50 1.0 4 50 _ ~i. Hcplvldichlor- 0.1 4 0 arsine 0.5 4 0 - i »* 1.0 4 50 PD 0.1 10 20 0.3 10 30 = i. 0,5 10 100 Table 7. Toxicity o if vapor of dielilorarsines for mice. All figure* are /i(7)io in mg min/I. Kxposure time = 10 min; observation period = 10 days. Agent Total exposure Methyldichlorarsine 2,7 (anal.)*3 Kl hvldirhlorarsine 1.555 (anal.)13 Kthvldichlorarsine 3.4 (nom. )«• Fropvldichlorarsinc 1.4 (anal.)13 Biitvldichlorarsine 1.8 (anal )« Hutyldichlorarsine 3.7 (nom.)130 Amyldiclilorarsinc 1.4 (anal.)1-1 Anivldiehlorarsine 3.7 (nom.)u7 Isoamyldichlorarsine 3.7 (nom. )’*■ 1 lexvldichlorarsine 1.5 (anal. )13 Fhenvldichlorarsinc” 3.4 (nom.)27; 3.3 (noni.)11* Lewisite 1.5 (anal.)*3 lewisite 2.8 (nom. )27 These data show that none of the dichlorarsines: tested are more toxie than L and that only amyldi- chlorarsine and PI) equal L in systemic toxicity. Thirty-five dihalogenated arsines and thirteen nmnohalogenated arsines were examined for vesi- cancy at the LKTL 6S without revealing any vesicant superior to lewisite. In general, the dichlorarsines are better vesicants than the monochlorarsines,79 and the simple alkyl- dichlorarsines compare favorably with L in respect to “absolute” vesieaney, i.e., when evaporation of the liquid from the skin is prevented by covering. The introduction of a single chlorine atom on the terminal carbon of a normal aliphatic substituent in a dichlorarsine or the use of branched chain sub- stituent groups results in loss of vesicant potency.27 Thus, ED is a more potent vesicant than L 27 when evaporation from the skin is prevented, and aim 1- diehlorarsine is a better vesicant than isoarnyldi- chlorarsine.271” Eve Effects The vapors of the chlorarsines are generally irri- tating to the eyes, leading to lacrimation and bleph- Examination of the data of Table 7 leads to the conclusion that all of the dichlorarsines tested, with the possible exception of Ml), have essentially the same toxicity toward mice. Fifty-three dihaloarsiaes were tested at the UC’TL and the conclusion reached that the members of the series vary in toxicity up to a maximum in the group that contains b, ED, and the homologous straight chain aliphatic dichlorarsines. Data have also been obtained for a number of monohalogenatcd arsines,6< but none of these compounds are superior to L. References to the vapor toxicities of other halo- genated arsines will lie found in Table 9. SECRET PHYSIOLOGICAL SECTION 97 arospasm which protect against further damage. Liquid Ml) produces a lesion in the rabbit eye which is less severe than one caused by L.2,9r Liquid KD produces a lesion in the rabbit eye which is compa- rable iu severity to that caused by L.2aal* UAL is effective in the prevention of eye damage from either MD or KD.2®*bc Assessment of the Military Value of Chlorarsines Olhir than I.. Of all the chlorarsines studied, only MD, KD, PD. hutyldiehlorarsine, and the ainyldi- ehlorarsines approach L in toxicity and vesicant potency. Of these, hutyldiehlorarsine is too un- stable 130 and the amyldiehlorarsines too difficult to prepare147 to lx considered as chemical warfare agents. Thus, after an exhaustive examination of many compounds, it appears that the l>est of the chlor- arsines other than L are those which were used (KD and PD) or considered for use (M D) in World War I. The status of MI). KD, and PD as military agents has recently been reviewed2I* with the following results: 1. MD. The vapor is so irritating that it is easily detected at low concentrations and would lead to prompt masking. The vapor is easily hydrolyzed and the dosage required for skin vesicancy so high that there is no hope of obtaining vesicant dosages of vapor in the field. The skin and eye effects of the liquid are not so damaging as those produced by L. 2. KD. KthyTdiehlorarsine is somewhat superior to M D but is inferior to L as a casualty agent. 3. PD. The vesicancy, systemic toxicity, and toxicity by inhalation of PD are equal to those of L, but PD penetrates clothing less effectively than L and the volatility of PD is so low that casual! ies from exposure to the vapor are hardly to be expected in the field. Like MD and KD, PD is easily hydro- lyzed. In view of these facts, it appears that tlie best of (he chlorarsines are inferior to I, and. since L itself does not appear to have any future as a chemical war- fare agent, it can lie assumed that the other chlor- amines will not be considered further as military agents for casualty effect. It is interesting to note, however, that the Allies captured a considerable number of German artillery shells charged with a mixture of mustard and PD. Whether this indicates that (lie Germans held a higher opinion of the effec- tiveness of PD than the Allies or the mixture was dictated by other considerations is not clear at the present time. 7.3..'5 Arsine and Nonhalogenated \rsine Derivatives A US IN K During World War I, the Allies did considerable exploratory work on the potentialities of arsine as a chemical warfare agent. In 1919 it was stated; 124 During I lie war many suggestions were made that arsine should t>e used The popular plan was to use magnesium arse- nide which would hydrolyze in moist air, setting free arsine. The experiments made by the Research Division showed that the hydrolysis does not take place rapidly enough under or- dinary conditions to give an efficient concentration of arsine. At the time of the armistice e\|)erimeiits were still under way to determine whether this material could be used effectively in the rain. While the use of magnesium arsenide or of any arsenide was not very promising, there seemed to l>e a distinct (jossibility of using liquid arsine ... If arsine is to be used in warfare, it seems probable that it must lie used as liquid. In 1939. the available data concerning arsine as a potential chemical warfare agent were summarized 124 with the conclusion that its value would depend on whether (he canister of the gas mask would afford sufficient protection against it under all conditions to which the canister might Ik* exposed. It was recognized that arsine might be useful as a casualty agent aside from its lethal effects and ac- cordingly studies of the toxicity and suitability of the, compound for chemical warfare use were reinvesti- gated by both the Americans and the British. Physiological Action. The physiological action of arsine has l>een well summarized in the open liter- ature.**® In vitro studies have shown that arsine is oxidized aerobically in aqueous solution, and that this oxida- tion is catalyzed by hemoglobin.*94* In the presence of arsine and oxygen, however, the hemoglobin un- dergoes destruction forming a number of compounds including met hemoglobin, and a tetrapyrrolie com- pound whose spectrum resembles that of sulfmel he- moglobin.2Mb3,M,lh During the reaction of arsine with hemoglobin about 40 per rent of the arsine taken up is held in a nondialyzable form, while the remainder is mostly arsenite with a small amount of arsenate. There is no reaction 1x9ween arsine and hemoglobin under strictly anaerobic conditions.2®41* Arsine is a strong hemolytic agent in vivo; and in vitro under aerobic conditions only.294b In view of the known oxidation products of arsine, experiments were carried out to determine whether the hemolytic effects of arsine were due to arsenite or arsenate rather than to arsine per sc, but with negative re- sults.®3 SECRET 98 VHSEMCA LS The action of arsine on tissue slices has been stud- ied and compared with that of arsenide, with the con- clusion that the effect of arsine in reducing the oxygen uptake of kidney slices is similar to that of arsenite.93 3041 HAL protects kidney slices against the effects of arsine but not of arsenite.93 The action of arsine on liver slices is not identical with that of arsenite since the toxicity of arsine increases more rapidly with increasing concentration, and liver slices treated with arsine change color, suggesting a reaction with heme compounds that does not occur with arsenite-treated liver slices,303'- Toxicity. Available data on the toxicity of arsine by inhalation have been summarized.-16 The data cited are quite variable both for exposures of a given species and for different species. The LCM for mice has I>een determiner! as of the order of 0.250 mg 1 for a 10-minute exposure; 121 2,6 3031 but studies at the U(TL23 resulted in a figure_of 0.520 ± 0.100 mg I (analytical), with no apparent explanation of the discrepancy. There do not appear to be any satisfactory data for the LC-m for dogs with 10-minute exposure, but 0.35 mg I for a 30-minute exposure is said to be the LCsn,,M and lethal concentrations for various ex- posure periods have been compiled.*'* Rabbits are apparently less susceptible to arsine than mice, the LCu> for 10-minute exposure Ixdng estimated to lie between 0.65 and 0.96 mg 1.176 No satisfactory LC;„, has lieen reported for cats, but 0.80 mg 1 for 10 minutes caused the death of 3 4 cats within 18 hours (G-2 Report No. 1322216), whereas cats exposed to 4.1 mg I for 1 minute did not die.23 The LC-3n for rats on 10-minute exposure is of the same order as that for mice, being between 0.39 and 0.66 mg 1 (G-2 Report No. 1322 *«). The LCht) for goats on 10-minute exposure is estimated as being between 1.0 and 2.2 mg 1 (G-2 Report No. 1322216). Four of fi\ e monkeys died after exposure to 0.45 mg 1 for 15 minutes.3031 No data exist for the AC50 for man, but the mini- mum disabling concentration has lieen estimated as 2.0 mg I for 2 minutes or 0.2 mg 1 for 30 minutes.'*8 Henderson and Haggard state that exposure to a concentration of arsine lietween 0.051 and 0.191 mg I would lie dangerous after 30 minutes, whereas ex- posure to 0.798 mg I would be fatal after 30 min- utes.328 British estimates based on the assumption that 2 mg kg of arsine would lie fatal to man put the casualty-producing Ct at 14 mg min 1 for a man at rest and at 4.66 mg min 1 for a man working; and the fatal Ct at 28 mg min I and 93 mg min I for a resting man and working man respectively.3" Marly British results indicated that for the effect of arsine on mice the product CH rather than Cl was a constant, hut later investigation showed that for concentrations greater than 0.5 mg 1, Cl was constant, whereas for concentrations less than 0.5 mg 1. C'!l was constant.3,Mf On the grounds that the incapacitation of troops may he as valuable as their death in most military situations, and that the incapacitating dose of an agent may l>e quite different from the lethal dose, studies were carried out on rabbits to examine the possibilities.178 The results indicated that exposure of rabbits to 0.05 mg 1 for 10 minutes caused significant changes in the oxygen-carrying capacity of their blood, but that the effect was transient. After 10- minute exposure to concentrations between 0.13 and 0.20 mg 1 the rabbits were no longer able to maintain a relatively high red blood cell count, and the de- crease in oxygen-carrying capacity of the blood was severe in about half of the animals, whereas with 10-minute exposures to concentrations between 0.234 and 0.40 mg I a marked decrease in hemoglobin was invariably noted. A similar decrease in the hemo- globin content of human blood might be expected to cause severe but sublethal casualties. Therapy. J idhiul compoimds are effective in the treatment of arsine poisoning, although BAL-ethyl ether (2,3-dimereapt opropy I ethyl ether) is more effective than HAL itself.**8 Since BAL-ethyl ether is tolerated by human Ixangs in therapeutic dosages without toxic symptoms, the compound appears to be suitable for the treatment of arsine poisoning in man.-’1® Assessment of Valor as a Chemical Warfare Agent. The conclusion of (he United States Chemical Warfare Sendee in 1939 was that the value of arsine as a chemical warfare agent would depend on the question of canister protection.'25 The British in 1941 concluded that the only potential method for the liberation of arsine would lie by high-capacity bombs and that the only possible advantage over gases of the phosgene type would be that its detection at low concentration is more difficult. In order to utilize low concentrations of arsine, however, exposure must be prolonged and this is very difficult to obtain short of excessive effort, so that on the whole arsine should not merit any particular consideration as an offen- sive weapon, provided respirator protection is ade- quate.236 The question of canister protection against arsine SECRET PHYSIOLOGICA I. SECTION 99 has been summarized as follows; “At one time arsine was thought to lie a very promising war gas because it penetrates humidified unimpregnated or copper oxide impregnated charcoal very readily. With the introduction of silver impregnation, however, the protection against arsine was made almost compa- rable to phosgene. .. .” 100 The weight of arsine that would have to be ex- pended to produce a lethal concentration is theoreti- cally about 10 times as great as the weight of phos- gone required for t lie same purpose.236 Since, in ad- dition, modem res) lira tors give adequate protection against it, arsine shows little promise in chemical warfare. Noxhalogexatkd Arsixe Derivatives A numher of tertiary arsine derivatives have been examined for toxicity. Data for 51 such compounds were obtained by the UCTL,** and reference to these and to other tertiary arsines are listed in Table 0. Tabi.e 9. Arsenical compounds examined as candidate chemical warfare The com|Miumls in Table It are arranged in the following categories: 1. Derivatives of arsine. 2. Derivatives of primary arsines. 3. Derivatives of secondary arsines. 4. Tertiary arsines. “ 5. Quaternary arsenic derivatives. 0. Arsenic analogs of hydrazine. 7. Derivatives of arsenic oxides, sulfides, and amines. 8. Halogen and oxygen derivatives of tertiary arsines, 9. Derivatives of arsenic, arsenic, and arsinic acids. — 10. Arsenic derivatives of uncertain constitution. British reports describing the examination of com pounds marked with an asterisk are not Centigrade scale is used throughout the table. agents,- all available. Reference _ _ to Compound synthesis Physical properties- 1{efc™ toxicity Properly Reference data Derivatives of arsine 1. Calcium arsenide — — 311 d1* 2.5 311 311 2. Arsine 296b, 311 d” 1.44 296b 23, 311 " nip 110.1116.0° 296b *'P 62.8° 296b 3. Arsenic tnflnoride 311,333 d* 2.6659 298a 68, 311 — nip 8.5° 298a . . . i>p 60.4° 29Sa • . . vol 152 311 4. Arsenic trichloride* ~ ... NdIU 1.6009 311 252 ,p« 2,163 244 - _ .... inp 13° 311 bp7*" 129-130 244 ... voP6 84 311 5. Arsenic trichloride - dioxane complex* 227 6, Arsenic trichloride — thioxane complex* 227 7. Arsenic pentafluoride 342 mp 80.4° 298a Derivatives of primary arsines bp 52.8° 298a ... 8. Mcthylarsine " 5 bp T 5 9. Melhyhlifluorarsine* 296c, 298a d 1 9725 29Sa 298a nip 30° 296c, 298a ■ ‘ ’ — - - . . . bp 76° 296c, 298a 10. Mcthyldichlorarsine* 32, 200j, 311 n i/8 1.5588 32 27, 43,68, 311 rp° 1.8358 32 ■ ... mp 42.5° 32 bp7*" 132.5° 32 • vol1" 74.4 311, 70 * — 68.3 11. Chloromcthyldichlorarsine — 47 bp1® 53° 47 27, 68 ... vol 135 27 SECRET 100 ARSENIC ALS Compound Heference to synthesis Physical properl ies Properly Hefcrenct Reference In toxicity data 12. 2-Chlorovinyldifluorarsine* 113, ISO, 290c J* 1.97 ISO 121 — mp 20° 180 bp" 4 bp vol2* 13.5° 105 110° 31.77 296c ISO 180 13. 2-Chlorovinyldichlorarsine* See llibli- Hu5* 1.6073 43 See llibli- Lewisite (isomer 1) ogrnphy d24 1.879 27 oKraphy ' mp 2.4° 27 bp10 75° 43 bp16" 1 !KJ° 27 Vol-° 2.3 311 VoI‘° 4.47 70 Lewisite (isomer 2) See Hibli- II I.51 1.5900 27 See Hibli- o^mphy d2* 1.8681 27 ography bp10 02.8° 27 . . . 14. 2-( dilorovinvldkhlorarsine-dioxanc complex* ... bp76” 150.2° 27 227 15. 2-Chlorovinvidibromoarsinc* 231 bp17-1* — 106-107 231 231 in. 2-Hromovinvldibromoarsine* 231 bp”* 132 137° 231 231 17. 2,2-1 Mchlorovinyldichlorarsine* 311 .... 311 IS. Elhvlarsine 39 tP* 1.217 “ 39 68 bp73* 35 36 39 19. Kthyldifluoroarsine* 112, 296c d 1.743 296c _-ii7 ‘ ... — nip 38.7° 296c bp 94.3“ 296c 20. Ethvldichlorarsine* (KD) 5, 32, 4S, n uH s 1.5588 311 27,43, t»S, 58, 311 cP" 1.6595 32 79,311 bp734 153 155° 5 _ . voli0 21.9 127 — ... Vol29 30.2 70 21. Et hyldibromoarsiue 58 n if* 1.6405 58 68, 79 (P‘ 2.103 58 bp'6 87-88° 58 vol2* 5.72 70 22, 2-Chloroel hyldichlorarsine* 1,111, 114 bp1* 99.8-100° 1 27, 79, 116 23. 2-Hvdroxvelhyldichlorarsine* 227 24. 2-Methoxyethyldichlorarsine 1 (P“ 1.693 1 27, 79 bp* 94 95- 1 bp14 102 103° 1 ... 25. 2-El box vet hyldichlorarsine 1 iP« 1.605 1 27, 79 bp19 95-97° 1 20. Allvldichlorarsine 1,32,38, «D79 1.5702 1 105 ,p‘ 1.6294 32 bp4-* 42° 32 68, 121, 27. 3-Chlorallvldichlorarsine 301c bp18 104 105° 301c 79, 120 227 28. Fropyldichlorarsinc* 32 no28 1.5297 32 43, 68, 79 ,P« 1.5380 32 mp 28.2° 32 — bp7* 99° 32 ;;; bp76" vol20 175.3° 12.4 32 70 29. Propyldibromoarsine* MO. Propyldicyanoarsine * -rr. . 227 3!) nip 82-86° 39 68. 79 31. Isopropvldichlorarsine* 227 32. 3-Chloropropyldichlorarsine* 311 311 33. 3-ChloromethoxypropyIdichlorarsinc 5 bp* 130-137° 5 27, 79 Table 9 (Continual). SECRET PHYSIOLOGIC.*I, SECTION 101 Table 9 (Continued). Compound Reference to synthesis Physical properties Property Roferenco Reference to toxicity data 34. 2-('hlor»>-3-(2-chIorocthyltliio)-l-bulenyldi -hlor- nrsine* 35. Butyldichlorarsine* I, 32, 130 d3" 1.4064 32 7~ :N -r 1 N bp7** 194’ 32 121, 130 vol3* 6.3 1.30 36. Bulyldibromoarsine* 37. Hutyldicyamiarsinc 58 inp 61-63° 5S 227 68, 79 38. sw^But-yldichlorarsinc* 32 nua 1.5245 32 27, 68, 79 ,p« 1.4128 32 bp7** 182° 32 — bp11 39“ 32 31». 2(or 4)-( 'ldoro-3-methyl-1,3 (or l,2)-bul-adieayl- dichlorarsine* 227 40. 4-Pentenyldiehiorarsinc 32 »■>** 1.5698 32 27, 68, 79 . *.’ d» bp5* 1.453 102.5-105.5 32 ° - 32 ■ ■ - ■ bp!‘ 111 114° 32 vol 0.12 27 — 41. 2-Chloro-l-| >oiitonyIdichlorarsinc 38 bp54 130 1330 38 68, 79 42. A my la rsi ne ‘ 58 bp'3" 125-127° 58 68 43. Amyldiclilorarsiuo* 28, 32, 58 ntr* 1.5177 32 27, 43, 157, — (P'< 1.4035 32 79 _ bp3* 118°- 32 44. Amlydiliromoarsino 39 bp34" an11* 213° 1.5760 32 39 68, 79 lP» 1.8804 39 bp18 125.5 127' 39 ... bp734 248° 39 vol 0.399 70 45. Amyldicyanoareinc 58 nip 6(b 69.5° 58 68, 79 46. Isoa my id ichl< >ra rsine 28, 32 an3* 1.5157 32 68, 79, 157 -( ’hloroacetylphcnyldiclilonirsiiie* 227 91, p-Xenyldichlorarsine* 227 92. 2-{4-C'hloroacetylphcnyI)phenyklichlorareine* 227 93. 2-Fluorencdich It >rarsiuc * 227 94. 2-Fhlorenonedichlorarsine* 227 95. o-Benzoylphenyldichlorarsinc* 227- 96. 2,2'-his(Dichloroarsino)still(cne* ... - 227 97. 2- Xa ph 1 hyldichlorareine 73 mp 74.6-75.1° 73 68, 79 98. 2-Furyldichlorarsine* l,25,301d, 311 no1** 1.6092 25 27, 311 erl ies Projierty Reference Reference to toxicity data 100. 2-Dichlorarsinophenoxthiin 84K nip 64 -65° 101 d 68, 79 110. 6-I)ichlorarsino-2-phenvlben/thiazole hydrochloride 84h 111. l»i(t(2-Dichlorarsinoclhvl)Kiilfonc 1 mp 70.5 80.5° 1 68 112. o-Phenvlerie-bbKdichlorarsinc)* 227 113. «(-Phcnvlcne-6/s(dichIorarsine)* 227 114. p-Phenvlcne-fc/s(diehlorarsine)* 58, 311 d 2.15 311 68, 79, 311 - mp 97 98° 58 bp” 200° 311 115. 1,\-hiM L)ichh)rarsino)-2-nil rolienscne 58 nip 72.6-74.3° 58 68. 79 116. p-Dichh)rostibinophenyldichlorarsine* ... 227 117. 2-I)lchlorarsiuudiphenylchlorarsinc* 227 118. 6is(/i-I)ichlp 160-161° 58 124. Dimel hylthiocyunoarsinc 73 nii” 1.6100 73 68, 79 1.4695 73 bp25 106-107° 73 125, 6i«(Chloromet hyl)chloroarsinc 47 d ca. 1.85 27 27, 68, 79 bp10 75° 47 -s.-. 126. Reaction product of mercury chloroacctylidc and ar- senic trichloride* 227 127. 6(’s(2-ChlorovinyI)chlorarsine*(lewisite II) 311 nlt" 1.6096 311 235, 311 d" 1.7047 311 bp'“ 112° 311 bp30 136° 311 128. 6i)i(2-('lilorovinyl)cvanoarsine* . 227 120. 5is(2,2-Dichlorovinyl)chloroarsine* 311 .... 311 130. h/s(2-Hromovinyl)hromoarsinc* 231 bp’6 153-158° 231 231 131. bis{ 1,2,2-TrichlorovinyDchloroarsine* 231 bp5 141° 231 231 132. Diet hvlchlorarsinc 58 n o2; 1.5080 58 68, 79 d“ 1.368 58 ... bp" 52-54° 58 . bp™ 156° 58 133. Diethylcyanoarsine 58 Bn” 1.4863 58 68, 79 d!h 1.238 58 bp11 80-81 ° 58 lip737 190-191° 58 ~ . . . 134. Kthylpropylchlorarsine 39 tP” 1.330 39 68, 79 bp-" 82 85° 39 bp7” 176° 39 135. I't hylpropylcyanoarsine 58 n ir7 1.4838 58 68, 79 <7* 1.194 58 bp77 110 113° 58 vol3" 1.45 70 130. Kthylpropylthiocyanoarsine 73 Hi)1* 1.5674 73 68, 79 1.286 73 - ... bp" 66 102-110° 73 137. Ethylbutylchlorarsine 58 no3' 1.5025 58 68, 79 d36 1,272 58 bp” 89 92° 58 ..... SECRET 104 ARSE MCA I.S Tabi.K 9 (Continued). Compound Ilefcrence to synthesis Pro Physical proper! |x‘rty ics Hefprrnct* Reference to toxicity data 138. Kthylbutylcyaniarsinc 58 n i,-'_ 1.4828 58 68, 79 iP‘ 1.152 58 139. h(x(2-Chh>ro-3-(2-chloroethyllhio)-l-butcnvl) chlor- bp»» 112-112.5° 58 arsine* 227 110. F )ihntvlu«hmrsine 841. 68, 7'.* 141. hidCvclohexyl tehlora rsine* 227 142. Methvlphcnylarsine 73 (P 1.31 73 bp57 108-111° 73 bp" 128-129° 73 ' 143. Methvlphcnylehlorareine 58 «n31 ~ 1.6022 58 68, 79 ■ , .. * ,P< 1.449 58 bp” 127° 58 14 I. Mel hylphenyliodoa rsine* 145. Melhylplienylcyanoarstne 58 n n76 1.5812 58 68, 79 1.372 58 bp=" 147-148° 58 14*1. Met hvlphenvlthiocvanoa rsine 58 n n” 1.6577 5S 68, 79 1.433 58 bpI4~'8 176-179° 58 147. m-Chlorophenylmelhylchlorarsine* ... 227 148 Methvl-w-nitrophenvlehlorarsine 73 n,r 1.6272 73 68, 79 roa rsine* 227 163. Diptienylchlorarsine (DA)* 73, 311 n D» 1.6429 73 79. 68, 311, 249 — 1.413 73 ' • mp 38° 244 bp" 7 157 160° 73 vol50 <0.0001 . 311 164. 2-('hlorophenylphenylchlomrsine* 301e mp 30-35° 301c 227 165. 3-Chlorophenylphcnylchlorareinc* 227 166. 4-ChlorophrnyIphcnylehlora rsine* .. r. 227 167. 2-Nilrophenylphenylehlorareinc* 227 168. 3-Xitroplienylphenylclilorarsine* 227 169. 4-Nitropheiivlplienylchlorarsine* 227 170, h/>(2-Chlorophenyl)chlorn rsine 30Ie mp 73 75° :301c 227 171. 5/>(4-Chlorophenyl K'hlorarsine* 227 172. fc/«(3-Xitri»plienyllehlornrsine* 58 mp 112-113° 58 68, 79 173. 2-Phenvlchlorarsinoaniline hvdroehloride* .... 227 174. 3-Phenvlchlorarsinoaniline hydrochloride* —r. . 227 175. 4-Plienvlchlorarsinoanilino hydrochloride* 227 176. 67*(»i-Aminophcnyl)chlorarsine dihydrochloride* 227 SECRET PHYSIOLOGICAL SECTION Compound Reference to synthesis Physical properl ies Projierly Hcferi-noe Reference to toxicity data 177. 6(«(/*-Aminiiphcnvl)chlorarsine dihydrochloride* 227 178. hix{ ;>- Met hoxv phenyl lehlorarsine* 227 179. 1 Jiphenylhromoarsine* 341 . .77 121 iso. Diphenvlevanoarsine ( DC)* 311 n a50 1.6254 244 311 J* 1.3327 244 inp 31.2° 244 - - Veil*" 0.0001 0.00015 311 1KI. 2-Chlorophenvlphenvlevanoarsine* 301e nip 40-42' :30ic >27 182. 3-( ’hlorophenylphenylcyanoarsine* 227 183 l-Chlorophenylphenylcyanoarsine* 30 le mp 102 301c 227 184. 2-Nilroplienylphenylcyanoarslne* >27 185. 3-X it rophenylphenylcyanoarsine* 227 ISO. 4-Nitrophenylphenylcy»noarsine* 227 187. bm(‘2-( 'hlorophcnyl )cyanoarsine* 30 le nip 85-87° 301 e 227 188. ftj*(4-Chlorophenvl)cyanoar>dne* 227 189. bi»( 3- Nil ropheiiyl )cyanoarsine* 58 nip 151-152 58 68, 79 190. 2-Phenvlevanoarsi noaniline* .... 227 191. 3-PhenvlevaiUKirsinoaniline* 227 192. 4- Phen vie vain mrsinoaniline * . . . 227 193. hixfin-Aminophenyllcyanoarsine* 227 194. Diphenylt hiocyanoarsiue* 58, 301 j 1.6766 58 68, 79 • 1.379 58 bp*-* 217 219° 58 195. hix{ m-N i I rophcnyl it hit icy anon mine 58 mp 103-105° 58 68, 79 196. Phenvl-o-tolylcvnnoarsine* 301e ■227 197. o-Chlorophenyl-o-tolyfcyanoarsine* . 227 198. p-Chlorophenyl-o-tolylcyanoarsine* 227 109. o-Nitrophenyl-o-lolylcyanoarsine* 227 200. i/i-Nitrophenyl-o-lolylcyanoarsine* . 227 201. ;j- N i I rophcny 1-o-toly Icy anoa rs i i in * — . T.- 227 202. Phenyl-w-tolvlcvanoarsine* 227 203. Phony 1-p-tolylchlorarsinc* 30 le -... 227 204. Phenvl-p-tolvleyanoarsine* 30le 227 205. o-Chldrophenyl-p-tolylcyanoarsine* .... ‘227 206. «i-Chhirophonyl-/>-tolyleyanoarsine* 227 207. p-Clilorophenyl-p-tolylcyanoarsine* . - - 227 208. p-Nitrophenyl-p-toiylcyanoiirsine* 227 209. m-(Pheiivlcyanoarsino)bcni!aldehyde* 7. . 227 210. o-( Phony lohlorarsinolbenzoic acid* 227 - 211. Methyl o-(phcnylchlorarsino)l)enzoate* 227 212. w-( Phenvlehlorarsino)bonzoio. acid* :301b, 301 j mp 134 136° 3011. 301 h 213. Methyl m-{phenylchlorarsino)benzoate* 301b, 301 j ,. . 301 h 214. Methyl »i-(phonylcyanoarsino)l»eiizoate* 301b, 301 j _ 30 Ih 215. ;>-(Phenvlehlorarsiiio)l)enzoic acid* 301b, 301 j mp 115 117° 3011. 301 h 216. Mcthvl p-{plieiiylchlorarsino)bcnzoate* 301b, 301 j • ... 301 h 217. Methyl p-(phenylcyanoarsino)benzoate* 301b, 301 j .... 301 h 218. 2,4-1 )i methyl phony Ipheny Icy anoarsine* 227 219. his{ o-Tolvl )cyam mrai ne * :301c mp 74° :30ic 227 220. bi»(o-i ’a rlxiinel hoxvphcnyl lehlorarsine* 227 221. o- T ol v 1-m-l ol vlcvanoarsi ne* 227 222. o-TolvI-p-tolylcyanoarsine* 227 223. bix( m-T i >lyl )cyanoursine* . • • 227 224. t/i-Tol \ 1-p-t«>ly leva noa rsi ne* 227 225. bi.i( p- T i dyl leyanoarsine 30 le mp 62“ 301 e 227 226. /«-(Phcnylehlorarsiin»)acetophenone* 301 j mp 71 72° 30 Ij 227 227. it) A Phenvlcyalioiirsiiio)«celophenonc* 30 Ij mp 57-59° 301 j 227 228. /«-( Phony Ichlorarsinol-uM’hloroa cot ophenone* 301 j 227 229. ;«-( Phony loyanoa rsi no)-t^-chloroacetophenono* 301 j 227 230. 2.1-I)i met hylphenyl-«-loly leyanoarsine* ...- 301f 231. 2,4-l)imothvlphonyl-//-tolylcya noarsine* 301 f 232. tr-N.aphl hylpheny lehlorarsine* .,. „ . 227 233. d-N aph t hylpheny lehlorarsine* 227 Table 9 (Continued). SECRET 106 ARSENICA LS Tvhi.e 9 (Continued). Compound Reference to synthesis Physical properties Pro|.erty Reference deference to toxicity data' 234. a-Xaphthylphenylcyanoarsine* 301e mp 98 99° 30 le 227 235. 0-Xaphthylphenylcyanoarsine* 227 236. a-Xaphthyl-p-tolylcyanoarsine* 227 237. (8-Xaphlhyltp-lolylcyanoarsine* 227 238. 6is(nr-Xa|ilithyl)ch!orarsiiu- 301 j mp 163-165° 301 j 239. h.s-or-Xaphthylcyam.arsinc* 301 j mp 191° 301g 301g 210. 6(s-0-Naphthylchlorarsine* 227 241. b.K-d-Xaphthylcyanoursine* ...— 227 242. Phenyl-(xV-thienylchlorarsine* 301 e hp0* 150 156° 30 le 227 243. Phony l-fxj-thicnylcyanoarsine* 301c mp 49-51° 30le 227 bp" * 168-174° 301o 24 1. Phem’l-3-pvridvlchk.rarsine hydrochloride* 227 245. Phenvl-3-pvridvlchh.rarsine melhiodule* 227 246. Phenyl-3-pvridylcyanoarsine hydroohlorido* ... 227 217, 5-Phcnylchlorarsino)-2-chloropyridinc hydrochloride* 248. 5-( Phenvlehlorarsim.)-2-amin. .pyridine dihydrochlo- •••»- 227 ride* 227 “ 249. 6i8(or-Furyl)clilorarsinc* _ 1,25, 30Id nn2S 1.6082 25 68, 79 " 1.5909 25 : bp* 122 127° 25 250. 6i*(o-Furyl)cyanoarsine* 39, 30 lo tivr* 1.5749 39 68, 79 ...

1.4857 39 . .5 l.p51 142° 39 251. fcfs(a-Thienyl)chlorarsine* T.. 227 252. ft i»(a-Th io ny 1 )cya noa i> i 110 * 30 le mp 51 55° 301e 227 bp1’6- 180-182° 301 e 253. his(3-Pvridyl)chlorarsine dihydrochlorido* 58 mp 270-273° 58 68 254. bis( 3- Py ridyl )cyanoarsi no * 227 255. p-Phonylenechlorarsino 1 mp ca. 145° I 256. 1-Chlorars indole* 257. 2-(Dielhylaminomethyl)-!,3-dichh.rarsindole hy- 227 drochloride 73 mp 204.2 205.2° 73 68 258. 5-Chlorodihenzarsenole* 5, 3071. mp 161° 5 68 i.p’-4 230° 5 259, 5-(’lil<.ro-3,7-dinitrodil.enzarsenole* — 227 260. 5-Chlor.>3-amini slit .enzarsen. tie* 227 261. 5-Chloro-3,7-diaminobenzarsenolc hydrochloride* 227 262. 5-Cvanodil.enzarsenole* 5 mp 178° 5 68 263. 5-l«dodibcnzarsenole* 227 264. 4,1 '-Diphcnylenechlorarsine* 227 265. 5-Chloro-5,10-dihydroacridarsinc* 39, 3071. nip 113 114° 39 227 266. 2,5-Dichlomd>,10-dihydroaeridflrsine* 307m mp 116° 291e, 307m 29 ic 267. 5-Cyano-5,10-dihydr. laoridarsino* 307k mp- 114-115° 307k 285. 291.1 268. 2-Chloro-5-cyano-5,I0-dihvdroacridarsine* 307m mp 113-114° 307m 285, 291c 269. 5-Chloro-10-oxo-5, lO-dihydroacridarsine* 227 270. 6-C 'hh>r<>-2-mct hyl-5,10-dihydroacridarsinc* 307j mp 87° 307j 285 271. 5-Cvano-2-melhvl-5,10-dihydroacridarsine mp 87° 29 If 285, 29If 272. 5-Chtoro-3-met hyl-5,10-dihvdroacridarsine* mp 65.5-66.5° 307n 29 le 273. IO-AcetyI-5,10-dihydrophenarsazine* 227 274. 10-Acel vl-5,1O-dihvdrophenarsaziiie piorate* 227 275. 10-Trichloroacetyl-5,10-dihydrophenarsazine* 227 276. 10-Chloro-5,10-dihydrophonarsazine (DM )* 38. 73, 311 >r-" 1.648 311 68, 79,311, 104 mp 189-190.4° 73 vol;o 0.00002 31 277. 5-AcetvHO-chloro-5,IO-dihvdrophcnarsazine* ♦ . . 227 278. 5-Propionyl-IO-chloro-5,IO-dihydrophenaraazine* 227 279. 5-Benzoyl-10-clJoro-5,10-dihydrophonarsa2ine* 227 280. 10-Hromo-5,10-dihydrophoua rsazino* 227 281, 10 lodo-5,10-dihydrophenarsazine* 227 282. 10-Cvano-5,10-dihvdrophenarsazine (Cyan DM)* 107, 108 121 283. l(>Thiocj'ams5,10-dihydrophonarsazine* 227 SECRET PHYSlOLCXaCAI, SECTIO> 107 Table 9 (Continued). Compound Reference to synl hesis Physical proper! ies Property Referenc* Reference to toxicity data 2SI. l(or 3),10-I)ichloro-5,10-dihydropheiiamazine* 227 285. 2,10-Dich!oro-5,10-dihydrophenarsazine* 227 280. 10-Chloro-5,10-dihvdro-4 (?)-nitrophennrsazine* 227 2S7, l(or 3 ),2,10-Tricliloro-5,1O-dihydrophenarsazine* 227 288. 1,3,10-Triehloro-4j,l O-dihydrophenarsazine* 227 2S!». l,9(or 3,7),10-Trichloro-5|10-dihvdrophenarsazinc* 227 290. 2,8,10-Trichloro-5, lO-dihydrophenarsazinc* 227 291. l,2,3,10-Tetnichloro-5,10-dihydropheimrsnzine* 227 292. 2,4,t5,8,l0-Pentahromo-5,10-dihydropl»enarsazine* ■ 227 293. 2-Aminu-10-ehloro-5, 10-dihydrophenarsazine hydro- chloride* 227 291 l(or 3)-Amina-10-chloro-5,10-dihydrophcnarsazine hydrochloride* 227 295. 1-Amino-lO-ehIoro-5,10-dihydrophenarsazine hydro- chloride* — 227 290. 10-( ’hloro-5,10-dihydro-l(or 3)-methyIphenarsazine* 227 297. IO-(.'hloro-5,10-dihvdro-2-melhylphenarsazine* .... — 227 298. 5- Ace t y I- 10-cl ik m >-5,10-dihyd ro-2-met hy Iphena rsa- zinc* 227 299. lO-Chloro-5, lO-dihvdro-4-mcthylphenarsazine* 227 300. 1 (or 3), 10-Dichh>ro-5,10-dihydro-6-methyIpheimr- suzine* . — 227 301. 4-Amino-10-ehloro-5,10-dihydro-7-methylphenar- sazine hvdn >chh iride * 227 302. 10-Chloro-5,10-dihydropheiiarsazine-l(or 3) car boxy- — lie acid* 227 303. 10-('liloro-5, 10-dihydrophenarsazine-4-carhoxylic acid* ... 227 304. 10-Chloro-5,10-dihydro-l(or 3)-0-dimelhylphenar- sazine* — 227 305, 10-('hloro-5,10-dihydn>-2,8-dimcthylphenar8a*ine* 227 300. 5-Aeetyl-10-ehloro-5,10*diHydro-2,8~4limet hylphenar- sazinc* __ 227 307. 1 (or 3 )-Acetyl-1 0-chloro-5,10-dihydrophenarsazine* ~ ~ 227 308. l(or 3)-Acelo-10-hromo-5,l 0-dihydrophenarsazine* 227 309. 10-C'hloro-5,10-dihydro-l ,4,7-t rimet hylphcnarsazine* 227 310. 10-Chloro-5,10-dihydro- 2,4,7-t ri met hylphenarsazine * . r. 227 311. 10-( ’hloro-5,10-dihydro-l(or 3)-propionylpheuar- sazine* — 227 312. 12-Chloro-7,12-dihydrohetiz (a) phenarsazinc* ... 227 313. 7-C'hloro-7,12-dihys n ir’ 1.5665 58 68, 79 d* 1.473 58 bp18 97-103° 58 vol!" 1.72 70 335. 6t«{2-Chioroethvl)methylarsine* 307p bp** 50-55° 307p 227 336. bis(2-Iodoet hyl )met hvlarsine 307p bp11 68-69° 307p 337. ft/s{2-F.thoxyethyl)melhylarsine 58 «n“ 1.4664 58 68, 79 ... — d* 1.100 58 hp** 124-125° 58 bp“« 230° 58 338. triM.2-Cl.lorovinvl)an«ne* 73 227 339. (riX2,2-T)ichlorovinyl)arsine* 227 340. Triethylarsine 102 68, 79 341. 2-Chlon.ethvUlicthvlarsine* 307p bp10 53° 307p 227 342. 2-llronmctl.vldiel hvlarsine* 227 313. fcis(2-ChlorocthyIJethylarsine* 307p bp11 64° 307p 227 344. Ir/sfCvclohexyllarsim* 39 bp! 187-189° 39 68, 79 315. Trioclvlarsine 101a 0.9357 101 a 68, 79 bp‘» 238-240° 101a ' bp1 181-185° 101a 346. Phcnvlarsenophosijcne 84 i ... 347. 2-(p-Dimethylarsinophenyl)quinoliue 84 j 348. 6/*(2-Chh»r«vinyhphenylarsii.e* 1, 231 tp" 1.384 1 68, 231 bp18 166-171° 231 319. 2-('hloroviiiyldipbcnylarsiiM>* 1 d* 1.327 1 68 350. Triphenylarsine 73 nip 58- 60.5° 73 351. tris( w-X it rophcnyl )arsinc 352. Ins(p-Dimethylaminophenyl)arsine sulfur monochlo- 7 ride addition product* 227 353. Tn-p-lolvlarsine* ... ... 227 354. Dimet hyl-2-pyridylarsinc 84j 68. 79 355. Difurvlmcl hvlarsine* 301e 267 356. 2-Chlorovinvl-5/s(2-furvl)arsii.c 267 »>pj 127° 267 267 357. fri *( 2- Fu ry 1 )arsi ne * 25, .30Id phena rsazine )* 227 383. 10, I0'-his(5-.\cet vl-5,IO-dihvdroj»henarsazine)* 227 384. I0,10'-6i»(5, IO-l)ihydrophenarsazine) sulfate* 227 385. 2,2',4,4',6,6-ltexanitroarsenobenzene 7 386. 4,4'-Dihydro.xy-3,3'-dinitroarsenolienzene 7 387, 4,4'-1>ihydroxy-3.3',5,5Metranitroarsenol;>enzene 7 Derivatives of arsenic oxides, sulfides, and amines 388. Arsenic oxide* — _ _ 227 389 Kthoxvdichlorarsine 841 68, 79 390. I timet hylaminodifluoroarsine ... - 68 391. Isopropoxydichlorarsine 84 m bp7*1 155-156° 84m 392. 2-( 'hloro-4,5-dihydro-l ,3,2-oxthiarsenolc 84 in «n2< 1.6690 84 m 68, 79 ,r.i 1.988 84m bp"-* 72-73° 84m 393. I tiethoxychlorarsine 841 68. 79 394 . 6/«(2-('hloroethoxy)chlorarsinc 47 lip1 * 112 118° 47 68 395. Diisopropoxyfluorarsine 84 j 396 tris( 2- KI uoroet hoxy (arsine 86 68 397. t r/*( 2-C h 1 o roe t h oxy )arsi ne 47 bp10 160-170° 47 68 398. trisi2-( ’hloroel hylt hio(arsine* 84 r 1.5972 S4r 68 399. /ris(Phenylthio)arsine S4o nip 92-94° 84 o 68 400. Mel hylarsine disulfide* 227 401. Phenyl met hoxychlorarsine* 227 402; F’henvlet hoxychlorarsine* 227 403. Phenvl-2-chloroelhvllhioehlorarsine* 297a 227 404. o-IIydroxychlorarsinolicnzoic anhydride* 58 mp 146.5-147° 58 OS hp” 233-230° 58 405. o-Phenylenediarsine oxychloride* 73 nip 150.5-151.5° 73 68 406. Methylarsenic oxide* 311 mp 95° 311 311 bp ca. 275° 311 407. Methylarsenic sulfide* 227 408. Met hvldimel hoxvarsine* 227 409. Melhyldimethvllhioarsine* __ 227 410. Methyhliethoxvarsine* 227 411. Methyl-5»s(X,X-diethvldithiocarbamvl) arsine* 227 4 12. Met hyl-5/x( N,S-bis(2-hvdroxvethyl (dithioear- liamyl (arsine* 227 413. Methyld iphenvlt hioarsine* 227 414. Metliyldi-p-loly It hioarsine* 227 415. 2-( ’hlorovinylarsenie oxide (various isomers)* 12, 311 Physical properties vary with method of prepa- ration 122, 12 68, 79 416. 2-Chlorovinylarsenic sulfide* 227 417. 2-( 'lilorovinvlarsenie selenide 67 08 418. 2-Chlorovinyldimcthoxyarsine 68, 79 SECRET ARSENIC A LS Compound Reference to synthesis Physical proper! ics Property Reference Reference t toxicity data 419. 2-Cblomvinyldimelhyhhioan.ine 68, 79 420. 2-Chlorovinyldiethoxyarsine* 30 bp17 ,s 84 85° 30 68, 79 421. 2-Cblorovinyl-5is(2-chlor.)cthylthio)arsine* 84p " ir" ./-" bp1 1.6100 1.610 SI 86° Sip 84p 84 p— 68, 79 422. 2-( 3dorovinvl-feis( 2-ethoxyet hoxv )arsiiH»* 227 423. 2-Chlorovinyl-fc.a{2-hydroxycthyIthio)arsine 33 bp* 83.1 .86.7° 33 424. 2-Cldorovinyldintlyloxyarsine* 227 425. 2-('hlorovinyldiisopropoxyarsinc* 227 426. 2-ChIorovinyldi|>entoxyar8inc* 427. 2-( 2-Chl«rovinyl )-5,5-6/s( hydroxy met hyl)-l,5-dihy- 227 dro-l,3,2-dithiarsin 33 mp 127 5-128 5° 3.3 68 428. 2-Chlorovinvldiisooctvloxvars.ne* 227 429. 2-Chlorovinyldiphenylthioarsinc*_ 430. 2-Chlorovinyl-5is(N,X-iliethyldithioc»rhamyl)ar- 227 sine* 227 431, Elhylarsenic oxide* 103a, 31) 1.5821 311 68, 79 — !t 1,4715 58 68, 79 ,r-‘ 1,335 58 437, .Vrnylarsenic oxide 35 bp13 120 123° 58 438. Isoamylarsenic oxide 35 439. Ilcxvlarsenic oxide 35 440. Phenylarsenic oxide* 39, 311 mp 118-120° 39 68, 291a 441. e-Xit ro phenyl arsenic oxide 7 (Rut varies with method of preparation) 311 442. w-Xitrophenylarsenie oxide 443. 2,4,6-Trinit rophenylarsenic oxide 444. 2,4,6-Trinilrophenylarscnicdinitrate 58 7 7 mp 184.5-187.5° 58 68, 79 445. PhenvT5ix{2-ehloroelhyllhio)arsine* 297a 227 446. p-IIydroxyplienylarscnic oxide* 227 447. w-Aminophenylarscnie oxide* 448. p-Dimcthylaminophenylarscnic oxide* 449. 3-Ainino-4diydroxvpheny)arsenic oxide hvdroehlo- 7 227 2911. ride (Mapharsen) 450. o-Arsenosol ten zoic acid* Commercial .... 334 227 451. m-.\rsenoeohcnzoic acid* 452. 3-Pvridvlarseuic oxide* — 227 227 453. 2-f’h!oropyridine-5-arsenie oxide* 227 454. 2-Dihenzolhienylarseiiie oxide 84f 455. 4,4 '-biM Arscno8o)biphenyl* 227 456. 5(*(Dimethylarsenie) oxide (cacodyl oxide) 8, 38 bp 149 151° 38 68, 79 457. 2-Chloroethylt hiodimelhylarsine 84 m bp1* 94 95° 84 m 68, 79 458. bix{ Dimethylarsenic) disulfide* 227 459. his( Diethylarsenic) oxide 17 460. 6i*(hi>-2-Chlorbviiivlarsenic) oxide 235 461. 6is(2-Chlorovinyl)-2-cl.loroelhylihioarsine- 84., W I)*6 1.6085 84 q 68 bpni 128-129° 84q 462. 6i«(2-Chloroviiiyl)-l,3-diehloropropyl-2-thioar8ii>c S4s bp*-* 148-151° 84s 68 Tabi.k 0 (Continual). PHYSIOLOGICAL SECTION 111 Table 9 (Continued). ('on»|H>und Reference to synthesis Physical properties Property Reference Reference to toxicity data 463. Melhylphenylmethoxyarsinc 58 nu" 1.5613 58 68, 79 tP' 1.295 58 bp"- 101 102° 58 464, Mclhylphenylacetoxyarsinc 58 nn" 1.5612 58 68, 79 oxvphenyl) hvdroxyarsinc anhydride* . 227 472. bi»{o-Carl«)xypln nylpheiiylarsenie) oxide* 227 473, hiM /> (. ‘arboxyphen vlphenvlarsenic) oxide* •227 474. p-Phenvlene-6(s(phenylarsinc) monoxide* — 227 475. Acetoxydithienylarsine* 227 476. 1,3-Dihvdroxvars indole* . . 227 477. bid Dilienzarsenyl-5) oxide* 227 478. b;.<(3,7-D'nitr<>diben*arsenyl-5) oxide* ... ... 227 479. b ts( 3- A tn i in td i be n*a rseny 1-5 ) oxide* 227 480. bis( riienoxarsinvl-10) oxide* 288a mp 182“ 227 481, 10-Chloroe(hvlthio-5,10-dihvdrophenarsazine 84 n mp 128-153° 84n 482. ?»>(5,10-Dihvdroplienarsatiiiyl-101iixide* 227 483. bis( 5, UP Dihydrophenarsazinyl-10) sulfide* 484. his(b, 10-Dihvdrophenarsazinyl-10) oxide. 10-Chloro- ••• 227 5,10-dihydrophenarsazine (basic DM)* 227 485. 6/«(5-Acctvl-5,10-dihydrophcnars«zinyl-10) oxide* 227 486. b/.s(5-llen/,ovl-5,IO-dihydrophenarsazinyl-IO) oxide* . .-r- 227 487. 5,10-Dihydroarsa n 1 hrene-5,10 monoxide* 488. 5,10-Dihvdro-5,10-dioxvstibarsanthrene-5,10 mon- 227 oxide* _ _ Halogen and oxygen derivatives of tertiary arsines 227 489. Triphenyldichlorarsine* 227 490. Tri-wi-nitropbenyldibromoarsinc 7 491. Tn-p-tolyldichlorarsine* 227 492. Tri-m-nitrophenvlarsenic oxide 7 493. Tri-m-nitroplienvlarsenie dinit rate 7 mp 147-148° 7 -.. 494. o-Carboxypbenylplieiiylmelliylareenic oxide* 495. 7-Plienvlmethvloxidoarsino-2-naj»lithalcne8iilfonie •227 acid*. Derivatives of nrsenie, arsonic, anil arsinir arids - ■” •227 496 o-Nitroanilinimn arsenate 7 mp 146-147° 7 497. w-Nitroanilinium arsenate 7 mp 114 115° then 7 — 147-148° 7 498. p-Nilroanilinium arsenate 7 mp 77-78° 7 499. Sodium metbanearsenate hydrate 69 08 500, 2-Cliloroethvleiiearsonic acid* 69 mp 129° 69 68, 79 501. Kthanearsonic acid fit) mp 90° 69 68 502. Benzenearsonic acid 58 mp 165° 58 68, 79 503. o-Nitrobenzenearsonic acid 7 504, Cadmium o-nitroliemsenearsonate 34 505 Ijead o-nit rolK-nzenearsonate 7 506. Hi-Nitrolienzenearsonic acid 7 . ... 507. I .ead m-nitrobenzenearsonate 7 508. p-Xilrohenzenearsonic acid 7 509. Sodium 2,4-dinitrobenzenearsonale • 7 510. Magnesium 2,4-dinit rohenzenearsonate 84a 511. Potassium 2,4-dinitrobenzeneareonalc S4a ... SECRET 112 ARSENIC\LS Table 9 (Continued). Compound Reference to synt hesis Physical properties Property Reference Reference to toxicity data 512. Manganous 2,4-dinitrolienzenenrsonate 84a 513. Ferric 2,4-dinitrolienzenearsonate 84a 514, Cohalt 2,4-dinitrolienzenearsonate 84 a 515. Nickel 2,4-dinit rohenzencarsonate 84a 510. Cupric 2,4-dinitrolienzenearsonate S4a 517. Cadmium 2,4-dinitrolienzenearsonate 34 518. Stannic 2,4-dinitrolienzenoar8onate 84a 510. Barium 2,4-dinit robenzenearsonate 84a 520. Mercuric 2,4-dinitrolienzenearsonate H4a 521. I-ead 2,4-dinitrobenzenoarsotmte 101 b 522. 2,4,0-Trinitrobenzenearsonie acid 7 523. Sodium 2,4,0-1 rinitrolienaenearsonate 84a 524. Potassium 2,4,6-trinitrobenzeneansoiiate 84a 525. Calcium 2,4,0-trinitrobenzenearsonatc 84 a 520. Cupric 2,4,6-trinitrolienzenearsonate 84a 527. Cadmium 2,4,6-trinitrobenzencarsonate . - 34 52H, Stannic 2,4,0-1 rinit ml x-nzenoarsonate 84 a .... TT." _ 529. Barium 2,4,6-trinitrolienzenearsonate 84a 530. Mercuric 2,4,6-trinit rohenzencarsonate 84a ... 531. la-ad 2,4,6-1 rinitmlicii/.enearsonate 7 5142. p-Ilvdroxylx-nzenearsonic acitl* 227 533. 4-Hvdroxv-3-nitrobcntsefiearsonic acid 7 534. la-ad t-hvdroxv-3-nitmlienzenearsonate 7 535. 4-llvdroxv-3,5-diniirolicnzenearsomc acid 7 530. Cadmium 4-hydroxv-3,5-dinitrobenzencarsoiiale 34 537. Lead 4-hvdroxy-3,5-din rtrobenzenearsonate 7 538. ('admium 2,4-dihvdroxy-3,5-dinitrol>enzenearsonale 34 539. o-Arsanilic acid* 227 540. 2- \lnino-5-nitrobenzenearsonic acid* 227 541. p-Arsanilic acid picrale 7 mp 109-170' 7 542. 4-Amino-3,6-dinitmhenzenearsonic acid 7 543. Lead 4-ami no-3,5-dinitmlienzenearsonate 7 544. o-Tolucnearsonic acid* .... ... 227 545. 2-Hiphenylarsonic acid* .... . . ._ 227 540. 4,4'-14iphonvldiar»onic acid* 227 547. 2-Benzophenonearsoiiic acid* 84f 227 548. S-Metlivl(juinoline-5-arsonic acid 549. 3-Dilienzothiophenearsonic acid 84 f 550. X-Kthvlcarbazole-3-arsonic acid Sle 551. 1,3-Dihydroxv-l-oxyarsindole* 227 552. 5-llvdroxv-5-oxvdilienzarseiiole* mp 7250 5 227 553. 5,lli-Dihvdro-10-hydroxy-10-oxyphenarsazine* 227 554. 5,10-1 )ihvdro-10-hvdroxv-l0-oxypl«*narsazine hy- drochloride* 227 555. 10,10-Dili vdroxv-10-ethylplienoxarsine* 227 550. 5,10-Dihydn>-10,10-diliydroxy-l0-ethylphcnarsazine* 227 Arsenic derivation of uncertain constitution 557. Chlorination product of Propane- 1,3-diarsonic acid* 558. Bv-producl of the preparation of dimethylaminodi- 227 fhioroarsine (iS 559. Anhydride from o-livdroxvphenylarsenie oxiile* 227 560. Di(fn>(.5,10-dihydrophenarsazine l0)oxalate)-awfate* 227 Xo compounds of unusual toxicity were discovered in this class, and, although some of the memliers a,>- pearetl to offer potentialities as irritant smokes, none showed evidence of lieing significantly Itetter than the standard irritants DM. DA. and DC- T.:t.t Irritant Arsenical Smokes Certain arsenical compounds which arc relatively nonvolatile and of fairly low toxicity are, neverthe- less, highly irritating to the respiratory tract when dispersed as a cloud of very fine particles. Further- SBCRET PHY SIOLOOICAI, SECTION 113 more. such particles will i»enctrate gas mask can- isters unless the canisters are fitted with an efficient particulate filter. Such filters were not available in World War I. but have since boon developed and are standard equipment of all nations. Diphenylchlorarsiue (DA) was introduced by the Germans in 1917 as a mask-breaking irritant which was expected to produce temporary casualties and to cause troops to unmask and expose themselves to the effects of more lethal agents, usually employed simul- taneously. In 191S diphenylcyanoarsine (DC’) was used for the same purpose. The Allies claimed that these agents were not very effective but wen1 inclined to attribute their lack of success in part to the German method of dispersal of the agents.*20 The Americans produced a new respira- tory irritant diphenylaminechlorarsine (DM), usu- ally called adamsite after its discoverer. Dr. Roger Adams. DM was not used in World War 1. but was adopted by the United States as their standard irri- tant smoke and has Iieen found lobe useful in riot con- trol, since only temporary casualties are produced. During World War I. the method of dispersing the irritant smokes was in artillery shell employing a large burster charge. The particles obtained by this method are too large to obtain maximal effect from the agent. The method now used consists of volatil- ization of the arsenical in a cloud of hot gas produced in a t hermal generator. The hot arsenical vapor con- denses to a cloud of very minute particles on contact with the air. The Japanese used irritant smoke candles on a number of occasions against both (’hinese and Ameri- can troops, although the attacks were always on a small scale and were apparently undertaken by group commanders without the sanction of the high com- mand. 1 hivsiolootcal Action The physiological action of the irritant smokes has been summarized in the open literature.320 The effects of exposure consist of severe irritation to the nose and throat resembling that from a heavy cold. There is much sneezing, watering of the eyes, and flow of mucous from the nose. Headache, pain in the ears and gums, and nausea are frequently encountered. A feeling of depression often accompanies exposure to the arsenical smokes and is thought to lx* largely psychological in origin. Toxicity The /.(fUso’s by inhalation of DA, DC, and DM have not been determined with very great accuracy for many species, hut figures quoted*1* lead to the conclusion that the L{Ct)„0 for man would lx1 greater than 10 mg min 1. Since the particulate clouds are nonpersistent. death from inhalation of the arsenical smokes could be expected only under very unusual circumstances. British experiments247 have indicated that, men exposed to relatively high concentrations of DC (0.00265 mg I) for periods of 15 to o() seconds and re- exposnl to the same concentration four times at half- hourly intervals, do not show any cumulative toxic effect but rather develop some tolerance to the agent. A numlxT of compounds were examined by the British 273without revealing any more effective than DC, DA, and DM. From the effect of DC on rabbit eyes it has been concluded that a drop of <0.1 mm in diameter would probably not cause permanent damage to the human eye, but that greater contamination than this might exert a gross caustic effect which if untreated might lead to total loss of the eye.*70 Assessment of Military N am e The particulate filter of modem gas masks affords adequate protection against the irritant smokes. Such agents would only be of value, therefore, if they could lie used for surprise effect before the men could mask. Acting on the suspicion (hat the effects of exposure to the irritant smokes was largely psychological, the British 251 carried out important experiments in 1942 in which a group of troops exposed to DA and a con- trol group exposed to a harmless smoke were put, through an assault course test. The troops were strongly motivated to turn in a good performance and were unaware of the fact that there was any dif- ference in the two types of smoke. The group exposed to DA remained in a cloud for 2 minutes or until the generator had burned out, and the mean concentra- tion of DA was 0.1)2(»6 mg 1. The results showed that the performance of about two-thirds of the group exposed to DA was hardly affected at all, that the performances of the remaining one-third over the assault course was definitely slower than the control group, and that about 7 percent of the men exposed to DA were unable to complete the assault course. In view of the proximity of the men to the DA gen- erator and the length of exposure, it was concluded that the dosage of DA was greater than could lie ex- SECKET 114 ARSEMCAI.S pected in the field Further experiment led to the following conclusions: 1. The effect on fresh troops of DA in concentra- tions practicable under active service conditions is almost negligible, apart from the fact that one man in ten would have great difficulty in keeping on his gas mask w hen doing heavy work. 2. Experiments on tired troops suggest that con- centrations of DA practicable under active service conditions give an average effect which is equivalent to making fresh troops wear their gas masks. 3. Hence, DA is not an effective weapon even when used against tired men. In view of these findings ami the failure to dis- cover an arsenical smoke significantly more effective than DA. DC, and DM, little interest remains in the irritant smokes as chemical warfare agents. T.t TABULATION OK \USENIC\LS EXAMINED \S CAN DID \TE CHEMICAL \\ \RK\RE AGENTS Table 9 comprises as complete as possible a tab- ulation of arsenical compounds that have been examined as candidate chemical warfare agents. Ref- erences to synthesis, physical properties, and tox- icological screening data are included. SECRET Chapter 8 ALIPHATIC MT ROSOC VRBAMATES AND RELATED COMPOUNDS" Marshall (Inks and liirdscy Ren show 8.1 INTRODUCTION AsrnvKV conducted by the National Defense Research Committee [NDRC] revealed that ethyl X-methvl-X-nitrosoearbamate (“nitrosometh- ylurethane”) was one of the most disagreeable and toxic commercially available compounds which had not received careful study in connection with chemi- cal warfare.2 Although it proved to I>e insufficiently toxic to compete with standard agents, synthesis and assay of related compounds revealed a number of highly toxic substances. 1'he most promising of these was methyl N-(0-chloroethyl)-X-nitrosocarba- matc (KB-Ki). KB-10 is a persistent agent with a volatility only slightly less than that of mustard gas (11). Its syn- thesis, although more involved than that of H. of lewisite (b), or of the nitrogen mustards, presents no great difficulty and the required starting materials are readily available. KB-10 came under investigation in 1942 at a time when the nitrogen mustards were being seriously considered. It was quickly shown that the compound possesses some of the desirable characteristics of me thy I -b is (/3-c h 1 oroe (by 1) a n lin e (HX2) — low freez- ing point, lack of pronounced odor, and effectiveness as an eye-injurant at low dosages. Interest was also aroused by the finding that, for mice it is three times as toxic as H. Subsequent investigations revealed that: (1) KB-16 possesses inadequate storage stability, and no satis- factory stabilizer has been found in spite of intensive search; (2) its eye-injuring potency is not of a differ- ent order from that of II or b/s(/3-ch loroethy I) amine (HXd); (3) although more toxic than II and the nitrogen mustards for mice, it is not so toxic as these substances for larger species (i.e„ dogs, goats, and monkeys); and (4) as a vesicant it is markedly in- ferior to H. Taking these and other findings into ac- count, assessment of the merits and limitations of KB-16 led to the conclusion that it does not possess the.general utility of the standard agent, H, or of the potentially available nitrogen mustard, IIX3. Ac- cordingly, KB-1G is not now seriously considered for use in chemical warfare. 8.2 SYNTHESIS VND PKOPKKT1KS 8.2.1 Synthesis '1'he aliphatic nitrosocarbamates tested during World War II (see Table 1) wore prepared by nitro- sation of the corresponding carbamates, which in turn were derived from the action of alkyl chloro- formates on amines. The synthesis of methyl N-(/J- ch1oroethyI)-X-nitrosocarbamate (KB-10), the only member of the series that has received detailed study, involves the following steps. 1. Preparation of N-(0-chloroethyl)earbamate. Thionyl chloride is allowed to react with ethanol- amine hydrochloride to produce /3-ehloroethylamino hydrochloride, which is then treated with methyl chloroforrnate. Alternatively, methyl chloroformate can be treated with ethanolamine and the resulting methyl N-(/8-hydroxyethyl)earbamate converted to the desired product by the use of thionyl chloride. The first of these alternatives is preferable (see be- low). Attempts to prepare methyl N-(0-chloroethy 1 )- carbamate directly by the action of methyl chloro- formate on ethyleneimine have not succeeded.4* H(X,H.CHiNH2HCl + son3—> arilsCHjNH* HC1 + ClCOOCHj I (la) CnCH,CH,NHCOOCH, HO('II3CH*NII, + C1COOCII, —> HOCII2CH*NHCOOCHa + sori2 (ib) I riCH*CH2NHCOOrHs Methyl chloroformate may lx* prepared in good yield either by the addition of methanol to an excess of liquid phosgene 2 43 or by the reverse addition of excess gaseous phosgene to methanol.23b The second alternative gives better yields based on methanol. An excess of phosgene is required to mini- • Hascd on information available to NDltC Division 9 as of November 1, 19-15, SECRET 116 ALIPHATIC MTKOSOCARBAMATES AND RELATED COMPOUNDS Table 1. Aliphatic nitrosocarbamates and related compounds examined a.s candidate chemical warfare agents. The compounds are arranged in four major categories in the following sequence: (1) nitrosocarbamates, (2) nitrosu- amides, (3) nitrosoamines, and (4) miscellaneous carbamates. The following abbreviations are used: no', refractive index at / C; earbamate 2 bp,,! 76-78° 2 10 a. Kthvl X-ethyl-X-nilrosoearbamatc Commercial lip35 80- 84 ’ 10 6. Methyl X-(£t-chhiroet hyl )-X-mtrosoearbamate 2,21a, 23c, 13 flu1' 1.4666 41 10, 41, 14 d,24 1 .3053 II bp*-* 72 76 2 -- . vol5" 0.600 11 7. Kthvl X-(d-cliliiroethvl)-X• nitrose>carbainate 2, 21a, 43 bp1" 02-03° 2 10 • vol111 0.426 11 8. /3-Fluoroethyl X-((Kchloroethyl i-X-nitrosocar- bamale 21b bp- 118-121“ 2le 10 9. Isopropyl X-(d-chlorocthyl)-X-nitrosocarbamate 2 bp* * 80° 2 10 10. Butyl X-O-chloroethyD-X-nil rosoearbamate 2 bp" 6 95* 2 10 11. Methyl X -(3-1 iromoe I hvl )-X-n it rosocarl m male 2Id, 54c bp1 110-115“ 21 d 10. 14 12. Mcthvl X-(d-hvdroxvetliv 1 )-X-nitrosocarbamate 2 bp" • 00 95“ 2 10 13. Methyl X-(3-chloropropyl)-X-nitriKsocarbamate 2 bp* 3 * •* 75-80° 2 10 4 4. Methv l-N-bwtvl* X-n i t rosoca rba mate 2 l'l>- 70-72° 2 10 15. Met hyl X-8-{fl'-ehloroet hyl t hio)-et hyl-X -nil roso- ca rba male 44 .16. Methyl X-phenethyl-X-nitrosocarbamate 21d Cannot lie distilled 2 Id 10 AT ilrosoaniides ~ . 17. X-(3-(’hIoroelhvl)-X-nitrosoformamide 2 bp* * 78-80“ 2 18. X-(3-Chloroethyl )-X-nitrosoacetamide 2 bp" s 70 72° 2 10 10. X-Met hyl-X-nit rosofluoroacet amide 51 bp11 84° 51 50 .V it rnsoa mines 20. X-Xit rosopiperidine 2 bpu 94-06° 2 10 21. X-Xit rosomorpholine 2 mp 28° 2 10 bp14 105 107° 2 22. X,X '-Dinitrosopiperazine 2 mp 155 157“ 2 23. d-Chloroethvlmelhvimtrosoamine 54b ♦ . . 44 24. hist3-Chloroelhvl(nitrosoamine 2 Cannot lie dist died 2 10, 44 25. 4-Methvl-4(methylnitrosoaminoVpcnlanone-2 10 26. X,X '-Dime!hyl-X,X '-dihitroso-p-phenylene- diaminc 15 nip 149-150° 15 It) 27. X,X '-6/s(3-(bloroethyl)-X,X '-dinitroso-/>-phenyl enediaminc 15 mp 106.5° 15 10 MisreUancous carbamates 28. Methyl X-fd-cliloroethvlVX-nilrocarbamate 2 bp"* 95-100° 2 10 vol20 0.138 11 20. Methvl X-d-ehloroelhvIca rba mate 2 no50 1.4575 27 10, 4 4 . bp14 100° 2 30. Kthvl X-isobutvlearbamate 15 bp“ 04° 15 10 31. Kthvl X-isoamvlcarbamate 15 no20 1.4333 15 10 • bp** 109° 15 32. Kthvl X-methoxycarhamate 10 SECRET 117 Tabi.e 1 (Conti mint). Compound Reference to synthesis Physical properties Pro|ierty Reference Reference to toxicity data 33. Mcthv! N-ethylthioIcarbamale 12 «.r’s 1.4078 12 10 50 12 10 dIJ 1.067 12 — bp-4 109.5-110.5° 12 bp37 110-121.5° 12 — 35. Mi'llivl X-et li vldil liioearbamate 12 «irr 1.6130 12 10 -r-4 1.151 12 : bp* * 121-122° 12 SYNTHESIS AND PROPERTIES mize formation of methyl carbonate. Distillation of the crude methyl ehloroformate is not necessary.49 2. Pivparation of HR-16 hy nitrosat ion of methyl X-{0-ehloroe(hyl)earbamate. This step may l»e ef- fected hy nitrous acid in solution or hy nitrous gases either with or without a solvent. The action of ni- trous gases on methyl X-(/3-ehloroethvl)carhamate in the absence of a solvent is the most rapid and convenient. — UNO. or (It IU II XH('(XX H, nitrous gases (’1CH,( I I2X (XO)COOCH* (2) Reaction (la) was employed in the original lal>o- ratory preparation of KB-16.!*u/9-Chloroethylamine hydrochloride prepared essentially according to Ward fil from solid ethanolamine hydrochloride and thionyl chloride in chloroform was treated as a solid suspended in ether or benzene with aqueous alkali and methyl ehloroformate. The resulting methyl N-(0-el11oroethy 1)carl>amate was purified hy distilla- tion, diluted with ether or benzene, mixed with a so- lution of sodium nitrate, and nitrosated by the addi- tion of nitric or sulfuric acid. Overall yields of 62 per cent were obtained. Alternatively, N-(/J-chloro- el hyljcarbamate was nitrosated under anhydrous conditions by the use of nitrous gases. Flash distilla- tion was used to purify the final product and appears to be the only feasible method. The above procedures were used with little modification for the synthesis of the first samples investigated in Great Britain.54* The method is well suited for large-scale runs. Preparation of KR-16 by the alternative procedure utilizing reaction (lb) is also convenient for labora- tory scale work and can be carried out in overall yields of 65 per cent.4143 It is less readily modified for use on a larger scale because the hydroxycar- bamatc must be distilled and the conversion of this intermediate to the eh lorn compound has not been achieved in yields greater than To per cent. The first met hod,-as modified for production on a larger scale, has been simplified by: (1) elimination of the isolation of ethanolamine hydrochloride and of d-chloroethylamine hydrochloride, both of which are hygroscopic; (2) combination of the hist three steps into one; (3) reduction of the large excess of thionyl chloride and sodium nitrite; (4) the use of a single solvent (chloroform or benzene) in reduced quantity throughout the reaction steps; and (5) elimination of all distillations except that of the methyl N-(0- chloroethyl)carbamate.9 233 49 A brief description of the modified process follows. Ethanolamine in chloro- form,49 in benzene,23® or in the absence of a solvent 9 is treated with dry hydrogen chloride to produce ethanolamine hydrochloride. Thionyl chloride is then added directly (if no solvent was used in the first step, benzene is added at this point), and the mixture is heated to convert the ethanolamine hydrochloride to 0-chloroethvlamine hydrochloride. The mixture is then diluted with water, and caustic alkali and methyl ehloroformate are added. After the acylation is complete, the organic layer is separated, washed, dried, and stripped of solvent. The crude methyl X-(d-ch 1 oroethyI)call>amate thus obtained is then distilled under diminished pressure. It has been ob- tained in yields of 77.5 per cent in runs utilizing 24-5 lb of ethanolamine.23® If nitrosat ion is carried out by slowly acidifying a mixture of the carbamate in benzene or ether with an aqueous nitrite solution, the reaction is slow and a large excess of sodium nitrite is necessary.*-23® When the reverse addition is used and (he reaction mixture is strongly acid, a slight excess of sodium nitrite is sufficient and the reaction proceeds rapidly.4* SECRET 118 \LIPIIATIC MTROSOCAUB AMAXES VM) RELATED COMPOUNDS Aqueous nitrosations were used in all investiga- tions where scaling up the synthesis of KB-16 was tried, but it was subsequently shown that the re- action of nitrous gases with methyl 0-chloroethyl- carbamate in the absence of a solvent is quantitative and almost instantaneous.21' 2*r This variation pos- sesses a number of practical advantages. 1. Solvent is eliminated and effective reactor ca- pacity is thereby increased. 2. The purity of the final product is sufficient to obviate the need for flash distillation. 3. Equipment is simplifier! and the total time cycle is reduced. 4. The method should allow preparation of the agent in inlu shortly before use, or in shell subsequent to firing. This would eliminate the problem of storage stability ami greatly lessen the hazards involved in synthesis. Preliminary design data and cost estimates for a plant to produce KB-16 at the rate of 500 tons i>er month have been submitted. The calculations were based on the use of aqueous nitrosation in the final step.* N-(0-Chloroethy!)-N-nitrosoacetamide, a highly toxic analog of KB-16, has been preparer! on a labora- tory scale by nitrosation of an ethereal solution of N-(/3-ch!oroethyl)acetamide with oxides of nitrogen. Yields of 60 per cent of material purified by flash distillation were obtained.* 8.2.2 Physical Properties KB-16 is usually obtained as an orange-red limpid oil of limited thermal stability. It is soluble in water to the extent of 0.7 g 100 g, and is completely misci- ble with ordinary organic solvents. Although it has not been obtained in crystalline form, it assumes a semisolid state at —65 C; at —25 (.' it is a viscous oil. The density of KB-16 is 1.3053 g ml at 25 C,41 the refractive index 1.4085 at 25 and the boiling point 100 (’ at 15 mm, 89 (’ at 6.5 mm, 86 C at 5.5 mm, 82 at 1 mm, and 75 C at 2 mm.43 The volatility of KB-16 is 0.87 mg/I at 25 C, slightly less than the corresponding value of 0,96 mg 1 for f«s(0-chloroethy 1) sulfide (H). Several de- terminations of the volatility (or vapor pressure) as a function of temperature have been made.2" 53 The vapor pressure at temperatures in the range of inter- est for chemical warfare is given by the following equation:11 log p (mm Hg) = 8.91282 — — The standard free energy of formation of KIM6 and several thermodynamic constants of the inter- mediate carbamate have been calculated from the results of a series of calorimetric and equilibrium studies.7 Chemical Properties KB-16 decomposes within 48 hours in water or in aqueous bicarbonate solutions.’ In the former case about 40 per cent of the nitrogen appears as nitric acid, the remainder disappearing from the reaction mixture. Only 5 per cent of the chlorine appears as chloride ion. In bicarbonate solution more than 00 per cent of the nitrogen is lost, presumably .as nitro- gen gas, and carbon dioxide and methanol are pro- duced. About 80 (x-r cent of the chlorine appears as chloride ion; the remainder is bound to carbon, pre- sumably in the form of ethylene chlorohydrin. The production of chloride ion at pH 8 is not significantly altered in the presence of substances which react with the 0-chloroethyl groups of (he sulfur and nitro- gen mustards. The decomposition of ethyl N-(/J-chIo- roethyl)-N-nitmsocarbamatc in aqueous solutions is similar to that of KB-16.* One of the most characteristic reactions of KB-16 is its rapid and complete decomposition with evolu- tion of nitrogen when treated with alcoholic ammonia or primary aliphatic amines.* Solutions of ammonia or ethanolamine in ethylene glycol have therefore been recommended as personal or laboratory decon- taminants.2 However, the distinct possibility that substances of the nitrogen mustard type may be formed by this reaction should lx* considered in the choice of a decontaminant (see below). Secondary amines and primary aromatic amines react relatively slowly with KB-16. In aqueous solutions, the reaction with ammonia and with primary amines is slower, perhaps because of the low solubility of the nitrosocarbamate. At pH 8, the main reaction with primary amino groups is carboalkoxylation, as has been demonstrated by the isolation of methyl and ethyl N-benzylcarba- mates as products of the reaction of bcnzylamine with methyl and ethyl N-(0-chIoroethyl)-N-nitroso- carbamates, respectively.* Secondary amines (di- ethanolamine) are also carboalkoxylated. In ethereal solution, reaction with bcnzylamine leads to methyl N-benzylcarbamate and N,N'-di- benzylethylenediamine, (he latter probably arising through bcnzyb/S-chloroethylamine its an inter- mediate.’ SECRET SYNTHESIS VM) PROPERTIES 1 10 The amino groups of a-amino acids also react with KB-16, but the reaction is more sluggish than those of primary aliphatic amines and does not appear to go to completion. With cysteine, the reaction pro- ceeds along several lines; both amino and sulfhydryl groups disappear. 6/s-S-(Cys(einyl)ethane, probably arising through the intermediate S-(d-chloroethyl)- cysteine, has been isolated as a product of this re- action.* Nitrosomethylurethane also carbethoxylates the amino group of cysteine, but is far less active toward the sulfhydryl group than is KB-16.* In solutions containing egg albumin KB-16 reacts /3-chloroethyl group into amines and into the sulf- liydryl compounds. The intermediate products are of the sulfur and nitrogen mustard type, and undergo further reactions characteristic of these substances (see Chapter 19). With regard to loci of action in tissues, it may be noted that reactions of the sulfur and nitrogen mus- tards involve a process of thermal solvolytic activa- tion in water (see Chapter 20). On the other hand, the alkylation of benzylamine by KB-16 in ether so- lution demonstrates that this agent need not lie so activated. As a result, it is possible that KB-16 can ru' h. ■('H;- \( n o) • co • or n> i ~~ ' n + II:0 at /(II 8 + RNH, i + HS■ CH2 -('II(NI!;)• COOII \ i i i r ] CK H: CHj\H + IK)CO (M il, ClCIIrCIIrMI + R-NHCOOCHa + HSCIUCIUCOOH I ! II- NO ~ NO NO NH-COtX'Hj - H.O - H.O ~ - H-0 t T 11 CICHjCHN, CX), + IKK'Il, CICH-CIIN. ClCHjCHN, ' - ” k + 11,0 + R-NH- | + TIS.CIl,.CH(NH,) COOII ClCH, CH,.OII + N, C1CH,.CH. NH-H + N, CICI1,.CH2 S CII2 -f N, + R-NH, 4- HS C'H, CH(NII,) COOII RNH CH, CH, NH R + HC1 COOH ('OOII CH-CH. S-CHj-CIIj-S.CH., i'll + MCI xh, kn, slowly with the liberation of 1 mole of nitrogen per mole of nitrosocarbamate but no decrease in the amino nitrogen content of the protein occurs.*2S The reaction with hemoglobin is rapid 25 and in this case some amino groups disappear, possibly by carbo- methoxylation, although it has not been possible to ascertain the mode of reaction.* The following scheme of reaction, reminiscent of those of Klobbie &s and V. Pechmatin M for the breakdown of nitrosomethyl- and nitrosoethylur- ethanes. has been proposed to explain many of the observed reactions of KB-16.* Thus KB-16 can be- have as a chloroalkylating agent, introducing the react in fatly phases of tissues, whereas reactions of the nitrogen mustards in these loci are not equally probable.* i!.2.V Detection and Analysis KB-16 reacts with the 1)11-3 reagent to produce the characteristic blue color. Samples as small as 25 //g may be detected by use of the DB-3 tube of tin* United States Army M-9 Detector Kit according to the standard procedure. By heating to 200 C, the sensitivity of the tube can be increased sufficiently to permit the recognition of I Mg.'*’* in the absence of a guard tube, the spotted dick test of the British SECRET ALIPHATIC MTROSOC \RB A \1 AXES AM) RELATED COMPOLNDS Vapor Detector Kit gives an overall blue color.41 Acidified iodoplatinate paper is bleached by the vapor of the agent. Other procedures for detection involve the use of the diethylamine and diphenyl- benzidine reagents or the Liebermann reaction. Positive reactions given by the decomposition products of KB-16 limit the usefulness ot these methods. The most useful method for the analysis of KH-lti depends upon the quantitative evolution of nitrogen which occurs when the compound is treated with primary amines ,6i **r or with alcoholic alkali.41 This method is suitable for use as an assay method or for analysis of samples collected in chamber or field tests, anil has the advantage of specificity to the ex- tent that it measures only the nitrosated material. The Griess reagent as used for nitrites can be em- ployed for the field or chandler analysis of this agent.4* A more detailed discussion of the detection and analysis of the nitrosocarbamates will be found in Chapter 34. 8.2.5 Stability KB-16 and its homologous esters are thermally un- stable. Decomposition with gas evolution occurs at rates which make storage impractical.2-41 In steel containers with 25 per cent void, the pressure in- crease per day is appreciable at temperatures as low as 4 C and amounts to about 2 psi at room tempera- ture.3 At high temperatures the decomposition be- comes even more rapid, the pressure increase in glass with 50 per cent void amounting to about 4.5 psi per day at (50 C.41 No significant difference between the rates of pressure development in glass and steel con- tainers has lieen observed (unpublished data), even though steel appears to be attacked.41 The purity of (he sample has a considerable effect on the rate of thermal deeomposition, carefully purified material decomposing at a lower rate than crude material.2*11 The stability of preparations made by nit-rogation with nitrous gases is as good as or better than that of flash-distilled samples. Decomposition is accelerated by acidic and phenolic substances and by zinc and magnesium oxides.2 41 There is disagreement as to the effect of traces of water, weak bases, or contact with metals other than steel.2-41 The gas produced during !Ik- decomposition of KB-16 consists principally of nitrogen; oxides of nitrogen, carbon dioxide, and hydrogen chloride have also been identified.I*h 2*c-41 In spite of intensive searches for a stabilizer to prevent or minimize the spontaneous decomposition of KH-16, little success has been achieved. Few of the tested substances were of any value and none pro- duced a pronounced increase in storage stability. The tested compounds include organic and inorganic bases, acids and derivatives of acids, hydroxy and mercaptan derivatives, oxidizing agents, inert liquids and solids, salts and complexes of heavy metals, and numerous miscellaneous compounds.2*"41 Few reliable data are available on the stability of KH-16 to detonation in munitions.18"1 30 31 11 The re- sults of a field trial with 105-mm shell supply no definitive information.3" In a small chamber, detona- tion of a 75-mm shell charged KH-lti resulted in more or less complete destruction of the agent;w in similar tests ethyl-6fx(/3-ehlonx‘thyl)amine (HNl) was also destroyed but /n«{0-chloroc(hyl)amine (HNS) was not. It may lx* noted that (he conditions of these tests were more severe than would 1m> en- countered in the field, and that HNl can effectively lx* dispersed from M47A2 bombs.-' KH-lti is not de- stroyed to any great extent by the milder explosions that occur when it is dispersed from glass bottles either in the field by means of a standard detonator 31 or in a 2-cu m chamber by means of a blasting cap or detonator.18"1 31 K.2.6 Decontamination Rapid surveys of the reactions of KH-lti, with emphasis on reactions of possible use in decontamina- tion. have been carried out both in this country and in Great Britain.20 47 The reagents examined included bleaching powder, chloramides, a number of inor- ganic salts in solution, mineral and organic bases, at least one strong oxidizing agent, and reducing agents. Solid bleach or lime slurry would appear to be suit- able for field use. Caustic soda or alcoholic ammonia has been recommended for laboratory use, and aque- ous ethanolamine for personal use. In line with these recommendations, groups concerned with the synthe- sis of the agent have used solutions of ammonia or ethanolamine in alcohol or ethylene glycol for per- sonal, laboratory, and pilot plant decontamina- tion.32,11 As stated above, ammonia or primary amines should be used with caution because of the possibility of producing toxic intermediates.* For treatment of eyes contaminated with KH-lti, mild alkalies and reducing agents (e.g., HAL) should be more effecthe than in the case of nitrogen mus- tards.2*-47 SECRET CHEMICAL STRICTURE IN RELATION TO TOXICITY 121 8.2.7 Protection The canisters of modern gas masks afford ade- quate protection against the vapor KB-16.13 41 For details the reader is also referred to the Summary Technical Report of NDRC Division 10. Tlie chloramidc impregnation of clothing would appear to offer little resistance to KB-16. because (his agent fails to react with chloramine-T or with various impregnites.2" It may lx4 assumed that cloth- ing containing activated carl ion would effectively exclude the vapor of the agent. a..i CHEMICAL STRICTURE IN RE LATION TO TOXICITY In Table 2 are presented data on the toxicity for mice of compounds in which the N-nitroso, N-(/J- Table 2. Toxicities of X-subst it uted aliphatic carbamates for mice. Most of the data arc taken from reference If). The mice were observed for 10-15 days after exposure for 10 the stated nominal concentration. In the case of one compound, methyl N-methyl-N-nitrosoearbamatc, the obtained from reference 48 ami relate to rats exposed for 30 minutes. minutes to data were Mortality Structural Nominal cone. for 10-min ('ompound formula (mg 1) The prototype Compound Methyl N-(3-chloroethy 1 )-X-nitrosocarbamate (KB-16) (’ICIT/H, o \ !! —- - - x—r—och, 0.03(5 /^’in Effect of replacement of the \~nilroxo group —~ Methyl X-(d-chloroethvl)carbamate cich.ch, o _ — \ 1! X—C -OCH, 1.0 0 20 11 Methyl X-(d-chloroethyl)-X-nit rosocarbamate nciijCH, o \ 1! - N—C—OCH, / 1.3 0 20 OtN Ethvl X-{0-chloroethyl)-X-nitro8ocarbumate C1CH,CH, O V 11 . . N—C—OTjHi 0.075 — Ethyl X,X-f>/s(/t-chloroethyl)carlwmate ricHjCii, o \ 1! ' X C—OC,H, / 0.4 0 40 CICHiCHi X-(3-chli iri«-thvl)-X-n it rosoaeet amide CICHjCH, O — \J--nu on/ O.Olfi Li ’fcO — -. X, X -bix( /3-ch loroet hy 1 facet a midc CICHjCH* O \ !! - X—6—CH, 0.5 0/10 / CICHjt'H, Effect of replacement of the \ -{ff-chloroelh yl) group — Methyl X-met by 1-X-nil rosocarbamate (II, O \ . !! N—C OCH, 0.20 (30 min) !4 (rats) 1 X \ 0.13 (30 min ) 34 (rats) Methyl X-(d-bromoethyl)-X-nit rosocarbamate BK'HjCH, O \ li X C -OCH, 0.2 0/10 ' / ON 0.82 10/20 SECRET 122 ALIPHATIC MTHOSOCAKBAMATKS AM) BELATED COMPOUNDS Table 2 (Continued). Mortality Structural Xominal cone. for lO-iniu Compound formula (niR 1) exposure Mcthvl X-(/3-hvdroxvcthvl)-X-nitn>socarbamatc HOCHCH. o \ II X C OCH, 0.3 0/20 OX Methyl X-(d-chloropropyl)-X-nitTOsocarbamatc CIIjCHCICHi () — \ II X—C—OCH, 0.3 0/10 OX Mi thy 1 X-butyl-X-uitroeocarbamnte CIliCIIjCH-CH* O ■' ” y NT—C OCH, 0.3 1/20 / OX Methyl X-phcnethyl-X-nitrosooarbamatc (VIC CH,CII, o -\ It / OX X ('—OCH, 0.97 0 20 Kffrrl of rtplocrmrnl of the mi lhuxy group Ethyl X-d-chloroethy 1-X-nil rosoearbanmtc CK’TIjCHi 0 II X -C—OCH2C-Hi 0.07a IJ J} - / OX - (3-Fluorocthvl X-(^-chlorcx'thvl)-X-nitrosoc!irbaniate CK'HjCHj O 0.1 11/15 \ 11 0.2 11 20 X -C OCHjCIIjF 0.5 10 20 —■/ OX Isopropyl X-(P-ehloroethvl)-X-nit rosoearbanmtc CK'ITjCHj o 0,1 0 20 \ \ II 0.12 20/20 X - C—OCH(CHj), 0.2 16/18 ■ / OX Butyl X-(/3-ehloroctbyl)-X-nit rosocarbamate cicH.cn, O \ 11 X -C OC.1E 0.10 U'm ’ '• - ■_ / ON 0.41- LC'* X-0-<-hloroethyl)-X-nit rosoai-ct amide CICHjCHj 0 \ II X-€ -CHj 0,040 — / OX chloroethyl), and methoxy groups of KB-16 are re- placed by other substituents. On (he basis of these data, and subject to their limitations, the following conclusions can be "drawn. 1. The N-nitroso group is essential for high toxic- ity. Its replacement by another group has always re- sulted in at least a 30-fold reduction in toxic potency. 2. The N-(/3-chloroethyl) group is essential for highest toxicity, but moderate toxicity is possessed by some compounds in which it is replaced by an alkyl group (e.g., ethyl N-methyl-K-nitrosocarba- mate apparently possesses one-tenth the potency of ethyl N-(jJ-chloroethyl)-N-nitrosocarbamate).. 3. The methoxy group, although optimal, is not essential for high toxicity. Its replacement by a methyl group (to form N-(/3-chIoroethyl)-N-nitro- soacetamide) results in an insignificant decrease in potency, and the ethoxy analog is about one-half as toxic as KB-16. Toxicity data for other species, vesicancy tests, and determinations of eye-injuring potency arc not sufficiently complete to permit analyses of the rela- tive potencies of members of this series. Such data as are available indicate the relative superiority of KB-16 and are not inconsistent with the other con- clusions drawn from (he toxicitv tests with mice. SECRET 123 TOXICOLOGY Compounds which possess a fluoroacetate-Hke toxic- () H ity by virtue of the presence of an FCH-C— group are an exception to this generalization (see Chap- ter 10). 8.4 TOXICOLOGY Of the following toxicological sections, those on detectability by odor anti sensory irritation, vesi- ca ncy, and eye-injuring action bear most directly on the evaluation of KB-16 and related compounds as chemical warfare agents. 8.4.1 Detectability by Odor and Sensory Irritation The vapor of KB-16 has a pleasant odor, some- times described'as sweet or fruity, which can be de- fected by smell only at concentrations several times greater than those required for H. Men exposed to relat ively high concent rations (i.e., 70 jug I nominal) for 30 seconds detect the odor but experience no sensory irritation,41 and concentrations as high as 0.2 41.4 mg 1 elicit no signs of irritation in animals.1*1’ Those properties of KB-16 vapor, considered in rela- tion to its eye-injuring potency and the delayed on- set of the injuries caused by casualty-producing dosages (see the following section), confer upon it some insidiousness. However, in this regard it is not notably superior to some of the nitrogen mustards (e.g., HNS), which are probably less easily detected by odor and not notably inferior in eye-injuring potency (see Chapter 6). Laboratory determinations of the median de- tectable concentrations in jug/1 of KB-16, II, ethyl-6w03-chlorocthyl)amine (HNl), and HNS arc tabulated below. Attention is directed primarily to the relative values, inasmuch as the absolute values arc not necessarily of significance for field conditions. Agent Mg/1 Reference II Plant run Levinstein 0.6 34 Pure thiodiglycol 1.8 35 KB-16 _ 7 ± 16 1,41 HNl Plant run 13 34 Pure _ 17 33 1IN3 Plant run 15± 37 The vapor of N-(/3-ehloroethyl)-N-nitrosoaeeta- mide does not possess the insidiousness of KB-16.16f l?i 8.4.2 Toxicity Inhalation- Toxicity In Table 3 data on the toxicity of KB-16 vapor Tabi.e 3. Inhalation toxicity of Kli-16 in comparison with mustard gas (H) and /mO-chlorocthyl)aminc (11X3). Approximate nominal L('.„, in mg 1 for 10-tnin exposure and 15-day ohservalion [icriod.* S|iecics KB-16 11 MX3 Mouse 0.(130 (259)t41« 0.12"*1,1 0.12§ls‘ Hal Guinea pig Rabbit Gat Dog Goal Monkey 0.1-0.2 (19)"“> 0.1“'.•» o.2 0.035 (60)i 0.2 ± (17),c,‘ 0.21*1 >0.216* >0.2 (7)*0.281® 0.14'“ 0.1-0.2 (11 )'«'■•* 0.07'*' 0.08,4k o 0.1 (H)i«'*.i.ud 0.07161 o.l">k‘*“ 0.3 (6)'T1 0.19t« 0.2-0.5 (6),Tt* 0.08"4’ .... ■ * In the ease of KB*16 some deaths occurred among the larger «|M*eit's after observation periods as long as 15 30 days and were includcii in inti- mating the Lf.’se’a. t The figures in parenthesis give the nutnlier of animals U|»oii whieh the estimated LfW« are based. X Analytically determined concentration. § The analytical LCh* is about 0.055 mg 1. for various animal species arc set forth in comparison with corresponding data for 11 and HN3. One of the early observations arousing interest in KB-16 was the discovery that for mice it is several times more toxic than H. When tests were made with other se- cies, however, no such differential in favor of KB-Hi was found, except possibly in the ease of the rat. In- deed, it may be questioned whether KB-16 is as toxic as H for (he larger mammalian species which have been studied. The only evidence bearing on the relation of tox- icity of KB-16. to exposure time is the demonstration that the L{Ct)io for mice is approximately the same for 30-minute exposures as for 10-minute expo- sures, IBhand the result of a single experiment in which a dog succumbed 23 days after exposure to a total vapor dosage of approximately 1,100 mg min/m* administered during three 8-hour periods on suc- cessive days.17,1 During exposure to KB-16 vapor at concentra- tions as high as 0.2 0.4 rng/1, animals exhibit no signs of discomfort or irritation.161’ Concentrations considerably in excess of the 10-minnte LC„o s occa- sionally caused closing of the eves, but the irritation was mild and not accompanied by profuse lam- ination. The development of symptoms after gassing with KB-16 is usually delayed for 12-24 hours and follows the same general pattern in different speeies.41*,’-nd Respiratory distress becomes prominent. The ani- mals appear depressed and stop taking food and water; as a consequence weight loss may be precipi- SECRET 121 ALIPHATIC MTKOSOCARUAAtATKS AM) RELATED COMPOUNDS (ous. Severe eye injuries also develop (see Sec- tion 8.4.4). In nonfalal eases the symptoms slowly subside. In fatal cases respiration may become la- bored and terminate in death after 3-10 days, or the animals may slowly waste away and die as late as 3-4 weeks after exposure. The principal pathological changes occurring in animals gassed with KB-16 are found in the eyes (see Section 8.4.4) and the respiratory tract.4 In mice the most severe changes are confined to the nasal and nasolaryngeal mucosa, and an exudate, first fluid and later mucopurulent, is often produced in sufficient amount to block the air passages. The trachea and bronchi show much less damage. The lungs may become hyperemic but pulmonary edema is minimal and pneumonia does not ordinarily de- velop. In larger species, perhaps liecause the larger size of the air passages permits further penetration of the vapor, nasal injury is accompanied by severe pathological changes in the deeper parts of the respir- atory system. The larynx, trachea, and bronchi are severely involved, and pulmonary injury with con- solidation occurs. The pneumonia often appears in focal patches around the bronchi. Degenerative ma- terial cast off from the larger respiratory tubes often blocks some of the larger and smaller bronchial pas- sages. In general the bone marrow is little affected.4 6 Atrophy of the lymphoid organs with rhoxis of the lymphocytes of the thymus gland and the splenic follicles has been reported in some species ,7g but was not found to he conspicuous in another investiga- tion.4 Hematological studies fail to reveal the con- spicuous changes in numbers of circulating white blood cells which characterize severe intoxication with the sulfur and nitrogen mustards, although a rise followed by a fall in lymphocyte count has been reported in mice exposed to about eight L(Cl)M dosages of KB-16 vapor.17* In some instances limited degenerative changes, possibly secondary to respira- tory embarrassment, have been observed in the liver and kidney. In mice and rats the digestive tract from upper esophagus to anus is often markedly distended with gas but no hemorrhages or perforations have been observed;4 the distention is probably caused by swallowing of air, which occurs because of mouth breathing and difficulty in respiration. The available data suggest that the principal pathological effects of ethyl N-(/J-chloroethyl)-N- nitrosocarbamate and of N-(0-ehloroe(hyI)-N-nitro- soaeetamide are similar to those of KB-16.®,®1*1" Toxicity through the Skin In its actions on and through the skin, KB-16 is relatively ineffective as a lethal agent when com- pared with H, HNl, or HNS. In spite of the high sensitivity of mice to the inhaled vapor, exposure of only the bodies of animals of this species to KB-16 vapor at a nominal dosage of 6,900 mg min m3 (/ = 10 min) caused no deaths within 15 days; 11,300 mg min m* killed 1 5 unshaved mice; and 13,000 mg min m3 killed 6 0 mice with shaved backs.16,1 For other agents -approximate nominal L(Cl)b0’s {I = 10.min) for mice upon body only exposuiv are: H, 1.000 mg min nv1; HNl, 5,000 mg min m3; HNS, 2.000 mg min m* (analytical value — 1,000); and b. 2.100 mg min m*.u The toxicity of liquid KB-16 applied to the shaved skin of mice is also low when inhalation of vapor is minimized. Necrosis, of the skin and ulcer formation occur at the site of appli- cation but a minimum value for the /,/>j0 is believed to be 62 mg kg.4 Corresponding values for other agents are: II, 92 mg kg; HNl, 13 mg kg; HNS, 7 20 mg kg (see Chapter 22). It may be noted that all of these values are high in comparison with the per- cutaneous /.Dio’s for some of the compounds con- sidered in Chapter 9. Toxicity by Injection Parenteral injections, although they have no direct bearing on chemical warfare, supply useful informa- tion concerning the toxicological properties of KB-16. //tin's upon intravenous injection are: mouse, 0.45 mg kg; rat, 1.1 mg kg; and rabbit, approximately 2.0 mg kg.4 The subcutaneous LD,n for the mouse is 9.0 mg/kg;4 and those for the rat and rabbit ap- proximately 8 and 20 mg kg, respectively.41 Even large doses are without immediate pharmacological effects, and subsequent developments reveal no neu- rological injury, central nervous or gastrointestinal action, pronounced leueopenic action, or significant changes in the total number of circulating white blood cells.4 The conspicuous pathological changes occur in the lungs, which become distended, moist, and hyperemic. They sink in water and on cutting a pinkish, foamy fluid runs from the lungs and trachea. A small amount of pleural fluid accumulates. The heart is often dilated but gross pathological changes elsewhere are conspicuously absent. Venous con- gestion of the liver and myocardial injury with focal necroses are occasionally but not constantly ob- served. The thymus gland and spleen are usually but not markedly decreased in size — probably (he re- 8 EGRET TOXICOLOGY 125 suit of a nonspecific lymphoid involution. In some instances (e.g., in rabbits receiving large doses) there is evidence of lymphocytic fragmentation in the spleen, lymph nodes, and thymus. The bone marrow usually appears normal, although evidence of leueo- blastic stimulation apjiears in some rabbits.4 It has been reported that one of two dogs receiving 18 mg kg intravenously died in 4 days with aplasia of the bone marrow and drastic leucopenia, involving both lymphocytes and granulocytes,* It levs been concluded that intravenously injected KB-16 causes death by producing fatal pulmonary edema, which develops slowly over a jieriod of 2 8 days.4 5 Injections by various routes demonstrate that KB-16 reacts with lilx-ration of gas (presumably X2) in the first capillary bed it reaches.4 Circulatory stasis may occur, in some eases possibly because of -vessel spasm or thrombosis, so (hat contact w ith the tissue may lie. prolonged. These observations give an explanation for the finding that the principal patho- logical changes following inhalation or intravenous injection occur in the lungs. The liberation of gas, which occurs in the tissues to which injected KB-16 is first carried and which also occurs when KB-16 is added to tissue suspensions, undoubtedly contributes ischemic injury to the chemical injury produced by the direct reactions of KB-16 with tissue compo- nents. The liberation of gas following inhalation of KB-16 vapor is presumably not sufficient to be sig- nificant. Toxicity by Mouth KB-16 is moderately toxic when administered by stomach tulie. The hi)-,o’s are in the order of 20 mg kg for the rat and 15 mg kg for the rabbit.41 The substance is immediately irritating, as evidenced by vomiting in dogs, and it produces severe esophageal and gastric damage.17® In the rat, vesicles similar to those produced by lewisite oxide have been found in the stomach at autopsy.41 Lung pathology has been observed in some cases,7* and the absorption of KB-16 from the gastrointestinal tract has been demonstrated by the appearance of gas bubbles in the hepatic portal vein.4 Death occurs after from one-half day to many days and is preceded by pro- nounced weight loss when survival is prolonged.17e 41 That KB-16 presents some hazards as a water con- taminant is demonstrated by the virtually 100 per cent mortality of mice, rats, and dogs whose supply of drinking water was contaminated with 0.5 1.0 mg ml.17' Only few deaths occurred when the water was contaminated with 0,1 mg ml. In most of the experiments the contaminated water was freshly prepared each day. In spite of the fact that aqueous solutions of KB-16 decompose within 48 hours (see Section 8.2.3), mice whose drinking water was con- taminated with 1.0 mg ml of KB-16 at the beginning of one experiment died almost as quickly as those whose contaminated water supply was freshly pre- pared at daily intervals. K.t.3 Vesicant Action In comparison with H, the vesieancy of KB-16 is of a low order 11 41 46 and screening tests indicate that none of the related compounder is more potent.14 A direct comparison of “absolute vesieancies,” de- termined by application of agents diluted with ben- zene and covered to prevent evaporation, reveals that KB-16 is about 1 ;1 to 1 4 as potent as II.41 As shown in Table 4, small doses of liquid KB-16 applied For I. arc made of the of 63 Table 4. Vcsicancy of K B-16.14 the sake of comparison, data obtained with H and included. All applications of the vesicants were during winter weather (January 1943) to the skin forearms of human subjects at room temperatures 72 F and relative humidities of 14-37 per cent. Days after Dose applica- Agent (mb) tion Erythemas Blisters KB-If) 200 2 7/28 (4 mm) 0/28 7 7/9 ... 1/9 (2 mm) II 65 2 112/119 (7 mini 70/119 (5 mm) I- 95 2 285/290 (8*mm) 279/290 (6 mm) to tlie skin in the usual way (i.e., undiluted and with evaporation permitted) produce far less injury than do II or L. 'I'ested more realistically in relatively large doses (drops t.l mm in diameter), it produces injuries which after 3 days are no more severe than those elicited by HN3 or IIN2.46 It is known from other data (see Chapter 6) that, under the moderate conditions of temperature and humidity prevailing in the above test, these nitrogen mustards are no more than one-fourth as vesicant as H. All observa- tions14414652" indicate that skin injuries due to KB-16 require considerably longer (i.e., 5 6 days) to attain maximum severity than do those usually produced under similar conditions by II, L, or the nitrogen mustards. It should be noted that all of the above observa- t ions were confined to applications of the liquid agent to the not visibly sweating skin of physically inactive subjects at moderate temperatures and humidities. SECRET 126 ALIPHATIC NITROSOCARBAM AXES A ND REL ATED COMPOUNDS No determinations have been made of the vesicant potency of liquid KB-16 on hot, sweating skin, of the vapor under any conditions, or of the effectiveness of either the liquid or the vapor through ordinary or protective clothing. B i t Eye-Injuring Action Numerous observations on the effect of KB-16 vapor on human and animal eyes demonstrate that the agent is an insidious and potent eye-inju- rant.16* h r g,h i k l7*,d *‘ f i i 24* “6 S2'44’5'* h As has been mentioned, no exposure symptoms are produced in animals by even high concentrations (i.e., 0.2- 0.4 mg 1), and exposures entirely undetected have sufficed to produce moderately severe injuries in laboratory workers.The onset of injury and ac- companying symptoms is more delayed than in the ease of H. and much more delayed than in the east* of the arsenieals. There is an asymptomatic latent pericxl of many hours. Maximal damage develops after from two to several days, and recovery is pro- tracted. Corneal edema, opacity, and delayed but extensive vascularization are the most prominent symptoms. The conjunctivas are also injured, al- though less extensively than in the ease of H. Iritis occurs but is not so conspicuous as in eyes exposed to IIN2 or H. Delayed relapses such as occur in the case of H have not been observed. An interesting preliminary report lfi,‘ indicates that, in addition to the injuries just described, severe retinal damage can be produced in animals by ex- posures to relatively small dosages of KB-16 vapor which produce only moderate conjunctivitis and slight and transient superficial keratitis. Changes in the retinas of eats examined 3 14 days after exposure of the animals to O.Oii mg I for 10 minutes consisted of: (1) slight increases in glial cells and perivascular macrophages, with hyperchromatieity of ganglion cells; (2) restricted zones of perivascular cuffing with leucocytes, resembling a periarteritis; and (3) intense chorioretinitis with subhyaloid hemorrhages, migra- tion and phagocytoses of pigment, and extensive chromatolysis and destruction of ganglion cells. ( om- parable exposures to II Nl produced no morphologi- cal changes in the retinal ganglion cells, although clusters of leucocytes adhering to the endothelium of the blood vessels represented a difference from the normal retina. Exposures to H vapor (0.04 mg 1 for 10 minutes) likewise produced the clustering of leuco- cytes, and itt addition isolated small patches of cho- rioretinitis; however, changes in the neural elements wore absent or at most mild compared with those produced by KB-16 at the slightly higher dosage. Data to be summarized in the following paragraphs lead to the conclusion that KB-16 vapor is a dis- tinctly more insidious eye-injuring agent than II vapor but not necessarily a more potent injurant when assessed on a dosage {Cl) basis. In this respect KB-16 is similar to HN3 (see Chapter 6). Exposuke of 11 rsiAS Eves to Small Dosages ok KB-16 Vapok The eye injuries produced by the vapor of KB-16 at small and minimal dosages may l>est be illustrated by citation of the ease histories of accidentally ex- posed laboratory workers. In one case 38,1 some KB-16 was splashed on the left side of the face. It was immediately decontaminated and the liquid presumably did not enter the eye, as the worker was wearing glasses. Nevertheless as a precaution the eyes were quickly washed wit h water. There were no ocular symptoms out he day of the accident. On the following day both eyes were slightly sore but normal duties could be carried out. Ophthalmic ex- aminations 3-22 days after the accident revealed the following effects. J days. The conjunctivas were hypercmic, the injection being more marked in the palpebral aperture than elsewhere. The cornea did not slain with fluorescein but scattered epi- thelial cells showed hydropic degeneration. Tin-pupils were normal and there was no iritis. 4 day*. The eyes were more un- comfortable and the patient experienced slight difficulty in keeping them open. The conjunctivas were more hyperemic and there was epithelial bedewing all across the palpebral ajierture. All the limbal blood vessels were congested. The substantia propria of the cornea was normal. The lids were slightly swollen. There was no eheinosis and no iritis. 5 days. The symptoms were worse and an attack of blepharospasm and photophobia occurred. Suhepithelial cellular infiltrates could be seen in the left eye. V days. The congestion was worse and the left cornea still 1 axle wed. 7 days. The patient felt that his sight was worse. Visual acuity was reduced from 6/12 (on the fourth day) to6/24. The congestion was more marked and the superficial layers of the substantia propria were densely infiltrated with celts but not edematous. 9 days. The lids were slightly sticky and puffy, the conjunctivas very injected. The interior of the eyes was normal. 11 days. The limbal loops showed great activity and appeared to be advancing on to (he corneas from all meridians. 16 days. The right eye was slight ly lx*tter, the left showed further roughening of the epithelium and slight edema of the substantia propria. The limbal loops were advancing. 20 days. Photophobia persisted and the eyes appeared worse. Conjunctival injection was marked. The margins of both corneas were invaded with a rich superficial vascular net. The corneas were full of cells at all levels. 22 days. The limbal ls were still extending. The symptoms were somewhat alleviated but definite objective improvement had not started. Comment. The main points of interest are the absence of immediate symptoms, the long latent period, and the delayed recovery, even though the dose was insufficient to produce pupillary contraction or iritis. The prognosis was SECRET TOXICOLOGY 127 considered good in view of the course of the case next to he descril>cd. In a second case a chemist hud been working with KB-lti for 3 days during which time tie smelled nothing and experi- enced no sensation to suggest that he was being exposed to the vapor. On the second day his eyes were slightly bloodshot but not painful. On the evening of the third day of work he had a severe headache and pain in the eyes, and on awakening during the night found himself unable to keep his eyes open. He was examined at 3-22 days after tic commenced Ids work. 3 days. The lids were only slightly swollen but the patient was unable to keep his eyes open. There was lacrimafioii but no discharge. The conjunctivas were not very congested and there were no hemorrhages. In the paljiebral aperture there was a band of epithelial edema and punctate staining. The pupils were small and their reaction to light poor. The patient had nasal discharge. .{ days. The eyes were still closed and the pain, now a gritty feeling, was relieved by phenacetin. The corneas appeared improved. The lids were slightly reddened. ■> days. The gritty feeling jiersisted. There were no signs of iritis but the limbal loops were beginning to encroach on the corneas, d days. The eyes were much (letter and could be kept ojien for periods of an hour or more, with attacks of blepharo- spasm lietween. The conjunctival injection was almost limited to the paljiebral apertures. The epithelium was bedewed but an ocular infiltration was lieginning under Bowman’s mem- brane. The deep structures were normal. 7 days. The eyes could lie kept open much better but lacriniation jiersisted. There were infiltrates throughout the substantin propria. days. Photophobia persisted and there was a slight whitish discharge, the epithelial bedewing in the jialjjehral ajiertures was less pronounced. The conjunctivas were still slightly in- jected. 11 days. The eyes were about as above except that new vessels wore extending in ojien loops onto the corneas. Photo- phobia jiersisted. 15 days. The newly formed sujierficial vessels on the cornea were beginning to empty. 20 days. The photo- phobia had jiraclieally disapjieared and the eyes were nearly normal on macroscopic examination. 2 i days. The eyes wore practically symjitomlcss. The limbal loojis had extended onto the cornea all around in both eyes but wore mostly empty and disappearing. Suliepithelial infiltrating cells were fewer. A few" hydropic cells remained in a line on the jialjicbral fissure of one eye. Comment, the main points are that the exjmsure was unsusjiecled and symptoms delayed for 2 days. They then became severe. Virtually comjjlcte recovery had occurred within 22 days. A number of investigators working with KB-16 and presumably receiving minimal vapor dosages have developed mild ocular changes.16* The conjunc- tivas showed at most only mild congestion. Exami- nation of the corneas revealed superficial punctate nebulae largely peripheral in location and almost al- ways confined to the interpalpehral area. The nebulae sometimes escaped detection on slit lamp examina- tion but were seen after fluorescein staining. Clini- cal notes also mention a Stahli’s line, unduly prominent corneal nerves but no alteration in comeal sensibility, and fine punctate hyaline areas best seen with lateral illumination or rctroillumination and disappearing within 2-3 weeks. The observed changes were of a type commonly observed in various non- specific irritations of (he eye and are difficult to evaluate. ’1 hey were insufficient to produce signifi- cant subjective symptoms or loss of visual acuity. Nevertheless, in view of the slow development, of the pathological changes caused by KB-16 vapor, it has been recommended that individuals showing such lesions avoid any possibility of further exposures for 1 2 weeks.-6 Comparison- of KB-16 with Other Agents ox Basts of Effects of Relatively Large Vapor Dosages on Animal Eyes Quantitative comparisons of the potencies of dif- ferent agents in terms of the dosages necessary to produce eye injuries of casualty severity are difficult at l>est and for KB-16 there exist no detailed quanti- tative studies such as have l>een made with H.-9 Of the numerous interim studies with animals, two m permit a more or less direct semiquantitative com- parison between (he potencies of KB-16 and II. Both studies (Tables 5, 6, and 7) indicate (hat the poten- Tabus 5. Effects of the saturated vapors of KB-16 and H on the eyes of rabbits: effect of exposure time on sever- ity of injury.3,“ The eyes were protopsed and exposed for the stated limes at 22-24 C to vapor cups containing KB-16 or H. At 23 ( ' the volatility of KB-16 is approximately 0.8 ing/1, that of It approximately 0.0 mg/l.” The severity of the resulting lesions is tabulated. Exposure time Agent (sec) KB-16 II 15 Mild conjunctival le- Minor conjunctival Ic- sion, slight corneal lc- sion. Rapid recovery. sion. Rapid recovery. 30 Injury variable. Rare Injury variable. Com- perforation, occa- plcte recovery in sional complete recov- most cases. cry. 60 Mild permanent dam- Moderate permanent age. Perforation in damage. No cases of some cases. perforation. 120 About same as 1-min- Severe damage. Many ute exposure. cases of perforation. cies of the two agents are of (he same order of mag- nitude, the principal difference being that the onset of damage and possibly the rate of recovery are more delayed in the case of KB-16. In so far as this con- SECRET 128 ALIPHATIC MTKOSOCAHBAAIATES AM) RELATED COMPOI ADS Table (i. Effects of the saturated vapors of KB-16 and H on the eyes of rabbits: tabulation of types and relative severities of injuries.51* The eves were protopsed and exposed for 1 minute at 22-24 C to vapor cups containing KB-16 or II. At 23 C the volatility of KB-16 is approximately 0.8 tng/1, that of 11 approximately 0.9 mg 1." Characteristic of Agent injury KB-10 II Latent jieriod for severe injury 18-36 hr 6 16 hr Conjunctival reaction: 4- 4-4-4- - Redness 4-4-4- 4- 4- Cheinosis 4- 4-4-4* 1 lemorrliagic necrosis 0 4-4- Ischemic necrosis 0 — 4-4-4- Corneal reaction; 4-4-4- 4-4- Edema 4-4-4- 4-4- Vascularization 4- 4- 4- 4" 4- 4- I'lceration 4- 4- 4- Residual opacity 4- 4- 4- Purulent discharge + 4- 4- Iritis 4- 4-4- Relapse 0? - -4-4- Table 7. KfTeets rties which presumably under- lie the necrotizing action of KB-16 and the less toxic related compounds were reviewed above (Sec- tion 8.2.3). In resume two types of react ion have been demonstrated to occur with substances of biological interest in aqueous solutions at pH 8. First, a gen- eral pro)MMlv of N-alkyl-N-nit rosocarbamic acid esters is the capacity to transform HNH2 groups into H• XII CO ()Br groups (carbomethoxylation, carbethoxylation, etc.). This reaction characterizes not only KB-16 and the corresponding ethyl ester, but also u'trosocarbamates (i.e., ethyl N-methyl-N- nitrosocarbamate) which do not contain a /3-chloro- ethyl group attached to nit rogeh. Second, interaction of KB-16 or the homologous ethyl ester with o-amino acids results in the disappearanee of amino groups, and, in the ease of cysteine, of the sulfhydryl group as well. Substances analogous to the “one-armed” sulfur and nitrogen mustards are presumed to lie intermediates in these reactions, and conceivably may be toxic by virtue of the alkylating power of their 0-chloroethyl groups. The /S-chloroethyl group of KB-16 itself is relatively unreact ive and neither of the above-described reactions corresponds to the principal mode of interaction of the sulfur and nitro- gen mustards with amino, sulfhydryl, and other physiologically important groups (Chapter 19). The difference in mechanism is further emphasized by the fact that KB-16 reacts in nonaqueous media with the amino group of benzylamine, whereas reactions of the sulfur and nitrogen mustards dejxmd upon a preliminary solvolytic activation in water. The reaction of KB-16 with hemoglobin in vitro supplies a model for possible react ions of t oxicological significance, and the absence of a comparably vigor- ous reaction with egg albumin suggests that the ef- fects of the agent in the cell may lie confined to only some of the biologically important molecules and re- active groups. Biochemical studies do, in fact, reveal that some enzyme systems are readily poisoned by KB-16, whereas others are not. In one study with enzyme systems in vitro, the effects of KB-16 were compared with those of H.'* The three tested systems were inhibited by KB-16, but not so effectively as by II; previously hydrolyzed KIM6 was without effort. Purified yeast hoxokina.se was inhibited 60 per cent by 0.006 M KR-16 and at) per rent by 0.003 M H. 1’hosphoereatinc phos- phokinaso was not significantly inhibited by 0.002 ,1/ and was inhibited 20 jxt rent by 0.006 M KB-16, whereas H at 0.001 .1/ produced an inhibition of 90 per cent. Inorganic pyrophosphatase was in- hibited 70 per cent by 0.001 M 11 and only 35 per cent by 0,002 .1/ KB-16. The respiration (oxygen consumption) of slices of tissue from a variety of organs was inhibited by treatment with 0.001 M KB-16.24 In general the in- hibition was greater (even complete) in the absence of added oxidizable substrates than in the presence of glucose, lactate, pyruvate, or other carbohydrate intermediates. The degree of inhibition increased with time in some instances. In contrast with its effect on “oxygen consumption, KB-16 had but a slight effect on glycolysis as measured by carbon dioxide output or lactic acid production. Some but not all aspects of the metabolism of pyruvic acid by tissue slices were markedly affected by KB-16. Oxi- dation of pyruvate (utilization in presence of oxygen) was inhibited, but considerable species and organ variation occurred. The disrnutation of pyruvate as measured by its utilization by chopped brain in the absence of oxygen was inhibited to a smaller extent, and its decarboxylation by dried yeast was unaf- fected. The synthesis of carbohydrate from pyruvate* by kidney slices (rat) was almost completely in- hibited by 0.001 M KB-16, but another condensation reaction, the synthesis of acetoacetate from pyruvate by chopped pigeon liver, was almost unaffected. Ex- perimenls with rat kidney indicated that the oxi- dative deamination of natural amino acids (i.e., glutamic) is greatly inhibited by KB-16 but that d-amino acid oxidase is unaffected. KB-16 had little effeet on the oxidation of citrate and fatty acids by various preparations. Cholinesterase (Stodman) was inhibited by KB-16 but the agent had no significant effect on a number of other enzymes including the following: carboxylase, succinic dehydrogenase, cyto- chrome oxidase, choline oxidase, pepsin, and urease. In summary, the primary effects of KB-16 seem to be flue to the inactivation of certain essential pro- teins. Prominent among the sensitive substances appear to be the activating proteins of pyruvic oxi- dase and /-amino acid oxidase. Inasmuch as the re- actions appear to be irreversible, the combatting of injury by KB-16 should be based primarily on pre- vention of the reactions. SECRET 130 ALIPHATIC NITKOSOC ARB A M ATES AM) RELATED COMPOUNDS Tabi.e 8. Properties of KIMfi, mustard gas (H), and fnsO-chlorocthyl)aniine (11X3) bearing upon their utility as chemical warfare agents. • Agent Property KIM 6 11 HN3 Storage stability poor good excellent Explosion stability questionable good good Factors influencing stability on moist terrain: Solubility in water (ppm at room temperature) 7,000 500 so Half life in water (min at 25 (') 8 ± 2.4 + Volatility (mg 1 at 25 (’) 0.87 0.00 0.12 Freezing point (C) <—50 14.3* - 3 5-9t Density (g/ml at 25 C) 1.21 1.27 I 24 Median detectable cone, (ugl) 7 ± o.tif 15± 1.8* Relative eve-injuring potency l± 1 1± Relal ive vesicant potency of liquid on not visibly sweating skin <0.25 1 0.25-0.5 Relative vesicant potency of vapor on sweating skin ? 1 O f. 0.9 * Parc It. t Ij-vinstrin H. _ As is the case with II and the nitrogen mustards, instillation of very small amounts of KB-16 into the eye results in an inhibition of mitosis in the corneal epithelium.341* This effect is exerted by less than one- thousandth of the minimal dose causing clinically visible lesions. R.5 EVALUATION AS WAR GASES KB-16 and the most toxic related compounds (i.e., ethyl X-(/3-chloroet 1 iy 1 )-X-ni trosoca rbamate and X- (0-chloroethyl)-N-nitrosoacetamide possess insuffi- cient storage stability to be seriously considered for large-scale manufacture for purposes of chemical warfare. It has been suggested that this difficulty might he overcome by nitrosating the stable inter- mediate, methyl X-(j8-ehloroethyl)carbamate, with nitrous gases just before use, or by development of a munition designer! to effect the nitrosation shortly before firing or even thereafter However, comparison of the other properties of KB-16 with those of such persistent agents as II and HX3 (see Table 8) leads to the conclusion that KB-16 does not possess suffi- cient general utility to merit such special treatment. Moreover, in the opinion of the authors, it would not deserve serious consideration even if a method for its stabilization should Ive forthcoming. KB-16 does possess certain desirable features — low freezing point, lack of pronounced odor, and effectiveness as an eye-injurant at low dosages. The available data do not permit the conclusion that the vapor dosages necessary to produce casualties among unmasked troops by eye or respiratory injuries would l>e of a different order than the dosages required in the cases of II and HN3. Given equivalent low vapor dosages, however, KB-16 because of its less pro- nounced odor would be a more insidious and there- fore more effective agent than H. On the other hand, it would not have this advantage over HN3, which is less odorous. Because of the necessity of assuming that enemy troops will be equipped with gas masks, current doc- trine giv es greater weight to the vesicant effects than to the eye-injuring potency or inhalation toxicity of a persistent agent not having either much less odor or much greater potency (or both) than II, KB-16, or 11X3. Thus, the relatively low vesicant potency of KB-16 places it at a great disadvantage in compari- son with H. SECRET Chapter 9 FLUOROPHOSPHATES AND OTHER PHOSPHORUS-CONTAINING COMPOUNDS By Marshall (inks and Binhey Renshaw 9.1 INTRODUCTION Approximately 200 phosphorus-containing com- pounds of widely varying structures were ex- amined as candidate chemical warfare agents during World War II, Only the few represented by the dialkyl fluorophosphates, the diamidophosphoryl fluorides, the alkyl cyanamidophosphates, and the alkyl duorophosphonates have merited detailed ex- amination. The individual compounds that have re- ceived most attention are: 2. Diary.idophosphoryl Jluoridcs. CH, \ N ) / \ / CH, \/ / P CH, /\ \/ F N / cn, hid I hmethylamido)phosphoryl fluoride (TL 792, T-2002) 1. Diailkyl Jluorophosphates. CH,—O O \ / P / \ — ch,—o f Dimethyl fluorophosphate (PF-1, TL 311, T-1035) CH, CH2—O 0 \ / 1* / \ CH, CH,—() F Diethyl fluorophosphate (TL 345, T-1036) 3. Alkyl cyanamiduphosphates. CH, \ “ N / \ O CH, \ / P / An ch,ch2—o Kthyl dimethylamidocyanophosphate (MCE, Tahiti), Lc 100, TL 1578, T-2104) CH, CH,CH, \ \ CH—O CH—O / \ o / \ o CH, \ / CH, \ / P - p CH, /\ CH5CH2 - /\ \ / F \ / F CH—0 CH—O / / CH, CH, 4. Alkyljluorophosphonales. CH, \ , CH—0 O J / \ / CH, P / \ / F H,C Isopropyl methanefluorophosphonate (MF1, Sarin, T-I44, TL 1618, T-2106) Diisopropvl fluorophosphate (PF-3, DPF, TL 466, T-1703, 1152) Di -scc-hutyl fluorophosphate (TL 1206, T-1835) H,C—ch2 / \ - - H,C CH—o \ / \ o 11*0 CH, \/ HjC CH* 1* / \ / \ H2C CH—O f \ / II2C CH, CH, _ \ CH—O O / \ CH, P CH,CH2 Isopropyl ct hanefluorophosphonate (TL 1620, T-2109) Dicyclohexyl fluorophosphate (TL94!, T-1840) SECRET 132 FUUOROPHOSPH VTES \ M) IMIOSPIIORUS-COXT VI \l \C COMPOUNDS The dialkyl fluorophosphates were described in the open literature in 1932. The British undertook their examination as war gases in 1911 and much work on them was subsequently carried out in the United Kingdom and United States* They are parasympathetic stimulants and cholin- esterase poisons of high potency. For some sjx'cies (e.g., the monkey), PF-3 and di-scr-butyl fluoro- phosphate are more toxic than any of the standard United States or British chemical warfare agents. At lethal concentrations they are” “quick-kill” agents, their action being only slightly less rapid than that of hydrogen cyanide (AG). However, their relatively low volatility, at 25 G 30 mg 1 for PF-l, S mg 1 for PF-3, and 1,8 mg 1 for di-xcr-butyl fluorophosphate. puts them in a class with the persistent agents and would render difficult the rapid administration of a lethal dose under field conditions. Chief interest in them has arisen from their action on the eye. They produce extreme constriction of the pupil, interfer- ence with the muscles of accommodation, potentially dangerous congestive iritis, and severe pain behind the eyes. PF-3 and di-scc-butyl fluorophosphate at a dosage of 50 mg min nr* produce pupillary constric- tion, and PF-3 at about 300 mg min nr* produces the other harassing symptoms just mentioned. However, by 1943 and 1911 careful assessments led to t he con- clusion that in practice these effects would be harass- ing rather than casualty producing. It is believed that troops supplied with gas masks would not he- come casualties from attack with the fluorophos- phates except under circumstances where standard non persistent agents would have equally or more severe consequences. A useful interim summary of work on the fluorophosphates was prepared by Di- vision 9 in 1944.37 The diamidophosphoryl fluorides proved to be about as toxic as the fluorophosphates but to be less potent in their action on the eye. Their chief point of interest is that they are extremely stable in water and upon oral administration are among the most toxic of the known synthetic compounds. The dialkyl fluorophosphates appear to be eclipsed in toxicological potency and potential value as chemical warfare agents by the alkyl cyanamido- phosphates and alkyl fluorophosphonates. These compounds, known collectively as Trilons (a name assigned to them by the Germans), first came to the attention of United States and British workers after the termination of hostilities in Europe in the spring of 1945. It was then discovered that the Germans had manufactured largo quantities of MCE for use in bombs and high explosive-chemical shell. They had been attempting also to prepare MFI on a large scale tint had I>een unable to overcome difficulties in its synthesis. The Trilons an* similar in mode of action to the fluorophosphates but are considerably more potent both in terms of inhalation toxicity and in the pro- duction of eye effects. For the monkey the L{Ct)„o’s of MCE and MFI are in the order of 250 and 150 mg min m3, respectively. In man MCE at the extraor- dinarily low dosage of 3.2 mg min m3 produces pupillary constriction. Dosages in the order of 15 to 20 proved to Im> definitely harassing because of ocular and systemic effects, and it would seem that 30 mg min m* might suffice to produce significant partial disability. Quantitative eye data on MFI are not available to the reviewers. Although MCE is some- what less volatile than mustard gas (H) and is sus- ceptible to hydrolysis, MFI has (he rather high saturation concentration of 16 mg 1 at 25 C and is quite stable. Moreover, it is virtually odorless. it would seem that the Trilons are the one new group of chemical agents discover'd during World War II which merit serious consideration for adop- tion as standard agents. Their use in high explosive- chemical shell, indistinguishable on detonation from ordinary high-explosive munitions, should 1k> care- fully evaluated and assessment made of the relative casualty-producing effects of (1) the initial cloud of droplets and vapor and (2) the subsequent vapor evolution from the contaminated terrain. Division 9 has participated in work on the Trilons only to (he extent of performing limited studies on synthesis, detection, and analysis. Most of the re- ports on work done by other agencies have become available during the period when the division was terminating its activities. Some of these reports may not have come to the attention of the reviewers. It has not been possible to render the review of the Trilons as complete as the discussion of the other agents of major importance. A summary of the field trials conducted at Raubkammer after the defeat of Germany has not been included, and a complete assessment of t he value of t heTrilons as chemical war- fare agents has not been undertaken in this chapter. 9.2 SYNTHESIS \ND PROPERTIES 9.2.t Synthesis Many methods have been used in the synthesis of the compounds listed in Table 1. The following dis- SECRET SYNTHESIS AND PROPERTIES 133 Table 1. Fluorophosphatcs, amidocyanuphosphates, fluoropbosphonates, and other phosphorus compounds examined as candidate chemical warfare agents. The compounds are arranged in the following general classes; (1) derivatives of phosphine, (2) derivatives of primary phosphines, (3) tertiary phosphines, (4) oxygen, sulfur, and nitrogen derivatives of trieovalenl phosphorus, (5) phosphorus pentahalides and related com|xmnds, (6) phosphoric' and phosphonic acid derivatives and their sulfur analogs, (7) quarter- nary phosphonium salts, and (8) miscellaneous compounds. The following abbreviations arc used; n'B, refractive index at / C’; int in V at /» mm Hg; vp', vapor pressure in mm Ilg at 1 C; and vol', saturation concentration (volatility) in mg l at 1 C. Centigrade scale is used throughout the table. Reference Reference to (’onqsnind to synthesis Physical pro|H*rlies Property Reference toxicity (hit a 1, Phosphorus trifluoride 10, 10tH>, bp 101.1° I06d 11, 106a 2. Phosphorus monochlorodifluoride 106d lll{> 151-5° 10< id 11 3. Phosphorus dichloromoiiofluoridc 11 4. Phosphorus trievanide 30d bp° 5 150' 30d ■>. Phenylphosphine 27g (sublime 11, 18 6. Kt hyldichlorophosphine 2 bpT«o 9-1-97° 2 11 7, Kt hyhlicyanophosphine 27r bp'" 94- 9(5° 27r 11, 60d S, Phenyldiehlorophosphine — !). Phenvldicvanophosphinc 27d tP; 1.1660 27d II. IS — — . . bp* 100 27d ... — bp20 145° 27d * mp 35 27d 10. Phenyhlithiocyanophosphine 27f 11, IS 11. p-Chloro|)henyldichIorophosphine 12. p-ToIyldichlorophosphine 13. a-Xaphl hyldichlorophosphine . T __ ■ —... 14. 2-Dibenzofuryldicyanopbosphine 27h 11 15. 3-( X-Kl hylcarbtizole) dichlorophosphine 16. 2-Phenoxthiindicyanophospltine 27h 27h ... — • • • • H 1) 17. Triehloromethylphosphinc —_ 27f, 77 11, IS 18. Triethylphosphine 27d bpj« 127.5° 27d 11 1ft. Tributylphosphine 27b 20. Trioclylphosphine 27.1 bp‘ 234-237° 27d 11. 18 - mp 30° 27d 21. Tridecylplaisphine 27h ... ■ 11, 18 22. Diethylphenylphosphine 27f II. 18 23. 1 tiallylphenylphosphine 27f 24. Dibittylphenylphosphine 27e p5" 185° 27e - — bp"* 116° 27e fp 25° 27e 25. difluoropb(>sphine 27n II 39. Dietbvlaminodichlorophospbinc 27d 1.196 27d 11 bp'* 72 75° 27d . . , ' — 189°/atmos. 27d 10 N,X-his((3-('blon>etliyl)aminodicliloropb<>.sphine 1. 27m II 41. Klhyl-(d-cbloroethylthin)ehlomphosphinc 27p bp" * 89-92" ' 27P 11, 18 42. DiplKMiyI-/3-chlorsphin® 82 bp' 74 75° 82 82 48. 5i>(T)imelhylaniino)fluorophosphine 10 iP* 0.975 10 11 — bp 120 10 40. KlbvW»is(£-fluoroethoxy)phosphinc 35a bp*-* 40 4'.)° 35a II 50. Pbcnyldiet hoxyphosphine 27g 11, 18 51. Phcnyl-Wsfo-ehlorophenoxy Iphosphiue 27h 11, 18 52. K(bvl-6)*(ifFchloroelliyllhio)phosphine -— 27p WO5* 1.5600 27p 11, 18 bp 115-120° 27p — ... 53. Phcnyl-5is(methylthio)phosphine 271. It. 18 54. 55. Phenyl-fnsfd-chloroethyllbiojphosphine p-Dimethylaminophenyl-bisfd-ehloruethyllhio)- 271 II, 18 phosphine rnonoelhylate 27n - .... 11. 18 50. Phenyl-fci*(d,p'-dichloroisopropylthio)phosphinc 27q II 57. Phenyl-5iK(hu1y!lhio)phosphinf 27h .... — 11, 18 58. Dimethyl hydrogen phosphite 27h .... • • 11, 18 59. fws(d-Fluoroelhyl) hydrogen phosphite bp" 109 110“ 104p I04p 00. 1 )iisopropyl hydrogen phosphite bp17 82.5° I04f 104f fil. Tri met hoxyphosphine 27h 11, 18 02. T riel hoxyphosphine 27c iP” 0.968 27c 11, 18 _ . . . l.p7'" I55-156“ 27e bp" 36-38° 27e 63. /rr.s(f)-Flnorriet hoxv )))hosphine 30(1 bp" 4 100-103° 30d II 04. tris{ d-Chloroethoxy Jphosphinc 27f II, 18 65. trfs( d-Rromocthoxy)phosphine 27m II, 18 66. Trihutoxyphosphinc 34h 1';" 98-100° 105e 104l> 129. Diisopropyl fluorophosphate (PK-3) Sec text 1.3780 105e Sec text hp-4 84-85° 10.5e hp:su 183“ (est) 105c fl> - 93° Vol*° 5.84 12 )30. btx(fi,ii1held,ireiis.ipn>|ivl) fluorophosphate 10.5k hp"-3 * 163 165” 10.5k 131. Dihulvl fluorophosphate 28h 105a 132". Di-sn’-hut vl fluorophosphate I05f, 10.51 hp- - 02-64° 105f Sea1 text voli0 1.12 12 133. Diamyl fluorophosphate ... ■ tOlh 13-1. Diisoamyl fluorophosphate I05f l»l»" 145 118° I05f 1041. 135. W«(o-Klhylpn>pyl) fluorophosphate 105k hps » 97-98° 105k 10-lj 13(5. Dieyelohe.xyl fluorophosphate 105i «l> 1.4558 281 See text. hp0-* 116° I05i voP» 0 0044 12 V • , 137. bix( 1,3-DimelhyllnilyI) fluorophosphate 105k 1»PIT 102 103° 10.5k UMj 138. hix(‘2- Methyleyclohexyl) fl\iorophosptuite I05p hp1" 120° 105p I05p 139. hlx(a-Ca els'! hoxvet hyl) fluorophosphate hp" 14 137° 105p ia5k hp" ‘ 126 128° 105k 10-lj 140. Di|dienyl fluorophosphate 2sj, ia*if bp" 106-108“ _ I0.5f lOlR 141. fus(Triethvllead) fluorophosphate 105c nip >200° 105e 105e 142. Diethyl chlorophosphate 11, 18, 104h 143. fcfsO-Fluor, tethyl) ehlorophosphale 30d i.p"4 108-112° 30d - 144. Diethvl evanophosphate J05r hp" 95 97“ 105r 105r 145. Diethyl lhioevanophosphate 105e hp14 115-125° 105e I04d 14R. Kthvl X-plienvIamidofluorophospliale 105n rap .50° — 105n 105n 117. Methyl N.N-diethvlamidoehlorophosphatc 25 «i> 1.4443 25 69d !4H. Kthvl X, X-di met hvlamidocya n. .phosphate hpn,*-*-i* 19 49.2° 25 (MCK) 21,25, 105r no 1.4243 25 See text iPn 1.077 25 hp"-* 56-58° 25 f|> .50.0° 60 vol*4 0.567° 69c vol“ 0.612 60 — 149. Methyl X, X-diet hylam idocyan. .phosphate 35b bp"4 65 66° 35b 69el 150. Kthvl X, X-diet by la midocy an..phosphate 151. Methyl X,X-f»/x(0-chloroethyl)amidocyanophos- 30c tide phatc 69el 152. hix(Dimelhvlami«lo)phosphoryl fluoride 10, 105m, 105o ip1 1.110 10 See text l.p14 86° 105m . . , _ ... . hp! 50° 10 vol.** 2.16 12 153. hix( ButvlnmiphosphoiyI fluoride 105m rap 59.5“ 105m I04ei 154. 6)«(Diethylamido)phosphoryl fluoride 105m m," 1.4321 28m lO-lo hp5 4 83 87° 28m hp5" 124.5-125.5 105m 155. bis(Morpholido)phosphoryl fluoride 105und synthesis Pr »|ierty Reference data 1(12. bix{ Diinethylainidoiphosph.iryl chloride 104r 1 03. Diethvl fluorothiophosphate 2Sk bp"' 55° 28k 11, 18 HU. Isopropyl inethaneflnorophosphonate (MFI) Sr-e text wna 1,37‘K) 09.1 .See text d1- 1.0941° 09.1 . ; , , . bp15 50.5 57 25 Vol2i 10.4 69e 105. Isopropyl ethanefluorophosphonate 22 »nsi 1.3872 22 See text «n55 1.3817 09e . v ■ £.* d» 1 .0552 09c bp“ 07-68° 22 .7. / vol;s 11.0 09e 1 (VO. 2-C’hlor.ihexenc-l-phosphonie achy 27k 107. Dimethyl met hanephosphonate 271 11,18, 09a 10S. bi«(d-(’hloroethyl) met hanephosphonate 271 —. . . . II, 18. 09a 109. Di-.w-bnl vl fluoromethanephosphonale ia5k lip3 90 100“ 10.5k KUj 170. Diethyl 2-flnomet hanephosphonate 105s bpH 200202 105s 405s 171. Diethyl 2-chloroothanephosphonate 27k II 172. Dimethyl propane-2-phosphonate 271 . . - 11, 18, 09a 173. hi.-i(J < 'hi. .methyl) pmpane-2-phosphonate 271 11, IS, 09a 171, Diethyl o-toluenephosphonate 105s hp" 155° 105s 175. Diethvl earbelhoxvmel hanephosphonate 27e d' 1.139 27e 11, IS bp 259’(a(inos) - 27c '•p'5 149-150° 27e 170. (l-C'liloroethyl diethyl phosphate bpw 144-145° 104f lOlf 177. frts(d-C'hloroethyl) phosphate 77 178. /ri«(d,/3'-Diehloroisopropy'l) phosphate ... 77 179. Irjs(o-Crcsyl) pliosphale 77 1 SO fns{2-MellivlJO-propylphenyl) phosphate 77 181. IriM. Ethvlt llio) phosphate bp" 172-174° 104f 104f 182. Diethyl amidophosphate bp" * 131 138° 104f lOlf mp 45.5° 104f . 183. Diethyl X-ethvlamkh»phosphate 30b bp° 1 90° ;ioi. 184. Diethyl N,X-diethylamidophosphatc 30b bp* 90° 30b 11, 18 185. Dime!hvl X,N-bisi/J-chlorocthyljamidophosphate 69d 180. Diethvl X, X-bix(jj-chh>roethylJamidophosphale 30c bp" 104-165.5° 30c 11 187. hi>(/J-Ch 1 oroethy 11 hin) X , X-h/.sfd-ehloroei hyDamido- phosphate 27o n,i* 1.5525 27o II, 18 d4 1.472° 27« bp""1 155 100° 27o 188. /m(Dimethylamido)phosphate 10 bp* 81° 10 11 189. Trimethyl thiophosphate 27h II, 18 190. Tricthyl thiophosphate bp19 100° 104k 101K 191. Phosphoniuin iodhle 27d sublimes 02.5° 27d 192. I(ir(ikis{( ’hloromet hyl )phosphonium chloride 27e nip 192-193° 27e 1 1, IS 193. ft-Chloroel hyltriet hvl phosphoniuin iodide ‘27k 11 194. fOHrmnocthyltricthylphosphoniuni bromide 27c mp 235°(d) 27c 11, 18 195. Triethyl phenyl phosphoniuin iodide 27h —... 11, 18 190. Triethyl-p-tolylphosphonium iodide 27h II, 18 I!I7. Triallylphenylpliosphonium bromide 27k 11, 18 198. Triphenylphospholietaine 27h 11, IS 199. jS-Chloroelhyltriphcnylphosphoninm iodide 27h 11 200. d-Ilromoethvltriphcnvlpliosph. mium bromide 27d mp 208° 27.1 11, 18 201. 3-Chloroacetoiiyltriphenylpliosphonium chloride 27h 11, 18 202. d-('hloroetliyl-een obtained using hydrogen fluoride as the fluoridating agent .m A well-eoolcd solution of phosphoryl dichlorofluoride in dry ether is treated with cyclohexanol. After complete removal of hydrogen chloride, the resulting dicyclohexyl fluorophosphale is isolated in 50 |>er cent yield by fractional distillation under diminished pressure,1061 Attempts to prepare this compound by the phosphite or the phosphoryl chloride methods have l)een unsuccessful. The following compounds have Ifcen prepared by this method: diethyl flnorophosphate ,“h dipropyl flnorophosphate lu6- diphenyl flnorophosphate l#5( Wsfethylthio) fluorophosphale ,osr W*(/>-fluoroethyl) fluorophosphale ,oy his{0-Chlorocthyl) flnorophosphate lu6k hidd-methyleyclohexyl) flnorophosphate ,05P dicyelohexyl fluorophosphale 581 >"*■ 2. Phosphoryl chloride method — synthesis of dimethyl flu- orophosphale (PF-!). Phosphoryl chloride is treated with methanol at — 78 C and the mixture is allowed to come to room temperature slowly. Hydrogen chloride is evolved rap- idly for a period of 2 hours. The mixture is transferred to a copper vessel and treated with hydrogen fluoride. Fractiona- tion following a crude distillation gives PF-1 in 34.5 to 38.8 per cent yield.2" The following compounds have been prepared by this method; dimethyl fluorophosphale !5r diethyl fluorophosphale ,8e diisopropyl fluorophosphale h/s(d-chloroethyl) fluorophosphale 281 A slight modification of the method has been used to pre- pare ethyl /8-ehloroethyl flnorophosphateISl and methyl ethyl fluorophosphate.” By using thiophosphoryl chloride as a starting material, derivatives of fluorothiophosphoric acid have been prepared, and, by the use of one mole of alcohol j»er mole of phosphoryl or thiophosphoryl chloride in the first step, derivatives of difluorophosphoric or diflnorothiophos- phoric acid.J8o *>,0i[ 3. Dialkyl hydrogen phosphite method — synthesis of diiso- propyl fluorophosphale (PF-3), A cooled solution of isopropyl alcohol in either carbon tetrachloride or ether is treated with a solution of phosphorus trichloride in the same solvent and then blown with air and treated with ammonia to remove hy- drogen chloride. Filtration and fractionation give diisopropyl hydrogen phosphite in 82.5-89 per cent yield. This material is then chlorinated in 71-80 per cent yield to give diisopropyl * Improvements on Lange’s preparation of ammonium fluorophosphate from ammonium bifluoride and phosphorus jjentoxide have Is-en reported by Marquina.118 SECRET SYNTHESIS AND PROPERTIES 139 cblorophosphatc, which is purified first hy blowing to remove hydrogen chloride and then by fractional distillation. Fluori- nation is accomplished by gentle heating of diisopropyl chloro- phosphatc in benzene with jiowdcred sodium fluoride. PF-3 is obtained in 84 |ht cent yield; the overall yield is thus in the order of GO per cent. Hydrogen fluoride can also l>e used as a fluorinating agent.58* Distillation of the intermediates can lie eliminated and the whole process carried out in the original solvent (carbon tetrachloride) without greatly decreasing the yield.1"’1' The following comjiounds have been prepared by this method: dimethyl fluorophosphate 4 diethyl fluorophosphate Ksa diisopropyl fluorophosphate 4-“.,IB* di-we-bulyl fluorophosphate ll4“ diisoamyl fluorophosphate ,"i( his( 1,3-dimet hylbutyl) fluorophosphate lu6k botfor-cartiet boxyct hyl) fluorophosphate ,l£‘k 5i«(a-ethylpropyl) fluorophosphate ll6k 6js(d,d'-dichloroisopropyl) fluorophosphate U6k fws(/*-fluordethyl) fluorophosphate ,®t The last step of the reaction can lie modified so as to replace chlorine by groups other than fluorine. Diethylthiocyano- phosphate and diethyl amidophusphate have been prepared in this way.1115* Semitechnieal .syntheses of PF-1 and PF-3 have been carried out by the dialkyl hydrogen phosphite method, but only with the latter compound have enough runs lieen made to standardize conditions. A description of the procedures used follows. I. Semi technical preparation of diisopropyl fluorophosphate (PF-3).* The main reaction was carried out in a 130-gallon I.astiglas-lincd jacketed vessel equipjied with a lined and coated gas inlet pipe, a propeller-type stirrer, a charging pipe, sight glasses, manometer connections, and a bottom outlet. Steam or refrigerating brine could be circulated through the jacket. The reactor was connected to a 10-foot steel tower, 6 inches in diameter, which was fitted with a lead coil condenser from which distillate could lie passed to either of two receivers. The bottom outlet of the reactor was con- nected to the top of a 40-gallon filter tank equipped for vac- uum filtration.of the slurry and return of the filtrate either to the reactor or one of the receivers. Since plugs developed at the Ixittom outlet in many runs, an additional connection lie- tween the gas inlet and the top of the filler tank was provided to allow transfer of the slurry by this route. All vacuum lines led to a 35-gallon separator tank which was connected to a three-stage steam ejector. Drain lines leading to a 40-gallon lead decontaminating tank were provided. A separate still was provided for benzene distillation. In a typical run, 212 lb (3.54 pound-moles plus 1 per cent excess) of isopropyl alcohol ( <0.2 per cent water) was cooled with brine to — 5C in the jacketed reactor. Phosphorus tri- chloride (100 lb, 1.16 pound-moles) was added gradually with cooling and stirring over the course of 4 hours, during which the temperature was not allowed to exceed 12 C. The system was kept under slightly diminished pressure (about 700 mm). The mixture was then stirred for \2 hour before applying the full vacuum of the steam jet. Chlorine was passed into the reaction mixture at a rate of 12 pounds per hour with contin- ued cooling. The end of the reaction {10 hours) was indicated by a drop in temperature, even though the rate of How of chlorine was increased. A total of 122 lb of chlorine (1.72 pound-moles, 48 per cent excess) was used. To remove excess chlorine, hydrogen chloride, and iso- propyl chloride, the stirred mixture was kept under vacuum for 2 hours, during which time the leni|ierature was gradually raised to 20 C by passing steam into the jacket of the reactor. Ten gallons of benzene was then added and distilled off under reduced pressure at a maximum temperature of 30 C. The last t races of hydrogen chloride were removed by adding an additional 10 gallons of benzene and distilling under reduced pressure at reactor temperatures not exceeding 50 C. After cooling to 20 C, 19 gallons of benzene recovered from a previous run was added. Dry sodium fluoride (95 |ier cent pure, 123.4 lb,-2.8 pound-moles, 142 [>er cent excess) was in- troduced into the reactor through an inlet line by means of a funnel. The stirred slurry was heated to reflux during I hour and held at reflux for 4 hours; it was then cooled and filtered. After washing the filter cake with three 5-gallon portions of benzene, the filtrate and washings were combined, collected in the cleaned reactor, and distilled under minced pressure. The lienzene forerun containing about 2 per cent of product was collected to lie used in the following run. One hundred and fifty-eight pounds (74 per cent of theory based on phosphorus trichloride) of FF-3 was obtained. The entire run required 44 hours. An additional 20 hours was necessary to decontam- inate and dry the system in preparation for the next run. Preliminary design and round cost estimates for a full-scale plant to produce 500,000 lb jit*r month of PF-3 by a batch process have been "drawn up using data obtained during oper- ation of this pilot plant. It is estimated that the capital cost of the complete plant would tie $700,000. Estimated manufact ur- ing costs are $0.37 per pound of product, $2,222,000 per man- ufacturing year.5 Hound cost estimates for a plant producing PF-3 by a con- tinuous process have also been-prepared.' Although fewer ex- perimental data arc available (the estimates are based on laboratory scale work only),4 a smaller capital outlay and lower operat ing costs seem possible. A total of 13 kg of PF-3 has been prepared at the British Research Establishment at Sutton Oak by a batch process resembling that just descrilicd.1"2 2. Pilot plant preparation of dimethyl fluorophosphate (PF-1). Three pilot plant runs on a process similar to that al- ready described for PK-3 have been carried out to produce a total of 35 lb of PF-1. This experience was not sufficient to allow standardization of conditions, but it was found that the temjieraturcs required, which are somewhat lower than those in the PK-3 process, could be maintained without difficulty. Because of mechanical difficulties, yields approaching those obtained in the laboratory (72 per cent) were not realized in these three runs.1 DIA M IDO PHOSPHOR VL F UOK IDES A number of compounds in this series have been prepared by Ibe application of the following more or less straight forward methods. SECRET 140 FU OROPHOSPHAXES AM) I’llOSPUOUUS-CONT VIM M; COMfOlADS 1. The action of amines on phosphoryl dichloro- fluoride. 2. The controlled action of amines on phosphoryl chloride followed by fiuorination. 3. The action of amines on phosphoryl fluoride. The first of these methods appears to be general. It is carried out by adding a solution of phosphoryl dichlorofluoride in ether, benzene, or toluene to four moles of the amine in the same solvent. After filtra- tion from the precipitated amine hydrochloride, the product is isolated by distillation or crystallization. The following compounds have been prepared in this way; dianilidophosphoryl fluoride ,nSf 6j«(dimethylamido)phosphoryl fluoride 1 " fe/s(diethylamido)phosphoryl fluoride fcf.v(butylamido)phosphoryl fluoride iu:,m bbv(cyclohcxylamido) phosphoryl fluoride ,95m 6/»(met hy lanilido)phosphoryl 11 uoride 'li5,u 6/*(benzylamklo)phosphoryl fluoride l05m A modification of this method involving prior treatment of phosphoryl dichlorofluoride with one mole of alcohol has yielded ethyl N-phenylamido- fluorophosphatc.H*“ The second method also appears to be general, and avoids the use of the difficultly available phosphoryl dichlorofluoride. The fiuorination of fns(alkvlamido)- phosphoryl chlorides proceeds somewhat less readily than that of dialkyl chlorophosphates. The method has been used successfully with 6/«(anilido)phos- phoryl fluoride and with 6 rs(di me thy lamido) phos- phoryl fluoride.1050 The third method suffers from the disadvantage that a large part of the fluorine is wasted. It has Wen used to prepare 6/s(dimethy!arnido) phosphoryl flu- oride.19 Alkyl Cyanoamidophosphates Although vague but persistent rumors of a new German gas, Trilon, reached Allied hands from time to time during World War II through intelligence channels, no reliable information as to the nature of this gas or gases became available to the Allies until the spring of the German surrender, when German munitions charged with a new agent were captured. The agent was very quickly identified as ethyl di- mefhvlamidocyanophosphale (AICE) and an in- tensive study of it covering all phases of interest to chemical warfare was started. About the same time an intelligence team interviewing members of the staff of the I. G. Werke, Elberfcld, reported that this compound had been discovered in 1937 by 1. G. Elberfcld du ring a search for new insecticides, and that in the following year an even more toxic and in- sidious substance, isopropyl methanefluorophospho- nate, had been discovered.72 Both compounds had lx*en imported to the War Ministry under its standing order to the German chemical industry regarding the reporting of toxic substances. The laboratory method of synthesis of ethyl di- methylamidocyanophosphate (MCE), disclosed in detail by (he 1. G. representative's, made use of the following steps.72 1. The interaction of t wo moles of dimethylamine and one of phosphorus oxychloride, first at 30 (' and finally at 120 C, to produce dimethylamidophos- phoryl chloride in 93 per cent yield. 2. The action of sodium cyanide and ethanol on dimethylamidophosphoryl chloride to give MCE in 90 per cent yield. This procedure has lieen checked in at least two laboratories in this country 24 25 and the German claims substantially confirmed, although the yields obtained were not so high. No detailed study of the reactions was carried out. A novel alternative method for laboratory preparation of the agent has Ix-en used in Great Britain.195 In this procedure, diethoxy- phosphorus chloride is allowed to react with dimethylamine and the resulting diethoxydimethyl- aminophosphorus is treated with cyanogen iodide to give MCE directly. In 1939-40 the Germans began pilot plant produc- tion of MCE at Munsterlager, near Bremen, and ex- perienced no difficulty in the manufacture of 50 tons of the material. Construction of a large plant at Dyhemfurth near Breslau was begun in January 1940, but production did not begin until April 1942.‘2‘3 In the plant process, chlorobenzene was used as a reaction medium in the final step. Initially the product was stripped to a content of approxi- mately 5 per cent of chlorol>enzene. Eater a product containing 20 per cent chloroltenzene was standard- ized. Both 105-rnm shells and 250-kg bombs were charged with the agent.7* A total of 10,000 to 12,000 tons ,3of MCE was produced. It is worth noting that the figure 12,000 tons represents 18 jkt cent of the, total German production of war gases of all kinds,113 which gives some indication of how largely this agent figured in the plans of the Germans. MCE is a high-boiling, fairly stable liquid pos- sessing a faint fruity odor. The pure material is color- less, but as technically produced MCE is dark brown. SECRET SYNTHESIS VM> PROPERTIES It boils at 85 C under 1.5-mm pressure, at 120 C under iO-mm pressure, and at 250 C with some de- composition at atmospheric pressure."-’ Its density at 20 C is 1.077 and its refractive index (//*„) is 1.4240.60 Its vapor pressure appears to be about one- half that of II "* and can 1m* represented as a function of temperature by tin* following equation:* 5,750 logio /'(mm) = 11.545 - _ ~ • Its volatility at 25 C is 0.567 mg I.**1 The tactical use of the agent as an aerosol produced by heavy- walled shell equipped with large bursting charges apjH*ars to have Ihmmi envisaged by the Germans. MCE is claimed by the Germans to bo the opti- mum compound of this scries as regards toxicological properties,7* but this assertion has not been verified in this country since no - comprehensive synthetic program was established to explore the field. A related compound of relatively high toxicity was encountered during an attempt to prepare the iso- propyl analog of MCE by the simultaneous action of sodium cyanide and isopropyl'"alcohol on dimethyl- amidophosphoryl chloride. In this case the cyano group alone was introduced, and dimethylamido- cyanophosphoryl chloride was obtained in 68 per cent yield. Its toxicity is approximately one-half that of MCE® Alkyl FLroKOPUoscnoNATKs Mention has been made of the discovery of this class in 1938 by members of the staff of I. G. Elber- feld. The optimum compound of the series, isopropyl methanefluorophosphonate (MFI), Ls several times as toxic for most species as is MCE, is more volatile, and is also more difficult to detect by odor. It aroused great interest among the Germans, but in spite of intensive efforts to develop manufacturing methods, production on a plant scale was never realized. As first reported to intelligence teams, the labora- tory preparation of MFI proceeded as follows.72 Dimethyl hydrogen phosphite is prepared in 90 per cent yield by the action of methanol on phosphorus trichloride, and is converted into dimethyl methane- phosphonate in 85 per cent yield by the action of metallic sodium followed by methyl chloride. Finally, methanephosphoryl chloride, produced by the action of phosphorus pentachloride on dimethyl methane- phosphonate, is converted to MFI by the simultane- ous action of sodium fluoride and isopropyl alcohol. The yields in these steps are 90 and 82 per cent re- spectively. Attempts by both American and British groups to use this scheme without modification were not en- tirely successful. In tins country yields greater than 14 per cent w ere not obtained in the nv*t Inflation step even w hen methyl iodide was substituted for methyl chloride or when the reaction was carried out in an autoclave at 125 C. By using dimethyl sulfate, how- ever, yields of 77 per cent were obtained in this step.21 British workers were able to carry out the methyla- tion step in 59 per cent yield by using a modification of the original German procedure in which dimethyl- hydrogen phosphite was alkylated by treatment with sodium sand in dry ether followed by methyl chlo- ride.10* Neither group obtained greater than 42 per cent in the final fhioriuation and esterification. After this work was well under way, additional in- formation became, available from intelligence sources to the effect that the Germans had used sodium methoxide in methanol instead of metallic sodium in the methylation step 72 71 and that two alternative methods for the final fiuorination. one using sodium fluoride and the other hydrogen fluoride, were pos- sible, The use of hydrogen fluoride made possible operation at lower temperatures but introduced cor- rosion problems. Few details on the actual operation of the final step are available.7* The substitution of higher alcohols for methanol in the first step of this process appears to be advan- tageous. Dimethyl hydrogen phosphite is rather unstable, is water-soluble, and its"sodium salt is in- soluble in organic solvents. Diethyl hydrogen phos- phite has given better results in the hands of British workers, particularly in the methylation step,251"4* whereas the use of butanol to give dibutyl hydrogen phosphite followed by methylation with dimethyl sulfate and sodium methoxide was adopted as opti- mum for a simplified process suitable for pilot plant use by NDRC workers.21 In the latter example, the solubility of sodium dibutyl phosphite in organic solvents appears to be distinctly advantageous. By this method methanephosphoryl chloride can be obtained in 79 per cent overall yield.24 Other improve- ments made during this study were substitution of a water-wash for filtration to remove sulfate salts after the methylation. and combination of the first three steps to eliminate all distillations except that of methanephosphonyl chloride. The isomerization process of Arbusow ns has also been used to prepare dialkyl methanephosphonates. Dimethyl methanephosphonate is obtained in 95 per cent yield by heating trimethyl phosphite with SECRET " FLLOROPHOSPHATES AND PIlOSPHORtS-CONT VIM NO COMPOl' \DS methyl iodide,105* whereas a similar reaction using tributyl phosphite yields 89 per cent of dibutyl methanephosphonate.24 A novel process well suited for conversion to plant scale operations has been developed on a laboratory scale for the synthesis of the ethyl analog of MFI. In this process tetraethyllead is allowed to react un- der nitrogen with phosphorus trichloride to give 89 to 96 per cent of the theoretical yield of ethylphos- phorus dichloride, which is then converted in 85 to 95 per cent yield to ethanephosphoryl chloride by the action of sulfuryl chloride. Treatment with sodium fluoride and isopropyl alcohol converts this substance into isopropyl ethanefluorophosphonate in 72 to 85 per cent yield. The fust two steps can be carried out in the same vessel.” The resulting ethyl analog of MFI has about three-fourths the toxicity of MFI itself. No attempt has been made to synthesize MFI by a similar process using tetramethyllead, w hich is reputed to lx* much less easily handled than tetra- ethyllead. Pilot plant production of MFI has not Ireen under- taken in this country. The efforts of the Germans to produce this substance on a plant scale were not suc- cessful. Although intermediates for the material were made in substantial quantity (300 tons of dimethyl hydrogen phosphite, 5 to 10 tons of dimethyl meth- anephosphonate, and 1 to 2 tons of methanephos- phoryl chloride were produced), not more than }/'i ton of MFI itself was produced.73-111 Corrosion ap(>eared to have been the principal source of difficulty. Equip- ment shortages necessitated the use of resin-coated equipment where stainless-steel or glass-lined equip- ment would ordinarily have been used. Silver-lined equipment was resorted to in some cases.72 73 MFI is a colorless, almost odorless liquid broiling at 59 C at 8 mm of mercury. Its volatility at 25 C is 16.4 mg/1.*9" It is less stable than MCE, but can be stabilized by the addition of 0.5 per cent of diethyl- amine. 9.2.2 Chemical Reactions, Detection, and Analysis Studies on the chemistry, detection, and analysis of phosphorus compounds as candidate chemical war- fare agents have Ireen limited almost exclusively to PF-3, certain of its close relatives, and MCE. Dialkyl Fluorophosphates Solutions of PF-1 in 0.9 per cent saline lose virtu- ally all toxicity in 3 hours. This deterioration is re- tanled by buffering the solutions near neutrality but is markedly no (‘derated by buffering at /d 1 9.7.'®*' PF-3 is hydrolyzed slowly at room temperature by water to give fluoride ion and diisopropyl phosphoric acid. This hydrolysis is less than 50 i>er cent com- plete in 15 hours and is still incomplete after 25 hours.* In neutral aqueous solutions at body temper- ature the half-hydrolysis time is about 9 hours.46 In 2 per cent aqueous alkali PF-3 is rapidly hydrolyzed at room temperatures, although more concentrated alkalies appear to retard this hydrolysis.IOSe his( I )i- methylamido)phosphoryl fluoride appears to be con- siderably more stable to hydrolysis than PF-3.** In contrast to the ease1 with which fluoride ion is freed by aqueous alkalies, the isopropyl groups of PF-3 are very resistant to alkaline hydrolysis. For example, no isopropyl alcohol can be detected after refluxing with 10 per cent sodium hydroxide for 72 hours.*'* Advantage is taken of this resistance to hydrolysis in several of the analytical procedures for PF-3 based oir determination of fluoride ion, the titration of which is interfered with by phosphate ion but not by alkyl phosphates.7,1*-1*51 The kinetics of hydrolysis of PF-3 have been stud- ied in several laboratories.In addition to the marked catalysis by alkali already noted, the reaction is also acid-catalyzed, and thus m pure water is auto- catalytic. In buffered solutions the hydrolysis is pseudomonoraoleeular. The observation of a pro- nounces! acceleration by phosphate ion suggests that the decomposition may be subject to general base catalysis as well as acid catalysis, although acetate ion is the only other anion which has been observed to have an accelerating effect.20 When hydrolysis of PF-3 is allowed to proceed in acid solutions, the course of the react ion may become complex. For example, in some experiments, acetone and isopropyl phosphorous acid were formed in addi- tion to fluoride ion, and no phosphate ion could Ik* detected. Other dibasic acids were likewise absent. Acetone is also formed when acid solutions of diiso- propyl phosphoric acid are treated with sodium flu- oride. It has not always been possible to reproduce these experiments, however, and the mechanism by which acetone and isopropylphosphorous acid are formed is not yet clearly understood.20 PF-3 does not react with sodium hypoiodite to give iodoform and does not react with thiosulfate ion.s,b c Methods for the detection and analysis of com- pounds of the fluorophosphates series are summa- rized in Chapters 34 and 37. The following general SECRET SYNTHESIS AND PROPERTIES 143 remarks may be supplemented by reference to these chapters. 1 he fluorine atom of PF-3 and related compounds is readily converted to fluoride ion on hydrolysis and any detection methods depending upon (he recog- nition of fluorine ion are thus applicable to these com- pounds. The ability of fluoride ion to bleach metallic lakes of certain dyes or its etching effect on glass has l>een utilized for recognition.14 41 •42-5s 04 M10*c A device making use of the etching effect has I>een examined by (he British.95-9®07 The decomposition of volatile fluorine compounds by hot platinum filaments or hot platinized silica gel to produce hydrogen fluoride is applicable to mem- Imts of the fluorophosphate series.14-M Detection of PF-3 collected upon plain silica gel tulies can lie accomplished by testing either for fluoride ion or for phosphate ion after suitable treat- ment. The DB-3 reagent may also be used.13 Chemical methods for the detection of fluorine compounds, including PF-3, in water have been de- veloped.4142 Use of the miosis produced by PF-3 as a method for detection of this agent in water has also been proposed. It is claimed that 25 to 50 ppm can bn detected in 3 minutes by this method without injury to the eye.40 The analysis of PF-3 has been accomplished by volumetric, colorimetric, or gravimetric determina- tion of the fluoride ion produced by alkaline hydroly- sis, Alternately, phosphate ion can l>e determined colorimetrically after vigorous acid hydrolysis with hydrobrornic, hydriodic, or sulfuric acids.7-*’50-80-81 10" 10ie,t,10«f Methods suitable for use in field and chamber analyses of PF-3 have been descril>ed.3 5,161 Ethyl 1)imkthvlamidocvanuhhosi*hate (MCE) MCE is readily dest royed in either acidic or basic solutions.*1-"2 In alkaline solutions, cyanide ion is liberated rapidly even in the cold, the half life at 25 C twang 5 minutes at pi I 8.5 and 30 minutes at pH 7.5.61 In acid solutions rapid liberation of dimethyl- amine occurs, the half life in solut ions of pi I 1 being 2 minutes, that in solutions of pH 3, 90 minutes. The substance has maximum stability at pH 4.5, where its half life is 7 hours with respect to both cyanide ion formation and dirnethylamine liberation. Solu- tions of maximum stability result from hydrolysis in unbuffered solutions, since the hydrolysis products are acidic and self-buffering in the range pH 1 to 5.*1 In solutions of high acidity (i.e., 3 normal), hydro- gen cyanide as well as dimethylamine is liberated rapidly but complete degradation to phosphoric acid results only from boiling the substance with mineral acids.1 “ Bleach and chlorinating agents react readily with MCE to yield CK.70112 MCE is extremely hygroscopic, and moist solu- tions of it slowly liberate AC."1-6*1! Its faint fruity odor cannot be relied on for detection.91 1,4 Its median detectable concentration as determined with the osmoscope is 2.2 pg l.Mh The standard liquid vesicant detectors, l»oth Brit- ish and American, give positive reactions with MCE. This is true of the II papers of the kit, food testing, and of the M-6 paper, M-7 crayon, and M-5 detector paint of the United States Chemical Warfare Service, and of the British Detector, Gas, Ground. The Brit- ish differential detector powder gives a yellow color with the agent.***-70-112 The black dot (AC) tube of the M-9 detector kit has about the same sensitivity for MCE vapor as it has for AC itself (20 pg) but is considerably less sensitive than the German AC tula* (sensitivity 2-3 gg). The red dot (nitrogen mustard) tube gives a nonspecific test.70 The British pocket vapor detector gives no reaction with the agent.112 The ready production of cyanide ion and a volatile amine on alkaline and acid hydrolysis, respectively, together with the production of phosphate ion on ultimate hydrolysis, can be taken as confirmatory identification."2 For field or chamber analysis, MCE can be col- lected in 1.25 normal sodium hydroxide and titrated with silver nitrate,23 62 *** or (for small amounts) esti- mated colon metrically with sodium pic rat e.23-6*k -6*1 Phosphorus colorimetry using molybdivanadophos- phate is also suitable if the sample, collected in al- kali. is fumed with perchloric acid or otherwise completely decomposed. The sensitivity of this method is several times as great as that of those already described.23 62 Attempts to adapt the DB-3 method to the analysis of MCE have not been en- tirely successful.6*11 9.2.3 Stability 1 )IALKVL FH OKOI’HOSPHATKS PF-3 is stable when stored in glass at 25 C. When stored in steel at 65 C, slight decomposition lakes place as indicated by sludge formation. This decom- position continues at an increased rate when the sample is removed and stored in glass at 25 C. This SECRET 144 FLUOROPHOSPHATES AND PHOSPHOIU S-CO\T\[M\C COMPOUNDS effect may be due to the action of light and dissolved iron salts.* In the presence of steel at 58 Gl) C, diethyl fluoro- phosphate appears to stable for several months.'-8® Both PE-1 and PE-3 are resistant to flashing. No temperature has been found at which dimethyl Hu- orophosphate flashes; PE-3 can l>c made to Hash feebly over a narrow temperature range.-60 Ethyl Dimkthylamidocyanophosphate (5ICE) Technical MCE containing 20 per cent mono- chlorobenzene is reported by the Germans to be stable even on prolonged storage.” It is also claimed by them that MFI, when stabi- lized with < 1 percent of diethylamino, can be stored in iron anti that it is stable in methanol solution. It was supposed to have been used in such solutions.71 9.2, t Decontamination Dialkyl Elcohophosphates Bleach suspensions and dry bleach react vigor- ously with PE-3 and presumably with other Huoro- phosphates, and normal field decontamination, pro- cedures as used for vesicants should be effective. The chloramides S-4GJ ami S-32S do not react with PE-3 or diethyl Huorophosphate, nor do dilute solu- tions of calcium hypochlorite.3 -" 04 The ease of hydrolysis of the fluorine atom of the dialkyl fluorophosphates by water alone varies con- siderably with structure. PE-1 is 72 per cent hy- drolyzed after standing I hour in water at 21 C; the diethyl compound, 21 percent; and the PE-3, 1 per cent. However, dilute alkalies at room temperature produce rapid hydrolysis of all three esters.3 Lime slurry should thus be an effective decontaminant. Dilute solutions (approximately 0.4 per cent) of sodium hydroxide have been proposed for skin de- contamination. Mere hosing of contaminated areas with water should mitigate the vapor hazard produced by PE-3, since it is soluble to the extent of 1.5 per cent in water.04 Ethyl Dimethylamidocyanophosphate (MCE) In the Dyhernfurth plant of the Germans, equip- ment used for the synthesis of MCE was decontam- inated by steam and ammonia. Surface decon- tamination, in the absence of steam, was done by solutions of ammonia or of amines.72 Alkalies or bleach and water have been recom- mended by the Chemical Warfare Service for decon- tamination, but it is recognized that the production of CK by flip action of bleach on MCE might prove hazardous under some conditions.70 1.2.5 Protection Dialkyl Fliorophospiiatks Adequate protection against dialkyl Huorophos- phates appears to be provided by United States. British, German, and Japanese canisters, and it is doubtful whether canister [>enetration by these agents will over be a significant problem. Repre- sentative United States, German, and Japanese canisters have been tested against PE-1, PE-3, and methyl ethyl Huorophosphate; ail afforded good pro- tection.57 The standard United States Navy can- ister provides complete protection against PE-3 as does the British l.t. Mk. II canister.75111 Ethyl D im kth v l a m i di>cy a nophi»e11ate (MCE) Completely adequate protection against the vapor of MCE isafforded by American, British, and German canisters 62 70 72 90 "2 and it is implied in intelligence reports that the German canister gives adequate protection against MEL72 American canisters (M-ll and M-lOA-1) give adequate protection against ethyl dimethylamidocyanophosphate as an aerosol (particle size 2 w, concentration 100 Mg 1, How rate 32 1pm), but the Canadian canister, which has a resin wool pad-type filter, allows serious }>ene- t rat ion after 5 minutes.70 It is to be noted that the tactical use of the agent contemplated by the Ger- mans was as an aerosol. Combined activated carbon-aeration treatment of water contaminated with MCE gives excellent re- moval of cyanide ion, odor, and color but does not remove organic phosphorus if the water has been standing more than 15 hours after contamination.6®' k 9.3 TOXICOLOGY 0.3.1 Detectability by Odor and Other Physiological Signs The Triions and fluorophosphates may be detected by (1) odor, (2) a feeling of tightness in the chest and or throat, and (3) pupillary constriction. MCE and PE-3 have faint, sweetish odors. The available osmoscopic data for these and other agents are presented in Table 2. It is apparent that the Huorophosphates are relatively . odorless. Crude MCE (German shell filling) is more readily detected but does not possess so pronounced an odor as II. MFI is said to be odorless, or practically so.10" SECRET TO\IGOLOCV 145 Table 2. Detectability by odor of MCE, phates, and other representative agents as (let: the osmoscopic leeliniqiie. flnorophos- ermined by Median detectable Agent cone, (gg 1) Reference MCE (German shell filling) 2.2 66h PE-3 36 lit Dimethyl fbiorophosphate IS 49 1 bet hyl fluorophospbatc la 49 II (plant-run Ix'vinstein) Of. 51 11 (pure thiodiglycol) 1.8 65 HN3 (plant run) to 66m AC 34 39 CG 1.4 38 mask donned before a large dosage had reached the eyes or lungs. Sufficiently low concentrations to escape these means of detection would he revealed after some minutes or an hour by pupillary constric- tion, and the mask applied if more prolonged ex- posure were unavoidable. Thus, except upon very sudden exposure to high concentrations of vapor and aerosol, dosages sufficient to produce systemic ef- fects would seem to be theoretically avoidable. It is much more difficult to detect exposures to small dosages sufficient to produce miosis and the other harassing but not disabling symptoms descrilted in Section 9.3.2, and it is in this sense that MCE and PF-3 may be considered insidious. Accidental ex- posures to undetected dosages that resulted in these symptoms are reviewed in the next section. It has lx*en emphasized that PF-3 is readily absorbed by lacquer, rublxT, clothing, and hair. The gradual de- sorption of vapor can result in obtaining, within con- fined spaces, concentrations which suffice to produce eye effects but which may remain undetected until these effects appear.*7 The lack of odor of MFI may not prove to be so great an advantage as would appear at first sight if throat irritation and feeling of chest constriction should prove to be definite indications of the inhala- tion of very low concentrations. 9.3.2 Eye Effects The vapors of the fluorophosphates and Trilous are absorbed directly by the eye and produce con- traction of the pupil (miosis) and interference with the muscles of accommodation. As a consequence harassment due to poor dim light vision in dim light and to pain and difficulty of focusing is experienced. A potentially dangerous congestive iritis can develop and pain behind the eyeball frequently becomes very severe. These ocular symptoms can l>e relieved by (repeated) instillations of a mydriat ic (e.g., atropine) but the subject is left with a dilated pupil and para- lyzed accommodation. The concomitant systemic effects often include a feeling of tightness in the chest, nausea, and vomiting. No data are available concern- ing the exposure of human subjects to dosages suffi- ciently large to produce more severe disability. Studies ox Aximals Tests of the effects of dialkyl fluorophosphates on the eyes of animals originally served (I) to demon- strate beyond reasonable doubt that cautious trials with human volunteers (see the next section) could In man-charnber experiments a German shell fill- ing containing MCE with 20 per cent monochloro- benzene was detected at a concentration of 1.6 Mg 1 by 2 of 10 subjects.M The pure agent seemed to be more odorous and was detected at a concentration of 0.35 ag 1 br each of 4 subjects.'18 It is possible that the Germans considered decreased detectability by odor to be one advantage of the addition of mono- chlorobenzene to MCE. In one of the man-chamber experiments with PF-3, concentrations of 37 to 70 Mg 1 remained undetected by odor.42 In a field (an- nulus) test PF-3 could lie detected at an average concentration of 0.5 Mg 1 but the odor waanorisUffl- ciently characteristic to t>e easily identifiable. Man-chamber experiments indicate that throat irritation and a feeling of tightness in the chest are apparently more sensitive indicators of exposure to MCE and PF-3 than are the odors. In the case of MCE, 6 of 10 observers exposed to the German shell filling at a concentration of 1.6 gg I experienced the feeling of chest constriction, as did each of, the 4 who were exposed to 0.35 Mg I of pure MCE.** In the case of PF-3 each of 18 subjects exposed to 8.2 gg 1 — not detected by odor — experienced throat irritation and a feeling of chest constriction within 60 to 90 seconds.7* Pupillary constriction to pin-point size1 develops within a matter of minutes upon exposure to mod- erate dosages of MCE and PF-3, although it is longer delayed at the minimal effective concentrations (see the following paragraph). The foregoing suggests that troops having masks available could protect themselves against danger- ous dosages of MCE and PF-3 if they could take note of odor, feeling of chest constriction, and pupil- lary size. High concentrations could lx* detected quickly by odor or chest and throat signs, and the SECRET 146 FLl OROFHOSFH VTF.S AM) FHOSFHORIS-COXTAIMAG COMFOl NDS be carried out without risk of causing permanent eye damage, and (2) to determine the relative miotic potencies of some of the compounds. Various observations have demonstrated that pupillary constrict ion is produced in rabbits and mon- keys at dosages considerably smaller than those re- quired to cause permanent ocular injury or marked systemic cffects.2*r *®* Ma'49 79 10u,h The factor of safety in the case of PF-3 is most strikingly illus- trated by experiments with rabbits. Instillation into the conjunctival sac of a nearly lethal dose of the liquid (i.e., 1.15 mg kg) and repeated instillations of smaller doses, while eliciting intense miosis, lacrima- tion, and a transient increase of intraocular pressure, caused no permanent ocular injury.*** Similarly, al- though vapor dosages of less than 1.000 mg min m* sufficed to induce marked pupillary constriction, dosages of 15,000 mg min m* caused no permanent damage.*®*10411 In the case of both PF-1 and PF-3 the vapor dosages necessary to produce miosis in the rabbit are considerably smaller than those required to kill.104* With the monkey, a species that is excep- tionally sensitive to the lethal actions of PF-3 and di-sec-butyl fluorophosphate, the difference between dosages producing miosis and serious systemic poi- soning may be smaller,-’8"1 p q -r■M *4 as may also he the ease with man (see next section). Tests with rabbits have demonstrated that PF-3 is a markedly more potent pupillary constrictor than are (he dimethyl, diethyl, dipropyl, or diallyl es- ters.49'0411 It not only produces constriction at lower dosages, but also for longer times.49 7M04b Illustrative data are presented in Table 3. ating the relative potencies of this compound and of PF-3 ait* not available. Dicyclohexyl fluorophosphate also appears to be an effective miotic that produces pupillary constriction after a somewhat greater la- tency than characterizes the compounds just men- tioned.'041 Its potency relative to that of PF-3 is not known. The high miotic potency of MCE is illustrated by observations on animals 69,-*s but quantitative com- parisons with the fluorophosphates are not available. At high doses MCE can produce conjunctival hemor- rhages. ** Observations ox Human Subjects MCE appears to l>e considerably more potent in producing eye effects than any of the_fluorophos- phates.** Although a dosage of 0.7 mg min m* (t = 2 was without effect on the eyes. 3.2 mg min m* it — 2 minutes) produced slight but definite miosis. Dosages of I t to 21 mg min m5 produced a seven* harassing effect of several days’ duration. The action of these* dosages was characterized by the fol- lowing symptoms, not all of which were observed in all the subjects: pin-point constriction of the pupils, lasting for several days; severe frontal headache; retrobulbar pain, tightness in (he chest, and cough- ing; pain on focusing on near objects; slight blurring of l>oth distant and near objects; slight blurring of peripheral visual fields; nausea and vomiting; en- gorgement of the bulbar conjunctival, anterior cili- ary, and radial iris vessels, and of (he vessels at the base of the iris; acute ciliary tenderness; and fall in intraocular tension. This symptomatology was usu- ally almost completely relieved within an hour after the instillation of either atropine or hyoscine solu- tion, but the effects of the treatment did not persist. In the absence of treatment the symptoms became most harassing 24 to 4S hours after exposure and persisted in gradually decreasing intensity for several tiays thereafter. In the case of one observer exposed to 30 mg inin/m* (/ = 10 minutes), the harassment was very severe and, in addition to the effects men- tioned above, visual acuity was markedly reduced and had not returned to normal 17 days after ex- posure. Data on the eye effects of MFl are not available. PF-3 produces symptoms similar to those caused by MCE but is definitely less potent. From the data presented below it would appear that exposure to 10 mg min up of PF-3 vapor produces about the same effects as exposure to 3 mg min/m* of MCE Table 3. Relative miotic effects of several dialkyl fluoro- phosphates in rabbits."’41. The animals were exposed for 3 minutes to nominal con- centrations of 1/50,000 (0.11 to 0.16 nig/1). Dialkyl ester of fl iiorophospln >ric acid Average prr cent of initial pupil diameter After After After 10 min 100 min 300 min Dimethyl (PF-1) 32 82 100 Diethyl 27 58 85 Dipropyl 45 68 96 Diisopropyl (PF-3) 16 31 52 Diallyl 27 46 67 That di-scc-butyl fluorophosphate is a very potent miotic is revealed by the production of marked pupil- lary constriction within 10 minutes after the exposure of monkeys to 50 mg min m* (I = 2 minutes).*5 Animal data adequate to provide a basis for evalu- SECRET TOXICOLOGY 147 vapor. At larger dosages, 200 100 mg min/m* of PF-3 may correspond roughly to 14 to 20 mg min/m* of MCE, PF-1 and diethyl fhiorophosphato are definitely less potent miolies than PF-3. 6/.s( Dime thy lamido)- phosphoryl fluoride is also less potent, probably much less so. Di-sec-butyl fluorophosphatc appears to he somewhat more potent than PF-3 but defi- nitely less potent than MCE. The observations on which (he above statements tire based may lx* abstracted as follows. 1. Ethyl dimelhylamidocynriophosphate (MCE). The results of one controlled laboratory study are available.*8 In addition there have been accidents which demonstrate that exposures to undetected concentrations of (he vapor can produce ex- treme pupillary contraction and in addition congestion of the eyes.**!'51 In other instances a feeling of tightness in the chest has accompanied and given warning of the exposure. Four subjects exposed in a man-chamber to 0.7 mg min nf1 (I = 2 minutes) detected the odor of the agent and cxjierienced a brief feeling of tightness in the chest. They developed no miosis. Ten additional subjects were exposed lo a dosage of 3.2 mg min /m5 (I = 2 minutes). Only two noticed any smell. Six ex- jiericnced a very slight feeling of constriction in the chest. Slight miosis develop'd in all after 140 to GO minutes. Ten subjects, some of whom had liccn exposed 4 hours previously in the preceding group, were exposed to 14 mg min/m* (I = 2 minifies). The gas was detected faintly by smell and those not previously exposed felt a slight tightness in the ehest. Soon after exposure all subjects had contraction of the pupils which persisted for 48 hours. Severe headache and pain in the eyes followed unless atropine was administered. Vascular injection of the eyeballs was present. Difficulties of focusing were experienced. Vomiting on the day after exposure occurred in four of the subjects. Three additional subjects were exposed to 14 mg min/m* (t = 10 minutes). The odor and a feeling of tightness in the chest were detected. Pupillary constriction, headache, rhinor- rhea, nasal congestion, and other symptoms developed rapidly and jiersisted for several days in the aliscnce of treatment. Visual acuity at moderate illuminations was not markedly affected. Five additional subjects were exposed to 21 mg min/m* (I = 10 minutes). They became severely harassed by (he symptoms (hat developed. The symptoms and their limes of onset (minutes, in parenthesis) were tightness in the chest (1.5 to 8), coughing (1.5 to 0), pin-jsiint pupils (10), lamina- tion (2 lo 10), retrobulbar pain (8 to 19), conjunctival con- gestion (2 to 10), “tingling” of the eyelids (6 to 10), rhinor- rhea (fi to 120), frontal headache (13 lo 18), difficulty of seeing distant objects (11,14 — two cases), difficulty in seeing near objects (15 — one case), and constriction of the |ieripheral visual fields (15 — one case). One subject with one eye protected was exposed to 30 mg min in5 (I = 10 minutes). The protected eye was unaffccted- The pupil of the exposed eye began lo contract within 4 min- utes and had become fully contracted within 12 minutes. Visual acuity in dim light had markedly deteriorated within an hour and had not fully recovered 17 days later. Moderate conjunctival and severe ciliary congestion had develop'd within 3 hours. The subject was unable to sleep for two nights because of severe pain above and behind the exposed eye. 2. Dimethyl fluoraphosphate (PF-t). At low dosages PF-1 is not so potent a harassing agent as PF-3, nor does the pupil- lary constriction which it induces persist as long. Although this ester is considerably less readily detected by odor than PF-3, it is more irritating to the throat and chest. In subjects expost“d to nominal concentrations as low as 5.7 jig/1 (1/10*) it producer! a tightening sensation in the throat.111** No eye effects were noted when subjects were exposed, presumably for short times, to this concentration or to one four times as great. At a considerably higher concentration, li t 1 (1 50000), the throat, sensation was not more marked but eye effects were produced: an exposure of 30 seconds’ duration (Cl - 57 nig min ni5) produced in five of seven subjects some pupillary constriction and discomfort but no spasm of the muscles of accommodation; exjmsures of 1 to 5 minutes’ duration (Cl = -111 to 570) produced within 5 to 10 minutes pupillary constriction lasting for an hour or more, and, in 60 jier cent of the subjects, a marked spasm of the muscles of accommo- dation. 3. Diethyl flnorophnsphale.'3 This ester also apjiears to be considerably less potent than PF-3. In twelve subjects 2-min- utc exposure to a nominal concentration of 139 eg/l (Cl — 278 mg min m*) produced throat irritation within 10 to 30 seconds, then a painless tightening sensation in the chest, and finally coughing toward the end of the exposure. Within 30 to fiO minutes the pupils had partially contracted and their reflexes to light and accommodation were absent. There was no significant alteration in visual acuity in daylight or in sim- ulated twilight, although the sensitive Rangefinder Test revealed harassment. The size and reflexes of the pupil had returned to normal within 18 hours. At no time was there more than minimal congestion of the iris in any of the subjects. 4. Diisopropyl fluorophmphate. (PFS).**'’ *•• ,8- *’• s:-:*- 71, M. 10)t, lute a. Find (preliminary) British examination.'"* Ten minutes after exposure of two subjects to a nominal dosage of 24fi mg min m* (0082 mg 1 for 3 minutes), the pupils lie- gan to constrict and subsequently were reduced to pin- point size, with the result that the lalwratory appeared dim. The olwervers exjicrienccd difficulty and pain in focus- ing, eye ache, and headache. A I wok could lie read only if held within a few inches of the eye. The miosis and diffi- culty of accommodation jiersisted for 2 to 3 days in the case of the older volunteer (over 60 years of age) and for almost a week in the younger (28 years). The report does not mention extraocular symptoms. Upon exposure of two additional subjects to a nominal dosage of 82 mg min 'nr1 (0.0082 mg I for 10 minutes) the effects did not develop for about 30 minifies but then ap- peared as descrllied above and persisted for 3 days. The subjects could read only with pain and difficulty. Vision in dim light was poor. Distant vision was. impaired but re- covered sometime liefore near vision had returned to nor- S ECU FT F LUO R OPII OSH 11 AXES AM) PHOSPHORUS-COATAIMING COM POL M>S mal. The eyes of one observer were congested for about a day beginning I day after exposure. b. Preliminary American observations.**-33 1 pon exposure of only the eyes of several subjects to a nominal dosage of approximately 300 mg min m* (0.1 mg 1 for 3 minutes) only one subject reported slight subjective eye irritation during exposure. Miosis became maximal within 20 to 30 minutes and persisted for about a week. The subjects ex- |s‘iienced difficulty in accommodation and found reading painful during the first 2 days; they had less difficulty after 1 to 2 days in spite of the maintenance of pupillary con- traction and the development of irritation (congestion), eye ache, and headache. There was only a slight decrease in far vision; Snellen charts could lie read ulxiut as well as be- fore exposure. The subjects reported that their night vision was poor. Atropine and adrenaline instillations gave relief but had to lie repeated daily. In two men accidentally ex- cised for several hours to low and undetected concentra- tions. a viewed object first appeared clearly but then rapidly liecame blurred, accommodation was slightly re- duced, and nearsightedness apparently increased.4’ Four men whose eyes only were exposed to approximate dosages of 111 to 210 mg min m3 (0.037 0.070 mg 1 for 3 minutes) detected no odor and experienced no discom- fort during exposure. They subsequently developed miosis, difficulty of focusing and blurred vision, eye ache and head- ache, conjunctivitis and a gritty sensation in the eye, and twitching of the eyelids. Visual acuity as tested by Snellen charts was not reduced. Four men accidentally exfwsed to low, undetected concentrations experienced eye effects as just described and, in two cases, nausea. There is some evi- dence, that in two cases visual acuity in dim light was reduced.53 c. Scrawl British examination.7* Subjects were exposed to nominal concentrations of vapor in a large man-chamber and subsequently examined for pupil size, pupillary re- flexes to light and accommodation, and acuity of near and distant vision iis tested with Jaeger and Snellen Test Type indices both in daylight and in simulated twilight (approxi- mately 0.4 footcandle).h The general condition of their eyes was also examined and their performance on the Rangefinder Testc in ordinary daylight and simulated twilight b determined. All six subjects exposed to 41 mg min/m3 (0.0082 mg 1 for 5 minutes) complained of throat, irritation about 1 min- ute after the start of the exposure and of “tightness in the chest” after about 1.5 minutes. Three hours later (he pupil was only slightly contracted and pupillary reflexes to light and accommodation were present and normal. The tests for visual acuity revealed no significant deterioration either in ordinary light or in simulated twilight. The aver- age degree of harassment for the group as measured by the Rangefinder technique was 36 |>er cent in ordinary light and 50 per cent in simulated twilight. At no time was there congestion of (he iris and no subject experienced headache or other discomfort. I'pon exposure to 00 mg min in3 (0.033 mg 1 for 3 min- utes) all of 18 subjects experienced throat irritation after about 50 seconds of exposure and complained of “tighten- ing of the chest” within about 1.5 minutes. It took 4 to 6 hours for maximal miosis to develop, at which time pu- pillary reflexes were absent. The tests for visual acuity (as described) revealed no significant change although the Rangefinder test indicated It |«*r cent harassment with ordinary lighting and 100 jw-r cent in simulated twilight. Congestive iritis with accompanying headache and photo- phobia developed within IS to 21 hours. Atropine sulfate (1 per cent) proved more effective than homatropine (I jier cent) in dilating the pupils and relieving the iritis. Of 12 subjects excised to 328 mg min m* (0.164 mg I for 2 minutes) all experienced throat irritation and a feel- ing of tightness in the chest within 30 to 105 seconds. Them was also some coughing, but no eye irritation, Incrimalion, or blepharospasm occurred. The pupils were only partially contracted 30 minutes after exposure but bad contracted nearly to pin-point size within 3 hours. Pupillary reflexes were then absent. A definite deterioration in acuity for dis- tant vision bad develo]>ed within 30 to 60 minutes after exposure and was not notably more marked when tested in simulated twilight (see preceding paragraph) than when tested at higher levels of illumination. The individual alter- ation in near vision was variable til'both tested levels of illumination but for the group as a whole there was definite deterioration. Twenty-four hours after exposure distant vision had improved but near vision had deteriorated fur- ther. The subjects without exception complained of head- ache, the pain being referred to above or behind the eyes and being sufficiently intense to interfere with sleep. There was well-marked congestive iritis and conjunctival con- gestion but no edema of (lie lids, conjunctiva, or cornea. The average degree of harassment for the group as meas- ured by the Rangefinder technique was 63 |ior cent in or- dinary light and 100 jier cent in simulated twilight. Granted that the symptoms caused some discomfort and, at night, visual harassment, they were not considered to lie of a disabling nature in the recorded opinion of the British Ophthalmic Panel and Medical Subcommittee."" d. Third British examination,84 The eves only of sixteen sub- jects were exposed for 5 minutes in a const ant-flow device to analytically determined dosages of 40 to 250 mg min/m3. Subsequent clinical examination included observations on pupil size, visual acuity at high and relatively dim illumi- nations, the near point of accommodation, the threshold of scotopic vision, and the condition of the conjunctiva, cornea, and iris. In summary, with dosages up to 101 mg min/m3 the effects produced by the vapor on pupil size and (he accom- modative mechanism were not considered of serious con- sequence. However the three subjects exposed to 250 mg min m3 developed a congestive iritis associated with pain- ful symptoms and consequently were considered to 1k> partially disabled for 3 to 6 days. The pupil had contracted to a minimum diameter of 1 to 2 mm within less than 1 day at all dosages and within I hour at dosages above 116 mg min/m3. The miosis l«‘gan '• This level of illumination was far greater than would l>c encountered at night and the tests therefore give no adequate measure of the handicaps which the subjects would have experienced in night fighting. r This technique has been dcscrilHMp* and critically dis- cussed."1’ SECRET TOXICOLOGY to abate after 2 days at the lower dosages and after 3 days at the higher ones, but was not completely relieved for 5 to K days. During the first days prolonged dark adaptation resultixl in no pupillary dilatation. Visual acuity tested with Snellen charts in bright light (17 footcandles) showed practically no deterioration. In- deed, the uncorrccted vision of myopes was improved, as a result of the small pupil size. Visual acuity in relatively dim light was determined by lowering the illumination of tho Snellen chart until the smallest type which the subject could read at high illumination was no longer legible. Be- fore exposure the average illumination recorded for the twelve eyes of six observers was 2.8 footcandles (range 0.32-10.5). Twenty-four hours after exposure to 40 or < 80 mg min/in 5 it was 0.4 footcandles (range 0.32-17). This change was not considered consistent or marked It must Ik- emphasized that the tested range of illuminations was sufficiently high that cone (not rial) vision was living measured, and that the results throw no light on the im- pairment which may have been produced in night vision. Among subjects exposed to 40 to 191 mg min m* the near |x»int of clear vision was brought in, indicating in- creased ciliary tension. However the absence of serious im- pairment of distant vision indicates that any existing spasm uf the muscles of accommodation was not a serious handicap at the light intensities employed in the tests. From the changes in pupil size caused by the PF-3 vapor one might have expected as much as a 16-fold rise in sco- topic visual threshold. Actually the change in threshold brightness level, as measured with the Craik Adaptometer 1 to 2 hours after exposure to 116-191 mg min/m*, was only 2- to 10-fold (average 5+ fold in six subjects). Subjects exposed to 100-191 mg min m3 generally de- veloped conjunctival hyperemia 2 to 3 days after exposure. At 250 mg min/m* the hyjiercmia was much more severe and developed within 24 hours. In addition a marked and potent ially dangerous congestive iritis, accompanied by painful symptoms, made its appearance. Examination with the slit lamp revealed no corneal changes, nor were changes noted upon opt halmoseopic ex- amination in instances where miosis had been abolished with a mydriatic. Among the subjective symptoms reported by the sub- jects were mistiness before the eyes, eye ache, and diffi- culty of seeing in the dark. Instillation of homatropinc (0.43 minim) had to lie re- peated three times at hourly intervals in order to obtain significant pupillary dilatation in two observers who de- veloped congestive iritis following exposure to 250 mg min m*. After the third instillation the congestive symp- toms were relieved but paralysis of accommodation oc- curred and the observers became partially disabled because of blurry vision, c. American assessment.^• ” -*71’.>■ One subject was exposed in a man-chamlier to a dosage of 181 mg min m* (I = 0.7 minutes), eight subjects to 165 mg min in* (/ = 8.7 min- utes), one subject to 2140 mg min in* (I — 10.7 minutes), and six subjects to 244 mg min m* (1 = 9 minutes). All the men exposed to 165 mg min m* experienced a slight feeling of tightness and constriction in the chest, apparent 1 2 hour after exposure and particularly noticcable several hours later. Instances, of rhinorrhea, diarrhea, nausea, and vomiting (one case) occurred but, except for the rhinorrhea, may not have been due to the effects of the PF-3. Xo muscle tremors —a sign which might be ex- pected to herald serious systemic poisoning were ob- served. Of the men exposed to 211 mg min m* (0.027 mg 1 for minutes), five of six experienced a fleeting feeling of chest constriction while in the chandler. This returned and per- sisted for 2 days, being accompanied by coughing in two cases. All the subjects developed rhinorrhea within an hour after the exposure. Only one developed nausea, and he vomited twice. There were no abdominal eramps or muscle tremors. One volunteer exposed to 290 mg min in* de- velops! constant nausea for a day following exjsisure, ex- perienced abdominal cramps, and exhibited increased nasal secretion, lie had no muscular tremors and felt no chest constriction. The majority of men in both groups had diminished dis- tant vision, which was caused by a spasm of the muscles of accommodation. Although the resultant false myopia measured between 1.75 and 0.5 diopters, because of the small pupil sine the visual acuity was not diminished greatly. The greater part of the diminution of distant vi- sion had developed within 45 minutes after exposure. Fur- ther deterioration occurred at 3 hours in some cases. Re-” covery occurred at 2 to 7 days, being slightly more rapid in the subjects exposed to the smaller dosage than in those exposed to the larger. Maximal miosis developed within 10 to 15 minutes after exposure. Among the men exposed to 105 mg min in3, the pupils liegau to relax after 1 to 3 days and attained normal size and activity after 3 to 9 days. Among lhose exposed to 244 mg min m* relaxation did not begin until after the” third day and complete recovery required 5 to 11 days. All the volunteers showed a diffuse conjunctival injec- tion which required 5 days to clear up. Concurrently with the development of pupillary con- striction and spasm of accommodation, the near point of accommodat ion moved to within 3 to 6 cm from the cornea, and it became increasingly difficult for the men to focus after gazing into infinity. Small type could be read but several seconds were required before it could lie seen clearly. Without exception the men complained of intense pain when they attempted to perform visual tasks within IS inches. Recovery of the untreated eyes gradually occurred over an average of 3.5 days after exposure to the lower dosage and 4.5 days after the larger. Except for one man who exhibited a transient rise in in- traocular tension, all displayed a subnormal tension for several days. A performance test (in daylight) showed no decrease in efficiency of marksmanship and all of the men felt that they could competently discharge such military tasks as guard duty, vehicle driving, and rifle firing. The reports state that prolonged questioning failed to elieit any symptoms of defective night vision, all the volunteers feeling that their visual acuity at night was proportional to (hat in the day. This statement is at variance with the results obtained in other tests and observations. It would not be anticipated that the vision of men with maximally SECRET 150 FLUOKOPHOSPH \TES VXD PHOSPHORUS-CONTAINING COMPOUNDS Compound Mouse HCl)ia in mg min/m3 (10 min nominal) Uange for all Monkey tested s|M‘eies Increase of /dm-with increase of exposure time LDiV Rabbit, intra- venous (mg/kg) Mouse, percu- taneons Kthyl dimethylamidocyanophosphate (MCE) 380 250 200 1,000 Slight 0.1 ± 1 f- Isopropyl methanefluorophosphonate (MFI) 250 150 100-300 Definite 0.02 1 ± Isopropyl elhanefluorophosphonate 330 200 150 700 ± Definite 1.7 Dimethyl fluorophosphate (PK-1) 2,000 2,500- > 12,000 Marked 3 30 Diethyl fluorophosphate 8,200 7,000- > 11,000 ? 35 Diisopropyl fluorophosphate (PF-3) 5,900 000 000 >8,000 Slight 0.4 72 Di-wr-hutyl fluorophosphate 5,200 250 ± 150 250->18,000 ? Dievelohewl fluorophosphate 1,100 i ,ooa 8,ooo 9 6(s(Dimethyiamiilo)phosphoryI fluoride 950 950- > 4,000 ? 3 + Table 4. Summary of toxicifics. contracted pupils would l>e normal at illuminations suffi- ciently low to confine visual function to the rods. The serum cholinesterase concentration of all the sub- jects was reduced by the exposure to PF-3 to 1 to.') per cent of the normal value. f. Additional accidental exposurtWorkers acciden- tally exposed to undetected concentrations at the American pilot plant developed extreme miosis of 1 week’s duration. Difficulty of night vision was stressed.* In a report on a similar incident at the liritish pilot plant emphasis was placed on pupillary contraction, blurring of vision, especially in artificial light, headache, and light- ness in the chest.100 In another incident 7* undetected cx- posures to vapor produced miosis, pi sir vision in dim light, difficulty in focusing, twitching of the eyelids, nasal dis- charge, and (in some cases) conjunctivitis. There was no mention of headache or chest symptoms. 5. Di-scc-bulyl fluorophosphate.'^1 This ester has been given only preliminary tests with four human subjects. L'pon exposure to a nominal concentration of I/10* (Cl = approxi- mately 15 mg min m3) all noticed a tightness across the chest but three of the four fell that it. was not sufficient to call for a respirator. About 5 minutes after the subjects left the cham- ber, miosis set in, became intense, and persisted for 5 days. Comparison with the results obtained with PF-3 at compa- rable and somewhat greater dosages T,-,ov would indicate that the di-scc-butyl ester may be the more potent miotic agent. (5. hint Dimdhytnmido) phonphoryl fluoride.,IMo Exposure of four volunteer subjects to about 45 mg min m1 (7.3 Mg 1 or 1/10*, for 5 to 7 minutes) produced no observable ocular or systemic effects. This compound is therefore less effective, perhaps very much less effective, than PF-3. 9.3.3 Toxicity Inhalation and Injection Toxicitiks Toxicity data for the more intensively studied Tri- Ions, fUiorophosphales, and dialkylamidophosphoryl fluorides are given in Tables 5 through 17 and are summarized in Table 4. It is apparent that the Tri- bal.- arc the most toxic volatile agents considered in Table 5. Toxicity of ethyl dimcthylnmidoeyanophos- phale (M('K) by inhalation. The animals were totally exposed to the vapor of the agent. Concentrations were nominal except when other- wise designated. Exposure N umber time UOtho of Species (min) (mg min/nr1) animals Reference Mouse 2 100 100 C9d 5 385 100 69d 5 500-750 22 104s 10 380 400 69,1,e 10 220* 140 87 10 500-750 IS 104s 20 420 100 69d 30 420 100 69d 00 670* 120 87 120 840* 60 87 Hat 5 750-1,000 14 104s 10 750-1,000 10 104s 10 500-1,500 14 69c,e 10 304* 2:40 87 20 385* 54 66k 60 620* 60 87 120 1,200* 120 87 Guinea pig 5 1,000 ± 6 104s 10 1,000-2,000 6 104 s 10 393* 81 87 10 500 1,500 12 69e,c 00 740* 50 87 120 1,500* 48 87 llahhit 10 1,000* 15 87 10 >4,000 6 104s 10 >2,000 4 69c, e 10- 62 840* 55 G6h Cat 7.5 300 800 3 104s 10 2.50 8 69e,e I tog 10 400 4 69c,e Goat 10 700* 9 87 14-23 400-700* 10 66h 13-120 765* 30 66k 20 1,400* t 38 66k Monkey 5-10 400 ± 5 104s 10 250 4 69e,c 10 180* 3 87 * Analytically determined concent ration t Angora goal*. SECRET TOXICOLOGY Table 0. /./>„o’s of ethyl d imcthy la midi >eya qo| >hns- phate (MCK). The figures in parentheses arc the ininiber of animals used. Route of Approximate administration S|tecies /.Din (mg kg) Reference Intravenous Mouse 0.15 (15) 69c _ Rat 0.006 (35) 66h Rabbit 0.0025 (40) 6011 0.18 (14) 87 0.125 (15) 104s DoK 0.084 (20) 06i 0.116 (0) 69c Subcutaneous Mouse 0.4 (25) 87 Rat 0.3 0.4 (42) 87 Guinea pig 0.2 (20) 87 -Rabbit 0.5 (30) 87 Percutaneous Mouse 1.0 (70) —69c >4 (20) OOg Rat 18 35 (47) 87 Guinea pig 35 (43) 87 Rabbit 2.5-3.0 (5) OOg 3.3 (00) 09h 35 (19) 87 Dog 30-50 (4) 69c Goat >5 (2) OOg 1.1 (21) OOh - 3 (17) 87 Monkey 0.3 (0) 69c I’er oh Rat 3.7 (107) 66ii 8 (20) 87 Rabbit 10.3 (51) OOh Dug 5-11 (12) OOh Table 8. LI) (MFI): Tin* figures used. ,,,’s of isopropyl in parentheses methanefluorophosphonate are the numlier of animals Rout*? of administration S| levies Approximate 1.1I.J (nig kg) Reference Intravenous Hat 0.0-15 (50) OOi Rabbit 0,0 Hi (14) OOi Percutaneous Mouse 1.08 (40) OOd Rabbit 0.025 (10) (ilii Per ok Rat 0.55 (00) OOi Table 0. Toxicity of isopropyl elhanefluoruphosphonate by inhalation. The animals were totally exposed to the vapor of the agent. All concentrations were nominal. Kxposure Number _ time /.(Oh, of Sjreeies jiniii) (rnjj min in') animals Reference Mouse* 5 245 80 title 10 330 120 60d,c 10 350 1,000 8 104t 30 570 60 title Rat 10 260 6 69e 10 <350 4 Kilt Guinea pig 10 >210 6 60e 10 350 1,000 4 1041 Rabbit 10 230 4 60c 10 350-1,000 4 104t Cat 10 170 6 title Dog 10 230 4 60c Monkey 10 210 3 60e * SuhoutaiM’tms LDw (8 mice) “ approx. 0.4 L/)w (40 slaved mire) =1.7 mg/kg.1^ mu, kg. '*** Percutaneous Table 7. Toxicity of isopropyl imThahcfluoroptios- phonate (MFI) by inhalation. The animals were totally exposed to the vapor of the agent. All concentrations were nominal. Species Exposure time (min) urt),a (mg min/in’) Number of animals Reference Mouse 5 230 100 GOe 10 250 lOf) title 10 1.50-230 22 104t 15 34.5 120 69e 20 360 100 Otte 30 420 100 title Hat 10 300 IS 6tld,c 10 150-250 12 1041 Guinea pin 10 ISO 18 69d,e 10 150-2.50 12 104t Rabbit 10 120 6 Gtld.c 10 1.50 2.50 5 104t Cat 10 100 10 69d,e I)oK 10 100-150 8 09d,e Monkey 10 150 5 G9d.i- cate Unit the species variation in susceptibility is not prononneed. On the other hand the animal species exhibit considerable variation when MCE, anti par- ticularly tiie fiuorophosphates, come into considera- tion.1* This variation makes estimates of I lie human lethal dosage precarious. In the case of PF-3 com- parison of the systemic effects produced upon ex- posure of human subjects (see preceding section) and of monkeys2SI|,r'63 64 83 to dosages in the order of 300 to 400 mg min m* indicate clearly that man is the more resistant species. Man was affected but not prostrated,-whereas the monkeys were severely pros- trated and some were killed. How much more re- sistant man is to PF-3 than the monkey is not known, nor does the same relationship necessarily hold for the Trilons. this volume. The fiuorophosphates are considerably less toxic, although the potency of PF-3 and di-scc- butyl fluorophosphate for the monkey approaches that of tho Trilons. The limited data for MFI indi- '* The high L{Ct)j,of PF-1 and PF-3 (possibly also MCE) for the rabbit are due largely to inhibition of respira- tion. \\ hen inhibitory respiratory reflexes are suppressed, or when t lit* agents are injected, the rabbit is not found to lie excessively resistant.**•'4.“ SECRET 152 FLI'OUOPIIOSPII \TKS V ND IMIOSPIlORLS-CONT VIM NO COMPOUNDS Tarle in. Toxicity of dimethyl fluorophosphate (PK-1) by inhalation. The animals were totally exposed to the vapor of the a ({cut. All concentrations were nominal. Injection and tiercHlaneous toxicity figures are Riven in the footnotes. Hxeept for 1 the mouse /,((V )..„’s, the figures are very rough approximations based on only a few animals of each sjH'cies. Kx|>osure time unu S|ierics (min) (mg min m*) Heferenee Mouse* 1 1,200 201 2 1,740 20h 10 2,550 11 10 3,000 — 40 30 5,000 ± 26f 120 >5,000 20f Hat 1 1,800 ± 26f,l 10 3,000-0,000 20a, I04b Guinea pig 1 7,000 + 20f,l 0.5 4 8,(XK) ± 20f Habbitf 1 > 12,000 20f, 104b ('at t 1 0,000 + 201 r>o*s 1 6,000 + 201 Goat 3 5 20,000 + 26f House fly 10 <30 201 Mosquito ' 10 <30 201 » Mouse ii.tr: sve non* LI) w » 0.15 mg kg/** Mouac infra peritoneal me : kg ** Mouse jx-r cutaneous LI)ao (70 mice) » 0.72 mg animal, or approx. 3tt mg kg.4* t Habbit infra ivcnous LDu = 2 I mg kg > t ('at int ravel hour LD*o = 1.5 mg kg."1 § I>-« intravc nous /,Os. = 12 mg kg.5* || Antra urr/yjJi. Table 12. Toxicity of diisopropyl fluorophosplmto (PF-3) by inhalation. The animals were totally exposed to the vapor of the agent. All concentrations were nominal. Kxeept for the mouse and rat s, the figures are rough approxi- mations based on relatively few animals per species. A total of 39 monkeys were exposed. _ Exposure time ««)». Species (min) (mg nun nr') Reference Mouse 1 4,000 I04e 2 3,800 104e 5 2,700 I04e 10 3,500 I04e It) 5,500 49 10 5,900 26c,p — 30 4,500 1 Ole 100 >0,400 — 26p Ral 1 4,200 104h 2 3,000 I04h 5 2,850 10 th 10 2,800 104h 30 4,500 I04h Guinea pig 10 >8,200 104b Rabbit 10 (8,000 ±) 104b Dog 10 5,000 + 8.3 Goat 10 6,000 7.000 S3 Monkey 2 500-800 261 2-15 500 ± 63, 64 10 800 + 83 100 1,000-2,000 26p,r Table 13. LD. •u’s of diisopropyl fluorophosphate (PF-3). Route of Approximate administration Species L/)jo(mg/kg) Reference Intravenous Rabbit 0.3-0.4 — 07b 0.4 + 53 0.5-0.75 32a Cat <3 33a Goat 0.8 + 53 Monkey 0.1-0.2 48, 0«a, 07f Intramuscular Rat 2- 321. Rabbit 0.75-1.0 67b Sulieutancous Mouse 4 ± 80, 104e Rat 3 + 80 Rabbit I + 86 • Dog 3 ± 80 Goal 1 + SO Percutaneous Mouse 72 + 40 (1.45 mg mouse) Per os Mouse 30.8 08a 2 + 80 Rat 5-10 20r 6 + 80 Rabbit 0.8 08b Ry eye Rabbit 1.4 32a Table 11. Toxicity of diethyl fluorophosphate by inhalation. The animals were totally exposed to (he vapor of the a Kent. All exposure times were for 10 minutes and all concentrations were nominal. Kxcept for the mouse /.(COjo’s, the figures are very rough approximations based on 6 to IS animals per speeies. UCt)„ S|ieries (mg min/m3) Reference Mouse* 8,200 II ■ ‘ - ' - 4,100 - 40 4,000-6,000 104b Kat 7,000-14,000 20b, 104 b Guinea pig 7,000 14,000 26b, 104b Rabbit >14,000 104b * Mourn* pcrrutapwHis /-/>** (00 mice) ■** 0.70 i 35 me k^.** mg,animat, or approx. As indicated by (he data of (he toxicity tables, the “rate of detoxification” as measured by the increase of the lethal dose with increase of time of its admin- istration. is marked for PF-1, moderate for MFI and isopropyl ethanefluorophosphonate, and slight but definite for MCE and PF-3. More detailed data hearing on the rate of detoxification as determined by inhalation and injection experiments will Is1 found in the references cited in the tables. Other references are also pertinent.l#f * w “ “ Both the Triions and the fluorophosphates are TOXICOLOGY 153 Tabu 14. Toxicity of di-scc-hutyl fluorophosphale by inhalation. The animals were totally exposed to the vapor of the agent. All concentrations were nominal. Except for the mouse L{Ct)i,’s, the figures are very rough approxima- tions based on 2 to 23 animals of each sj)ecies. Exposure time UCtu Species (min) (mg min/in’) Reference Mouse 10 5,140 20r 10 5,400 104i Rat 10 4,000 10,000 20r, 104 i Guinea pig 10 >18,000 20r, 104i Rabbit 10 5,000-10,000 20r, I04i Gat 10 0,000 ± 2Gr Dog 10 4,er sixties. UClU SjH'ries (nig min/nv1) Keference Mouse 800 1011 I,I00 26n If at 1,200 ± 26n, Kill (Iniitea pig 6,000-10,000 ion Hal.bit 1,2(Xh 2,800 2fin, 1011 Dog 1,000 1.100 201, 20u Table 10. Toxicity of 6i«(diinethylami4,000 20j, 104o 2 4 f 80 Rabbit > 2,000 104o 3±_ 0 + 3 + 80, lOlo Cat 2 t- 80 Goat 2 + 80 Monkey >1 or 2 SO “quick-kill” agents. Although occasional deaths are delayed for 1 or 2 days, most lolhally poisoned ani- mals die within 2 hours after exposure, and the ma- jority during or within a few minutes after exposure. Detailed statements may be found in the references cited in the toxicity tables. A special study has shown that PF-1 and PF-3 are only slightly slower in speed of action than hydrogen cyanide (AC), although the lower volatilities of the Huorophosphates would make high concentrations relatively difficult to attain in the field.44 Symptoms and Pathology The symptoms produced by exposure to the Tri- Ions and fluorophosphates are those which character- ize the nicotinic and muscarinic actions of parasym- pathomimetic agents in general. There are also evidences of central nervous stimulation. Although there are variations according to species, agent, and dosage, frequent mention has been made of the fol- lowing: lacrimation and salivation; apprehension; coughing, dyspnea, and gasping; hyperexeitability, incoordination, and ataxia; tremor, muscular twitch- ings, and convulsions; sometimes bronchospasm, pilomotor stimulation, urination, and defecation; general weakness ami depression; and finally cessa- tion of respiration. Detailed descriptions for the various agents and species may be found in the refer- ences cited in the toxicity tables (see also the refer- ences given under the section “Protection and Treat- ment”). Respiratory failure is probably the usual primary cause of death.*3 However, the action of the agents as revealed, for instance, by a study of PK-3,33* clearly involves most of the important systems in the body and the weakest link in the chain of events leading to death is questionable. One point of view is that bronchospasm may be important in some species, including man. This is not t rue in the cat.33 Because of the early time of death, pathologi- cal changes frequently are not conspicuous at au- topsy.261** '£1X7-,(M Dicyclohexyl fluorophosphale, which seems to act somewhat more slowly than the other Huorophosphates, has berm found to produce in rabbits a marked pulmonary edema and edema of the perivascular connective tissue, marked pulmo- nary hyperemia, large areas of ataleetasis, hepatic congestion and incipient central atrophy, and slight lymphorhexis.1*'" The action of 6fs(dimethylamido)- phosphoryl fluoride is slower still ami the pathologi- SECRET 154 Fl.l OHOPHOSnr ATES V\'D PHOSPHOR17S-CONT M M NO COMPOl M)S Table 17. Toxicity of vapors through the ski n (body only exposures) F,X|sisiire Dot age (Cl in mg min in’) I ime Nominal Analytical Agent Species (min) cone. cone. Mortality Reference Dimethyl fluornphosphate (l’F-1) Mouse 10 124,000 122,000 0/6 20*1 10 51,600 48,200 3/6 20*1 15 13,000 12,000 0/6 26*1 Kthvl dimelhylamidoevannphosphate Mouse 10 3,850 2,500 UCt)u 00c, d (Mt'K) 10 1,000 750* Uri)M 00c no 0,000 3,000 UCl)m 00,1 Guinea pig 10 11,200 0,100 0 6 69c Dog 10 11,200 0, UK) 0/1 69c 210 45,400 20,000 0/1 69*1 227 360 80,000 ± urnM 00k (8 animals) Hablut 77-282 10.000 + UCDu, Oflli (30 animals) Isopropyl methaneflnorophnsphonate MFI Mouse 10 8,720 1 20 69e 6/s(jM-'hloroelhvl) sulfide (II) Mouse 10 3,500 UCI),a 15 Habhit 13.5 2,000 0/1 15 18 4,000 0/1 32 5,800 i/i 35 8,(*)0 i/i - — 00 13,400 0/1 80 20,500 —1/1 " rh>g 00 0,550 0/1 15 - CO 0,000 1/1 - 30 15,400 1/1 00 17,000 1/1 00 24,000 1/1 Iris(,%{ ’hloroet hvDamine (H X3) Mouse 10 800 UCl)M 15 Rabbit 47-140 >5.500 urt)M 20t,u Dog 30 13,300 0/1 15 45 14,500 0/1 75 21,400 l/l 100 51,000 1/1 * Skin of miee shaved. cal changes are somewhat different. Attention has been directed to marked pleural effusion, pulmonary edema and hyperemia, and inflammation of the sub- mucosal layer of the tracheal and broncheal epi- thelium.36'ksB The references cited in the toxicity tables shoijld lx* consulted for more detailed patho- logical information. Effects on and Timorr.H the Skin Neither the Tritons nor the fluorophosphat.es exert a vesicant action.ll !*d s', *6h k e S|7,9° With regard to absorption of the agents through the skin, the • lata given in Table 17 suggest that vapor dosages reasonably attainable in the field would not produce significantly severe systemic effects percutaneously in the cases of the larger animals nor, presumably, in man. Moreover, ordinary clothing can be expected to afford some protection against the vapors; C(-2 impregnated clothing, considerable protection; and carbon clothing, virtually complete protect ion.6#Jr On the other hand, liquid contamination of the skin with MCE or MFL is potentially very dangerous (see Tables (i and 8).**h' In the ease of MCE rapid removal of the liquid by blotting is effective treat- ment. Apparently chloramides do not react readily with MCE. Consequently antigas ointments are of limited value except in so far as their application can facilitate the removal of the agent by solvent or me- chanical action. In experiments with rabbits it was found that interposition of a single layer of plain herringbone twill increased by 6- to 8-fold the dose of MCE that must lie applied to the skin to cause death. A single layer of CC-2 impregnated cloth in- creased the dose 10- to 12-fold; two layers of this cloth, 20-fold; and one layer of carbon cloth, 15- fold.M,, i Thus clothing, particularly protective cloth- ing, is of considerable value in preventing the absorption of lethal doses of this agent. Protection and Treatment Numerous substances and procedures for prophy- laxis and therapy of the systemic effects of fluoro- SECRET 155 TOXICOLOGY phosphate and Trilon poisoning have been inves- tigated.4*4 * b *-32 33*-44 *8* b.*.h. i,k,«7».b.r.M,*!to.h,t04h.n.« Qf these the injection of atropine and magnesium sulfate seems to offer the most promise and has been recom- mended for use in the event of human poisoning.**3®7* These therapeutic agents suppress the autonomic symptoms. Injection of Nembutal in addition will control the convulsions which occur in MCE poi- soning,*"1 •S7 However, in severe poisoning the action of these drugs will merely delay but not prevent death. In any event it is essential that therapy be instituted promptly. Adequacy of protection by the gas mask has been mentioned, as have Ix'en methods of I renting eye effects and of preventing percutaneous absorption of liquid contamination. Physiological Mechanism In 15)41 and 1912 British workers reported that the dialkyl fluorophosphales are very potent inhibi- tors of cholinesterase.1"3 1043 h Since that time exten- sive studies have been made on the clinical pathology and biochemistry of action of these compounds,-*' *• m a,uj moro recently of the Trilons, which have also proved to be potent antieholines- terases.M« '' ' «7.6oe,f h i 'i')ie results have already begun to appear in the open literature and need not be re- viewed hem. It is obvious that the agents will In* of great value as tools in physiological and biochemi- cal research. Their possible use in the treatment of myasthenia gravis has also been under investi- gation. SECRET Chapter 10 METHYL FLU01U)ACETATE AND RELATED COMPOUNDS* Brnhey Ren show and Marshall dates lo.i INTRODUCTION Rkhokts that methyl fluoroacetate is highly toxic were received from Polish investigators by the British in 1912 and prompted extensive stud- ies in the United Kingdom and United States. Flu- oroaeetie acid and many simple derivatives including salts and esters, 0-fluoroethanoI and its esters, anti salts and esters of y-fluorobutyric acid, y-fluoro-0- hydroxybutyric acid, and y-fluorocrotonie acid, proved to l>e highly toxic by inhalation, injection, and ingestion. These compounds produce death, usu- ally after a latency of one-half to several hours, by action on the heart or central nervous system. Compounds of this group are not seriously con- sidered for large-scale use in chemical warfare at the present time because: (1) although very toxic for some species, (he human lethal and incapacitating doses are believed to be comparable to, or consider- ably greater than, those of the current ly standardized persistent and nonpersistent agents; (2) the stable derivatives do not possess sufficiently high vapor pressures to be dispersed from available munitions in high concentrations as nonpersistent agents; and (.3) the gas mask affords adequate protection. The silts of fluoroacetic and related toxic acids are nonvolatile, stable in aqueous solution, and ap- proximately as toxic when administered orally as when injected. They are, therefore, potential water poisons in warfare and are proving to lx1 highly ef- fective bait poisons for rodents. The compounds of this group selectively poison enzyme systems and as inhibitors will 1h» of value in the study of intermediary metabolism. 10.2 Synthesis and Properties Approximately 160 aliphatic fluorine compounds have U’en prepared by NDRC and British investi- gators for evaluation as eliemieal warfare agents. The compounds, their physical properties, and refer- ences to their synthesis and toxicity are listed in Table I. 10.2.1 Synthesis In general, the syntheses have Ixx-n effected by the fluorination of corresponding chlorine and bromine compounds by treatment with metallic fluorides, usually anhydrous potassium fluoride, less frequently silver fluoride, mercuric fluoride, or antimony fluo- ride. according to known procedures.19-'22■“•w.ioi.im.im Application to the fluorinated compounds of standard synthetic methods has resulted in a variety of derivatives including representatives of most of the common aliphatic types. The methods may lx- illustrated by the following examples. 1. Methyl fluoroacfiate '».**•>»*.!«» has been pre- pares! on a large laboratory scale (50 lb) by heating methyl chloroacetate under pressure with anhydrous potassium fluoride at 220 (' for 5 hours. The product is distilled directly from the pressure vessel and is purified by fractionation. Yields in the neighborhood of 75-77 per cent are obtained. The compound is a colorless mobile liquid with a faint ester-like odor. Unlike the other haloacetates, it has no lacrimatory properties. It boils at 104.5 C, freezes at —35 C, and is soluble in water to the extent of about 15 per cent. Its physical properties have been thoroughly investigated.u-*7-*Stt 2. Sodium fluoroacrlate, l9-22-92<‘ because of its prom- ise as a rodent icide, has been prepared on a much larger scale than has any other member of the fluoro- acelate series. Complete pilot plant conditions were worked out during the course of preparing 1,000 lb 22 for use in experimental rodent-control projects. It is prepared by saponification of ethyl fluoroacetate " Rased on information available to Division 9 of the Na- tional Defense Research Committee [NDRC] as of October 1. 1945. Attention is directed to a recent paper hy ,J. S. C. Marais, entitled Monofluoroaretir Arid, The Toxic Principle nf "Gif- bhmr" Dirhapetnlnm rymosnm (Hook) Emjl., Ondcrstepoorl Journal of Veterinary Science and Animal Industry 20, 67 73 (1944). The early Dutch settlers in South Africa gave the name “Gifhlaar” to a plant the leaves of which are poisonous to livestock. A numlxT of toxicological and chemical studies have Ix-cn made in South Africa since about 1900, and it is apparently a remarkable coincidence that the active principle was lx-ing identified there at the same time that fluoroacetic acid derivatives were being actively studied as potential chemical warfare agents in the United Kingdom and United States. Although the South African literature corroborates many of the chemical and toxicological findings summarized in this chapter, a cursory survey fails to reveal data permitting an independent estimation of the human lethal dose. SECRET 157 INTRODUCTION Table 1. Aliphatic fluorine compounds examined as candidate chemical warfare agents. The compounds are arranged in three major categories in the following sequence: (1) compounds containing not more than one fluorine atom attached to any carbon atom; (2) compounds containing two fluorine atoms attached to any one carbon atom; and (3) compounds containing three fluorine atoms attached to the same carbon atom. Within each major- category compounds are arranged in sequence according to the following types: hydrocarbons, alcohol derivatives, amines, carbonyl derivatives, and acid derivatives. The following abbreviations are used; n’n, refractive index at 1 C; 1.708 52 52 mp 12 18° 52 bp“ 55-5G° 52 vol54 70.1 _ 52 - ... 4. Icd-Butvl fluoride 3, 19 bp™ 14.5-16° 3 13 5. /J-K!uoroethanol 19, 92c nn!“ 1.3618 14 13, 91c, 92c -- — _ _ iP* 1.0913 14 bp7** 99 100° 14 r~. vol5* 74.8 14 , . . 6. Methyl j3-fluoroethvl ether 91 f bp ea. 60° 91f _ 91 f 7. Chloromethyl 0-fInoroethyl ether 19 bp5i 35-40 19 13 8. 0-Chloroethvl 0-fluoroelhyl ether 19 bp” 55 58° 19 13 9. /3-Fluorocthvl d-hydroxycthyl ether 19 n u!S 1.1050 19 13 bp" 01-62° 19 10. Methvl £-fluoroethoxyacetate 19 bp1* 8.5-88“ 19 11. d-Fluoroethyl phenyl ether 92f mp 41 92f 01 h _x_ ■ “ ... bP>7 92.5° 92f 12. 2 '-Flin>ro-2,4-dinitrophenetolc 19 mp 89-91° 19 13 13. 0-Fluoroelhvl d-naphlhvl ether 92g mp 49.5 50° 92g 91 h 14. b/»(d-Fluoroeethvl X-nit roso-X-(d-chloroethyl)carlmmate 19. 30 bp5 118-121° 19 13 27. d-F’luoroethyl glycine hydrochloride 92j mp 150.5° 911 911 28. (J-Fhioroethyl betaine hydrochloride 92j mp 122° 911 911 29. 0-Fluoroethyl nitrite 19 "o50 1.3589 14 13 p‘*° 128° 14 vol5* 47.0 14 SECRET 158 METHYL FLL’ORO ACETATE WO RELATED COMPOUNDS Compound Reference to synthesis Physical pro|ierti* Projierty R 'ferenee Reference to toxicity data 33. bi.s(/3-Fhioroethyl) carltonale 19 >p‘ 71-72° 14 Vol*4 1.15 14 34. U-Fluoroethvl chlorosulfonate 19. 92g •>p!" 85 86° 10 13, 91f 33. b/«(d-Fluoroethvl) sulfate 19, »r2g «ir* 1.4177 14 13. 91 f — d?4 1.3678 14 bp? SO-81° 14 vol54 0.425 It 36. fris(tf-Fluoroethyl) arsenite 19 bp! 4 132 134° 19 13 37. Mrakis(P-Fluoroethvl) silicate 19 bp"4 102 104.5° 19 13 38. trix(H-Fluoroethvl) borate bp 192° 91 o 91o 39. d-Fluoroethoxydichlorophosphine 19, 31 e •»P” 50 31e 13 . * . - bp™ 140-145° 31 e 40. fcjsfd-FluoroethyD hydrogen phosphite 92n bp15 109 110° 92n 91 h 41. his( Diethvlamino)-/S-flii(»roelhoxyphosphine 19 b|'a 108-111° 19 13 42. Kthvld>f*(f3-fluoroethoxy) pTiosphine 19 bp1'-4 4(F49 19 13 43. trisfp-Fluoroethyl) phosphite 19 bp11-4 100 105° 19 13 44. his(d-Flu• 1.3759 14, 24q 13, 91c — dP 1.0826 14, 24q bp™ 114-118“ 14, 24q ' vol5* 68.57 14, 24q 79. Klhvl dichlorofluoroacctate 47h bp7J0 131.5-132° 47b 13 SO. d-Fluoroethvl fluoroaeelate 19, 92d 1.3802 14, 19 13, Old bp« 85°— 14, 19 — vol54 7.81 14, 19 81. 0-Clilorocthyl fluoroaeelate 19, 92d it'-" 1.3160 14 13, 9lc _ bp51 86“ 14 Vol=* 3.55 14 82. Allvl fluoroaeelate 19, 921 n ir" 1.4063 14 13, 911 ... iP" l.OOtil 14 — bp50 64.5-65° 14 - vol5* 31.69 14 . . . 83. Propyl fluoroaeelate 92e bp 135 137° 92e 91c 84. Isopropvl fliloroacetate 19. 92e n n50 1.3804 14 13, 91c (Pa 1.033 14 bp™ 121-123° 14 -=- vol5* 62.31 14 85. /J-Ethylhexyl fluoroaeelate 59a, 59c 86. Plicnvl fluoroaeelate 19 mp 01.5-63° 19 13 87. /)-('hlorophenyl fluoroaeelate 19 mp 52-54° 19 13 88. Cliolestervl fluoroaeelate 92f mp 144 144.5° 92f 91 h 89. Met hvlenc-4«',>t(monofluoroaeel ate) 921 mp 57“ 921 t>21 90. Gl ve< >1 bts( m< >n< (fluoroaeetate) 92f bp17 110-141° 92f 13, 91 h 91. Fluoroacetvlcholine chloride 7T. 92. Fluoroacet vlsalicylic acid 19, 92j mp 141-144° 19, 92f 13, 91b — mp 131.6° 93. S-(J-chlorocthvl fluorothiolaeetate 19, 92j bp10 80 81° 19 13, 911 94. Phenyl fluorothiolaeetate 92f mp 36.5-37.5° 92f 91h bp'* 132° 92f ... 95. Methyl fluoroselenolacetate 11 nu-“ 1.4879 14 13 iP" 1.573 14 — bp7” 130-132° 14 Vol55 69.95 - 14 96. Fluoroacet vl fluoride 19, 92e bp70" 35-40° 19, 92e 13, 91f 97. Flui >r< meet vl ehloriile 19, 92b »i.5“ 1.3831 14 13, 91c iP" 1.3530 14 ... bp75* 69 71° 14 . . . — vol5* C>07 14 98. Fluoroaeetonitrile 19, 92« m.50 1.3324 14 13 bp™ 78° 14 vol5* 260 14 99. Fluoroacet vl isot hioevanate 19 nu5“ 1.5327 14 13 ,P« 1.3527 It — bp*° 76° 14 vol5* 15.51 14 ... 100. Fluoroacet ic anhydride 92e bp15 88-89.5° 92c 91c 101. Fluoroacet amide 19, 92h mp 108° 19, 92b 9lc 102. X- M et hy Ifliu iroaeet a mide 92b nip 64° 92b 103. X - Xi t roso-X-mclhy lflu< >roaeel a mide 92e bp1* 84° 92e 91c SECRET 100 METHYL FLL’ORO ACETATE AND RELATED COMPOUNDS Tabus 1 (Continued). Compound Hcference to synthesis Physical properties Property Hcference Reference to toxicity data 104. X-/J-Chlor«)cthylfluoroacet amide 19, 92e mp fi5° 19, 92e 13 bp" 3 77° 19, 92c 105. X-0-Tlydroxyethylfluoroacetamide 92e mp ca. 21° 92c bp"1 114° 92e 106. X.X-Diethvlfluoroacctamide 19 bp" 86° 19 13 107. X,X-6fs(d-Chlor(>ethyl)fluoroacctamide 92e nip 64.5° 31a, 92e bp“-°* 102° 31a, 92c 108. a-Flnoroacetanilide 23 mp 73 74° 23 20 109. Fluoroacetvlglvcinc ethvl ester 92f mp 50 50.53 92f 9lh J 110. Fluoromethylfluoroacetvlurea 921 mp 84° 921 921 / 111. 2-Fluorocthane-l-sulfonyl chloride 92g bp13 81.5-84.5° 92g 91 h 112. Methyl o-fh i«.ropropiona t c 92c bp 106.5-108.5° 92c 91 f 113. F.thvl jl-fluoropropionatc 19 bp‘* 65-68° 19 20 li t. Diethvl fluommalonatc 9lc bp" 84 86° 91c 91c 115. S«>dium y-fluorobutyrale 19 13 110. Methyl y-fluorobulyrate 10. 19 «ir° 1.3887 10 13 1.0662 10 — — , ..... lip1"0 79° 10 — ... vol3* 39,6 10 ... 117. Methvl nr-fluoroisobutyrate 92c bp 108-1014° 92c 91c 1 IS. Ethvl a-flu■»*.»?<■ has been prepared on a large laboratory scale (50 lb) by heating anhydrous potassium fluoride with ethylene ehlorohydrin at ISO (’ for 4 to 5 hours. Yields of 53 per cent are ob- tained. Except for the difference in temperature the reaction is carried out as described for methyl fluoro- acetate. During the heating ethylene oxide is formed almost quantitatively from the ethylene ehlorohydrin but appears to he the product of a reversible side re- action. Attempts to produce 0-fluoroelHanoi from ethylene oxide and hydrogen fluoride have been un- successful. /3-FIuoroethanol is a colorless liquid with a pleasant alcohol-like odor. It boils at 102.5 C at atmospheric pressure and is completely miscible with water. Several of its physical properties have been deter- mined.14-*Se 4. Mrlhyl y-fluorobulyrate,019 is prepared by treating trimethyleneehlorobromide with sodium cyanide to produce a mixture of -y-chloro and -y-bromo- butyron it riles. The mixture is then heated under pressure with anhydrous potassium fluoride at 200 C to give -y-fluorobutyronit rile, which is converted to SECRET 162 METHYL KLCOROACETATE WD BELATED COMI’Ol ,M)S the corresponding methyl ester by treatment with methanol and acid. The overall yield of methyl y-fluorobutyrate obtained in preparations on a large laboratory scale has been about 25 per cent for the three-step process. 5. Methyl y-Jlnoro-0-hydroxybutyrnte, methyl fi- chloro-y-fluorobutyriile, and methyl y-fluororrotu~ nnte 1019 are prepared as follows. Epichlorohydrin when heated under pressure at 225 C with potassium fluoride is converted into epifluorohydrin. Treatment of the latter with anhydrous hydrogen cyanide and a small amount of sodium cyanide gives y-fluoro-/3- hydroxybutyronitrile in excellent yield. This inter- mediate is converted to methyl y-fluoro-jS-hydroxy- butyrate by treatment with methanol and acid. Methyl /J-chlorb-y-fluorobutyrate is produced by the action of thionyl chloride and pyridine on the hy- droxy compound. Methyl y-fluorocrotonate may then he formed by dehydrohalogenation of the /it- ch loro compound with triethylamine. Yields are good except in the case of the first step involving the eon- version of epichlorohydrin to epifluorohydrin. It has not yet been possible to raise the yield of this step above 40 per cent, although 70 to 74 per cent of the unconverted epichlorohydrin is recovered in a form suitable for re-use. The overall yield based on the amount of epichlorohydrin utilized is 46 per cent for methyl y-fluoro-/3-hydroxybu(yrate, 39 per cent for methyl /3-chloro-y-fluorobutyrate, and 33 per cent for methyl y-fluorocrotonate. All three esters are stable colorless liquids. An alternative method not requiring high-pressure equipment has been developed for the preparation of methyl y-fluorocrotonate on a laboratory scale.19 Methyl y-bromocrotonate, prepared by bromination of methyl croton ate with N-bromosuccinimide, is re- fluxed at atmospheric pressure with anhydrous po- tassium fluoride. The product is slowly dist illed from the mixture as the reaction proceeds. Yields of ap- proximately 10 per cent are obtained. In addition to the synthetic procedures already described, a variety of methods has been used to pre- pare other fluorinated aliphatic compounds. .Men- tion may be made of (he preparation of difluoro- and trifluoroaeetic acids by the oxidation of 1,1-dichloro- 3,3-difluoropropene and l,l,2-trichloro-3,3,3-triflu- oropropene, respectively 15 (see Chapter 40); the preparation of several ethers of /3-tetraHiioro- ethanol by the addition of alcohol to tetrafluoro- cthylene; the synthesis of /3-fluoroethyl thiolacetate, from which /3-fluoroethylmercaptan may be obtained by hydrolysis, by the peroxide-catalyzed addition of thiolacetic acid to vinyl fluoride;33 the synthesis of tetrafluoro-1,2-dinit methane and chlorotrifluoro- 1.2-dinitroethane by the addition of nitrogen tetrox- ide to (he eorresponding halogeiuited olefines; and the synthesis of derivatives of t-fluorocaproic acid from eyelohexanone through t-hydroxycaproic acid and the corresponding bromo-eompound, which is treated with silver fluoride.wk l».2.2 Chemical Properties Methyl fluoroacetate has l>een the subject of most of the work on the chemistry of fhoaliphatic fluorine compounds considered in this chapter. It is readily hydrolyzed to fluoroaeetic acid and methyl alcohol, the half life of the ester in water buffered at pH 7 being less than 1 hour.21 On the other hand, the flu- orine atom can be removed from the molecule only by relatively drastic treatment. No reagent has been found which will bring about rapid replacement at room temperatures. As an example, no fluoride ion is produced by heating methyl fluoroacetate for 5 minutes with 20 per rent alcoholic potassium hy- droxide, although more prolonged heating (18 hours on steam bath) does result in the incomplete libera- tion of fluoride ion.95d Concentrated acids at steam bath temperatures hydrolyze the fluorine atom at unspecified rates.1*0 Under physiological conditions of pH and temperature, no fluoride ion is liberated in 72 96 horn's in the presence of any of a variety of ni- trogen bases, sulfur compounds, and inorganic salts.2" A further example of the chemical inertness of the fluorine atom in fluoroacetates is given by the follow- ing comparison of the rates of replacement of halogen by sulfite in the ethyl esters of fluoroaeetic, cliloro- acetic, and bromoacetic acids; 99 Temp Bi molecular Compound C _ velocity constant Kthyl bromoacctatc 25 18.3 Ethyl chlonmoetate 25 0.13 Ethyl fluoroacetate 45 4,5 X 10-» Limited data on the storage .stabilities1’ of both h A recon) report from the Chemical Warfare Service {TCI H 345, Surveillance of Fluorine Compounds, September 5, 1015) testifies to the stability of methyl fluoroacetate and re- lated compounds with respect to pressure development as follows; (I) methyl fluoroacetate and 0-fluoroelhanol arc stable in 75-mm steel shell with respect to pressure for at least 1 year at tin C; (2) methyl 7-flnorobutyrate docs not de- velop pressure in fi months at fi5 C when in contact with a steel strip in glass apparatus; and (3) methyl -y-fluoro- d-hydroxybntyratc develops a pressure of about 120 psi at 35 per cent void in glass apparatus at 65 C in either the pres- ence or absence of a steel strip. CHEMICAL STKICTIHE IN RELATION TO TOXICITY 163 methyl fluoroacetate and sodium fluoroacetate also illustrate the high stability of these com]K>unds. Methyl fluoroacetate undergoes no visible change on storage for S months at 00 (’ in glass containers in the presence of varnished steel, but a slight deposit of silica forms in the presence of bare steel.* Sodium fluoroacetate undergoes no visible change, loses no weight, and does not alter in fluoride content on storage for 30 days at 65 (’ in tin-plated cans; the tin surfaces show no change.47'- Methyl fluoroacetate resists oxidation by aqueous permanganate or chromate. In the presence of chromic acid plus concentrated sulfuric acid, pro- duction of hydrogen fluoride occurs, slowly in the cold and rapidly on heating.so Methyl fluoroacetate exhibits a thiosulfate de- mand on heating at 100 C.sl There is no evidence that other members of this scries differ strikingly from methyl fluoroacetate in the stability of (he fluorine atom, or that- they exhibit peculiarities in the reactions of the more common functional groups. 10.2.3 Detection and Analysis The fluorine atom in compounds of the fluoro- acetate type is too stable toward hydrolysis to make practical the use of this reaction for purposes of identification. Therefore, recourse is had to oxidative or thermal decomposition producing hydrogen flu- oride. which is then detected by its etching effect on glass or by its ability to bleach metallic lakes of ap- propriate dyes.,7i5i so.s'.ssi, y device making use of the etching effect to produce a nonwet table surface in small glass tubes has been examined by the Hrit- ishd” v-■!W Hot platinum filaments and hot platinized silica gel both decompose volatile fluorine compounds and both have been utilized in experimental appar- atus designed for field use.17 " Satisfactory tests for fluoroacetate ion in water have been developed.4,,M All quantitative methods for determination of fluorine in compounds of the fluoroacetate type have involved the conversion of the organically bound fluorine to fluoride ion, which is then determined by one of the standard methods. The detection and analysis of aliphatic fluorine compounds are reviewed in more detail in Chap- ters 34 and 37. 10.3 CHEMICAL STRUCTURE IN RELATION TO TOXICITY 24i 44 , i , In Table 2 are listed representative compounds which do and do not possess to a marked degree the Table 2. Aliphatic fluorine compounds illustrating Ihc relationship between molecular structure and toxicity. - Compounds exhibiting definite fluoro- Compounds exhibiting no or only acetate*- or 7-fhiorobut yra t e-like slight fluoroacetate- or 7-fluoro- ■ toxicity* licfcrence bulyrate-Iikc toxicity* Reference ’ - — „ .4 rids Fluoroaeetic acid 91 e, 92c Difluoroacelic acid 13 Salts Sodium fluoroacetato 13,20,34a Sodium chloroacetate 13, 38c Sodium y-fluorobutyratc 13 Sodium bromoaectatc 13, 3Sc Sodi 11 in 7-fliiet hy 1 7-fluon >bnt yra te Sec Table 4 Ethyl i-fluoro valerate 91 r d-Fhioroethyl 7-fluombutyrate See Table 4 Ethyl oj-fl uorohendcca noa te 9lr Methyl 7-fluorolhiolbutyratc Sec Table 4 Methyl Sec Table 4 Methyl 7-fliioro-/J-hydroxybutyrate See Table 4 * SECRET 164 METHYL FLUOROAEETATE AM) RELATED COMPOUNDS Table 2 (Continued). Compounds exhibiting definite fluoro- (omiKiunds exhibiting no or only slight acetate- or ■y-fluorolmtyrate-like fluoroacetate- or >-fluorobutyrate- toxicity* Reference like toxicity* Reference Methyl y-fluoro-d-hydroxyt hiolbutyratc See Table 4 Methyl -y-fluorocrotonate See Table 4 Diet hyl fluoromalonatc 91c Ethyl t-fluorooaproate !Mo (3-Fluoroethyl t-fluorocaproate 91r Ethyl uf-fluorocaprate !)lr _ Anhydrides Fluoroacetic anhydride 91c, 95c Nitriles * - Fluorojicctonit rilef 13. 91c — y- Fl u orobu tyron i t rile f 13 y-Fluorocrotoni t rile t 13 Trifluoroacctonit rile 13 Aldehydes Fluoroacetaldchvde 91 o Amides Fluonwcetamidc 91b, 92b X-d-chlon*et hvl fluoroacet amide 13 X -ni t r< iso-X -met hvl flm >n meet amide 91c ; Arid halides — Fluoroacetyl chloride 13, 91c Acetyl fluoride 13 Eluoroaeetvl fluoride 13, 91f ('hloroacetyl fluoride 13, 91c — - ‘ Hutvryl fluoride 13 Crotonyl fluoride 13 — ,4 Icohols 3-Fluoroethanol See Table 4 •y-FluoroppopanoI 59b Esters of Fluoroethanol mnnoUi-FluorOflhyl) derivatives 0-Fluorocthyl chloroformate 13 d-Fluoroethyl acetate 91 f 0-Fluoniel hvl fluoroacetate See Table 4 d-Fluoroethvl ehloroaeetate 91c, 92g - - nitrite 13 . Dichloro(/S-fluorocthoxy)phosphine 13 3-Fluorocthyl sulphury! chloride 9lf — hid 0-FIuoroel hyl) derivedives his( 3-FI uoroel h yl) carbonat e 13 bis(3-F1 nonx>thy 1) fluorophosphate 91 f bisfd-Kluoroethvl) ehloromaleate 13 6/s(3-Fluorocthvl) sulfate 13, 9lf,92g — Di-3-fluoroelhyl hydrogen phosphite 91 h Ethyl his(3-fluoroethoxy )phosphinc 13 — ' lris{ d-Fl uoroel hyl) derived ives _ - tn'M3-Flu-fluorobulyrate-likc toxicity would, for any specie* at doses equal to or slightly greater than those listed for methyl fluoroacetate or methyl >-fluorob«tyn»tr in Tables 3 and 1, produce the characteristic symptoms after the usual latent period (see below), and at least some deaths within 2 days. — f May posse** .slight activity, hut markedly less than corresponding esters. t Produce methyl fluoroacetate symptoms but only at somewhat higher concentrations. SECRET TOXICOLOGY 165 Table 3. Toxicity of methyl fluoroacetate. With the exception of the entries market! with an asterisk, the figures are approximations based on limited numliers of observations. Species LC\„ (mg/1) f = 10 min Reference Intra- venous Reference LD,„ (mg kg) Subcutaneous Reference Oral Refer- ence Dog 0.025* 24t 0.08 241 0.1 0.2 73 0.1-0.2 73 Cat (0.025-0.05)t 24 i 0.2* 40b 0.3 73 0.3 73 Rabbit 0.065* 241 0.33* 241 0.3 0.5 24 i, 73 0.5 73 Guinea pig 0.15 24d, Die 0.2 73 0.5 73 Goal 0.2 76 <2.0 51 1.0 73 1.0 73 Hat 0.3 24d, 73. 76 2.5 73 3.5 73 Mouse 3,2* 24f,t 17* 2 it 5-20 24i, 73 (5-0) 24 j, 73 Rhesus monkey 0.8 2.0 73, 76 5-15 51 10 12 73 10 12 73 Ccrcopitheeus monkey * , . >50j 01 q Krog 100 200 40a t Hstimatr based on susceptibility h> £pftuort*‘lhanol. X Intraperilotiral injection. ' characteristic toxicological properties of methyl fluoroacotate and methyl y-fluorobutyrate, Com- ])oiiuds which produce the characteristic toxicological actions fall into the following categories; 1. The following acids, in some cases tested only as salts and esters: fluoroacetic, y-fluorobutyric, y-fluoro-d-hydroxybutyric, /1-ehloro-y-fluorobutyric, y-fluorocrotonic, t-fluoroeaproic, and w-fluoroeaprie. 2. Other simple derivatives of the above acids and their thiol analogs, including anhydrides, amides, aldehydes, and acid halides, but not the nitriles. 3. /J-Fluoroethanol, its esters, and certain other derivatives. The following compounds do not evoke the char- acteristic toxic effects; 1. Di- and poly-fluoro derivatives of the toxic mono-fluoro compounds. 2. Chlorine, bromine, and iodine analogs of the toxic fluorinated derivatives. 3. Fluoride-liberating compounds such as acid fluorides. 4. Derivatives of aliphatic acids in which the fluorine atom is not in the terminal position (i.e., methyl a-fluoropropionate, methyl o-Huoroisobutyr- ate, ethyl a-fluorobutyrate, and diethyl fluoromalon- ate). 5. w-Fluoro derivatives of aliphatic acids with an odd number of carbon atoms (e.g., ethyl 0-fluoro- propionate, ethyl 5-fluorovalerate. and ethyl «-fluoro- hendecanoate). Thus, the F-CH»-group appears to be essential. Its presence is not sufficient, however, and presum- ably it must form the end of a chain of an even num- ber of carbon atoms. It is also necessary that the proper group, usually an oxygenated one, form the other end of the chain (e.g., methyl fluoroacotate is highly toxic, l-ehloro-2-fluoroethane is less so, and fluoroacetonitrile Ls relatively nontoxic). That other features of the molecules play a role in determining the degree of toxicity by inhalation is also revealed by the large differences which exist between the pre- cisely determined LCi0'* for a number of related de- rivatives containing one and twoF ■ CHi-groups,241 •"44 and by the large differences in toxicity which are associated with various in methyl y-fluorobutyrate (see Table 4). tot _ TOXICOLOGY id.1.1 Toxicity for Animals The toxicity of methyl fluoroacetate for animals is set forth in Table 3 and may be evaluated in com- parison with hydrogen cyanide, the LDi0 of which is in the order of 1 mg 'kg for most species, including man (see Chapter 2). It is noteworthy that: (1) the species variation is unusually large — the dog is ap- proximately 100 times more susceptible than the mouse or monkey and two tested species of monkeys show considerably different susceptibilities; and (2) the compound is approximately as toxic when administered by mouth as when injected intrave- nously or subcutaneously. The toxicity of /3-fluoroethanol for various species is comparable to that of methyl fluoroacetate; d-flu- oroethyl fluoroacetate, the most toxic member of the fluoroacetate group, is somewhat more potent (Table 1). Methyl y-fluorobutyrate and related com- pounds (Tables 2 and 4) produce toxic effects similar in a general way to those of methyl fluoroacetate but exhibit less pronounced species variation, principally SECRET 166 MKTim. KLUOUOACET \TE \M> RELATED COMPOUNDS Table 4. Inhalation toxicides of fluorinaled aliphatic compounds. With the exception of the mouse LCsn’s, the figures are approximations based on limited data. LCao (mg/1, nominal, for I — 10 min) Monkey Guinea Compound (Rhesus) Mouse Rat P'g Rabbit Cat Dug Methyl fluoroacetate 0,8-2.O'*-18 3.2“** 03>*i,n.7« 0.15**1 0.065*** 0.025s 0-Chloroelhyl fluoroacetate 0.7Me 0.2 ± ’*<’ 0.15+**•'•*“ o.r*- (i-Fhiornet h vl fluon meet a t e 0.63s** 0.2 ±,,J 0.07s** »w Q.0591,1 fJ-Fluoroelhanol 1.5»M-« 1.2s-" 0.2+5M.76 0.15s'-'-*"- 0.025s*1* 0.03.VXI (0.007) Methvl y-fluorobutyratc 0.5S4b 0.12s*' 0.35+slh 0.07s1«• 0.035s**1 0.0351*** 0.05s!"> Met liyl -,-fluorol hiolbut yr- ate 0.004-11 d-Chloroet hvl y-fluorobut yr- j ate >0.3-0 0.051s1) 0.1 + s'i .... d-Kluorocthvl y-fluorobulyr- 9 ate o.5+s*b 0.077s4' 0.2 s lh 0.035s*1* <0.075=* 0.025s**1 0.025s* Methyl d-ehloro-y-fluorobu- tvratc 0.16s** Methyl y-fluoro-^-hydroxy* - butyrate 0.2-|B 0.023s**" . .T. <0.063s*1' 0.P <0.0(43-* Mel hvl y-fluoro-jj-met hoxy- butyrate .... >0.1“" >0. !«'■ Met hvl y-fluoro-0-hydroxy- -r ; thiolhutyrate 0.2-1P <0.03S4P ...» <0.063s*1, 0.063s “ /S-Chloroethyl y-flumo-d-hy- — — droxybulyrate 0.048s*’ Met h vl y-fluoroerotonatc <0.5Mh 0.080s*1 0.15s**1 .... Ix'cause of a much greater toxicity for mice. When tested on monkeys, the members of this group are more toxic than methyl fluoroacetate but not so toxic as either mustard gas or phosgene. Methyl fluoroacetate, and presumably also (he •y-fluorobutyrate derivatives, are detoxified in the laxly, but only at a slow rate.24'1* '-403-73-76'911’'4 Changes in with changes in exposure time over the range I to 100 minutes have been observed in experiments with methyl fluoroacetate, 0-Huoro- ethyl fluoroacetate, and /3-fluoroethanol, but the effects are not large.24'1*'73 76 The L{Ct)„o of methyl ■y-fluorobutyrate for mice is the same for exposures of 1, 10, and 100 minutes,24* and that of methyl y-flu- orocrotonate may not be significantly different for exposures of 10 or 100 minutes or for two fractional exposures at a 24-hour interval.241 Summation of the effects of multiple sublethal doses of methyl fluoro- acetate administered at daily intervals by mouth, injection, or gassing has been observed but some de- toxification occurs and, with sufficiently small incre- ments, the equivalent of several lethal doses can l*e tolerated.40m’73'il,f However, species differences appear to exist; successive small doses produce a more pro- nounced cumulative effect in guinea pigs than rats, the latter species probably developing an increased resistance to the poison.9,1 Indeed, recent data dem- onstrate that a small dose (approximately 0.1 JJ)„o I administered orally or subcutaneously confers a sta tistieally significant, degree of resistance upon nib\ tested 24 hours later with an LD:,n administered; orally or intramuscularly.470 A similar phenomenon: has berm reported for orally administered sodium j fluoroacetate, but it would not appear that the ele-j vat ion of resistance is sufficient to affect the value of the salt as a rodentieide. A characteristic latency is associated with the visible effects of poisoning by methyl fluoroacetate and related compounds. Even 10 to 20 times the lethal dose produces sympt oms only after a minimum delay of 15 minutes.24'1 Survival times of animals dy- ing as a result of inhalation of median lethal dosages are almost always at least 1 hour, usually 2 to 12 hours, less frequently 12 to 21 hours, and rarely longer.24'1'91' The derivatives of y-fluorobutyric acid act similarly to the fluoroacetates but the latent period may be somewhat briefer and the recov- ery of sublethally poisoned animals more pro- tracted.24*11’4 There are two immediate causes of death in methyl fluoroacetate poisoning: action on the heart, culmi- nating in ventricular, fibrillation and circulatory failure; and stimulation of the central nervous sys- tem, producing convulsions, apnea, and death with- SECRET TOXICOLOGY 107 out severe cardiacr abnormalities.51 The relative severity of the two effects is not the same in different species: the cardiac action is the primary cause of death in monkeys, goats, and rabbits; effects on the central nervous system predominate in rats, cats, ami dogs.4"515sk 73 Transient but sublethal central nervous effects occur in some species (e.g., Rhesus) which eventually die with ventricular fibrillation, and cardiac effects in other species (e.g., the eat) which die of respiratory failure following severe con- vulsions.51 The poisoned heart has a decreased ex- citability and the effects are not due to diminished coronary blood How.51 In experiments on three monkeys methyl *y-fluoro- butyrate produced cardiac depression and arrhyth- mias, as well as marked parasympathetic symptoms, but ventricular fibrillation has not lieen observed.58,1 /3-Fluoroethyl y-fluorobut vrate, likewise tested on only three monkeys, produced effects similar to those of methyl fluoroaeetate but was more toxic.58,1 Both compounds produced effects similar to methyl fluoro- acetate in the cat and rabbit.681 The symptoms associated with poisoning by methyl fluoroaeetate and related compounds have been deserilied in detail for various species ?4', E h1'1, 5i.58k,73,stia and can lx* interpreted as resulting from the actions of the poisons on the heart or nervous system, or both. Pathological studies «».J h.7j »ik.., jn animals dying acutely from single doses reveal no significant changes other than signs referable to venous con- gestion. In animals exposed repeatedly to sublethal doses until death ensues,911 ,q there are found the sequelae of protracted venous congestion att ributablc to heart failure, definite abnormalities in the myo- cardium, changes in the kidney which may or may not be secondary to disturbances in the metabolism of other organs, and changes of doubtful significance in some other organs; no unequivocal pathological changes have been observed in the nervous system. in.t,2 Physiological Mechanism A number of clinical pathological and biochemical studies have been made to throw light on the cellular mechanism of action of methyl fluoroaeetate and re- lated con Ipou lids. l8"4,11' 2H,34,38.40,51,58l,.f .j.k.l, 73. 9U.li.g.j, m.n.q.n.ss xiipir heterogeneous character precludes a review of all the isolated facts whieli eventually may prove to he of significance. The evidence is strong that methyl fluoroaeetate does not owe its toxicity to the liberation of fluoride ion at critical loci in the body. In accord with chemi- cal studies on the stability of (lie fluorine atom (sec* Section 10.2.3), none of a large manlier of biochemi- cally important substances,*1 including some with a high reactivity toward organic halogens, liIterates fluoride from methyl fluoroaeetate at physiological conditions of yd I and temperature; -8 383 nor is fluoride ion liberated when the ester is incubated with rat tissues.18 Moreover, methyl fluoroaeetate does not show a marked tendency to inactivate enzymes which arc highly susceptible to fluoride.38" It has been proposed as a working hypothesis that all the lexicologically active compounds under con- sideration may be the precursors of some common toxic material, possibly the fluoroaeetate ion, which could l»e. produced, for example, by hydrolysis of esters, oxidation of /3-fluoroethanol, and /3-oxidation of the 7-fluorobutyrates.91' Although this hypothesis, which conceivably could explain the facts set forth in Section 10.3, “Chemical Structure in Relation to Toxicity,” has not as yet been submitted to sys- tematic test, (lie following findings may be cited as bearing upon it. 1. Sodium fluoroaeetate, fluoroacetic acid, and fluoroaeetamide possess approximately the same toxicity as methyl fluoroaeetate and produce symp- toms after a comparable latency.51 01,1 The latency in poisoning by the ester is not, therefore, determined by time for hydrolysis. However, this does not imply that hydrolysis of the ester may not be a necessary prelude to the initiation of toxic action. Tissues and blood contain a methyl fluoroaeetate esterase,18 38* which in the rat is sufficiently active to afford the ester a half life of not more than a few minutes — a fraction of (he usual latent period for symptoms.18 2. That the characteristic effects of methyl fluoro- acetate and sodium fluoroaeetate on the myocardium do not require in vivo chemical changes in other organs is suggested by experiments on eviscerated rabbits 61 « Tt may !«■ noted that the conclusions concerning cardiac effects have been based on detailed, continuous electro- cardiographic observations,** and that species which exhibit central nervous stimulation concomitantly develop abnormal electroencephalograms in the absence of notable cardiac irregularities.578 d Arginine, serine, histidine, tyrosine, proline, asparagine, glutamic acid, lysine, tryptophane, alanine, glycyl glycine, imidazole, guanidine, cysteine, glutathione, S-allyl thiourea, d-mercapt oet Hanoi, 2,3-dimerca ptopropani >1, carbobcnzoxy methionine, Ihiodiglycol, benzylamine, triethanolamine, tetra- cthanolarnnionium chloride, hexamethylenetetramine, and d-aminolienzoic acid; or sodium thiosulfate, sodium sulfide, sodium bisulfite, or sodium iodide. SECRET 168 MKTin L ELI OROACETATE AND RELATED COM 1*0 U NDS and proved by tests with isolated, perfused hearts of the cat.51 rabbit,91 ,n and guinea pig,31" and with the isolated papillary muscle of the cat.51 A similar con- clusion with respect to effects on the central nervous system is suggested by the finding that local appli- cation of methyl fhioroacetate to one cerebral hemi- sphere produced convulsive discharges after the usual latency for symptoms; although the convulsions were generalized, the effect of the poison on the treated hemisphere ajqx’ared to be greater than on the con- tralateral areas.31* 3. On the contrary, it may be necessary for /3-flu- oroethanol to undergo chemical change, possibly by oxidation to fhioroacetate in the liver. This is sug- gested by the finding that the alcohol exerted no effect on the isolated, perfused heart when tested at concentrations at which methyl fhioroacetate pro- duced marked decreases in rate and survival time.31*" 4. If the toxicity of the y-fluoro butyrates and other longer-chained nominated aliphatic acids de- pends on the production of fhioroacetate by 0-oxida- tion, their relatively high toxicity for some species (Table 2) would require that they l>e concentrated to a greater degree than the fiuoroacetates at critical loci in the body. That the /3-oxidation of 7-Huoro- bot y rate is not prerequisite for all its actions upon biological systems is indicated by evidence that, methyl 7-fhiorobutyrate is not converted to Huoro- acetate by rabbit kidney cortex in vitro, in spite of the fact (hat both compounds markedly inhibit the oxidation of acetate by this preparation.38* Substitu- tions on the /3-carbon atom are, however, important determinants of inhalation toxicity, as is revealed by the widely differing toxicities of a number of the butyric acid derivatives listed in Table 4. Changes indicative of a derangement in carbo- hydrate metabolism in methyl fhioroacetate poison- ing in various mammalian species are increases in blood sugar,58,173 non protein nitrogen,40*-75 inorganic phosphate,58j lactic acid,40*-5801 pyruvic acid,5Kb and lactate-pyruvate ratio.586 In rabbits there is a marked reduction in liver glycogen 58i and, in the heart, marked decreases in total acid-soluble phosphorus and organic soluble phosphorus.5** Serum potassium and calcium show only minor increases.588 75 Negative results have been obtained in many but not all the studies on the effects of fhioroacetate on enzyme systems in vitro and on the metabolism of tissues obtained from poisoned animals or treated with the poison after isolation.18-34 3*-40-58b f-,-3!* 38 Illuminating experiments have been performed with an isolated skeletal muscle, the sartorius of the frog, hut have not lx*en extended as yet to cardiac muscle.18-401’The resting oxygen consumption and the contractility of the* unfatigued sartorius are not, affected by the poison at a concentration of 0.005.1/, but the extra oxygen consumption following activity is strongly inhibited.18A01’*1 The inhibition is associ- ated with a greatly decreased resynthesis of phos- phocreatine1X and abolition of the delayed heat production normally associated with aerobic re- covery of stimulated muscle.401’r Similarly, the extra oxygen consumption produced by pretreatment of the isolated muscle with the stimulants caffeine and dinitrophenol is essentially abolished by fluoro- acetate.1*-40' Similar changes are produced by sodium azide but the mechanism of action is different: azide inhibits cytochrome oxidase and adenyl pyrophos- phatase, whereas methyl fhioroacetate has no in- hibitory action either upon these enzymes or upon cytochrome reductase.18 The possibility jhat fhioroacetate inhibits lactic acid dehydrogenase is suggested by the findings that the isolated frog sartorius utilizes pyruvate (also acetate) but does not oxidize added lactate,40'' and that the effects of fhioroacetate upon the isolated guinea pig heart are counteracted by pyruvic acid derivatives but not by sodium lactate.31'" An iiuiitm study on lactic acid dehydrogenase likewise revealed an inhibition of the enzyme prepared from yeast,40,1 although in another experiment the enzyme pre- pared from heart muscle was not reported to lx* in- hibited by methyl fhioroacetate.3*' Data relating to the production of lactate by the stimulated poisoned muscle under anaerobic conditions are not con- sistent.18 401’ In the case of rabbit kidney cortex prepa- rations in vitro, however, methyl fhioroacetate in- hibits the oxidation of glucose and certain intermedi- ates of carbohydrate metabolism but it has no effect on the anaerobic phase of carbohydrate degradation resulting in the formation of lactic acid. The latter findings have led to the suggestion that a locus of action may be at one of the steps in the dehydro- genation of pyruvate via the citric acid cycle.18 The effect of fhioroacetate on the oxygen con- sumption of stimulated skeletal muscle has been found to be reversible,1* offering some hope that methyl fhioroacetate poisoning may eventually be subject to treatment. Moreover, if the oxygen con- sumption of heart muscle should prove to lx; as easily inhibited by methyl fhioroacetate as that of stimulated skeletal muscle, the possibility would SECRET TOXICOLOGY 1G9 exist that a therapeutic agent could be found in a carbohydrate intermediate the oxidation of which is not strongly inhibited.1* The slight therapeutic value of procaine and p- amiuobenzoic acid in fluoroacetate-poisoned monkeys and the absence of a corresponding effect with other antifibrillatory drugs (see Inflow) suggested the al- ternative possibilities that p-aminolx*nzoic acid might l>e fluoroacctylated, thereby detoxifying the poison, or that the toxicity of the fluoroacetate might be associated with an inhibitory action on normally occurring acetylations. However, experiments reveal that (he monkey does not acetylate, and therefore probably does not fluoroacetylate, p-aminobenzoic acid,5*1 that the acetylation of p-aminohippuric acid by rabbits is not markedly affected in fluoroacetate poisoning,341, and that fluoroacetate does not inhibit the acetylation by liver slices of sulfanilamide, p- aminoljenzoic acid, or choline.3** h On the other hand, fluoroacetate does produce some inhibition of the utilization of acetate in vitro by rabbit heart and kidney 18 preparations and by rat kidney, liver, and heart slices.3*1’ In (he case of Corynebaeterium rrca- tinovorar and of yeast, the inhibition is almost com- plete.3** These and other findings have led to the sug- gestion that fluoroacetate may produce a profound alteration in the metabolism of carbohydrate by vir- tue* of a specific inhibitory effect on the oxidation of acetate.3*0 However, poisoned caffeine-stimulated muscle does utilize acetate.4110 The resting potential of frog peripheral nerve is sensitive to concentrations of methyl fluoroacetate as low as 0.001.1/.340The potential was reduced in poisoned nerves by a period of anoxia, and the oxi- dative recovery was little affected by addition of acetate or acetyl phosphate, but was counteracted by addition of pyruvate. to.t.:t Therapy * No satisfactory procedures for the treatment of fluoroacetate poisoning have been discovered. Tests have been made of substances and procedures de- signed to prevent convulsions, to stimulate respira- tion, to stimulate diuresis and excretion of the poison, to prevent ventricular fibrillation and otherwise* re- store the failing heart, to promote detoxification by fluoroacetylation, to compete for enzyme systems with fluoroacetate, and to supply necessary metabo- lites the formation of which may lx* cut off by the action of the poison on enzyme systems.1* •*4r 151 ■ 4fte,e,i,i,k,l,73.»l«.i.in Intracardiac inject ions of procaine accompanied by artificial respiration and cardiac massage through the thoracic wall temporarily restore an organized beat to the monkey heart fibrillating as a result of methyl fluoroacetate poisoning; but fibrillation recurs and eventually proves fatal in spite of continued treat- ments and tlie presence of subcutaneous deposits of procaine.51 ssd Administration of large doses of sodium p-amino- ben zoa t e t o a n esl h e t i zed monkeys{Rhesus and Afrits) poisoned with one LDMOdose of methyl fluoroacetate corrects the cardiac disturbances and saves the ma- jority of animals.5** * However, (he value of this treatment is limited inasmuch as it does not save monkeys poisoned with larger doses5*' or rabbits poisoned with four /,/>i0’s;-*1"' nor does it combat the lethal action of methyl fluoroacetate in the rat, a species which dies of central nervous lather than cardiac effects.581 Large concentrations of sodium acetate (0.1 per cent) prolong the survival of the isolated rabbit heart perfused with depressant concentrations of methyl fluoroacetate, but the acetate ion has exerted little or no protection when administered to the poisoned animal.4*1 Similarly, the sodium salt and other derivatives of pyruvic acid protect the isolated guinea pig heart poisoned with methyl fluoroacetate but have little or no therapeutic value in vivo A"" Various anesthetics have been shown to l>e effec- tive in controlling the convulsions associated with methyl fluoroacetate poisoning,-’41 51 -91*-' but even when combined with respiratory stimulants they do not decrease the mortality.*1* Sodium pentabarbital is contraindicated because it increases the mor- tality.81' The following additional substances and proce- dures have been without significant value under the tested conditions in saving the lives of animals poi- soned with methyl fluoroacetate or /3-fluoroethanol: artificial respiration;91* artificial respiration plus sodium phenobarbital;-4f 73 oxygen plus carbon di- oxide;73 urethane, paraldehyde, or chloral hydrate, with or without theophylline or coramine;9lE the- ophylline; Mr,91i bromide;91* Dilantin (sodium di- phenyl hydantoin); 73 91* morphine hydrochloride;91* ' quinidine, digitalis, quinidine-digitalis, or caffeine;5’9' papaverine;5*' yohimbine;581 anticholinesterase drugs or aconite;5** atropine;’40 5*0 ephedrine;iHF0 thia- mine;5*' glucose;54* potassium salts;5,e*,m calcium salts; 240 •Mc barium salts;5** 2,3-dimercaptopropanol (HAL), acetophenone, or cobalt acetate.91'" SECRET 170 METHYL FLL’OIIO ACETATE AMI RELATED COMPOUNDS 10.4.4 Toxicity for Man Alan is among the species which are relatively re- sistant to methyl fluoroacetale. Direct evidence comes from the results of ingestion of the compound by a British volunteer.91' Upon taking an oral dose of 0.1 mg kg in water he experienced no symptoms other than a slight, possibly psychogenic, feeling of unsteadiness upon standing up l}4 hours after the dose was taken. Similar ingestion of 0.65 mg kg pro- ducer! no symptoms other than a feeling of unsteadi- ness for a few minutes 1 hour after the dose and a slight malaise 5 hours later; however, the subject continued work in the lalxiralory with no obvious loss of efficiency and his electrocardiogram and elec- troencephalogram, recorded at frequent intervals, showed no deviation from the normal, ft is to lx> noted that the dose ingested was greater than (he LDhp for guinea pigs, rabbits, cats, and dogs. Various lines of evidence 73 80 suggested that the lethal dose per os is in the order of 6 8 mg kg. Ex- posure of workers for prolonged periods to low con- centrations of the vapor9* produced marked weak- ness, reluctance for any physical effort, and strong mental depression with periods of nervous irritation difficult to control, followed by physical and mental exhaustion, drowsiness, and giddiness; a few days’ rest resulted in marked improvement.e Assuming (1) that the above estimate of the lethal dose per os for man is correct, (2) that the toxicity of methyl fluoroacetatc is more or less independent of the route or rate of administration, and (3) that 100 per cent absorption of inhaled vapor occurs, one may calculate that the lethal vapor dosage for a 70-kg man breathing 10 1pm (relative inactivity) would lx* 50,000 mg min m3, corresponding to a 10-minute LCm of 5 mg 1; for a man breathing 40 1pm, corresponding to exercise intermediate bet ween a walk at 5 mph and a slow run,- the figure would be 12,500 mg rnin m3, the equivalent of 1.25 mg I for 10 minutes. Although the validity of this method of calculation has been questioned,7* it has been shown to yield good approximations when applied to the flog and rabbit, the only larger species for which both the /.('.mi’s and LI),n’s have lx*en determined with precision.*4* For defensive purposes the British have estimated the L(Ct)-M at 4,000 and 7,000 mg min in3.76-79 For most species the margin between the convulsive and lethal doses is small. The mild, indistinctive odors of methyl fluoro- aeetato and 0-fluoroethanol make it possible that large vapor dosages could lx* inhaled undetected. It has liven reported that methyl fluoroacetale at 0.05 mg 1 is just detectable, that at 0.2 0.3 mg 1 it would easily lx* overlooked, ami that at 0.1 mg 1 it possesses a fruity smell and may produce a slight feeling of tightness in the chest.79 a,<- Most of the 7-fluorobutyrates are probably somewhat more odor- ous than methyl fluoroacetafe but it has Ijoen empha- sized that methyl y-fluoro-d-hydroxy butyrate pos- sesses only a very slight odor, similar to but much fainter than that of ethyl lactate.-'1’ A prominent symptom in severe poisoning is the occurrence of repeated and severe convulsions indis- tinguishable from status epileptieiis;73 less dramatic symptoms may include nausea, vomiting, dizziness, and fall in body temperature.*1" In addition to the symptoms, tests with urine mar aid in the recog- nition of fluoroacetatc poisoning, for a toxic, lluorinc- containing substance not present in normal urine is excreted.91* ' n q The fluorine may be converted to fluoride and detected by chemical test 9"’ or the urine given to rats by stomach tube, the character- istic symptoms of fluoroacetale poisoning73 then Ix-ing produced.*1" q In the absence of specific therapeutic procedures for fluoroacetale poisoning, cases can at present only be treated symptomatically. Morphine has been recommended to allay distress, anxiety, and con- vulsions, but barbiturates (i.e., pentobarbital so- dium) are contraindicated. 9,*_ to.3 EVALUATION AS W AR GASES Evaluation of the potentialities of methyl fluoro- acetafe and related compounds in terms of available data and present concepts of chemical warfare indi- cates that none of the derivatives possesses the gen- eral utility of currently standardized gases. They remain a subject of some military concern, however, in view of (heir potential use as food and water poisons (see Section 10.6) or for other -penal pur- poses. For man methyl fluoroacetatc is not appreciably more toxic, and in all probability is considerably less toxic, than currently standardized gases. The lethal vapor dosage, calculated above on the basis of the, demonstrated low oral toxicity to be in (he order of 12,000 mg min m3 for ventilation rates correspond- ing to moderate physical activity and several times * These symptoms, experienced by Polish chemists, prompted the initial toxicological examination of the effects of methyl fluoroacetate on animals. SECRET EFFECTIVENESS VS FOOD AND WATER POISONS 171 this value for men at rest, may he compared with the minimum dosages of standard agents currently recommended as adequate for the following tasks:71 comparable to methyl tluoroacetate range from that of methyl tluoroacetate (i.e., 119 mg 1 at 25 (.’) down to very low values. Thus, agents of any desired de- gree of persistence are potentially available. Al- though the indistinctive odor and relative difficulty of detection by chemical means would confer upon these compounds a certain insidiousness, their lack of effectiveness on the eyes and skin renders them inferior in general utility as persistent agents to such vesicants as mustard gas and fiv.s(/3-chloroethyl)- amine (HN3). Except in drinking water, (heir decon- tamination offers no special problems.*0 Methyl tluoroacetate possesses excellent storage stability (see Section 10.2.2) and its explosion sta- bility is believed to be sufficient to permit its dis- persal from chemical munitions now in use.2,i It is potentially available in quantity. There are no data bearing upon the toxicity for man of the derivatives of y-fluorobu lyric acid. On the basis of the comparative toxicides of these de- rivatives and of methyl tluoroacetate for the monkey (Table 1), they would be suspected of being some- what more toxic for man than is methyl tluoroacetate; They are, however, less volatile (Table !) and nota- bly more difficult to manufacture. 10.6 POTENTIAL EFFECTIVENESS VS FOOD AND \\ \TEU POISONS IN \\ VRFARE112* 0-7*-7* * The chemical and toxicological properties of methyl tluoroacetate and related compounds make them potential water and food poisons. They are approximately as toxic when administered orally as when injected or inhaled; to a degree they may act as cumulative poisons; and they are not readily de- tected by smell or taste. Although methyl fluoro- acetate itself undergoes hydrolysis, the resulting fluoroaeetic acid is stable and toxic. At concentrations of 0.1 per cent or less in water, methyl tluoroacetate has no smell or taste; *6 ]/% 1 of a 0.1 per cent solution would probably be lethal for man (see Section 10,4.1). This concentration in milk is readily accepted by rats and dogs,1*2 although it is not freely accepted in otherwise pure drinking water by rats, nor are /3-fluoroethanol and sodium fluoro- acetate at 0.01 percent (100 parts per million) freely accepted by mice.21' - The effectiveness of sodium fluoroaeetate as a rodent bait poison is discussed below. Filtration of contaminated water with charcoal Dosage Task Agent (mg min nr1) To produce a large propor- Phosgene 3,2tK» tion of deaths or severe Hydrogen casualties in surprise at- cyanide 5,000 tacks with nonpersistent Cyanogen gases (dosages to lx- ob- tained within 2 minutes) chloride 11,000 To produce skin burns of Mustard sufficient severity to to- vapor 1,000(T > 80 F) tally disable 50 |ht cent 2,000 4,000 of masked troops not equipped with protective clothing (dosages to—he obtained within 4 hours) — (T = (10-80 F) To produce eye damage of Mustard sufficient severity to cause temporary blind- ness among troops not wearing gas masks vapor 200 In view of the relatively low toxicity for man, it is apparent from a consideration of (he physical proper- ties of methyl fluoroaeetate (Table 5), the most volatile stable compound of the group,1 that it would Tabus 5. Physical properties of methyl fluoroacetate and of currently standardized nonpersistent agents. Property Methyl fluoro- acetate Ilydro- gen cyanide Cyano- gen chloride Phosgene Liquid density (g/ml at 25 C) 1.17 0.68 1.2 1.36 Boiling point, C 104 26 12.6 8.3 Freezing |>oint, G — 35 -13.4 -7 -104 Latent heat of evap- oration, e;d, g 100 210 135 GO Vajxjr pressure. nun Ilg at 25 C 20 740 1,200 1,400 at -20 C ss 180 230 Volatility, mg/1 at 25 C 119 1,060 at -20 G 145 680 1,400 be more difficult than in the case of the standard nonpersistent gases to achieve in the field vapor dosages sufficiently large to be lethal in surprise at- tacks; and, in view of the effectiveness of the can- ister,*0 the breaking of the gas mask cannot be con- sidered a feasible task. The volatilities of /3-fluoroethanol and of various stable fluoroaeetate derivatives having toxicities 1 The more volatile p-ffiioroethyl nitrite, fluoroacetyl fluo- ride, and fluoroacetyl chloride arc chemically unstable. 172 METHYL FLUOROACETATE AM) RELATED COMPOUNDS removes methyl fluoroacetate but not the hydrolytic product, fluoroacetic acid.45 Filtration with charcoal plus pyridine is said to remove not only methyl flu- oroacetate but also, from neutral solution, sodium Huoroacetate as well.44 The detection of fluorine com- pounds in contaminated water is discussed in Chapter 54. 10.7 USE AS RODENTICIDES Sodium fluoroacetate was one of several substances studied in the chemical warfare program in the United Kingdom and the United States which were recommended by Division 9 of NDRC to/he Fish and Wildlife Service of the Department of Interior for test as rodenticides.4' Preliminary tests with small samples (200 lb) submitted by Division 9 were so successful that the division subsequently pre- pared an additional 1,000 lb - for large-scale field trials. Field campaigns in a number of states and in military establishments in this country and abroad were conducted by the Fish and Wildlife Service, by the Typhus Control Unit of the Public Health Service, and by the medical departments of the Army and Navy. The results demonstrate that sodium fluoroacetate, coded 1080, is one of the most promising available roden t icides.46d *0‘744 Sodium fluoroacetate possesses the following re- quirements of a good rodent icific: high toxicity and acceptability, stability, lack of volatility, lack of irri- tation and toxic properties for human skin, lack of inflammability, and potential availability in quan- tity at reasonable cost.464’ The oral lethal doses for various species of rats and other rodents of concern in public health and agriculture range between 0.1 and 5 mg kg.41*100 The substance is effective in baits at much lower concentrations than in the case of other rodenticides. Excellent results have been ob- tained in field trials utilizing 0 oz of sodium fluoro- acetate per 250 lb of cereal or ground meat bait.4"* Water solutions are also highly effective. A concen- tration of I 2670 has been recommended for general use,70 although concentrations ten times greater (i.e., 5 oz gal) are sufficiently acceptable to rats and have been used with good results.4"' As in the ease of other rodenticides, the possibility of accidental human poisoning cannot lie ignored, particularly in the absence of effective methods for treatment of fluoroacetate poisoning. However, it would appear that concentrations sufficiently low to make accidental human poisoning improbable may still be effective in rodent control. The human lethal dose is believed to be of the order of 5 10 mg kg (see Section 10.4.4). On the other hand, the oral lethal doses for eats and dogs are very low (0.1 to 0.5 mg kg) and, therefore, the likelihood of acci- dental poisoning of these species confers a certain disadvantage upon sodium Huoroacetate. The sodium salts of y-fluorobutyric, y-fluoroqS-hy- droxybutyric, and y-fluorocrot onic acids are several times as toxic for rats as is sodium fluoroacetate. However, in view of the already high toxicity of the lat ter, this apparent disadvantage is more than offset by the greater difficulty and expense of their prepa- ration. SECRET Chapter 11 CADMIUM, SELENIUM, AND THE CARBONYLS OF IRON AND NICKEL By John .1. Zapp ill INTRODUCTION IN tiik skakch for new chemical warfare agents, the toxic properties of certain metals were not neglected. The increasing use of cadmium in indus- try, for example, had revealed that the inhalation of finely divided cadmium metal, the oxide, or salts was capable of producing severe lung edema comparable with that produced by phosgene.**-*8 Selenium com- pounds showed similar properties,80 and, although somewhat less toxic than cadmium on an absolute basis, they produced physiological effects much more, promptly. Being inorganic, these agents offered promise for inclusion in burning-type munitions or incendiaries as well as for dispersion by high-ex- plosive shell. The carbonyls of iron and nickel aroused considerable interest not only liecause of their inherent toxicity, but also because they break down catalytically in contact with gas mask carbon, yielding carbon monoxide which is not absorbed in the canister but passes into the mask.9*-1’ Thus the carbonyls might be valuable in attacking either masked or unmasked troops, (’om pounds of mercury, thallium, tin, antimony, lead, chromium, and ger- manium were screened by the University of Chicago Toxicity Laboratory [LTCTL],7 but without reveal- ing any of special interest for chemical warfare purposes. The part of Division 9 of the National Defense Research Committee [NDHCj in the field of heavy metals was largely one of screening the toxicity of a great number of compounds, many of which were prepared under Office of Scientific Research and Development [DSHD] contracts. The detailed in- vestigation of the promising compounds, including investigations of cadmium and selenium and the carlHHivls, was carried out by the Chemical Warfare Service and by the Directorate of Chemical Warfare in Canada. 11.2 CADMIUM n.2.1 Physiological Action Cadmium, its oxide, and salts are toxic by any route of administration, but their particular signifi- ounce in chemical warfare lies in the fact that finely divided dusts can l>e set up either by thermal com- bustion of incendiary mixtures containing cadmium or by the explosive dispersal of cadmium compounds. These- dusts are quite toxic by inhalation,*4-*8 pro- ducing lung edema comparable with that observed in phosgene poisoning. Exposure to high concentrations of cadmium causes some early respiratory irritation, which pro- gresses to marked dyspnea within a few hours. Two cats exposed to a high concentration of cadmium oxide fume for 30 minutes 66 showed on autopsy ex- tensive acute pulmonary injury with edema, injury to the bronchioles and alveolar ducts, and acute alveolar emphysema. Liver and kidney damage was also found. Exposure to lower concentrations of cadmium fumes or dust results in a temporary irri- tation of the respiratory tract which disappears shortly after cessation of exposure only to reappear within about 12 hours with increasing severity ac- companied by general malaise. Within about 24 to 48 hours dyspnea is marked and cyanosis occurs prior to death.10-1™•“ On autopsy the lungs are found to lie firm, but with interstitial and perivascu- lar edema and extensive hemorrhage. Liver and kid- ney show evidence of fatty infiltration. Several cases of human poisoning by inhalation of cadmium have been reported. In one of these, re- porter! in 1858,60 three men were exposed to cadmium carbonate dust. Symptoms did not occur until sev- eral hours after exposure and then consisted of dyspnea, dizziness, vomiting, and diarrhea. One patient apparently cont racted pneumonia by second- ary infection, but all three recovered eventually. Fifteen cases of human cadmium poisoning from in- halation of cadmium oxide fumes, two of which were fatal, have been reported from Canada.67 In all these cases, dyspnea, which did not become severe until several hours after exposure, was the most prominent symptom, although the majority of cases also ex- hibited gastrointestinal symptoms. The two men who died showed congestion of the lungs, pulmonary edema, hemorrhage into the lungs, atelectatic areas, proliferative interstitial pneumonitis, and catarrhal SECRET 173 174 CVDMII M, SEIXML'M, AM) CARBONYLS OF IKON AM) NICKEL bronchitis. Liver and kidney damage was also present. 11.2.2 Toxicology The toxicity of cadmium oxide by inhalation is summarized in Table 1 for various species and ex- ditions where particles do not agglomerate,49 It is worth noting that prior to the Canadian experi- ments4'* there was a tendency to assume that the toxicity of cadmium oxide for man would closely re- semble that for the monkey, making the human L(Ct)„o of the order of 15 mg min 1. This estimate would seem to bo entirely too high. The true toxicity of cadmium oxide for man may be as great or greater than that of phosgene (see Chapter 3). The toxicity of cadmium metal itself ami of cad- mium compounds other than (he oxide by inhalation was tested at the UCTI,. The results are shown in Table 2. So far as mice are concerned, there is con- Tabi.k 1. Toxicity of cadmium oxide by inhalation. Kxposure T4Ct)H, time S|X'cies (mg inin/1) (min) Reference Mouse 0.5 15-30 60 0.87 10 17 0.58 10 15 0.34 10 7 Rat 2.0— 2 40 l.l 5 40 0.78 10 to 0.0 30 40 1.3-1.8 5-10 46 Guinea pig 3.0 15 30 60 Rabbit 3.0 15-30 60 >1.8 5 10 46 <5.2 10 IS Goat <1.6 5-10 46 Dog 3.0 1(T 45 Monkey 15-20 -40 45 - 15 ± 15 45 21 + ;«) 45 Man 1.5-2.0 75-90 49 Table 2, Inhalation toxicities of for mice.7 A = analytical concent ration; concentration. cadmium compounds X = nominal Compound Ct (us compound) (mg min 1) Ayr. Ct particle (as Cd) diameter (mg min/1) (m) Mortality Cadmium 0.38 A 0.38 <0.2 18,20 ('admium 0.17 A 0.17 <0.2 18/19 Cd oxide 0.31 A 0.30 <0.2 uctu Cd chloride 2.3 A 1.4 <0.5 lACtU.i, CM fluoride 1.8 N 1.2 ? 0/20 CM fluoborate 0.5 N 1.9 ? 8/20 Cd fluosdieale 0.7 X 2.1 ? 9/20 CM sulfide 1.35 A 1.05 <0.3 5/20 CMsclcnate 2 27 -X 0.03 ? 0/20 CM nitrate 3.85 A 1.4 <0.5 UCtu,, CM phosphate 6.5 X 3.07 ? 2/20 posure times. Some of the variability in results is un- doubtedly due to variation in particle size of the cadmium oxide. When disperser! from most incendi- ary munitions the median particle size is usually less than 1.0 n in diameter, but agglomeration of particles frequently takes place. This point has been particu- larly emphasized in the estimate of the L(Ct)i0 for man,49 in which the elementary particles were less than 0.3 n in diameter, but in which the cloud actu- ally consisted of large numbers of small agglomerates of 1.0 to 2.0 n in diameter, with a small number of agglomerates 40 n or greater in diameter. The L{Cl)i0 of 2,0 mg min 1 was based on analytical CV’s ob- tained in an experiment set up to duplicate condi- tions which resulted in two cases of fatal cadmium oxide poisoning in an industrial plant. There ap- peared to be every reason to believe that the concen- tration of cadmium oxide in the original accident was not greater than that obtained in the duplicate experiment, but the L(r/)50’s for rats and rabbits exposed in the duplicate experiment were about twice those previously obtained with arc-produced cadmium oxide fumes. The difference was attributed to the greater median particle size in the duplicate experiment and led to the hypothesis that the L{Ct)h0 for man might be as low as 1.5 mg min 1 under eon- siderable variability in I ho t oxicity of the difTercnt cadmium compounds even when dosages are calcu- lated in terms of the cadmium content of the com- pound. It is also of interest that cadmium metal itself is more toxic than any of its salts. In this in- stance, the combination of cadmium with anions which are themselves toxic resulted in decreased rather than enhanced toxicity. Unfortunately it is not possible from the data to assess the effect of part icle size on the different est imates of the o’s but taking the results at their face value it would appear that cadmium oxide is (he most toxic of the cadmium compounds. This fact is fortunate since the oxide is easily prepared in the field by the com- bustion of incendiary munitions containing cadmium metal.15'-4"*’4® 11.2.3 Assessment of Value as a Chemical Warfare Agent Cadmium appears to be a promising material for addition to incendiaries if toxicity as well as fire is SECRET SELENIUM 175 Tabi.k 3. Toxicity of selenium dioxide by inhalation. Kx) insure Time to Cl t ime death S|>ecics (ms min/I) (miu) Mortality (hr) Reference Mouse 2.30* 10 0 20 7 Hat 2:30t - 10 0/6 7 Rabbit o.89t 20 4/6 6, 6.5, 40, 132 27 6.59t 10 4 /6 3.8, 11, 13,32 27 13.1Ht 20 6/6 2.8, 3, 3, 5, 5.5, 8 27 Goat 5.89t 20 0.2 27 6.59f 10 2/2 5.5, 84 27* — S.83f 30 3/4 6, IS. 130 27 13.i8t 20 2/2 4,4.5 27 * Set i* di?»| termed by atomization of aqueous solution. - f SeO? formed and disperst-d by detoiuiti ion of Sr;liigh-explowive mixture, IVak range of particle size 0.0 to 1.0 p in diameter. desired. The cadmium oxide which results from the combustion of cadmium metal in incendiary mixes is odorless and probably not irritating enough in the presence of smoke and burning materiel to be de- tected by odor. Cadmium oxide smoke is brown in color, however, and may he detected by appearance after it has been used a few times. Cadmium chloride may also be dis- I>ersed from burning munitions IS:’V” and lethal con- centrations may lx* obtained in mixtures which are indistinguishable in appearance from harmless screening smokes. Attempts have been made to dis- perse cadmium compounds by the explosion of mu- nitions containing cadmium or its compounds, 5» 55,64 ]ulj this method of dispersal is relatively in- efficient because of the rapid .agglomeration and settling of the cadmium part icles. One drawback to the use of cadmium in offensive warfare is the delay in appearance of toxic effects, since, as has I teen pointed out, dyspnea does not usually become severe until at least 12 hours after exposure. If. however, incendiary at lacks are planned against industrial installations, large stores of ma- teriel, cities, and the like, such targets are usually well beyond the front lines and a delay in the appear- ance of toxic effects can lx* readily accepted. From the available data it would appear that cadmium might play a very important role if a military re- quirement for toxic incendiaries should arise. 11.3 SELENIUM A review of the toxicity of selenium as a potential indust rial hazard appeared in 1938.** At that time it was known that selenium compounds were toxic when ingested and that hydrogen selenide was toxic on inhalation. On this basis it was predicted that soluble dusts such as selenium oxides (SeO>, Se03, i CScOrt, I LSeO,) and certain halogen compounds might lx- toxic Ix'eanse of the ease by which they could lie absoii>ed from the lungs and gastrointestinal tract. These toxic dusts might he set up by the com- bustion of incendiary mixtures containing selenium or its compounds or by the detonation of explosives containing selenium. Hence selenium, like cadmium, was investigated as a possible chemical warfare agent. tl.3.1 Physiological Action The action of selenium appears to be similar to that of cadmium, with the exception that the onset of toxic effects is rnoiv rapid after exposure to sele- nium, Goats and rabbits exposed for periods of 10 to HO minutes to selenium oxide smoke showed dyspnea and tachycardia on removal from the exposure chamber. Animals receiving a fatal dose usually died within 21 hours and sometimes within 3 hours. On autopsy, pronounced pleural effusion and pulmonary edema were found, plus hemorrhages in the lungs, heart, and kidneys, and marked congestion of the glomeruli and spleen.27 H.3.2 Toxicology All workers agree that in absolute terms selenium is less toxic than cadmium. The toxicity of selenium oxide toward various species is shown in Table 3. Other selenium compounds were screened for toxicity at the UCTL without revealing any of greater in- terest or effectiveness than the oxide.7 11.3.3 Assessment of Value as a Chemical Warfare Agent Selenium oxide smoke differs from cadmium oxide smoke in the following respects; (I) it is less toxic than cadmium oxide; (2) it is acrid and irritating to SECRET 176 CAIIMIIM, SELEMIM, AND CARBONYLS OK IRON \M> MCKEL the respiratory tract, whereas cadmium oxide is odorless and relatively nonirritating; (3) it is white, whereas cadmium oxide is brown; (4) it kills or dis- ables more quickly than cadmium oxide. In contrast to cadmium, which was found to l>e most effective in incendiary munitions, selenium has been studied mainly in explosive-type munitions.57'*1-"-** Since selenium oxide is white, it would not lie de- tected by appearance alone if used in conjunction with ordinary screening smokes. On the other hand, its irritant properties might well load exposed troops to mask promptly. Whereas its relatively rapid action as compared with that of cadmium is a desirable feature, it is doubtful whether this out- weighs its lower absolute toxicity. The potential use- fulness of selenium oxide as a chemical warfare agent cannot be accurately assessed on the basis of avail- able information. If t here is a future requirement for this type of agent, further experimentation, and par- ticularly field trials, are in order. li t NICKEL CARBONYL \NU IRON CARBONYL Nickel carbonyl, Ni(CO)(, was discovered in 1890, and iron pentacarbonyl, Fe(CO);„ in 1891. Both eom- |mumls can 1m* made to dissociate into carbon mon- oxide and the pure metal under controlled condi- tions;-and this reaction forms (he basis for the com- mercial preparation of pure nickel (Aloud process), a method which is in use to the present day. Iron carbonyl was more difficult to prepare than nickel carbonyl, the yield l»eing only about 1 per cent of theoretical, so that the Mond process .was not eco- nomical for the preparation of iron on a large scale. In the 1920's, however, iron carbonyl found some use in Europe as an “antiknock” for gasoline, and more recently it has lieen an important source of the pure, finely powdered iron which is used in powder metallurgy. The toxicity of the metal carbonyls was recog- nized as early as 1891 7,1 and was extensively investi- gated and reported in 1907 1908.70 Chemical war- fare interest in the compounds arose primarily from two facts; (1) they are toxic enough to merit con- sideration as agents for use under certain circum- stances where they might not be readily detected, and (2) they dissociate readily in contact with the active carbon of the gas mask, releasing four or five volumes of carbon monoxide per mole of carbonyl. The carbon monoxide is not absorbed by the canister of the service gas mask. Therefore, the carbonyls provide an indirect way of bringing carbon monoxide into offensive chemical warfare. 11.1.1 Physiological Action When iron or nickel carbonyl comes into contact with moist air. dissociation into carbon monoxide and a finely divided metallic salt takes place. Tins salt appeal's to be a hydrated basic carbonate of somewhat uncertain composition. Thus, when a [Ma- son is exposed to an atmosphere into which iron or nickel carbonyl has been released, lie breathes a mixture of varying proportions of the metallic car- bonyl, carbon monoxide, and a dust of finely divided metallic salt. What part each of these components may play in the toxicological picture will be discussed, but for the moment the discussion will be limited to the overall effects of inhalation of an atmosphere known to contain originally iron or nickel carbonyl. Since the physiological action of the two compounds is essentially the same, they will lie discussed to- gether. - Armit in 1907 70 described the sequence of events in human cases of nickel carbonyl poisoning as fol- lows: ... immediately after having Iwcn exposed to air contain- ing plant-gas there was giddiness, and at times dyspnea and vomiting. These symptoms passed off rapidly ns soon as the patients were brought into the fresh air. After 12 to 30 hours the dyspnea returned, cyanosis appeared, and the temperature liegan to l?e raised. Coughing with more or less blood-stained sputum occurred on the second day or later. The pulse rate became increased hut not in proportion to the respiratory rate. Delirium of varying types frequently occurred, and a variety of other signs of disturbance of the central nervous system was noted. Death took plaee in the fatal cases lictween the 4lh and 111li days. The chief changes found post mortem were hemorrhages in the lungs, edema of the lungs, hemor- rhages in the white matter of the braifTfin one case this was very extensive), while some doubt exists as to whether any blood changes were present , 'Phis sequence of events parallels closely that of a fatal case of human nickel carbonyl poisoning de- scribed in 1934,72 in which death occurred on the seventh day following exposure. The reaction of mice, rabbits, cals, dogs, guinea pigs, and goats is similar to that of man.’h,,b-c-,,>I0-7u If masked troops are exposed to air containing iron or nickel carbonyl, (ho carbonyl is catalytically decomposed in contact with the active carbon of the mask, leaving the finely divided metallic salt and carbon monoxide. The metallic dust is efficiently retained by the particulate filter of the service mask, NICKEL CARBONYL AND IRON CARBONYL 177 but the carbon monoxide passes through. When mice were exposed to atmospheres which had origi- nally contained iron or nickel carbonyl but which had then I>een passed through active carbon, deaths which occurred were entirely due to carbon monoxide ami bore no similarity to those resulting from ex- posure to (he carbonyls peans and the investigation of its proper- ties from the standpoint of assessment as a possible chemical warfare agent were studied under Divi- sion 9 of the National Defense Research Committee [NDRC] during the |»eriod 1942-1915. Earlier work by British investigators had shown that ricin (com- monly coded as “W”) could l>e dispersed as a par- ticulate, nonpersistent, toxic cloud by explosion of bombs containing a susjiension of ricin in carbon tetrachloride. Notable progress was made by NDRC investigators in all phases of the work with ricin. Processes for the extraction of ricin from castor beans and cold-pressed castor bean pomace were the subject of laboratory and pilot plant studies.Timing the laboratory inv estigations the protein was crystal- lized; the crystals were not completely homogeneous but mpresent the purest ricin so far obtained. The pilot plant development culminated in a process of extraction of castor Itean pomace with water and purification of the toxin by two precipitations with sodium sulfate. A water solution of the purified toxin was spray-dried to give a dry product with a mass median diameter of 0 8 g. This was air-ground to give “disjiersible ricin” with a mass median diameter of 2.5-3.5 m. which was approximately half as toxic (by injection) as crystalline ricin. Early work on the physiological action of ricin resulted in the development of a bioassay procedure in mice which was used to determine the toxicity of various ricin preparations. Later studies investi- gated the inhalation toxicities of various ricin prepa- rations, which are a function of the intrinsic toxicity of the material and the particle size distribution in the inhaled particulate cloud. Toxoids have l>een prepared from ricin by various means, most successfully by treatment with formalin. The toxoid has been used to produce in horses and rabbits antiriein serums. These have been purified and concentrated as antiriein globulin fractions that were made available for therapy in case of accidental exposure. Immunization against ricin appears im- practical at present because of the short duration of passive immunity in animals, and the toxicity and local necrotizing action of toxoid preparations avail- able for use in inducing active immunity. The detection and assay of ricin in the field is a difficult problem. Sensitized guinea pigs afford the most sensitive, rapid, and specific means of detection through their anaphylactic response. Hemagglutina- tion and precipitin tests have been used; chemical tests are less specific. Determination of particle size distribution forms an important part of the assess- ment of ricin and all other particulate clouds-(see Chapter 15). Field trials employing these analytical means and animals exposed to (he clouds to deter- mine toxicity have been conducted to evaluate ricin as a war gas and determine the efficiency of various munitions for its dispersal. Hiein is most efficiently dispersed from small high explosive-chemical bombs as a suspension in carbon tetrachloride of the most finely divided material available. On the basis of airplane stowage such bombs are estimated to be seven times as effective as bombs charged with phosgene. Processing all of the castor beans used in this coun- try (based on 1911-1944 consumption) by the opti- mum procedure bast'd on pilot plant experience would yield approximately 1,000 tons of dispersible ricin annually at a cost of about $13 per pound. This is a significant quantity of a material which might be used as a unique nonpersistent agent in gas war- fare, difficult to detect and disturbing to morale be- cause of its delayed toxic action. Ricin has served as a model substance, presenting problems in prepara- tion, protection of personnel, detection, assay, and dispersal similar to those presented by other materi- als investigated in the field of bacteriological warfare. Some minor duplications appear in the subsections of this chapter, which were written by different authors. 12.2 PREPARATION OF RICIN b The isolation from castor beans of products con- taining the toxic principle known as ricin has been recorded many times in the open literature within the past 60 years.64 During World War I ricin was • By Arthur C. Cope. b By Joseph Dec. SECRET 179 ISO K1C1N examined as a candidate chemical warfare agent and its preparation was studied.21 The investigation of the preparation and properties of ricin pertinent to its use as a chemical warfare agent was renewed in Great Britain 53 early during World War II and in this country under NDRC’ Division 9 during the fall of 1942.’ 4 The objective of developing a process for the large- scale production of ricin in a form suitable for dis- persion from munitions was attained.” During the course of this development about 3,800 lb of ma- terial was products I on a pilot plant scale.1 1133 Also of considerable importance was the preparation of ricin in a crystalline form.2 for the first time. A complete review of the great number of products containing ricin whose preparation has Iwen recorded both in the open and classified literature is beyond the scope of this chapter. Emphasis is placed herein on the products studied most extensively during World War II and on the studies leading to their preparation. These include crystalline ricin; two products used in field trials with munitions, 470 BM 199 and 1,703; and the material used for the prepa- ration of toxoid, HI. A process for the large-scale production of ricin is outlined. 12.2.1 Crystalline Ricin The isolation during the late suminor-of-4643-of the material responsible for the toxicity of crude ricin preparations in crystalline form was a signal achievement.2 Neither the first crystals isolated nor any of the crystalline materials subsequently pre- pared 1216 could be shown to be single substances.21* Since the crystalline material was the most toxic fraction ever isolated from crude ricin, studies were initiated to determine its physical and chemical properties, composition, and physiological behavior. Properties The crystalline material is a protein of the globulin type,215 although the crude toxin shows albumin-like solubility behavior. Repeated crystallizations fail to increase its toxicity,212 which has been assayed to be 500 9 and 750 12 TU (depending on the method of evaluating TU; for definition of the toxicity unit known as TU see Section 12,5). The protein is soluble in acid or alkaline solution, is least soluble in the range of pH 5.0 to 8.0,2 12•’* and is more soluble in the presence of other proteins,21* Its ultraviolet light absorption spectrum is similar to that of a typical protein,21* and it has a specific optical rotation of — 26.3 UUraccntrifuge and electrophorcsis measure- ments showed tlie material to l>e fairly homogene- ous.2 3 However, solubility measurements indicated the crystalline material to consist of a solid solution of more than one component.21* On the basis of sedimentation and diffusion studies the molecular weight has been estimated at 36,000* and 77,000.* The rate of denaturation of the crystalline material in aqueous solution to a product insoluble at pH 5.1 has Iwen determined at 65.3, 71.5, 7S.1, and 86.5 (\ and from pH 2 to pH 11.14 The chemical composition of the crystalline ma- terial has been investigated, but not exhaustively. Evidence was obtained that the rf-amino acid content of an acid hydrolyzate of the toxin cannot be more than 3 per cent.16 On a moisture- and ash-free basis a sample of three times crystallized ricin was found to contain 16.23 + 0.4 per cent nitrogen.’* From the titration curve of crystalline ricin in water and in the presence of 8 per cent neutral formaldehyde the numbers of basic, amino, imidazole, and carboxyl groups were deduced.1* The amide nitrogen, alkali labile ammonia, hydroxvamino acid, arginine, his- tidine, aspartic acid, and glutamic acid 15 contents have been determined by chemical analysis. The amino acid analyses referred to account for 50 per cent of the weight of the protein and 60 per cent of the nitrogen. The protein was found to contain 1.34 per cent sulfur and less than 0.1 per cent phos- phorus. Preliminary to the first successful crystallization, ricin-sodium sulfate cake, an amorphous product (describes! in Section 12.2.5), was fractionated with ammonium sulfate at pH 6.8 to concentrate the toxin.2 The moist solid was dissolved in a minimum of water and allowed to stand at 5 C. A granular pre- cipitate formed, which gradually became crystalline on standing for several weeks. The crystals were isolated, suspended in water, and dissolved by the addition of a little hydrochloric- acid. The solution was adjusted to pH 6.8 and allowed to stand at 5 C. Recrystallization was complete in 2 or 3 days. Crystallization procedures more rapid and pro- ductive than the original method were developed.’*•’* Two extractions of the ricin-sodium sulfate cake with 10 parts of sodium sulfate solution (19 g Nar SOi TOO ml H20) were found to leach away many of the gummy low molecular weight impurities with- out appreciable loss of the toxin.14 One useful pro- cedure 15 involved extracting the residue with water and allowing the solution to stand overnight in a 181 PREPARATION OF RICIN refrigerator. The precipitate which formed was re- moved and dissolved in water with the aid of a little acid. The solution was neutralized, seeded with a few crystals, ami stores! in the cold for several days to yield a crystalline precipitate, which was separated and recrystallized. A modification of this procedure was performed starting with 1 kg of ricin-sodium sulfate cake.1*The yield was about 70 g of crystalline material, which is 7 per cent by weight or about 35 per cent of the toxin content of the starting ma- terial. Reerystallization was complete in 12 36 hours with an 85 90 per cent recovery. A 2- to 21 (-hour dialysis of a 20 per cent aqueous solution of ricin-sodium sulfate cake also served to remove the low molecular weight impurities.1'" The dialyzed solution after filtration and standing in a refrigerator yielded a crystalline precipitate. The percentage yields were comparable with those ob- tained in the procedure involving preliminary puri- fication with sodium sulfate solution. In an attempt to obtain a pure sample of ricin for an absolute standard, 60 g of 4 times crystallized material was extracted 25 times with 0.1 per cent sodium sulfate solution at /dl 7.0 and 10 C.1S The residue of al>out 6 g was recrystallized. Solubility studies on this product have not yet been made. Although the product is probably the purest sample of ricin obtained thus far, its allergen content has linen estimated at about 0.1 per cent on the basis of animal assay.12 - Numerous experiments were performed in the study of the crystallization of ricin which led to the procedures just described.21215 Flotation-purified ricin and the ball-milled and hammer-milled products (described in Section 12.2.5) were less satisfactory than ricin-sodium sulfate cake as the starting ma- terial;12 however, crystalline material has been ob- tained from flotation-purified ricin.15 Although a short dialysis of a solution of ricin-sodium sulfate cake is satisfactory for the preliminary purification, exhaustive dialysis is not.12 Some impurities can also be removed by adsorption on ('elite or floridin.12 12.2.2 Amorphous Klein Studies on the preparation of amorphous ricin have been extensive and a great number of products of varying properties and content of toxin, non toxic protein, proteose, and salt have been obtained.4-9,15 34 Crude ricin is soluble in water ami dilute salt solu- tions. In (he dry state the products are normally stable at room temperature and denatured at ele- vated temperatures.4-34**-33 The stability decreases with increasing moisture content.4 3#b Aqueous solu- tions are less stable than the dry product at both room and higher temperatures.415 33 Starting Material fob Preparation ok Hicin Samples of ricin prepared from castor beans of dif- ferent sizes and colors seem to be identical in physi- cal, chemical, and immunological properties.13 4 The maximum variation in toxin content of the different beans which were examined in one laboratory was 34 per cent.4 The beans contain about 50 per cent oil and the toxin is best isolated after removal of a substantial portion of this oil. The castor bean pomace which is obtained in the laboratory using a Carver press 4 or in industry using a hydraulic press 11 contains about 15 per cent oil and is satisfactory for the aqueous ex- traction of the toxin. A pomace containing 1-2 per cent oil can lie prepared by extraction of either ground castor beans or cold-pressed pomace with suitable organic solvents.4 34 If desired, the bean hulls can lie removed from (he pomace by flotation in or- ganic solvents.4 Hydraulir-pressed castor bean pomace is prepared commercially by castor oil producers. In one of the commercial processes the castor beans are ground, heated to about 60 C, and presses:!.11 This cold- pressed pomace is recommended as the starting ma- terial for the large-scale production of ricin.411 Com- mercially, this product is extracted four times with heptane at 82-87 C to obtain the remaining castor oil and then blown with steam to recover the residual heptane.” The latter step also serves to detoxify the pomace, which is sold as fertilizer. Tests on a labora- tory and pilot plant scale showed that no appreciable detoxification occurs during the extraction with hot heptane.4 11 Efforts to find an economical procedure for recovery of the residual heptane without detoxi- fication of the pomace were unsuccessful.11 Extrac- tion of the cold-pressed pomace with water at pll 3.8 to remove the toxin and subsequent solvent extrac- tion yielded castor oil containing free fatty acid.11 Extraction of Toxin from Bean Meal Among the solvents which have been used to ex- tract t he toxin from castor l>eans or the pomace arc water, dilute salt solutions, glycerol, ethylene glycol containing a little water, and diethylene glycol con- taining a little water. Water and dilute salt solutions are the most efficient and economical extractants for 182 RiCIN the toxin.4 Ten per cent saline is slightly more effec- tive than water; however, it also dissolves more non- toxic material, most of which is coagulable protein.4 15 About 3' i 4 parts of water at pH 3.S to 1 part of pomace seems to be most satisfactory. Less nontoxic protein is dissolved at pH 3.8 than at pH 7.0 and filtration is accomplished more easily.4 Extraction at temperatures approaching 70 C proceeds more rapidly than at room temperature but is accom- panied by denaturation of the toxin.4 Isolation of Toxin fhom Aqueous Extract of Pomace The toxin may !>e precipitated from the aqueous extract of pomace by nonaqueous solvents, by picric acid and similar precipitants, and by inorganic salts. Organic solvents such as alcohols and ketones pre- cipitate the toxin from aqueous solution but rapidly denature it at room temperature. At temperatures below 0 C acetone has been used to precipitate and wash the toxin.4 31 The use of ammonium sulfate,4 34 sodium chloride,4 "13 and sodium sulfate 3 4 "-'5 for precipitating and fractionating the toxin has re- ceived considerable study. Ammonium sulfate has l>een used for precipitating the toxin on a pilot plant scale.35 Sodium sulfate is now regarded as the best precipitant. It is sujrerior to sodium chloride because it gives better fractionation, is less sensitive to changes in pH, and precipitates the toxin more com- pletely.411 15 The importance of temperature control during precipitation, filtration, and drying when sodium sulfate is used have been st udied.4 Many data on the salting out of the toxin with different amounts of sodium sulfate and at different pH have been ob- tained.3 4 " 15 These data were useful in the develop- ment of the process for the large-scale production of amorphous ricin (Section 12.2.3)." Better yields of the toxin have been obtained in the laboratory than in the pilot plant.4 " 15 In the methods preferred by some investigators 415 slightly less sodium sulfate is used than in the proposed large- scale process and the first precipitation is performed at pH 3.8. In one laboratory run,15 during which the isolation of the toxin was followed by chemical anal- yses and toxicity determinations, the product amounted to 2.3 per cent of the pomace weight. It contained 10.4 jier cent nitrogen and 32.5 per cent inorganic material and had a TU value of 190. Of the toxicity present in the extract, 92 per cent was recovered. The product obtained at a similar stage in the pilot plant process amounted to 1.4 per cent of (lip pomace weight. Procedures involving a .single precipitation of the toxin with sodium sulfate yielded in (he laboratory products with TU values above 200,415 but these methods were not satisfactory on a pilot plant scale because of operational difficulties.41' Removal of water by lyophilizat ion of solutions of partially purified ricin yields products of good ap- pearance and stability.4 " Dialysis can serve to re- move much organic and inorganic impurity and in neutral solution leads to a precipitate of amorphous ricin.4 Comminution of Amorphous Uiriv Since the toxicity by inhalation of ricin aerosols increases with decreasing particle size,* considerable effort was directed toward developing a method to produce finely divided, readily dispersible material without concomitant denaturation of the toxin, '['he process involving spray drying and air grinding of partially purified ricin was the best solution found to the problem." Prior to this solution an appreciable number of other methods were considered and ex- plored.*11 - — The particle mass median diameter of freshly pre- cipitated crude ricin is 1 2 g, but as the moist filter cake is dried the particles agglomerate. The final precipitation was performed under various condi- tions with the objective of obtaining a product that could 1k» ground readily to fine particles." Among the conditions investigated were temperature of precipi- tation, agitation during precipitation, addition of sodium sulfate as a dry powder or from saturated solution, variation in amounts of sodium sulfate used, addition of colloids, addition of seeding agents, addi- tion of nonionic wetting agents, and transfer of the freshly precipitated product to a volatile liquid. A 2-hour ball-milling test was used for comparing all of the samples obtained in this series of tests. None of the experimental products showed significantly su- perior grinding properties. Lyophilizatipn of solu- tions of partially purified ricin proceeds without de- toxification to give a friable mat-like solid.4 " Ball- milling the solid reduced the particle size to a mass median diameter of 6 g in 33 per cent less time than that required with precipitated air-dried material." The detoxification which accompanied the ball- milling was 20 |M»r cent less than with precipitated air-dried material." Lyophilizat ion of a pomace ex- tract yielded a gummy product." Flotation-purified riein-sodium sulfate cake (de- scribed in Section 12.2.1) was used in ball-milling, PREPARATION OF RICIN 183 colloid-milling, and hammer-milling experiments.11 Hammer-milling gave products with particle mass median diameters no smaller than 20 g. Colloid- milling was even less effective. For about a year ball- milling appeared to be the most promising method for obtaining a finely divided material, and this method was investigated intensively.1148 *' The opti- mum conditions using an Abbe 4-jar mill fitted with It4 gallon “specimen” type porcelain jars, which were found to give a 4- to 6-ju product, involved (4) steel balls for the milling. (2) low milling temper- ature ( — 20 C), (3) low moist ure content ricin, and (4) milling a susjxmsion of ricin in carbon tetrachlo- ride." Factors affecting the ball-milling that were studied included the vehicle, grinding media, temper- ature, time, and moisture content of the amorphous ricin." The ball-milling time necessary to give a 4- to 6-n product, was proportional to the load of ricin in the jar, 1 lb of ricin requiring 8 hours. Ball- milling a high moisture content material at room temperature or in the dry state resulted in more de- naturation of the protein than otherwise. Even under the above optimum conditions at least 50 per cent detoxification accompanied ball-milling the material to a mass median diameter of 4-6 m " The toxicity loss was reduced somewhat by drawing off the fine particles as they were formed.51 A combination of spray drying and air grinding was found to give a product with a mass median di- ameter of 2.5-3.5 m with little denaturation of the starting material." A spray dryer was constructed and conditions for its operation investigated." Fac- tors such as type of nozzle, solution concent ration, atomizing air pressure, drying rate, drying temper- ature, and amount of drying air were studied. Under optimum conditions at an operating rate of 1 lb of product per hour the product has a particle mass median diameter of 6-8 m and is 95 per cent soluble in water. The spray-drying process is superior to the ball-milling method from the standpoints of low toxicity loss, processing time required, safety, and cost. Several types of air-grinding equipment were in- vestigated for the comminution of spray-dried ricin." A grinder previously developed by the Eagle Pencil Company was found to be the Ixst of the types ex- amined. Optimum conditions for its operation in a low humidity room were determined. Under optimum conditions the product with a particle mass median diameter of 2.5-3.5 n and a TU value of 225 is ob- tained at a rate of 1 lb per hour. A reduction in toxicity of about 5 per cent accompanies the air- grinding operation. I2.2.;i A Process for the Production of Finely Divided Klein 11 On the basis of considerable laboratory and pilot plant data a process for the production of finely divided ricin at the rate of 20 lb per day has been outlined. The equipment and manpower necessary for this scale of o|x* rat ions have been determined. The process involves extraction of the toxin with water from castor bean pomace, two precipitations of the toxin by addition of sodium sulfate, spray drying of a solution of the partially purified toxin, and air grinding of the spray-dried material. It was estimated that the cost of such a pilot plant would be approximately $125,000 and that the cost of pro- duction at the 20 lb per day rate would be about $10 per pound. The cost of operating a plant to pro- duce 2,000 lb of “dispersible ricin” daily was esti- mated to lx> approximately $13 per pound of product. The product has a particle mass median diameter of 2.5-3.5 ju and a toxicity value of 225 TU. The yield is 0,05 per cent basts! on (he pomace and would have amounted to about 1,050 tons annually during the years 1941-1944 if the castor beans crushed in this country during those years had been processed by this method." Reworking of the by-products from the spray-drying anti air-grinding operations and reuse of the nitrogen-containing sodium sulfate sepa- rated in the flotation step should increase the yield to about 0.85 jx*r cent. Starting Material The starting material for (his process is commer- cially available hydraulic-pressed castor bean pom- ace which has not been solvent-extracted to remove the residual oil and subsequently steamed. The pom- ace produced by one company averages 8.0-8.5 per cent moisture, 14.0-16.0 per cent oil, and 4.6-5.0 per cent nitrogen. The pomace is ground in a ham- mer mill prior to extraction. Extraction of Pomace The recommended conditions for extraction of the toxin from pomace are as follows: \\ ater for ext faction 350 per cent of pomace weight pi I 3.8 ± 0.1 Acid to adjust pH 5 per cent H2S04 Agitation time 60 minutes (not critical) SECRET 184 RICIN Temperature of extraction 25 C Filtration Continuous vacuum fil- ter Filter aid 7 per cent of pomace weight Water for washing 50 per cent of pomace weight Under these conditions at least 97 j>er cent of the extractable toxin is recovered. The amount of water used is the minimum necessary to produce a slurry that can be handled satisfactorily in plant scale equipment. Sulfuric acid is preferred over hydro- chloric acid because of lower cost and lower corrosion /rate. Continuous vacuum filtration at a higher pH y is not possible because of the changed physical char- acter of the slurry. The filter aid is necessary to in- sure a satisfactory filtration rate. Filtration with the vacuum filter proceeds about 30 times faster than with a recessed plate type filter. First Precipitation and Filtration The optimum conditions for precipitation of the toxin from the extract and subsequent filtration were determined to lie as follows: Salt usage 20 per cent Na2SO,,based on filtrate weight pH 7.0 Alkali to adjust pH 12 per cent NajCO* Temperature 25 C Time of precipitation 20 minutes Filtration Continuous vacuum filter Filter aid 4 per cent of slurry weight Wash solution 20 per cent of 16.7 per cent NaoSOi, based on weight of extract Under these conditions 50 per cent of the total nitrogen in the extract remains in solution and Is eliminated in the filtrate, whereas less than 2 per cent of the toxin is lost. Precipitation at pH 7-8 was found to remove 6-10 per cent more nontoxic nitrogen than at pH 3.8. Increasing the temperature from 25 to 35 C and varying the precipitation time from 15 to 60 minutes showed no appreciable effects. The rate of filtration with a vacuum filter was 3±2 times that with a plate and frame filter press, filter aid being neces- sary to obtain a satisfactory filtration rate in both cases. A full-scale pilot plant run was made to determine whether a single precipitation process would give a product suitable for spray drying.11 Filter aid was not used, because previously it had been found not possible* to reduce the sodium sulfate content of a product containing filter aid by a process involving flotation in carbon tetrachloride. Despite the absence of filter aid, which made the filtration very slow, the dried product separated very poorly in carbon tetra- chloride, The product, which amounted to 1.0 per cent of the original pomace, contained 11.0 per cent nitrogen and had a toxicity value of 200-250 ITT. The operational difficulties encountered indicated this one-step process to be unsatisfactory on a pilot plant scale. Second Extraction and Filtration The optimum conditions for extraction of the toxin from the ricin-sodium sulfatc-guhr moist filter cake were found to be as follows: Water for extraction 300 per cent of wet cake weight pH 3.8 ±0.1 Acid to adjust pH 5 per cent T USth Filtration Continuous vacuum filter Water for washing 25 per cent of slurry weight An additional 10 per cent (based on the pomace extract) of nontoxic nitrogen is removed during this operation. The pH was varied from 3.8 to 9.0, and it was found that 5 per cent (based on pH 3.8 extract) more nontoxic nitrogen is removed at pH 3.8 than at pH 9.0. The filtration is very rapid because of the large amount of filter aid present. Second Precipitation and Filtration The recommended conditions for the second pre- cipitation of the toxin and subsequent filtration are as follows: ~ Salt usage 20 per cent allow- ance being made for the sodium sulfate in the filt rate pH 7.0 Alkali to adjust pH 12 per cent Na*COg, or more dilute Temperature 25 C Time of precipitation 45 minutes 1* iltration Plate and frame filter press Filter aid None Washing None Drying of the filter cake can Ik* accomplished in 6-10 hours using a three-section hot-air dryer oper- ated at successively increasing temperatures from 55 (' to 75 C. The dried product is given a slight SECRET PREPARATION OF RICIN 185 grind, passed through a five- to ten-mesh screen, and slurried in five parts of carbon tetrachloride. The toxin is removed from the surface of the mixture and dried. The sodium sulfate which settles to the bottom is used in the precipitation steps. A quantity of partially purified ricin was produced by the process outlined except that the product was dried at 50 C. The product was obtained in 0.85 jx*r cent yield based on the pomace, contained 13.0 per cent nitrogen, and had a TU value of 250 3(H). Pilot plant tests indicated that a minimum of 20 lb of sodium sulfate is necessary to prevent loss of toxin. Approximately 3 (>er cent more non toxic nitrogen is removed at pH 7.0 than at pH 3.8. Operation at 35 C instead of 25 C removes 2 per cent more nontoxie nitrogen, but about 2 per cent more toxin is lost. Since filter aid cannot be employed in this step, the use of a vacuum filter, which requires filter aid, is not possible. However, the physical character of this second precipitate |iermits a satisfactory filtration rate with a plate and frame filter press. Washing the filter cake with sodium sulfate solution (19.5 lb NajSOj 100 lb H«0) does not result in .sufficient puri- fication to warrant a washing operation. The utility of a third precipitation of the toxin with sodium sulfate was invest igated. No appreciable purification was obtained without concomitant loss of toxin. Spray Drying and Air Grinding A 20 per cent aqueous solution of the above flota- tion-purified product is spray-dried under certain prescrilied conditions at the rate of I lb per hour to give solid particles, which are 95 per cent soluble in water and have a mass median diameter of 6-8 m- The solution for the second precipitation step can be spray-dried but it would contain about 50 per cent sodium sulfate. It was not found possible to separate the sodium sulfate from a spray-dried product by flotation in carbon tetrachloride. Air grinding of the spray-dried material is carried out under certain defined conditions in an air grinder, previously developed by the Eagle Pencil Company, at a rate giving about 1 lb of product per hour. This operation reduces the toxicity of the material about 5 per cent. The product has a particle mass median diameter of 2.5-3.5 m and a TU value of 225. 12.2.1 Four Vmorplious Ricin Products The four amorphous ricin products described in this section a re of particular interest because of the considerable extent of studies performed with them. The preparations known as (!) ricin-sodium sulfate cake, (2) 470 H.M 199. and (3) 1,703 represent suc- cessive stages in the development of an amorphous ricin product in a form suitable for dispersion from munitions, and (4) HI was used for the preparation of a toxoid. Ricin-Sodiim SriiKATE I’akk 1 A total of 1,550 lb of the product known as ricin- sodium sulfate cake was prepared on a pilot plant, scale at the request of NDRC Division 9,' and an additional 2,000 lb was prepared for the Canadian government.11 The method used in these operations, which was based on a procedure previously developed in another laboratory,4 utilized the facts that crude ricin is soluble in water and insoluble in saturated aqueous solutions of sodium chloride and sodium sulfate. Subsequent studies resulted in a marked improvement in the method of preparation (Sec- tion 12 2.3)." Castor beans were the starting material and an Anderson expellee was used for expressing the oil from the beans. From each ton of beans was obtained 810 lb of #3 grade castor oil. The expellee cake, which contained 13.1 [>er cent oil, 11.2 per cent moisture, and 4.6 per cent nitrogen, was ground in a hammer mill. Three parts of water at 15 20 C were mixed with the ground cake, the mixture agitated for 1 hour, the pH adjusted to3.8 + 0.1 with 5 percent hydrochloric acid, and the slurry filtered in a plate filter press. The filtered extract at pH 3.8 + 0.1 and 17 C was saturated with sodium chloride to precipitate the toxin. The precipitate was separated by filtration, sufficient guhr being used to insure a satisfactory filtration rate. V sample of dried filtered cake was found to contain 33 per cent guhr and 33 per cent sodium chloride. The wet precipitate was mixed with five parts of water and the mixture adjusted to pH 8.0 with 5 per cent sodium hydroxide solution. The mixture was agitated for 1 hour and then filtered to remove guhr and other impurities. The filtrate was saturated with sodium sulfate, allowance being made for the sodium chloride present. The mixture was adjusted to pH 7.0 and then filtered at 35 10 C. The filter cake, about I inch thick, was dried in trays for 6(U72 hours at a maximum temperature of 60 C and then packaged. About 55 per cent of the toxicity available in the starting material was present in the ricin-sodium sulfate, cake. The TU value '■* of the cake was 100- SECRET 186 RICIN 125. Analysis of the product showed 4.4 per cent moisture, 46.6 per cent ash, and 8.6 per cent nitrogen, of which 97 per cent was soluble and 45 per cent co- agulable.9 Electrophoretic and ultracentrifugal stud- ies indicated the cake to consist of several compo- nents with toxicity and hemagglulinating power associated with only the Bl fraction.3 Other studies indicated it to Ire composed of (1) the toxin, (2) a nontoxic protein otherwise very similar in properties to the toxin,3 16 (3) a dye derived from the bean shells, (1) an allergen, (5) an unidentified substance which tends to keep the toxin in solution at pH 7.0, (6) proteoses,14 mid (7) inorganic salts.12 Preparation 470 B.M 199 — About 100 lb of the product designated as 470 BM 199 was prepared 11 for field trials at Dugway Prov- ing Ground22 and Suffield Experimental Station, Canada.43 44 This ball-milled material was (he best available in sizable quantities from the standpoint of high toxicity and small particle size for the field tests held during the spring and summer of 1944. Ricin-sodium sulfate cake was the starting ma- terial for (he preparation of 470 BM 199. The cake was ball-milled for 15 minutes in an Abbe porcelain jar mill to yield a product that would pass through a 40-mesh screen and (hen slurried with 5 parts of its weight of carbon tetrachloride. The sodium sulfate tended to settle to the bottom of the mixture and the ricin concentrated at the Turface where it was re- moved by scooping with a wire screen. This flotation step reduced the salt content of the cake from about 45 per cent to 15-18 jrer cent. The flotation-purified ricin was suspended in carbon tetrachloride and the slurry ball-milled for 8 hours at room temperature in I1 4 gallon capacity Abb£ porcelain jar mill using 5,s-inch steel balls. The product was tray dried at 60 C for 2 hours and then at 82 C for 1 Yi hours, which gave a white friable cake readily disintegrated by ball-milling for 5 minutes. Considerable denaturation of prot ein accompanied the ball-milling operation. The TU values found for different samples of this material ranged from 00 to 100.1-9 Examination of a representative sample showed a particle mass median diameter of 6.3 n, 4.4 per cent moisture, 15.4 per cent ash, and 13.35 |>er cent nitrogen, of which 04 per cent was soluble and 14 per cent was coagulable.9 Product L703 11 A total of about 60 lb of spray-dried air-ground ricin was prepared.11 Eot 1,703 was examined in the laboratory fur toxicity by inhalation after dispersion as a dust 9 and similar lots L701 and 1,82(3 were tested in the field at the Suffield Experimental Station, Canada.46 The small mass median diameters, 3.1 n for 1,703 and 3.3 g for I.S'it),2'1'' are particularly note- worthy. The starting material for (he preparation of spray- dried air-ground ricin was (1) riein-sodium sulfate cake partially purified by flotation in carbon tetra- chloride, and included some (2) ball-milled and (3) hammer-milled products. Preliminary to spray drying, these materials were partially purified by another precipitation with sodium sulfate. The stal l- ing material was stirred with I parts of water, (he pH of the mixture adjusted to 7.0 ± 0.1, guhr added, and the mixture filtered at 30 C. Sodium sulfate (10.2 per cent of filtrate weight) was added to the filtrate. The resulting slurry was adjusted to a pir of 7.0 ± 0.1 and filtered at 30 35 C. The filter cake was dried at 00 C for 10 hours, ball-milled for 5 min- utes to pass a 40-mesh screen, and the sodium sulfate content was reduced by flotation in carbon tetra- chloride, Spray-drying 20 per cent aqueous solutions of this flotation-purified ricin gave materials with particle mass median diameters of 0-8 p. The spray-dried materials were processed in an air grinder to yield products with TU values averaging 200 and mass median diameters of 2.5-3.5 p." Analy- sis of lot L703 showed 2.0 per cent moisture, 19.7 per cent ash, 13.2 per cent nitrogen, of which 91 percent was soluble and 45 per cent coagulable, a TU value of 100, and a mass median diameter of 3.1 Preparation BU Preparation B1 is of interest because of its use for the preparation of toxoid. It was prepared as follows: 7* 2 K of ricin-sodium sulfate cake, which contained 71 mg of insoluble nitrogen and 050 mg of soluble nitrogen, was suspended in water and centrifuged. The precipitate was washed twice with 30 ml of water, to which was.added for the second washing about 0.1 g of sodium sulfate. To the solution and washings (300 ml) was added 175 ml of warm satu- rated sodium sulfate solution to precipitate the toxin, and the mixture was allowed to stand overnight. The precipitate was centrifuged and reprecipitated twice from a volume of 150 ml with 87.5 ml of warm (37 C) saturated sodium sulfate solution. Additional toxin can be recovered from the filtrates. B1 is about two-thirds as toxic as the crystalline material. The molecular weight of B1 was determined SECRET PHYSIOLOGICAL ACTION 187 to be 85,000 and the isoelectric point to Ik* 5.2. Crys- talline ricin and Bl seemed to differ only in toxicity, since by immunochemical, ultracentrifugal, and electrophoretic criteria they appeared to be identical. 12.5 PHYSIOLOGICAL ACTION' Systematic work on the use of ricin as a chemical warfare agent was begun in the United States during the fall of 1942. Its immediate objective was the pro- duction on a pilot plant scale of a sufficient quantity of an active product to make possible field trials of methods of dispersal of this novel type of agent. Such toxicological work as was done at this time was directed toward assisting in the control of (he plant process and toward (he accumulation of basic data on the inhalation toxicities of the product in various species of animal. When it Ix'came evident that the bulk production of a satisfactory material was feasible,1 1 u the ques- tion arose of the form in which it should be prepared for dispersal in the field. On the basis of experience in England,it was decided that it should be re- duced to a finely divided dry powder which could be introduced into munitions either in the dry state or in suspension in an inert volatile liquid. This decision made urgent the need for an extensive investigation of the relation between the particle size distribution in a toxic dust cloud and the inhalation toxicity of the cloud. Thereafter the chief emphasis of all as- pects of the program was on this complex problem. It was recognized that the significance of the pro- gram did not rest solely upon the potentialities of ricin as an agent for chemical warfare. Ricin was con- sidered, rather, as a readily available prototype of other unstable nonvolatile toxic agents of biological origin which might be exploited as offensive agents by one or other of the warring nations. The following subsection contains a summary of the available information on the parenteral and in- halation toxicities of standard preparations of ricin. This is followed by a review of the symptoms and pathology of ricin poisoning and a brief discussion of the mechanism of its action. 12,3.1 The Parenteral Toxicity of Ricin Details of methods of bioassay, of methods of field detection and assessment, and of the relation of parti- cle size to inhalation toxicity will be found in Sec- tions 12.5 and 12.0 and in Chapter 15, respectively. The summary which follows is concerned only with the toxicities for various species of standard prepa- rations of ricin under laboratory conditions. Ricin has been stated to be toxic for all verte- brates.5 Frogs are sensitive only if kept in a warm environment." Few invertebrates ap|x>ar to have been tested. The motility of a ciliate has been found to be arrested by low concentrations of ricin,5 but the relation between this effect of a preparation anti its toxicity for higher animals has not been established. The results of the few laboratories that have made comparative assays of a single preparation on a range of animal species are summarized in Tables 1 and 2. In Table I they are given as toxicities relative to the toxicity for the rabbit. The high sensitivity of the rabbit is well attested, but there is not full agreement on the order of sensitivities of other species. The ma- jority of the toxicities recorded in the literature have been based upon very few animals and are scarcely more than orders of magnitude. The most extensive series of observations are those made at the Uni- versity of Chicago Toxicity Laboratory [UCTL],* but even (best* can be accepted as precise only for the mouse and for the rabbit. Table 1. Relative LD- o’a (approximate) of ricin for dif- ferent sjx'cies * Author Osborne Field Hunt OSRD 5525’ Date 1905 HI 10 1918 1945 Reference til 56 2d 9 Route Sultou- lutrn- Subcu- Subcu- tancous muscular tancous tancous Rabbit I 1 1 1 Rat 1 1.5 Guinea pig 7 8 5 3 Mouse 8 S Sheep 2 D‘>g 7 16 2 Cat 2 16 10 Goal 30 ♦ An entry of 10 in this tabh hidicatea that for the species in question. ricin wan found to be one-tenth an toxic as for the rabbit. etc. In Table 2 some of the data on which Table 1 was based are given in absolute units. The preparation to which they refer exhibited about 28 per cent of the toxicity of crystalline ricin based on comparative assays on mice. Although the crystalline material is not believed to be molecularly homogeneous, it is definitely the most toxic material which has been prepared in contemporary work. It is suggested, therefore, that the best estimate of the attainable toxicity of ricin is obtained by dividing the LD:,,, for r By R. Keith Caiman. SECRET 188 R ICIN' Table 2. Kstimated LD ... (Mg /kg) Author Osborne Field OSHD 5525s (10-day observation) Crystalline Standard riein ricin* (computed) Rabbit 0.5 (7-day) 100 (2-day) 0.1 10 3 Hat 15 4 Guinea pig 3.2 (7-day) 100 (2-day) 0.8 30 9 Mouse 80 24 Sheep 20 7 Dog 500 (2-day) 0.6 20 7 Cat 100 (2-dav) 0.2 100 30 Goat • ... 3 • (3-day) ♦ The OSRD obwtvatiflns wort* made on the 1 pilot plant product (stand- ard i irtu). 1 ririn. tits had 28 per rent of the toxicity for mic c of crystalline tanoons and intravenous toxic! lies for I ho rabbit* are in the ratio of 1/5. • 2.3-2 Toxicity by Inhalation 1 he importance of the partic le size* distribution of the airborne toxin has been emphasized in the intro- duction. In one extensive investigation of this prob- lem,* two methods of varying the particle size wore user!. In one, animals were exposed to atomized aque- ous solutions of ricin containing varying amounts of glycerol. The mean particle size in the aerosol varied with the amount of nonvolatile solvent in the solu- tion. The other type of experiment was the exposure of animals to dust clouds generated from powdered standard ricin which had been reduced to varying degrees of fineness by milling or spray drying. The results are summarized in Table 3; standard ricin by 3.5. The figures given in the last column of Table 2 have l>een derived in this manner. The very high toxicities recorded by Field M for his preparation find no explanation. It is highly im- probable that they represent a product many times more toxic than crystalline ricin. On the other hand, his figures and those of Osborne do suggest that some of the early investigators of ricin succeeded in purifying the toxin to a degree approaching the purity of the crystalline material. The Relation of the Survival Time to the Dose The early investigators recognized that the time of survival of animals injected with ricin varied from a few hours to sc*vend weeks depending on the dose administered. This relation has been investigated for mice and, less extensively, for rats in several laboratories ».».■».«.« ;ind has formed the basis of the accepted method of bioassay (Section 12.5). The dose-survival time curves for mice obtained in one laboratory *‘l& have been found to approximate rec- tangular hyperbolas which may be represented by the-equations D{t — 11) = 430 (intravenous) D{t — 13) = 1,150 (intraperitoneal) D(t - 16) = 2,500 (subcutaneous) where I is the survival time in hours and D is the dose in micrograms of crystalline ricin per kilo body weight. Route of Injection The above results indicate that the relative toxic- ities for the mouse by subcutaneous, intraperitoneal, and intravenous injection, respectively, are (for the smaller doses) approximately 1 2.2 6. The subcu- A. Inhalation toxicitii Table 3 — s of atomized solutions of standard ricin. MMD (M) L(Cl )i„ (mg, min /in5) 1.4 4.0 fi.fl Rabbit 4 s 10 Guinea pig 7 15 Mouse— 9 40 45 Dog 24 45 Cat 24 50 Rat 50 120 Monkey 100 Preparation MMD(m) Rail-milled 10 0.3 Spray-dried 5.9 3.1 Atomized solution 1.4 ~ Relative lone Hies — Mice 3.5 2.8 0.5 0.5 100 Rabbits 5.7 5.3 30 100 % mass below 3 fi 7.5 10.0 3.0 15 loo? % mass below 2 fl 3.0 3.2 0.0 H 100? B. Inhalation toxieilies of dry dusts of standard ricin. lt would appear that the toxicity increased as the mass median diameter [.MMD] of the cloud dimin- ished. Indeed, then- is some justification for the eon- elusion. in the cases of mice and rabbits, that the toxicity was roughly proportional to the fraction of the airborne mass which was present in particle sizes smaller than 2 3 n in diameter. The reader is re- minded that the MM I) is an inadequate description of the characteristics of a dust cloud in which the particles differ in shape and density as well as in size and is referred to Chapter 15 for a discussion of the SECRET PfIVSIOLOGIC V1. ACTIO\ 189 relation of these factors to the probability that an inhaled particle will penetrate the nasal barrier. Although the most toxic aerosol was (hat with the MM I.) of 1.4 n it is improbable that this represents the maximum attainable inhalation toxicity. Some allowance for nasal retention and for incomplete re- tention in the lungs should probably he made. Even so, the inhaled doses of the 14-g aerosol for mice and rabbits, which may he computet! from the minute volumes of respiration and the L{Cl)50’s, are approxi- mately equal to the /./>.,o’s by intravenous injection.9 That is to say, Hein is at least as toxic hy inhalation as by vein. That it is probably more toxic in the lungs is indicated by the fact that the approximate when solutions were injected directly into the trachea of rabbits, was 0.5 Mg kilo. In cats, dogs, and rats it was about 5 Mg kilo.9 In contrast with these results were the very low toxicities resulting from the nasal instillation of ricin.9 When solutions of ricin are instilled in the eyes of animals in sufficient amount, enough may he al>- soribed to l>e lethal.-4 Only small amounts are neces- sary to produce serious local injury. The instillation of 1.5 Mg of crystalline ricin produced corneal damage in a rabbit’s eye which disappeared in 10 11 days.9 A particle of 100 m in diameter (0.5 Mg) implanted in the eye resulted in a conjunctival reaction persisting for a week. Corresponding lesions in the eyes of rats and guinea pigs required five to ten times this dost*. It must be remembered that only large particles will impinge in the eye from a cloud and that such parti- cles will tend to precipitate rapidly under wind con- ditions favorable for the persistence of a fine particu- late cloud. Clouds of fine dusts such as are highly toxic by inhalation would therefore be unlikely to contain a concentration of coarse particles which would present a serious hazard to the eyes. 12.3.3 The Toxicity of Ricin for Man The ingestion of two castor beans lias been fatal in man.24 54 6 8 It has l>een estimated that this corre- sponds with a lethal dose of about 0.3 mg of purified ricin per kilo. It has been suggested that ricin is about 100 times as effective by vein as by mouth.*" On this basis the intravenous lethal dose for man would lx* as small as that for the rabbit. Such com- putations arc highly precarious, hut other evidence has been advanced to indicate that man is quite susceptible to ricin poisoning.33 41 Elsewhere in this section are descrilied symptoms of mild poisoning in a number of individuals who had probably lioen exposed to low concentrations of air- borne ricin. It is significant however that no serious casualty has occurred in the pilot plant, in the ex- plosion pit at Dngway Proving Ground, or in labora- tories studying the dispersal of ricin. The atmos- pheres in all these places must have l>ecn contam- inated with ricin dust. It has also been suggested that the handling of solutions of ricin presents a skin hazard,4 hut the opinion of most investigators who have long worked with such solutions is that the hazard is small if ele- mentary cleansing precautions are taken. 12.3.1 Symptoms of Intoxication Laboratory animals show no evidence of intoxica- tion for several horn's after the injection of a dose which will kill them in 24 hours. Thereafter their fur liecomes ruffled, they grow restless, and refuse food. As the time of death approaches, diarrhea is frequent, breathing becomes dyspneic, (heir bodies feel cold to the touch, and their eyes may become sealed with exudate. Finally, the animals become moribund and die in coma or, more frequently, after a series of violent convulsions. With smaller doses the sequence of events is similar, hut their time course as well as the initial latent period arc more protracted. Some 150 cases of poisoning in man have been re- viewed.24 •64 M Most of these have been the result of the accidental eating of castor beans. In some cases weakness and prostration were the only symptoms. In more serious attacks, there was nausea and vomit- ing, epigastric pain, cramps in the limbs, a weak pulse, and a rapid respiration with a rise in body temperature. Fatal cases passed into collapse fol- lowed by convulsions. Symptoms might be delayed for 2 to 14 days, or, surprisingly, might be evinced within 1 hour after ingesting the t leans. Among the personnel working with ricin in the United States throughout 1943-45, there were no serious cases of poisoning, although there were a number of minor illnesses attributable to exposure. These were probably the result of inhaling airborne toxic dust. Two types of reactions among laboratory workers have !>een distinguished.9 One — the im- mediate reaction — resembles that of an individual sensitized to a foreign protein. The symptoms have varied from a protracted bout of sneezing to a severe asthmatic attack with violent coughing and retching. The symptoms disappeared within an hour. The second type of reaction probably corresponds to the toxic effect in animals. Symptoms were delayed for SECRET 190 HIC1N 4 to 8 hours. There was then a sharp febrile response, tight ness of the chest, tracheitis, aching joints, nausea, dyspnea, and coughing. Some hours later the onset of profuse sweating was commonly the signal of the alleviation of most of the symptoms. Somewhat similar observations have been made by the British, who have obtained local and general reactions by the intradermal injection of very small doses of ricin preparations 33 (see Section 12.4.3). I2.a .3 Pathology Accompanying the outward signs of intoxication in animals has been noted an early fall in body tem- perature,52 which may be preceded by a rise,57 In rabbits, it has been reported that the blood pressure falls from 100 to 65 mm of mercury at an early stage and remains at this level until death.7 There appear to l>e no notable changes in the blood picture.27 It is generally agreed 94 7 52 that about 20 hours after the injection of an dose there is a leucocytosis, with a simultaneous increase in both lymphocytes and polymorphonuclear leucocytes,10 A transient fall in red cell count has been recorded,9 10 but others report no change in red cell count, in red cell volume, or in sedimentation rate.52 Within 20 hours after an LD n dose the clotting time was found to increase to three times its normal value and remain at this level till death several days later.7 An extreme terminal hypoglycemia 7 and acidemia 62 have been observed in rabbits and in rats and a rise in blood phosphatase has been reported.52 Careful reports of the gross and microscopic pa- thology of animals dying after the parenteral admin- istration of ricin are found in the early literat ure.55-57 This information is reviewed and extended in Chem- ical Warfare Service Monograph 37, writ ten in 1918.21 Between this time and 1940, students of ricin became preoccupied with the chemical and immunological characterization of the toxin and with the hemag- glutmating activities associated with it. Little was added to our knowledge of the physiological action of the* toxin. During World War II, extensive patho- logical examinations of animals poisoned with ricin were made in England,*9 in the United States,91"-22 and in Canada.47 Some of these were confined to post-mortem examination of animals killed by the injection or inhalation of the toxin,9 22 39 whereas others relate to animals sacrificed at chosen times after the parenteral administration of lethal or sublethal doses.10 59 Bearing these differences in pro- cedure in mind, it may lx* said that there is substan- tint agreement between the laboratories referred to and the early reports in the open literature.45 57-6i It is possible, therefore, to summarize the situation in the following general conclusions. RahENTER AL A DM IXISTKAT Io\ 1. There is mild to moderate congestion and edema of the lungs. 2. There is mild degeneration of the intestinal epithelium at suprulethal doses only. 3. There is necrosis of the liver at and below IJ)Mi doses. t. There is hyixrphisia of the spleen at .sublethal doses and involution at higher dosage. 5. There is fragmentation and involution of the thymus at all doses. 6. There is congestion and delayed necrosis of the adrenal in rats but not in rabbits. The occurrence of pin-point hemorrhages through- out the body lias been emphasized by some 17 43 but minimized by others. Less consistent'findings have been necrobiosis of reticuloendothelial cells and hone marrow, cloudy swelling of the kidneys, and fatty degeneration of heart muscle. No differences be- tween the effects of crystalline ricin and of amorphous preparations have been observed 10 nor have any striking differences in the responses of different species been observed.* Exhalation The pathology is almost entirely confined to the thorax.9 '" The lungs are dark and greatly increased in weight and are filled with edema fluid. The ab- dominal organs are normal except, for some fatty de- generation and, occasionally, hyaline infiltration and necrosis of the liver. Ingestion The effects of ingesting the toxin have been in- vestigated in fatal cases of poisoning in man.24,54-** The chief post-mortem findings have been extreme congestion of the stomach and intestines. 12.it.a The Mechanism of Action of Ricin Such pathological work as was carried out in the United States in 1943-45 was incidental to the pro- gram outlined in the introduction. No systematic investigation of the mechanism of action of ricin was undertaken and our knowledge of this subject remains fragmentary. We are, indeed, as ignorant of the nat ure of the action of ricin as we are of the actions of those bacterial toxins which exhibit a sirni- SECRET IMMlNOLOGY 191 lar delayed effect and ill-defined pathology. Apart from revealing local effects depending upon the route of administration, pathological reports lie tray no characteristic lesions which would indicate the in- trinsic nature of the toxic action. The death of animals in convulsions is probably the result of hypoglycemia. It has been found (hat the blood sugar of rabbits and rats remains normal until a few hours before death, when it falls precipi- tously to convulsive levels.7 62 The toxic action, how- ever, is not primarily a reversible disturbance in carbohydrate metabolism. The liver glycogen is found to Ik- very low at death, but it has not been possible to induce glycogen storage in poisoned ani- mals by injecting glucose to maintain a normal blood sugar level. Nor has life been prolonged by this means.7 One of the earliest theories of the action of riein was that it was an enzyme. This was thought to ex- plain its great potency. It was also thought that its delayed action might plausibly lx attributed to the time required for the enzyme to build up a lethal con- centration of the hypothetical product of its activity. In this connection it should be borne in mind that several enzyme activities — phosphatase, lipase, esterase — are exhibited by extracts of castor beans. Purification of the toxin is not, however, accom- panied by enhanced enzyme activity. Indeed, it has been stated that crystalline riein is free from phos- phatase and lipase action.1213 Recently a Canadian laboratory has reported that riein preparations hy- drolyze adenosine triphosphate (ATP).43 60 They further observed that riein inhibited the I mat of the isolated frog’s heart and that the Ixat was restored by the addition of ATP. This would suggest that riein may act by interfering in those basic metabolic reactions whereby the energy of metabolism is con- veyed to the functioning structures of tissues. Data are, however, not yet available to indicate whether the concentration of crystalline riein which is re- quired for effective adenosine triphosphatase action is such as to make plausible the hypothesis that its lethal action is dependent on this property. More- over, in one investigation 7 the action on the frog’s heart was not confirmed. No increase in nucleotide in the blood of animals poisoner! with riein was ob- served. Riein did not cause a hydrolysis of ATP in the blood of dosed rats.62 It may lw submitted that it is just as plausible to attribute a disturbance in metabolism to the blocking or distortion by the toxin of the action of an enzyme native to the rolls of the animal as to consider it to be the result of the invasion of those rolls by a foreign enzyme in the form of the toxin. — An early theory of the action of rieiu was based on the hemagglutinating properties of riein prepara- tion.24*8 If (his action were manifested in vivo pro- found disturbances in circulation might be respon- sible for the toxic effect. Unfortunately the concen- trations of riein required to agglutinate red cells in vitro are greater than those established in body fluids by lethal doses of riein. Moreover, the agglutination of red cells is inhibited by serum 1 '* and crystalline riein is very much less potent as an agglutinin than are cruder preparations.216 Finally, the absence of thrombotic lesions would seem to deny the theory. Although the hypothesis has little to support it, it should lx* recorded that t issue cells as well as erythro- cytes have iKaai shown to be agglutinated by crude riein and, in the case of the tissue, cells, the action is accentuated rather than inhibited by addition of serum.** One investigator52 has drawn attention to (he similarity lietween intoxication by riein and circula- tory shock. He has found some evidence of dimin- ished blood volume in poisoned rats and of reduced peripheral circulation in the rabbit. The latter effect he was inclined to attribute to pooling of blood in the splanchnic area. He considered, but dismissed, (he thought that this condition might be due to capillary blockage resulting from agglutination in vivo. An in- cidental observation bearing on this question was that the rate of absorption of iron from the gut and the amounts deposited in tissues were increased in poisoned animals. He draws attention to a similar observation on animals in peptone shock.69 In conclusion it is worthy of remark that no effect of riein on unicellular organisms or isolated tissues has been clearly established. Much more work in this field is desirable as are more detailed studies of the time course of metabolic disturbances in poisoned animals and the level of differentiation of tissue or- ganization and function at which susceptibility to poisoning first becomes manifested. 12.t IMMUNOLOGY d In (he United States active research on the im- munology of riein was initiated in February 1943 by NDUC Division 9 (Section 9.4.2, Immunochemical d By Birdscy Uenshaw. SECRET 192 HK.1N Studies).3 ,8 Related work was subsequently taken up by other NDRC investigators,*141417 by the Chemical Warfare Service,2- 23 2,1 27 28 31 and by the Commit tec* for Medical Research.19 At the time the NDRC research began there were available, in addi- tion to the open literature* on riein, an account of studies carried out for the Chemical Warfare Service, during World War 124 and reports on preliminary work conducted in the United Kingdom during 1040, 1941, and 1042.33-3®'40'41 More recently Canadian in- vestigators have made a significant contribution.43 The principal objective was to provide and eval- uate immunological procedures for protection against and treatment of riein poisoning. With respect to protection, the aim — not yet attained — was the production of a toxoid which could practically lx* used to immunize troops. With respect to treatment, the problem — now satisfactorily solved— was the production and evaluation of potent antiricin serums and antibody globulin preparations. A secondary ob- jective was the study and evaluation of immunologi- cal methods for detection and estimation. By- products of the immunochemical work have been significant contributions to the purification ami physicochemical characterization of riein.31* For purposes of orientat ion it may be stated at the outset that immunological, ultracentrifugal, and electrophoretic studies on riein preparations from castor beans of different source and color have failed to reveal the existence of more than one heat-labile, toxic antigenic protein.318 On the other hand, a non- crystalline fraction (HI) from castor beans, which by these criteria is identical with crystalline riein, is not so toxic as the latter.319' Furthermore, solubility studies do not reveal the crystals to be homogeneous,2 It is also known that castor beans contain, in addi- tion to heat-labile toxic protein, one or more heat- stable antigenic substances of low molecular weight (allergen); small amounts of allergen appear to be present even in crystalline riein.14 43 12.4.1 Preparation of Riein Toxoids Incomplete success has attended efforts to produce from riein a toxoid possessing high antigenic potency coupled with negligible toxicity and skin-necrotizing properties. The available toxoids are satisfactory for eliciting vigorous antiricin production in animals. The best has been recommended for the active immu- nization of volunteers on an experimental scale but is not considered suitable for practical use in the routine immunization of troops. The most satisfactory toxoid has boon prepared by formalinization of the toxin as follows: 3 l8m riein at a concent rat ion of 0.5 nig riein nitrogen per milliliter in 0.15.1/ sodium chloride plus 0,02.1/ phosphate buffer at /di 7.4 is treated with 5 per cent formalin for 5 days at 37 C. Originally, partially purified pilot, plant riein (Hi fraction |Si-Vvas used. Recently crystalline riein has been utilized with sim- ilar results 1Ja and will undoubtedly be employes! in all future work. For best results the toxoid is pre- cipitated with alum or protamine. The resulting toxoid is about one-thousandth as toxic for mice as native riein.3-,8ml However, subcutaneous injection of as little as 0.1 eg of the toxoid nitrogen produces skin necrosis in some rabbits,19* and in the form of an aerosol the toxoid is only about 15 times less potent than native riein as a lung injurant.19* Some observers Ixdieve that formalinization in a more alkaline medium yields a better toxoid.14 Un- doubtedly a greater diminution of toxicity is effected under these conditions, but the indications are (hat antigenicity is more than correspondingly re- duced.3ISrtu Precise evaluations of toxoids prepared at pH values differing by only 0.1-0.2 unit are not available.21 The concentration of formalin is not critical; even high concentrations do not effect com- plete detoxification, and 0.5 per cent suffices to pro- duce a toxoid suitable for many purposes.3 Some consideration has been given to the chemical re- actions that occur during toxoid formation.1414 No success has attended numerous attempts to produce a toxoid more effective than that just de- scribed. Among the procedures to which riein has been subjected are the following: oxidation with chlorine or permanganate;40 ultraviolet irradiation at low intensities31St‘-f and for short times at high intensities; *IXu acetylation;3I8ik tryptic diges- tion;19a<' peptic digestion; 19b,‘ treatment with nin- hydrin;14 and heating.14A toxoid prepared by shaking riein with toluene showed some promise in pre- liminary tests but remains to be completely evalu- ated.191’ Injections of formalinized toxoid treated with normal serum and of specific precipitates of formalinized toxoid with antiricin rabbit serum proved unsatisfactory for active immunization.318' A few additional procedures have been suggested 21 but were not evaluated before the work terminated. A finding of significance is that a purified but non- crystalline fraction (HI) prepared from pilot plant riein is immunologically identical with crystalline riein but possesses only 00 per cent of the toxicity of SECRET 193 IMMUNOLOGY the latter.* 17b r l,“ This observation suggested (1) that some form of detoxified riein either exists in castor beans or is produced in the process of extraction and purification, and (2) that crystallization effects at least a partial separation of the toxic from the de- toxified material. That detoxified material immuno- logically indistinguishable from riein is indeed pres- ent in castor beans is suggested by the further finding that crude aqueous extracts from the beans also pos- sess considerably more immunologically active ma- terial per unit amount of toxic material than does crystalline riein.*asu.i**,!- ptp p, nmv p has been possi- ble to effect only a very incomplete separation of the toxic and nontoxic fractions.1** * However, further study of the conditions and factors responsible for the origin of detoxified riein in castor beans might lead to a solution of the toxoid problem.'19r In such work the changes which may take place in develop- ing and germinating beans should be examined. There is evidence that castor bean allergen *7 is not completely removed from the heat-labile riein by crystallization M or even by repeater! recrystal- lizations.14 Injections of a toxoid containing even small amounts of allergen conceivably might render men hypersensitive to the allergen contained in sub- sequently injected toxoid, and to sublethal dosages of airborne riein containing allergen. Some workers are inclined to minimize* the practical importance of this possibility; to others14 it has been a source of great concern. Animal experiments bearing on the point are reviewed in Section 12.4.4, and limited human data are presented in Section 12.4.3. 12.4.2 Antiricin Potent antiricin rabbit, horse, and goat serums have been obtained by a series of injections, first subcutaneous and subsequently intravenous, of riein toxoids.*1,28 Immunization can be continued with alum-precipitated but otherwise untreated riein. For therapeutic purposes the hyperimmune serums may be used as such, but the antibodies are preferably purified to lessen the likelihood of immediate reac- tions and scrum sickness. Standardization of Antiserums During World War 11 antiricin has been estimated with reference to an American Standard Antiserum arbitrarily assigned a potency of 100 units ml.!# 3,1 Each milliliter of this serum* contains antibody equivalent to about 7,500 mouse LDh«doses of riein; that is, by the toxicity test described below it neu- tralizes 200 ng of crystalline riein nitrogen1*1 or 500 ng of nitrogen of the relatively impure pilot plant preparation against which it was first tested.*11 Two tests have been developed for the quantitative assay of anti riein titer: M*® 1. Toxicity-neutralization. Solutions of known amounts of riein and of antiserum are mixed in 0.9 per cent saline, incubated at 57 C for x/i hour, and in- jected int rapedtoneally into mice. The minimum volume of scrum in the mixture for which mice sur- vive for 10 days is considered to he equivalent to the amount of riein used. The toxicity-neutralization test may be used to defect as little as 0.2 unit of anti- body and is the method of choice if time, permits. 2. Inhibition of hemagglutination. Portions of riein (e.g., 2 ,ug in saline) are mixed with decreasing volumes of scrum and saline is added to a volume of 0.8 ml. After incubation at 37 (' for hour, 0.2 ml of a 4 per cent suspension of washed human erythro- cytes of blood group () are added. The extent of agglutination is refolded after shaking and incubat- ing at 37 C for 1 hour. The minimum amount of serum which completely inhibits hemagglutination is considered equivalent to the amount of riein used. Because of nonspecific inhibition by normal serum,IM this test cannot be used to measure less than 5 units of antibody per milliliter.18*'1 In the choice of riein for use in this test consideration must be given to the fact that the hernagglut mating properties can be re- versibly masked under some circumstances.31518111’31* Potency op Antiserums The use of graded series of injections of riein t oxoid and/or native riein has yielded in rabbits antiserums having potencies as high as 250 imits/ml .* In horses serums possessing 150 units of antibody per milliliter have been obtained.* However, few animals have been observed with circulating antibody titers greater than that of the standard, and most animals in any series will attain titers more or less below it. Never- theless, the pooled scrum from a group of adequately immunized rabbits possesses what can be considered for therapeutic purposes a high and effective titer. Purification of Antiricin To reduce the possibility of reactions from the therapeutic use of antiricin scrum, methods which had been used for the partial purification of other antibodies were applied.* Sufficient experience has * Available at the Medical Research Lalwatory, Kdgewood Arsenal. SECRET 194 R1C1N been gained to make possible the production of con- centrated, partially purified horse or rabbit antiricin rapidly and on as large a scale as any program might require. Antiricin globulin from immunized rabbits was obtained in almost quantitative yield by 45 per ecut saturation of the diluted serums with sodium sulfate at 37 C. The precipitate was dissolved in water, merthiolate added as a preservative, and the solution sterilized by passage through a Chamberland fil- ter.18* 1 The ampouled material possessed an anti- ricin potency of 50 to 125 units ml and was pre- pared in sufficient quantity for distribution to the Chemical Warfare Service and NDRC laboratories engaged in work on ricin. Prompt intravenous in- jection of 25 ml was recommended in the event of accidental inhalation of ricin aerosols. Horse antiricin was partially purified by isolation of the pseudoglobulin fraction '*"' *,* h or by peptic digestion by the Parfentiev method.1*5 3lh The latter method was used to process Ifi 1 of horse plasma assaying 50 units of antiricin per milliliter. The yield was 1,090 ml of purified, modified globulin solution assaying 500 units/ml.1 * — Therapy with Anthucin Antiserum or purified antibody globulin is of con- siderable therapeutic value if promptly adminis- tered. Its effectiveness rapidly decreases with in- crease in the time between poisoning and therapeusis, and no benefit is obtained after the delayed symp- toms of poisoning have appeared.’18 31A0 Serotherapy is effective against injeetions of at least several lethal doses of ricin if sufficient antiricin is administered promptly.318 31 Typical data for mice are presented in Table 4.181 After exposure to airborne ricin the pathological effects occur mainly in (he lungs31' and the animals are not completely protected against pulmonary in- jury even by immediate therapy with injected anti- serum.31'1 11 However, the use of antiserum has defi- nite life-saving value up to 6 hours after gassing and is perhaps of limited lienefit even as late as 10 hours.3181"31'1 Illustrative data are presented in Table 5.18n The data reveal the desirability of utiliz- ing a large amount of antiriein. Equivalent amounts of rabbit antiserum, purified rabbit antibody globu- lin. and purified horse antibody pseudoglobulin ap- Table 4. Therapeutic use of antiricin in mice poisoned with riein by intraperitoneal injection.*"i Mice were injected intraperitoneally with about 20 lethal doses (2 of BI ricin nitrogen) and subsequently injected intraperitoneally with ten times the neutralizing equivalent of antiricin (rabbit antiserum). Mortality Treatment 0-24 hr 24-48 hr 2 10 da ys Total Xo serum 36 0 0 36/36 Serum 0.5 hr after ricin 0 0 0 0 37 Serum 2 hr after ricin 10 5 6/38 Serum 5 hr after ricin 9 3 9 21/37 Tabus 5. Therapeutic use of antiriein in mice poisoned with ricin by inhalation.”" — Mice were exposed for 10 minutes to an aqueous aerosol of ricin at a nominal concentration (about 8 neritoneally at various times after theexposure. Anliricin Time of admin- treat- Mortality istered ment 0 4 day! * 4-8 days 8-10 days Total 100 units None 20 0 0 - 20/20 1 hr 0 0 I 1/17 4 hr 2 3 0 5/19 10 hr 5 9 2 16/18 2-4 hr 17 3 0 20/20 10 units None 10 0 0 16/16 1 hr 2 4 1 7 20 4 hr 1 10 0 11/18 10 hr 11 7 2 20/20 pear to possess approximately t lie same therapeutic efficacy.*1*1' " Passive Immcnitv Passive immunity results from the injection of high-titer antiserum or partially purified antiri- cin.31s 3u b c *‘*f On the basis of these animal experi- ments, however, the protection cannot be expected to persist for more than 1 or 2 weeks at most. Thus passive immunization would have limited usefulness as a practical method for protecting troops. 12. t.3 Active Immunization against Ricin Injection or inhalation of native ricin in small doses evokes antiricin formation31*' and immunity2740 in surviving animals. The response is sometimes striking, particularly after repeated administration of the toxin. Practically speaking, however, active immunization must be attained the use of a toxoid. ( This material, put, up in amjxniles each containing 12 ml, is available at the Medical Research Laboratory, Eclgcwood Arsenal. SECRET IMMUNOLOGY Table fi. Resistance to airborne riein of rabbits immunized with six injections of formalinizcd ricin.19*' Tin; exposures in the gassing chamber were of 10 minutes’ duration. The 10-minute IAfor nonimrmmized rabbits was about 0.5 gg ricin nitrogen per liter. Thus the exposures were to approximately 4 and 20 times the A,(C/)5,i dosage. Interval between Serum antibody Cone, of ricin in Lung pathology Serum antibody titer in survivors last toxoid injee- tiler before eX|>o- gassing chamber Deat hs in 10 in survivors 14 days 14 days after tion and exjxjsurc sure (units) (#ig nitrogen /I) days after exposure. exposure 12 da vs 0.8 3.3 2.3 0 10 - to + + + 1-8 1.0-3.3 11.8 3/9 + to -f- T + 6.5-12 3 months <0.2 0.8 2.3 0/7 - to 4-4-4- 8->30 <0.2 0.2 0.8 4/7 + + to 4- 4~ + 12->30 5j months <0.2 0.6 2.1 3/8 — to 4- 4- 2 4 <0.2-11.2 11.0 5/7 -(- to 4-4-4- S- >30 Sn diks with Animals Several injection schedules have been used for studies with rabbits on the development of active immunity to inhaled ricin.3!9' The first schedule con- sisted of three subcutaneous injections at 5-day in- tervals of formalinized toxoid in the amounts of 2.5, 5, and 10 /ig of ricin nitrogen per animal, respec- tively. This schedule resulted in circulating antibody levels of 1-3 units per milliliter and many animals survived exposure to about 20 L{Ct)„0 dosages of air- borne ricin 10 days after the last injection. However, (he injections of toxoid produced severe skin re- actions with necrosis. A schedule of six injections conferred equal or greater immunity and the severity of the local reactions at the sites of injection was greatly reduced, although necrosis was not absent in all instances.19ac A dosage sequence of 0.1, 0.2, 0.5, 2, 10, and 20 of toxoid nitrogen is believed preferable to a schedule composed of doses of 0.1, 0.5, 2, 5, 10, and 20 gg. Some of the characteristics of the antibody re- sponse and protection effected in rabbits by six toxoid injections are illustrated by (he data of Table 6.,9r' Maximum antibody response and pro- tection is attained 10 to 20 days after the last injec- tion. At this time the circulating antibody levels are 1 to 3 units per milliliter of serum. The animals are immune to the lethal effects of at least several dosages of airborne ricin, but the development of lung lesions is not prevented. Circulating antibody titer then falls progressively to reach levels of the order of 0.2 unit per ml after 2 to 1 months.3 '■*' 3,‘- In spite of the low level of circulating antiricin, some resistance to airborne ricin porsis(s.,’,(i‘, t ,!k: After circulating antiricin has reached a low level, an injection of toxoid produces only a moderate in- crease in circulating antiricin.3 |Sr u In contrast, a very striking increase in circulating antiricin, to 5-30 Units per ml of serum, is produced by a single exposure to a sublet 1ml dosage of airborne ricin.,1lH" u- lih The effect is to be seen when the exposure follows by only 12 days the last of a series of toxoid injec- tions. If appears to be mow marked after longer times, when the circulating antiricin evoked by the toxoid injections has fallen to low values.1'1' A second exposure to airborne ricin does not elicit a pro- nounced further increase in the circulating anti- bodies.l8u These findings suggested that, for pur- 1 roses of active immunization, controlled exposure to aerosols of ricin toxoid might effectively reinforce the effects of toxoid injections. No studies on immuniza- tion by inhalation of toxoid have as yet been made, however, except for one experiment in which previ- ously unt reated rabbits were given a single exposure to airborne formalinized toxoid.,9a Twelve days later none of the survivors had develojred a circulating antiricin titer as great as 0.2 unit per ml. Challenge exposures to airborne ricin were not made. Actively sensitized guinea pigs possess consider- able immunity against the toxic effects of ricin.S18,, t Subsequent to exposure to 15 40 L(Ct)5n dosages of airborne ricin, a high proportion of the animals (i.e., 31 of 33) that recovered from the initial anaphylactic reaction survived indefinitely. This degree of re- sistance was present in the three animals tested as late as 116 to 173 days after sensitization. Although the results show that considerable re- sistance to inhaled ricin can be achieved by immu- nization with formalinized toxoid, it has been empha- sized that the resistance has been measured by a statistical increase in the number of animals surviv- ing challenge exposures and that the surviving ex- SECRET RICIN posed animals usually develop lung lesions.19*- Some of these lesions are severe and apparently predispose the animals to bronchopneumonia. Human Immunization With regard to immunization of large numl>ers of troops, representatives of the Surgeon General (Army) have indicated that practical considerations make it highly desirable to limit the numlier of in- jections of toxoid to one or at most two or three spaced over a period of 1 to (5 weeks.20 The animal experiments give no reason to believe that effective immunization can be obtained with so few injections of currently available toxoids at doses sufficiently small to preclude very severe local reactions. There is, moreover, no evidence that effective immunity, if once produced) would persist at high levels for more than a few months. No experiments on human immunization have been made. However, the use of formalinized toxoid at a dosage schedule similar to that employed for immunizing rabbits has been recommended as safe for test with volunteers. It was felt that data on the local effects produced and on the levels of circulating antiricin attained would help to orient the further course of the work. Numerous scrum samples from men working with ricin have been assayed for circulating antiricin by the toxicity-neutralization lest.3,,8dNo signifi- cant amounts of circulating antibody were found be- fore exposure to ricin. Considerable antiricin (i.e., up to 2.5 units ml) was found in the serums of men who had handled ricin at the pilot plant for several months or more. The highest levels were found in men having histories of either cuts and abrasions or symptoms traceable to ricin. There is no direct evi- dence as to the degree of immunity possessed by these individuals. In the experience of the University of Chicago Toxicity Laboratory two types of reaction to acci- dental exposure to ricin have been observed.9 17,1 One, a delayed reaction, sets in after a latent period of 5 to S houre. A febrile response then occurs, ac- companied by tightness of the chest, tracheitis, aeh ing joints, nausea, dyspnea, and coughing. In e viewed with suspicion in the case of detection and analysis of unknown ricin samples because of the possibility that hemaggluti- nating properties can la: masked.3-1518,1131* PnKOI CITIN' RKACTION Although the addition of ricin to normal serum produces a precipitate under certain conditions, much smaller amounts suffice to produce a specific precipitate with antiricin serum. Thus (he precipitin reaction is highly specific and sensitive; it is capable of delecting I pg of ricin nitrogen within 5 minutes, and much smaller amounts in longer times.3-18'1-1 Although less subject than hemagglutination to ex- traneous conditions, it must, be borne In mind that serum protein is precipitated by the ions of heavy metals which are present in smokes of various kinds.3 With the limitation that different ricin preparations possess different ratios of toxic potency to immuno- logical activity (Section 12.4.1), the quantitative precipitin test affords a very accurate method for the estimation of ricin.318p r■* Under optimal condi- tions it is accurate to about 1 per cent. Anaphylactic Responses of Sensitized Animals The anaphylactic response of actively sensitized guinea pigs appears to provide the most rapid, spe- cific, and sensitive method for the detection of air- borne ricin or ricin dusts that have settled on sur- faces.322 The animals must Ire watched, however, ami the possibility of desensitization guarded against.318,1 Passive sensitization was earlier em- ployer! by British investigators 33 40-41 but active im- munization has the advantages of inducing much more prolonged sensitization and. probably, greater sensitivity.3-,sik’n p-' The rapidity and sensitivity of the reactions was demonstrated by tests in which characteristic responses were evoked within 1-3 min- utes after exposure to ricin dust at the lowest nominal concentrations tested, 0.03 mK ricin nitrogen per liter.3Tin’s concentration was in the order of one-thousandth the 10-minute LCi0 for mice. Anaphylactic reactions of guinea pigs are known to 1m> highly specific. In the case of the studies with ricin, however, them has been debate, not yet re- solved. as to whether the reactions are due to sensi- tivity to the toxin itself or to contaminatings castor bean allergen. The evidence that guinea pigs can be sensitized to ricin itself, irrespective of the possibility that hj-persensitivily to allergen may also occur, may be summarized as follows: J. Guinea pigs passively sens!(ized by intravenous injections of rabbit antiserum to a fraction (Bl), which contained in relatively purified form most of the toxin in pilot plant ricin, were subsequently in- jected with fraction Bl and with fraction B3, a gummy fraction presumably containing much castor bean allergen but virtually free of ricin itself. Injec- tion of fraction Bl uniformly produced fatal ana- phylactic shock, whereas injection of fraction B3 produced much less severe reactions. The animals which received fraction’ B3 were fatally or severely shocked by subsequent injections of Bl.318k. 2. All guinea pigs that had been immunized by injections of ten times recrystallized ricin, of pilot plant ricin, or of alum-precipitated ricin toxoid showed anaphylactic responses when injected intra- venously with crystalline ricin or when exposed to airborne pilot plant or crystalline ricin at relatively low coticent rations.3 •17d 181 3. Guinea pigs injected with ricin develop con- siderable immunity to the toxic effects of ricin (Sec- tion 12.4.3). In general, immunity in guinea pigs goes hand in hand with hypersensitivity. Since the completion of this work, Canadian in- vestigators 43 have reported failure of attempts to elicit anaphylactic responses in guinea pigs sensitized with crystalline ricin and exposed to airborne pilot plant or crystalline ricin. Animals immunized with pilot plant ricin showed weak responses. On the other hand, animals given a single injection of castor bean allergen M-R7 reacted vigorously to crystalline ricin as well as to pilot plant ricin in low concentra- tions. These data, considered in conjunction with supplementary results obtained by the use of the Schultz-Dale technique, led to the conclusions that * The sensitivity chiimcd for the hemagglutination reac- tion in reference 27 appears to be in error. SECRET 198 iiir.iv the sensitization was to allergen rather than to toxin, and that allergen is present in crystalline ricin. Addi- tional evidence that a small amount of allergen is present even in many times recrystallized ricin has since l>een presented.14 Further work is required to clarify the apparent discrepancies. In any event, it is evident that guinea pigs can be prepared in such a way as to render thorn highly susceptible to anaphylactic shock upon ex- posure to very low concentrations of all known riciir preparations. The impract icability of employing (he reactions of animals other than dogs as routine methods of de- tection in warfare has often been emphasized. How- ever, general considerations as well as the results ob- tained during field trials with ricin 3 22 would indicate that sensitized guinea pigs could be of great value in the hands of special officers assigned the duty of checking upon the possible use of protein toxins by an enemy. 12.5 \SSA Vh It was early recognized that the toxicity of castor beans was associates! with the water-soluble, heat- coagulable protein of the beans.*4 55 The presumption was that the toxicity was the unique property of a single protein component, and this hypothetical com- ponent was designated ricin. In 1943, a crystalline protein was isolated from extracts of castor beans.5-15 Its toxicity was reproducible and was about twice as great as that of the most active amorphous product available.9-15 This result greatly strengthened the presumption that there is present in castor beans a single toxic protein component. However, although the crystalline product has met some, it has not met all, of the criteria of molecular homogeneity which are required of a single protein.2-315 The possibility cannot yet be rigorously excluded that the toxin is a complex whose components may. some day, be sepa- rated from one another and may then be found to be active only in association with one another. As long as this possibility remains, the only assay of the toxin of castor bean preparations to which no objection can l>e raised is an estimation of the toxicity under ap- proved experimental conditions. Theoretically, the measurement of any physical, chemical, or biological property which has !>een shown to l>e quantitatively related to the toxicity should serve as an assay. Un- fortunately, the demonstration of the existence of such a relation cannot he complete until the pure toxin has I>een isolated and fully characterized. A variety of properties of extracts of castor beans have been proposed as bases for assay and these will lie reviewed briefly. The problem of bioassay will, how- ever, lie considered first and in fuller detail. 12.3.1 Bioassay All vertebrates that have been tested are suscep- tible to ricin (see Section 12.3). Few observations have lieen made on invertebrates. It has been re- ported 5 that the motility of certain sfieeies of pro- tozoa is arrested by the addition of ricin to the medium, but it has not been established that the po- tencies of a series of preparations of ricin are propor- tional to their toxicities. Difficulties in controlling the action on the protozoa led to abandonment of the attempt to use these organisms as a means of assay. Ricin acts slowly on vertebrates. With minimum lethal doses, animals seldom die in less than 5 or <> days, and may survive for weeks. Assays based upon the estimation of median lethal doses are, therefore, protracted and require an arbitrary choice of obser- vation period. They also require large numbers of animals for statistical validity because of incidental variables such as casual infections. Where many assays must be carried out, there are obvious advan- tages in the adoption of a method which gives quick results and is economical of animals. The value of establishing a relation between the dose of ricin and the survival time in a given species as a basis of a method of assay was urged in 1918.24 In this country the problem has been investigated in three different laboratories.5 *15 ®1 Work was also done in Canada41 and in England.®-37 The mouse has been the favored animal, being preferred to the rat.12 Several homozygous strains have been used,4 9 the most popular one in the United States being the CFl strain of white mice developed by Carworth Farms, New City, Rockland County, New York.5 *-14 A method of assay based upon 24-hour mortalities has l>ecn adopted. Groups of five or preferably ten mice weighing 20-25 g are injected with a series of graded doses by the intraperitoneal route. The indi- vidual survival times of the animals are recorded and the mean survival time for each dose is derived. By interpolation in an accepted dose-survival time rela- tionship, a value for the dose corresponding to a mean survival time of 21 hours is, then, obtained for each experimental dose. This 24-hour lethal dose has 1 By R. Keith Canaan. SECRET ASSAY 199 been designated the toxicity unit [TU], Because of the skewness of the distribution of survival times, one investigator 4 prefers to convert each individual death time in a group to a TU and to average these to give the TU for the group. It has been customary to express the TU in micro- grams of the preparation per 20-g mouse though it is sometimes more useful to express it in terms of the total nitrogen or the eoagulable nitrogen present in the preparation. The desirability of making a simul- taneous assay of a standard product has l>een empha- sized by all laboratories concerned with the evalu- ation of ricin preparations.5 915 When this is done, one may then readily express the toxicity of an un- known preparation in terms of the per cent of the standard which it contains. Crystalline ricin is the logical reference standard. No difference in susceptibility to ricin of the two sexes of the CF1 strain of mice has been detected.9 In the cases of two other strains, small differences have lieen recorded.5-4- Fean mice seem to lie more resistant than fat mice of the same weight.4 42 This is probably because a greater proportion of the body weight of the lean animals is active tissue. Studies of the effects of diet 5 also have led to the conclusion that the toxicity is a function of the ratio of active tissue to body weight. Mice to be used for assay should he fiee from parasites. The susceptibility of young mice is found to be greater when the environ- mental temperature is elevated and is reduced at low temperatures.4-42 This is presumably due to corre- sponding changes in body temperature. Frogs are also sensitive to ricin only at elevated temperatures.*5 In the conduct of assays with mice, the temperature at which the mice are kept should be controlled. The preferred temperature has been close to 25 In one laboratory,5 automatic devices for the con- trol of temperature, the injection of the animals, and the recording of individual death times have been employed with the object of rendering the conditions of assay as uniform as possible. If the preparation of ricin is very active, it must be diluted to a concentration of 10 50 mg per liter be- fore injection. The danger has been emphasized 5 of losses by adsorption on the walls of the vessels in which the solution has Ix-en prepared. Adsorption is said to I>e significant even when paraffin-coated vessels are used. To reduce this error, a solution of 0.3 percent egg albumin has Ixren used as the diluent on the principle that the excess of inert protein will inhibit the adsorption of the toxin.5 Shorter sur- vival times are observed when this procedure is adopter!,5 * IS but there has Ihh-ii debate as to whether this was due to the suppression of adsorption or was the result of a synergistic action of the egg albumin. The modified technique has not found general ac- ceptance.9 14 When a simultaneous assay of crystal- line Hein is made and the result is expressed as the per cent of crystalline ricin in the product, it is prob- ably immaterial whether water or 0.3 per cent egg albumin is used as the solvent. On the other'hand, toxicity unit values derived from assays of egg al- bumin solutions of ricin arc consistently smaller than those of aqueous solutions and are not directly comparable with them. The Dose-Sihvival Time CTkve koh Mice For a given route of injection, this curve approxi- mates a rectangular hyperbola of the type repre- sented by the relation:5 9 15 if) - /)-) __ it ~ tm) where I) is the observed dose, I is the observed sur- vival time, and k is a constant characteristic of the preparation of ricin. Dm and tm are constants having the qualities of an extrapolated minimum lethal dose and an extrapolated minimum survival time respec- tively. Over a wide range of lethal doses a single pair of values of 1)m and f,„ fils the experimental observa- tions only very roughly. Over restricted ranges of survival times, however, values of the constants can l>e so chosen as to fit the data quite satisfactorily.* For short survival times, /)„. is small relative to D and may be ignored. Then, since the toxicity unit is the value of D when I = 24 hours, the above equa- tion can be rewritten in the form: TO-B? (< - This assumes that tm is independent of the nature of the material which is being assayed. If this assump- tion is not correct (and it has been implicitly ques- tioned) 5 the assay of an unknown product by in- terpolation in a standard curve would lie subject to error. However, extensive observations 915 have indi- cated that the use of tm = 13 hours satisfies the in- traperitoneal data for both crystalline ricin and standard ricin over a range of survival times of about 18 to 30 hours. These laboratories have, accordingly, adopted the following equation for general use: SECRET 200 RICIN TU = H D (f - 13)’ In order that the uncertainty of interpolation should l>e minimized, it is recommended that values of TU should lx* computed only from mean survival times falling within the limits of 21 and 28 hours. A summary of the data of one laboratory 9 on crystalline ricin and standard ricin is given in Table 7. In Table 8 will lx- found a comparison of the results M'iik Quantitative Precipitin Method3 Antiserums prepared by the injection of crystalline ricin have been found to precipitate from extracts of castor beans, and amorphous preparations generally, not only the toxin, but a noutoxie protein.* '6 Since this material is antigenically indistinguishable from the toxin, it may l>e called a natural toxoid. In the preparations that have been tested, the ratio of toxin to toxoid has varied from 1 1 to 2 I. Only by the process of crystallization has a separation of the two antigenic components been accomplished. If this limitation of the "method is borne in mind, the pre- cipitin technique (see Section 12.4) is a valuable method of assay. It requires only a few micrograms oLpurified material and gives a positive result within a short time. H KM AGGLU T l N ATI O N There is a wealth of evidence that the hemagplu- t mating activities of ricin preparations do not parallel their toxicities.*5-16 24 :t4 Crystalline ricin has, for ex- ample, only about 20 per-cent of the agglutinating power of amorphous preparations which are consid- erably less toxic.*16 The method can, therefore, have only limited use. A method of assay based on hemagglutination (sex* Section 12.1) has been proposed for use in the field.2-’ 23 26 Agglutination tests are rapid and require only small samples of material. However, the cus- tomary method of evaluating the agglutinating potency of a sample is only coarsely quantitative and depends on subjective discrimination by the ob- server. An attempt to increase the objectiveness and precision of the method has been described.16 Enzymic Activity The observation that crystalline ricin does not ex- hibit the esterase, phosphatase, and lipase activities of crude preparations eliminates these properties as means of assay.5 It has recently been reported 43,50 that ricin preparations hydrolyze adenosine triphos- phate. If it should be established that this activity is proportional to the toxicity in a representative series of preparations, a valuable alternative to bioassay may become available. Chemical Methods No chemical property of the toxin is known which distinguishes it from other heat-eoagulable water- soluble proteins of the l>ean. However, water ex- tracts from castor bean contain little coagulable pro- Tabu-; 7. Summary of a series of assays of crystalline ririn and of standard ricin carried out at intervals over a period of 13 months.’ The CFl st rain of mice was used. Solutions were prepared and diluted with water. Crystalline ricin Standard ricin Male Female Male Female Total number of assays 23 22 13 12 Mean toxicity unit pig of material — I>er 20-g mouse 2.00 2.04 0.06 7.01 Standard deviation 0.21 0.17» 0.7!t 0.72 Table S. Dose-survival time relationsJor different routes of injection. The CF1 strain of mice was used. Solu- tions were prepared in water and injected in a volume equal to I |xt cent of the body weight. Standard equation: 0(1 — l„) = k. Intravenous Intraperitoncal Subcutaneous k (rig-hours) 8.C 23 - 50 l„ (hours) 11 13 1C TU ng 20 k 0.0C “ 2.1 0.3 LD:o egkg 2.2 10.4 22.1 of intravenous, intrapeiitoneal, and subcutaneous injections.* The method of intravenous injection is technically too difficult for routine assays. All in- vestigators have agreed that intraperitoneal injec- tions yield more precise and reproducible results than do those by the subcutaneous route. Subcutaneous toxicities have been found to vary to a remarkable degree with the concentration of the solution which is injected.* 12.5.2 Alternative Methods of Assay A variety of properties of ricin preparations have l»eon proposed as bases for assay. These include the antigenic properties, the hemagglutinating potency, and various enzymic activities which have luxm found in crude preparations of ricin. The weight of evidence is that none of these are specific for the active toxin. Some of them are valuable for the com- parison of limited types of preparation, but must be supplemented by bioassays in critical situations. SECRET evaluation as a war <;as 201 tein other than the toxin and the toxoid.915 In such extracts, an estimation of the heat-denaturuble pro- tein gives a result only slightly greater than the esti- mation of the protein precipitated by antiserum.915 In extracts prepared with salt solutions, on the other hand, a large additional amount of coagulable pro- tein is present.15 54 Fortunately this is denatured at or below /dl 4. If such extracts are acidified and filtered, the soluble coagulable protein which remains corresponds closely to the sum of the toxin and tox- oid. The heat-coagulable protein has usually been estimated as the difference between the soluble pro- tein before and after boiling for 15 minutes to 1 hour at 100 C. Any acceptable met lux 1 of protein determi- nation may be used which is adapted to the amount of protein present.'5 I2.r>.;t Field Detection and Assay For the rapid detection of airborne ricin, the sensi- tized guinea pig is undoubtedly the most sensitive and specific*-" (see Section 12.4). The maintenance and care of sensitized animals in the field, however, present many difficulties. Moreover, there is some question whether the anaphylactic reaction is elicited by the toxin or by a so-called allergen which may be separated from it.5 53 Any other method of detection or assay requires the collection of samples adequate in amount for the test which is to be performed. Certain color tests have .been proposed and are both sensitive and rapid.5 In so far as they are simply tests for protein or for the carbohydrate commonly associated with protein, they are entirely nonspecific and are of value only in indicating the possible presence of ricin. More spe- cific, but less suited to field work, are the hemag- glutination and precipitin tests. Some limitations of the former have been mentioned. The precipitin re- action is decidedly more specific and more accurate. It has, however, been pointed out that the heavy metals present in some smokes will give nonspecific precipitates with serum proteins. Neither test gives an immediate response. An assay of toxicity is, of course, the most dilatory of all. It may lx* appropriate, in conclusion, to remind the reader that the hazard of exposure to a non-vola- tile airborne toxin cannot be evaluated simply from the time of exposure and the concentration of toxic material in the cloud. The inhalation toxicity is de- termined in large measure by the particle size distri- bution in the cloud. The relation of particle size to toxicity is discussed in Section 12.3 and in Chap- ter 15, w here methods of evaluating the particle size distribution in a cloud are reviewed. In contemporary field trials22 with standard ricin, it was found profit- able to assay the cloud not only for toxicity and for particle size but also for total protein and for heat- coagulable protein. The two latter estimations pro- vided useful information on the extent to which the method of dispersal resulted in detoxification and denaturation of the material with which the muni- tions had l>een charged. The results of field trials are reviewed in Section 12.6. , / 12.6 EVALUATION VS V WAR GAS1 The performance of field trials on munitions charges! ricin and the interpretation of (he results of these trials in terms of evaluation of the agent as a war gas rest in large part on the laboratory researches in the United States, Canada, and Great Britain on particulate sampling and bioassay (Chapter 15b The most significant criterion for effectiveness of ricin in the field was bioassay by animals exposed to the particulate cloud. Physical measurements were essential to an understanding of the reasons for poor or good results in the several trials and as a guide for design of subsequent trials. Low toxicity in the field could be associated with many variables, including large particle size arising from compaction and aggre- gation, thermal inactivation of the sensitive protein agent, inefficient munition functioning, and meteoro- logical conditions. The field trials also rested on the prior development of pilot plant methods for the preparation of finely divided ricin (Section 12.2). 12.6.1 Kelative Efficiency of Dispersion by Different Munitions The principal types of munitions and chargings which have been studied for the dispersion of ricin are the following: 1. High explosive-chemical bombs charged with a suspension of ricin in carbon tetrachloride. Bombs of this type, with steel casings and axial bursters, were employed in the British experiments carried out in 1941 ■i3 M and in the recent Canadian trials.4* Muni- tions of this type retain to a significant degree the effectiveness of ordinary HE fragmentation bombs. 2. Light-case metal bombs charged with dry ricin. The Canadian 4 lb L.C. bomb was a metal can hold- ing about 550 g of ricin and fitted with a small burster • By Stanford Moore. SECRET 202 IUC1N (e.g., 20 g of nitroguanidine and 70 g of sodium bi- carbonate).'”43 44 3. Base ejection bombs charged with dry riein. The U.S. M-71 10-lb tail ejection incendiary bomb was modified for use with about 385 g of dry riein."-24 4. Gas ejection bombs charged with dry riein. N DRC Division 10 carried out developmental work on a two-compartment bomb holding liquid carbon dioxide or compressed air in one compartment, which on functioning ejected the particulate charging from the second compartment." 5. Plastic and glass bombs charged with a susjkmi- sion of riein in carbon tetrachloride. Experimental munitions of this type developed by NDRC Divi- sion 10 were similar to (1) above but with plastic or glass casings instead of steel.44 The lines of investigation on dispersion of riein from the various types of bombs at the several field experimental stations have led to the same general conclusions. The results indicate that high explosive- chemical bombs charged with a 35 per cent suspen- sion of riein in carbon tetrachloride ate superior to the dry powder munitions in their ability to put up a cloud in which the volume mass median diameter is sufficiently small to pass the nasal barrier.®*-33-3*- 37-44 46 This conclusion confirms the earlier analysis of the problem made by British investigators in 19413,-36 on the basis of a less complete series of experiments. In the field trials plastic bombs have given results comparable with those obtained with steel bombs but the British 4-lb HE Chem Type F Mk I steel bomb, as used in the later Suffield trials, possessed the advantages of availability in standard design and durability in transport. The lower dispersion efficiency of the munitions charged dry powdered riein was largely the result of the formation of aggregates in the particulate clouds.22 Comparisons were based on parallel tests employing a given sample of powdered riein set up both in the dry and suspension forms.44 In trials with dry samples, aggregation of the initial particles of the riein charging to yield a cloud of larger mass median diameter was increased by increase in the moisture content of the charging or in the relative humidity of the atmosphere." In the 1941 British trials 33 36 it was concluded that bombs filled with riein suspended in carbon tetra- chloride were at least three times as effective as simi- lar bombs filled with a solution of riein in water. In more recent tests with plastic bombs the solutions in water were also found to lie loss stable to detonation than suspensions in carbon tetrachloride.17c-,i-f The munitions were functioned in a stainless-steel ex- plosion chamber at the NDRC University of Chicago Toxicity Laboratory and a material balance deter- mined. No measurable denaturation was observed in the ease of the suspensions in carbon tetrachloride, whereas a 40 per cent loss in toxicity occurred with the aqueous Hein solutions. Chamber trials on the plastic munitions were also carried out at the Divi- sion 10 NDRC Munitions Development Labora- tory.' 12.6,2 Comparison \silb Bombs Charged Phosgene On the basis of the early trials with suspensions of riein in carbon tetrachloride the British investigators concluded in 19413336 that bombs filled with riein were about as effective as phosgene bombs of the same size. With improvements made in the pilot plant manufacture of dispersible riein since that date, and progress in the testing of munitions, the relative effectiveness of riein has been increased to a position well above that of phosgene. The compara- tive data have been analyzed by the Suffield Experi- mental Station.46 From calculations of (he dosage contours from the field trial data the munition ex- penditures required for 80 per cent coverage of a target area with a riein dosage of at least 100 nig/ min m3 have l»een calculated. The L(Ct)ui of riein for man is not known. The results of the field experi- ments indicate that for goats in the field the L(Ct)w of the present pilot plant samples of riein dispersed by the 4-lb UK Chem Type F bomb is about 100 mg min nr*. For the present calculations it is assumed that this value holds for man. Employing (he meth- ods of calculation applied to the test data on phos- gene 33 it is estimated that for 500-lb clusters of Type F bombs an expenditure of 1.2 clusters (43 lb of riein) per 100x100 yard square would cover about 80 per cent of the target area with an L(Cl)t,o dosage on open terrain (neutral temperature gradi- ent; wind speed less than 12 rnph). For 500-lb bombs charged phosgene under the same conditions the estimated expenditure is 8 bombs (1,000 lb of phos- gene) pel- 100x100 yard square for coverage by a dosage of 3,200 rng/min/m* within 30 seconds or 4 Ixjmbs for coverage within 2 minutes. The com- parison is based on tests with a batch of spray-dried 1 These are reviewed in the Summary Technical Report of Division 10. SECRET EVALUATION AS A WAR GAS 203 air-ground ricin with a volume median diameter of 3.3 /i which yielded clouds of volume mass median diameter of about 15 g. From this it is concluded that ricin appears to !>e at least seven times as effective as phosgene on the basis of aircraft stowage when the comparison is based on a 30-second dosage of phosgene. If the L{Ct)so for phosgene is considered to be high by even a factor of two there would still l»e a margin in favor of ricin. Since ricin in carbon tetrachloride gives no detectable odor, the comparison on the basis of a 30-second dosage is suggested as the fairest compari- son. On the basis of weight of active agent employed, rather than the weight of munit ion, ricin has a superi- ority over phosgene of 40 to I from these data.4* 12.6.3 Ricin as a ar Gas Ricin is an odorless powder capable of hieing dis- persed as a particulate or dust cloud. The absence of odor and the complexity of the consequent detection problem in the field w ould render ricin more insidious than any standard U. S. or British chemical war- fare agent. Comparison with the German Trilons (Chapter 9) would present a closer differentiation problem. The physiological effects of ricin are de- layed. Lung injury, similar in character to that pro- duced by phosgene, can lead to deaths at from one to several days after exposure. Ricin can be dispersed in munitions not readily distinguishable from stand- ard HE bombs. For detection in the field attention has been given to hemagglutination tests and to the use of ricin- sensitized guinea pigs (Section 12.4). These methods are intrinsically more difficult in practice than the simple means for detecting such agents as mustard gas or phosgene by odor or chemical tests. The IT. S. and British gas masks, when well adjusted, give complete protection against any dosage of ricin likely to be produced in the field.37 The immunization of troops against ricin and serum therapy present diffi- culties, as outlined in Section 12.4. From the. few tests on (he persistence of ricin in (he field it has been concluded that the major part of the agent is rapidly dissipated in the particulate cloud. Only in the area immediately around the point of burst was ground contamination sufficient to be measurable. In tests in which sensitized guinea pigs were allowed to run through the brush in this area a possible hazard was detectable for about 3 days in dry weather.32 As a result of the progress made during-World War II on (he preparation and dispersion of ricin it must be considered that hrall-out chemical warfare it is possible that ricin could lx? employed in a prac- tical role in chemical Supply and manu- facture would place a ceiling on (he scale of use but, would not prevent the accumulation of significant quantities of this agent. It has been estimated that the cost of production of dispersible ricin on a large scale would be approximately $13 per pound (Sec- tion 12.2). In the course of the research during World War II the work on ricin has served to advance the knowl- edge on the general problem of particulate disper- sion. In some respects ricin has served as a model substance for work on the dispersion of agents of similar chemical and physical properties in the re- lated research in the field of bacteriological warfare. SECRET Chapter 13 AROMATIC CARBAMATES Arthur C. Cope 13.1 INTRODUCTION Beginning in 1913 under the auspices of the Na- tional Defense Research Committee [NDRC], search for a superior nonvolatile toxic agent was un- dertaken by several cooperating laboratories. Cri- teria for the agent sought were extreme toxicity on subcutaneous injection, rapid lethal action, ready availability through practical synthesis or otherwise, and sufficient stability for military use and storage. A survey of the open literature 17 and information currently available concerning the toxicity of chem- ical warfare agents guided the search. Among the more toxic classes of substances known, botulihus toxin, other bacterial toxins, and potent plant toxal- bumins (particularly ricin) were considered unsuit- able because of slowness of their toxic action, immunological characteristics, and in some cases in- adequate sources of material for possible use on a considerable scale. The alkaloids physostigmine and aconitine were high on the list of toxic substances. I I CHa CH, Physostigmine Aconitine is the more toxic of the two, but its com- plete structure is unknown, and search for a toxic agent among simpler related compounds is unprom- ising because minor structural modification of the toxic aconite alkaloids often destroys their toxicity. On the contrary, many synthetic N-alkylcarbamates related to physostigmine are highly toxic, and search for a superior agent in this class appeared more promising. For this reason, and after failure to ob- tain highly toxic compounds in several other classes, the investigation soon turned to a thoiough explora- tion of the carbamates. Similar studies were con- ducted at an earlier date in England by R. D. Ha- worth and his associates, and in Canada by Leo Marion and others. The following investigations of carbamates reported in the open literature preceded all the classified work. After establishment of the structure of physostig- mine. by Stedman and Barger,42 a number of syn- thetic analogs were prepared by Stedman. Several of his papers 13 47 describe the synthesis and mi- otic properties of such analogs, but contain no toxi- cological information. White and Stedman 4 ■ report a detailed pharmacological study of miotine, the syn- thetic miotic of choice from the group, including toxicity data for this substance and three related carbamates. Aeschlimann and Reinert49 report toxicity and other pharmacological data for physo- stigmine and 44 related synthetic carbamates, many of which had been prepared earlier by Stedman. Stevens and Bell tel ** also have investigated physo- stigmine substitutes, and report chemical and toxic- ity data for 27 related carbamates. The toxicity data for such compounds are recorded in the open liter- ature.484*’5’ As a result of the classified British and Canadian work, TL 1071 (British code T-1708) was the lead- ing candidate in the carbamate group. The com- pounds TL 1217 and TL 1299 proved to be the agents of choice on the basis of the NDRC work. (If, (iV)COXHCH, ([ V>COXIICH3 AoOONHCH, u u u NfCdLhCILI NfCdbhCIbCl X(CH3)jI Tt, 1217 TL 1299 11, 1071 (T-1708) Additional compounds that received more or less detailed study were: CII, /ytcoxiiCHi if Ycoxiich, NfCjHilsC'HjX X(CHj)jX TL 1217; X - I TI. 1071; X -1 TL 1299; X = Cl TI, 1236; X = Cl TL 1317; X = CILSO, TL 1185; X = ClIiSO. TL 1186; X = USO, (I \)('OXHCTb chv X(CH,).X TL 1210; X - I TI. 11.53, X = Cl TL 1188; X = CUiSO, SECRET SYNTHESIS 205 0OCON(CH,)j /Vk’OXHCH, X(CHj)jN[I J CII(CHi)t CH(CH,)t TL r,90; X - t TL 1327; X = I TL 1443; X » Cl TL 134.>; X - Cl In the following pages work on the carbamates which might be of some practical importance as toxic agents is summarized. Investigations which led to selection of leading candidates are mentioned briefly. 13.2 SYNTHESIS 13.2.1 m-Dielhylaininophenyl-N-methylear- hamate metbiodide ('LL 1217) The most practical preparation of TL 1217 is the following sequence: 0)11 CH.NCO- jj Vx'ONHC.'lb ri4 l> i[ \kqxhch, V - v NtC.IL), N(C.ITi). MtMLf.CH.I This process was operated successfully on a pilot- plant scale.15 Approximately 360 lb of methyl isocya- nate were prepared by reaction of methylamine and phosgene in the vapor phase to give methyl ear- Oamyl chloride, which was converted to methyl isocyanate by treatment with pyridine in toluene. The average yield was 81 per cent, w-Diethylamino- phenol (a commercial dye intermediate) dissolved in dry benzene was refluxed w ith an excess of methyl isocyanate for several hours, m - Die thy lam i nophenyl- N-methylcarbamate was isolated in yields of over 80 per cent by evaporating the solvent under reduced pressure, filtering, washing, and drying. TL 1217 was prepared by reaction of m-diethylaminophenyl-N- methylcarbamatc with methyl iodide in acetone un- der reflux. After addition of ethyl acetate, the prod- uct was recovered by filtration in yields of 79 to 86 per cent. The product so obtained was of high purity, as verified by elementary analyses, use of a special analytical procedure involving hydrolysis and determination of carbon dioxide and methylamine,13 and toxicity tests. Essentially this same procedure had been used earlier for the preparat ion of TL 1217 on a laboratory scale.1-25 This compound is among the group de- scrihed by Aeschlimann and Reinert49 and by R. D. Haworth.23 Prior to development of a practical syn- thesis of methyl isocyanate, m-diethylaminophonyl- N-methylcarbamate was prepared on a large labora- tory scale in yields of 71-86 per cent by reaction of m-diet In land nophenol with phosgene in the presence of diethylaniliuo, followed by reaction with methyl- amine.23 u This procedure was developed from a similar method used by Marion.29 32 is.2.2 m-Dielliylaininopbenvl-N-mcthyl- carbaiuatc inethochloridc (TL 1299) The most practical preparation of TL 1299 is the, following; CH.xro l^jOCONHt'H, N(C,IL), X(CiH»)jCHjC1 N(C|H»)jCHjC1 This process was operated successfully or a pilot plant scale.15 Redistilled w-dielhylaminophenol was treated w ith an excess of methyl chloride in an auto- clave at 100 C. After cooling and evaporation of the excess methyl chloride, the product was purified and isolated in 77 per cent yield by grinding, washing, and drying. Yields in this step on a laboratory scale were 95 per cent.,sm-DiethyIaminophenol methochlo- ride was converted to TL 1299 by reaction with methyl isocyanate in dimothylforraamidc as a solvent and a mixture of triethylamine and glacial acetic acid as a catalyst. Yields on a pilot plant scale were 91 per cent .15 This process was developed to a high state of perfection in an intensive laboratory investigation,18 in which yields of 94-97 per cent and 90-92 per cent in the two steps were obtained, or 86-5)0 per cent overall. A useful laboratory synthesis of methyl Iso- cyanate from methylamine and phosgene also was developed in this work,13 and was used until it was superseded by the pilot plant process 15 for this essen- tial intermediate. Prior to development of the above process, TL 1299 was prepared by a different procedure. m-Di- ethvlaminophcnyl-N-met hylcarbamate was | > repared first from w-diethylaminophenol by the phosgene- methylamine procedure, with diethylaniline as the acid acceptor (yield 78 per cent).2 314 This product was converted to (he methosulfate salt (TL 1317) by reaction with methyl sulfate (yield 75-79 per cent), and TL 1317 was converted to TL 1299 through reaction with anhydrous calcium chloride in methanol containing hydrogen chloride (yield 74 per cent).2 3 Earlier TL 1299 was prepared in high yields from TL 1217 and silver chloride.2 3 5 14 13.2.3 (2-Metliyl-5-diinethvlaminopbenyl)- N-metbvIcarbamatc methiodide (TL 1071) TL 1071 (British code T-1708) was commonly called the “Haworth compound” during the NDHC SECRET 206 AROMATIC CARR \MATES investigations, since it was the leading candidate from the British work. Details of Haworth’s method of preparation could not be obtained, but Canadian reports on the synthesis ->6-34 were available and served as a basis for further developments. The intermediate 2-methyl-5-dimcthy land nophe- nol was obtained from the National Aniline Division of the Allied Chemical and Dye Corporation, where it was prepared from p-toluidine by methylation, sulfonation, and alkaline fusion: (ML (ML C1L (ML 6-6-6"" - 6" XHi X(ClLh X((MLh X((MLh 2-Methyl-5-dimethylaminophenol was converted into the N-methylcarbamate by treatment with phosgene in the presence of diethylaniline, followed by methyl- amine (yield 75 80 per cent).14 With methyl isocya- nate available,14 this intermediate could lx? used in preparation of the N-methylcarbamate, which has Ix'en prepared in that manner in 85 per cent yield on a small scale.4 TL 1071 was prepared from the N- methylcarbamate and methyl iodide in acetone in 95 per cent yield,14 In a Canadian pilot plant opera- tion, 39 lb of TL 1071 were pro pans! from 2-methyI- 5-dimethylaminophenol by this process, with an overall yield of 39 per cent.33 Other quaternary salts differing from TL 1071 only in the anion were prepared. Among these were the methosulfate, TL 1185,14 which was hydrolyzed slowly with aqueous hydrochloric acid or water to the acid sulfate, TL 1186.14 The latter on treatment with calcium chloride yielded the methochloride, TL 1236. The preferred procedure for preparing this compound was to heat the crude methosulfate with an alcoholic solution of calcium chloride for 20 hours. Overall yields from the N-methylcarbamate were 77-87 per cent." Treatment of the N-methylcarba- male with methyl chloride also yielded TL I236.5 13 2.1 (1-Met In 1-3-diiuethy land nophenyl )- N-me th vicar ha male methiodide (TL 1216) TL 1216 was prepared during the Canadian work,27 and became known during the NDRC investigations as the Haworth isomer. It was synthesized in Divi- sion 9. NDRC. from 4-methyl-3-dimethylandnophe- nol, which was prepared by the National Aniline Division of the Allied Chemical and Dye Corpora- tion from o-toluidine: xil N((’iit)> n(cil)* x(cn,)s Bolh the phosgenc-mct hylaminc procedure "27 ami methyl isocyanate 4 were used in preparing (he N-mothylearbamate. The methiodide, TL 1216, was preparer! from the N-mothylcarhamateA 14 Other salts prepared were the methoehlorido * ('IT, 1453) and the methosulfate (TL 1 188). Synthetic metliods employer! paralleled those described for TL 1071. 13.2.5 (3*lsoj>ropvl-l-dimethylaminophenvl)« \, N-dimelhvlearhamate inetliir>t)irie (TL 599) TL 599 is the most toxic of the carbamates de- scribed by Stevens and Beutel.53 Its preparation on any scale is hindered by lack of a practical source1 or synthesis for w-isopropylphenol, the essential start- ing material. An investigation of.eight routes to (Ids compound was made,'* of which the most satisfactory started with benzoic acid and continued through methyl m-hydroxyl>enzoate and m-hydroxyphenyl- dimethylcarbinol, by way of the Grignard reagent. The remaining steps in the synthesis of TL 599 were the following:'* A„„ Aon _ Aon _ U °AJ«V CUfClbh CUCCILh (t l(Cli,)2 AOH (Cl.hNCOC, A<)fox«;i..). —-—> CIKClbh CtKClLh t| \)c;on((ti3)5 I(CH:Axily Cll(CILh Compounds in the corresponding N-monomothyl- carbamate series also were prepared (TL 1327, TL 1345).91* 13.2.6 Synthesis of Other Aromatic Carbamates for Toxicity Tests In addition to the carbamates described in the preceding sections which were the subject of rela- tively intensive laboratory or pilot plant investiga- tions, many similar compounds were prepared on a small scale for toxicity tests, in the search for the most toxic and readily synthesized agent in the SECRET TOXICOLOGY 207 group. The following inferences contain the results of such investigations, and an indication of the classes of compounds studied where that information can l>e stated concisely; otherwise they are classified as miscellaneous. Ref. Classes of Carbamates Described No. M iseellaneous; sulfur analogs 1 Derivatives of polyhydric plienols -1 Derivatives of 3-dielhylaminophenot, 3-dimethvl- amiiiophenol, 2-methyl-5-diinethylaminophcnol and 2-methyl-a-diethylaniinophcnol 5 Derivatives of 4-dimethylaminotbymol and 4-dimethyl- ammoeurvacrol 6 Hoinologs and analogs of Doryl (aliphatic carbamates) 7 Derivatives of p-aminophenol, 4-methyI-3-aminophe- nol, 3-meihyl-l-anunophcnol, 2-tnethyl-5-aminophe- nol . . S Derivatives of 5,6,7,8-tetrahydronaphthol-l; 3-iso-. pfopyl-4-aminophenol; miscellaneous ...... 9 Derivatives of 3,.5-dimethyl-t-aminophenol 10 Derivatives of 3-alkyl-4-aminophenols II Tb 1299, the corresponding X,X-dimelhylcarbamate methiodide (TL 1238) and methochloride (TL 1422) 13 Derivatives of 2-melhyl-5-diiuelhylaminophenol, 4- metbyl-3-dimethylaminophenol, 2-tnelhyl-5-die(hyl- aminophenol, m-dielhylaminopbenol 14 Derivatives of 3-isopropyl-4-aminophem>l, 2,6-diiso- propyl-4-aminophenol, 2-isopropyl-5-aminophenol, 4-lsopn»pyl-3-aminophenol, 4-isopropyl-2-aminophe- nol; arsenic analogs — . 19 Miscellaneous; toxicity, data only on compounds pre- pared by R. D. Haworth .... . . ~. ..... 23 Miscellaneous 24 Derivatives of m-dimelhylaminoplienol 25 Derivatives of 2-melhyl-5-dimethylaminophenol . . . 26 Derivatives of 4-metKyl-3-dimcthylaminophenol , . 27 Denvativc-s of m-diethylaminophenol ....... 2tl Derivatives of 2,4-dimet hyl-5-dimethytaminophcnol 31 Miscellaneous 34 Miscellaneous 43 Miscellaneous : 44 Miscellaneous . . . .~ 45 Miscellaneous 46 Miscellaneous 47 Miscellaneous 48 Miscellaneous 49 Miscellaneous 50 Miscellaneous 51 Miscellaneous . 52 Miscellaneous 53 13.3 STABILITY The toxic aromatic carbamates of possible practi- cal importance are reasonably stable at 65 C, show- ing little decomposition after 2 months storage.16 The two labile groups in such compounds are the carbamate and quaternary salt linkages. The carbam- ate group is subject to thermal decomposition to methyl isocyanate and the corresponding phenol, and to hydrolysis to the phenol, mothylamine, and carbon dioxide, or related products. The quaternary salt groups are subject to decom positiou at elevated temix’ratmcs to an alkyl halide and the correspond- ing tertiary amine. If the carbamates are kept dry, they have good thermal stability. The same pre- eaution protects them from hydrolysis. Hydrolysis is very rapid in alkaline solutions, and slow at an acid pH. As a precaution to insure stability, the carbam- ates may be crystallized from solvents containing hydrogen chloride. Alternatively, acidic stabilizers such as sodium acid sulfate or hydrazine dihydro- chloride may he added. A number pf the more toxic carbamates were ex- amined for relative stability 16 at a time when it ap- peared that stability might lie a decisive factor in choice of a superior agent. The following conclusion was reached concerning thermal stability: variation in the anion of the quaternary ammonium salt re- sults in (he following order of decreasing stability: methosulfate > methiodide > methochloride. Com- paring stabilities toward hydrolysis, two N,N-di- methylcarbamates were much more stable than two N-mcthylcarbamates (TL 1071 and Tb 1217), which in turn were more stable than two N-arylcarbam- ates. Ultimately the two agents chosen as superior on the basis of toxicity and ease of manufacture (TL 1217 and TL 1299) were determined to be sufficiently stable for any anticipated use. One factor with an important bearing on stability is the hygroscopic character of some of the carbam- ates. TL 1299 is quite hygroscopic in humid weather.* TL 1217 is not, and largely for this reason became the agent of choice. TL 1299 could be handled satis- factorily if it were needed on a large scale by con- trolling the humidity of (he rooms in which it would be crystallized, dried, and packaged. Whereas this could be done readily on a full manufacturing scale, on (he large laboratory and pilot plant scale it was much simpler to employ the nonhygroscopic methi- odide, TL 1217. 13.1 TOXICOLOGY A report has been prepared 12 which summarizes much of the toxicological work done in this country, in Britain, and in Canada on the aromatic carbam- ates. Tables from this report, reprinted as Table 2 of this chapter give toxicity data for the 319 aro- matic carbamates and closely related compounds known to have been tested. Aromatic carbamates prepared as part of the NDRC program were submitted to the University of Chicago Toxicity Laboratory for testing. There SECRET 208 AROMATIC C ARU VACATES they received a TL (Toxicity Laboratory) number, and were tested for subcutaneous toxicity to mice. Two to five mice were injected subcutaneously with doses of 80, 10. 20, 10, 5, and 1 mg kg of body weight, at dilutions such that each mouse received approximately I per cent of its body weight in a suitable nontoxic solvent (usually water). Any com- pound that killed at 1.0 mg kg was screened further and LDio determinations were made for all those killing at less than 0.5 mg kg. The data obtained are listed in Table 2, together with similar toxicity data obtained elsewhere for other aromatic carbamates. A number of factors influencing toxicity determina- tions made by injection were studied carefully for the more important aromatic carbamates. One of the most important was the animal species used in test- ing. The leading candidates were tested in several animal species, since the object of the search was to select an agent toxic for all species. TL 1217 proved to be very toxic for all species in which it was tested. TL 1345 is the most toxic compound tested in mice, but as Table 1 shows,12 it presents no marked superi- intraperitoneal (In the single comparison available for rats the in! ra peritoneal route was the more effective.) 3. Carbamates are relatively ineffective when ad- ministered by stomach 1ul>e. 25 to 500 times as much material being required to kill as by injection. The carbamates are toxic when administered by inhalation as aerosols,'- but do not show the ex- traordinary toxicity in comparison with standard chemical warfare agents which characterizes them when toxicitics determined by injection are com- pared. The aromatic carbamates are “quick-kill” agents capable of producing severe parasympathomimetic effects terminating in death. Death occurs rapidly, for example, in 5 to 20 minutes after subcutaneous injection in dogs. The symptoms produced are similar in all species which have been examined. They con- sist of salivation, evacuation of bowels and bladder, restlessness and incoordination, and fibrillary muscu- lar movements. Respiratory movements are quick- ened and labored. Coma is accompanied or preceded by convulsive movements. Respiration appears to cease first, the heart lieating, usually irregularly, for some moments after respiration has failed. Muscular twitching persists for some time after failure of res- piration and cardiac activity. The aromatic carbamates are powerful cholin- esterase poisons, and produce marked changes in the blood. Because of medical and toxicological in- terest in them, their physiological mechanism of action has received considerable study. Most of this work may be located through certain leading infer- ences,20 ■2U hd e Atropine or atropine and pento- barbital administered intravenously have been rec- ommended as antidotes for the carbamates.36 41 Anti- dotes can lx* demonstrated to be useful in animals, but must be administered quickly (at the onset of symptoms) because of the very rapid toxic action of the carbamates. For references to toxicity assays on the carbamates in addition to the summary previously mentioned 12 see the Bibliography.2211 *’<’ ‘12J 2S-3II S5 S7 I3,r. RELATIONSHIP BETWEEN CHEMI- CAL STRUCTURE AND TOXICITY Relationships existing between chemical structure of the aromatic carbamates and their toxicity have been pointed out in some detail.12 The following prin- cipal conclusions can be drawn from the available Table 1. 1345 for v Subcutaneous toxicitics of TL 1217 and TL a rious species. Sjiecies LT)a„ (mg kg) (| \)COXHCHj (|^OCOXIICH, V a(C",wv CIKCII.), TL 1217 TL 1345 Mouse 0.129 0.047 Rat ea. 0.400 0.103 Guinea pig 0.097 ca. 0.050 Rabbit ca, 0.150 ca. 0.075 Cat ca. 0.075 ca. 0.100 Dog ca. 0.075 ca. 0.100 Monkey ca. 0.200 ca. 0.150 ority over TL 1217 when other species are con- sidered. Other factors considered in precise toxicity de- terminations were the concentration of the solution injected; the strain, sex, body weight, and age of the mice used in LDh0 determinations; and the effect of the temperature of the environment of the assay animals. A number of compounds were tested by various routes of administration, and the following conclusions were reached.12 1. The carbamates tested were about twice as toxic intravenously as by any other route. 2. Subcutaneous injection was more effective t han SECRET CHEMICAL STRUCTURE V ND TOXICITY 209 toxicity data (figures cited refer to subcutaneous toxicity in mice). 1. The most toxic compounds contain both a carbamate and a quaternary salt group. 2. The carbamate group is more intimately con- nected with toxicity than is the quaternary salt group. This conclusion follows from several lines of evidence: a. The quaternary ammonium group can lie re- placed with sulfonium or arsonium without change in order of magnitude of toxicity. For example: - X(C2Hi)jCH,I \s(C4I).CHl Tl, 1217 TL 1306 TL 1501 - 0.120 mg kg LIh* =* 0.37 mg kg LIhu =» 0,5 mg/kg h. The 6/s-N,N-dimethylcarhaniate of catechol is highly toxic even though it contains no basic group; int roduction of a quaternary salt group in this com- pound results in diminished toxicity. OXX)X(CHi)j jj \x'ON(CHj), X’ON(CH,), I(CH,),nI!Jo( 0N(CH,)j TL 1015 TL 1165 Llha = 1.4 mg/kg Toxic dose > 10 mg/kg c. Quaternary salts derived from amiuophcriols are not very toxic, but the N-methylcarbamates de- rived from some of them am highly toxic. jj^jOCONIICH, NfC-HshCHjCl N(Cyis),CII,Cl TL 1300 TL 1200 Toxic dose about 40 mg/kg Lhut =• 0.09 mg/kg d. Structural changes in the carbamate group in related series of compounds may produce enormous changes in their toxicity. 0 OCONHCTI, /\sCOXHCI1j c”V N(CIIj),I X(CII,)jI TL 1216 TL 1230 LOfco = 0.17 mg, kg Toxic dose > 80 mg/kg 0DCOX HCITj (| \xX)N(CH,)» V X(CiHj)jCIIjI X(CjHi)jCH,T TI- 1217 TL 1238 /-/)» * 0.120 mg/kg LDu> » 0.175 mg/kg CH,CH, 0OCON CHj HC,Hj \hjCH, {J N(C,II,fcCHJ N(C.II4)jCH,,I TL 13(6 TI. 1-181 Toxic liosc about 1 nut k(t Toxic dose about 5 mg kg IK'(,11 HCdbOCH,-,, N(C’:Ilj)iCUjl X(C.H.U’UI TI- 1433 TI- 1412 Toxic dose > 8(1 mg kg Toxic dose > SO mg kg o. Changes in the quaternary salt group in a series iu whieh the N-methylcarbamate group is kept con- stant produce smaller changes in toxicity. OCOXHCIIj OCOXIICH, (S o N^xtdhhi c*n. TI, 1178 TI, 1323 LDw = 0.270 mg/kg /,/>» = 0.135 mg/kg OCOXIICH, OCOXHCIIj f) (S (C,Hi)j CIJjCHfzEIIi tl. 1217 TL 1435 LD& — 0.129 mg/kg LDm =» 0.102 mg/kg OCOXIICH, OCOXIICH, - (QLxCjHOjI (CHjIjCH, TI- 1434 TL 1259 LDw - 0.10 mg/kg LDx = 0.23 mg/kg OCOXHCIIj A - VfIl!l (CjHg), TI, 1324 /.Dm = 0.48 mg/kg II. In general, the N-methylearhamates are mom toxic than corresponding N,N-dimethyIcarhamates. Of 20 such pairs of compounds tested, (he mono- met hy lea rhamates were more toxic in I t eases (for some pairs they were 10 to 40 times as toxic); in (ho other G cases they were approximately equal. No other substitution on the carbamate nitrogen which was investigated led to compounds as toxic as the hi-methyl and N.N-dimcfhyl derivatives. SECRET 210 VROM \TIC CAR HAM AXES 4. With few exceptions, the most toxic compounds were those in which the X-methylearltamato and quaternary salt groups were in the meta orienta- tion. 0OCOX H( ’ll, /VxX)N IICH, X(CH»)jI {J \(CH,M Toxic dose 130 mu kg . TL 1178 Llho ■** 0.27 mg/kg OK'OXHCTb TL 1007 Toxic dose about 20 me kg 5. Methyl substitution in the nucleus ortho or para to the carbamate produces no great change in the toxicity of m-quatemary compounds, and may result in slight ly more toxic substances. Similar sub- stitution by bigber alkyl groups leads to less toxic compounds. Oil, OGC’DNHCHj a Xk'OXHCH, fi %HX)XncH, V CI,V N(C1I,U XfCHhl N(CIE)J TL 1178 TL 107! TL 1216 LDw = 0.27 mg kg £/>» «■ 0.111 mg kg LDm = 0.17 mg kg ti. The series with an alkyl substituent meta ami tlie quaternary salt para to the carbamate group contains some extremely toxic compounds. In the most toxic homologs of this type 11 the alkyl group is isopropyl. 0OCONIICIh j| \ K OX(CIb), UClb)A\J CH(('I!,), 011(011,), TI, 1327 Tl. 309 /.Dm = 0.0H7 mg/kc LOao =• 0.083 mg kg 7. The toxicity of aromatic carbamates'substi- tuted by quaternary salt groups resides in the cation. Of the various stilts, the chlorides have l>een found to Ik? somewhat more toxic than would be calculated on a molecular weight basis. Other salts with the same cation have toxieifies proportional to their molecular weights. Table 2. Toxicities of aromatic carbamates and related compounds. The following tables” contain the toxicity data available as of March 1945 for aromatic carbamates and closely related sulstances (319 in all). The tables are subdivided into IS structural classes, as follows; I Benzene compounds with one carbamate group, and no quaternary ammonium group. IT Benzene compounds with two carbamate groups and no other groups. III Benzene compounds with two carbamate groujis and other groups. IV Benzene compounds wit h 1 hree carbamate groups and no other group. V Benzene compounds with one carbamate group and one quaternary ammonium group in the ortho position. VI Benzene compounds with one carbamate group and one quaternary ammonium group in the ortho position and alkyl groups. VII Benzene compounds with one carbamate group and one quaternary ammonium group in the meta position. VIII Benzene compounds with one carbamate group and one quaternary ammonium group in the meta position and other substituents. IX Benzene compounds with one carbamate group amt one quaternary ammonium group in the para posi- tion (including thiocarbamalcs). X Benzene comjmunds with one carbamate group and one quaternary ammonium group in the para posi- tion and other substituents. XI Benzene compounds with one carbamate group and an alkyl side chain having a quaternary ammo- nium group. XII Benzene compounds with one carbamate group and two quaternary ammonium groups. - XIII Benzene compounds with one carbamate group and one sulfonium or arsonium group. XIV Carbamates of naphthalene derivatives. XV Carbamates of quinoline and isoquinolinc deriva- tives. XVT Carbamates of aliphatic alcohol derivatives, XVII Miscellaneous carbamates. XVIII Carbamides and carbazates. The tables represent a revision of a similar review issued on June 15, 1914,19 and follow the system of classification used in the earlier summary. Entries in the column headed “Code” have the significance noted in the Glossary. In the column headed “Route and Solvent” the following abbreviations are used: Sc.W. = subcutaneous injections in water. Se.P. = subcutaneous injections in propylene glycol. Sc.O. = subcutaneous injections in olive oil. Sc.M. - subcutaneous injections in mineral oil. Sc.Imp. — subcutaneous implantation of dry solid. Iv.W. = intravenous injection in water. Iin.Iinp. = intramuscular implantation of dry solid. Ip.W. = intraperitoncal injection in water. Oral W. = administered by stomach tul>e, in water. pll-t indicates that this acidity was achieved with Mcll- vaine’s buffer. Whenever the room temperature during the determination was recorded, it was listed immediately following the /.//,„ figure. SECRET 1. Benzene compounds with one carbamate group, and no quaternary ammonium group. Route — and Dose Code Xamc Structure solvent Species mg kg Kffect All-1 Carhamie acid, X-methvl- OCOXI1C1L Iv. Mice >50 U\« phenyl ester 0 TL-1113 Carhamie acid, X,X-dimcthyl- V OCOX(CHi) Sc.M. Mice 80 0/2 phenyl ester /\ 40 0/2 0 - 20 0/2 TL 1218 ('arhamthiolicacid, X-methyl- X/ SCOXTICHj Sc. 1’. Mice SO 2/2 p-lolyl ester /\ 40 0 2 o 20 0 2 V Cl I, TL-997 Carhamie acid, X-methyl-2- OCOXHCI1, Sc.Tmp. Mice 80 0/2 All 2 nitrophenyl ester Qxo, Tv. Mice :« /./),* TI, 948 Carhamie !acid, X-methyl-3- V OCOXHC1L Se.lmp. Mice 80 0 2 nitrophenyl ester /\ to 0/2 20 0/2 VNO* TI.-947 Carhamie acid, X-methvl-4- OCOXHCIL Se.Tmp. Mice 80 0/2 nitrophenyl ester /\ 40 0/2 () 20 0/2 \/ xo* - T 1,-980 Carhamie acid, X-methvl-2- OCOXHCIL Sc.Tmp. Mice 80 0/2 hydroxyphcnyl ester A 40 0/2 _ O’- 20 0/2 TL 1010 Carhamie acid, X,X-di methyl- \/ OC'OX(ClL)i Sc.W. Mice 80 0/2 2-hydmxyphenyl ester A 40 0/2 0m 20 0/2 TL-979 Carhamie acid, X,X-diethvl- \/ (X 'OX(CdL), Sc.Tmp. Mice 80 0/2 2-hydroxy phenyl ester A - 40 0/2 O’" 20 0/2 TL 1101 Carhamie acid, X,X-dimelhyl- V OC()X(CH3). Sc.O. Mice 80 0/2 2-allyloxvphenvl ester A 40 0/2 r jOCHjCl I CIT. 20 0/2 TL-1110 Carhamie acid, X,X*-dimethyl- V OCOX(CH.h Sc.M. Mice SO — 0/2 1-all vl-2-met la ixyphenyl A 40 0/2 ester o — 20 0/2 \/ C1I>CH—CUj CHEMICAL STRUCT THE VM> TOXICITY 211 SECRET 212 AROMVTIC CARBAMATES Tabus 2, Section I (Continual) Code Name Structure Route and solvent Species Dose mg/kg KfTeet TL-1111 Carbamic acid, X.X-dimethvl- OCOX(CHj)j Sc.M. Mice 80 0/2 2-mcthoxy-4-propylphenyl A 40 0/2 ester [ jOCIb 20 0/2 V CHjCIljCHi TL-lllG Carbamic acid, X.X-dimethvl- OCOX(CIL). Sc.M Mice SO 0 2 4-« llyl-2-mc t hoxy-5-n i fro- A 40 0 2 phenyl ester f jOCH, 20 0/2 o,x V ClijCH—CIIj IT. Benzene compounds with two carbamate groups and no other groups. TL-lOlo Benzene, 1,2-hia{ methyl- OCOXHC1L Sc. W. Mice 40 2/2 carbamyloxy)- 20 2/2 — [ jOCOXTICII, 10 0/2 5 0/2 TIi-978 Benzene, 1,2-hts(dimcthyl- OCON(CHj)j Sc.P. Mice 1.4 LD* «. carbamyloxy)- A -- 1 f jOCOXCCIbh TL-1118 Benzene, 1,2d>is{diel hylear- V OCOX(CdI.,b Sc.P Mice SO 0/2 bamyloxy)- A _ 40 0/2 1 r JocoxicdCb 20 0/2 TL-1119 Benzene, l,2-h/.s{X-penta- V OCOXCdl,,, Sc.M Mice SO 0/2 met h vlenecarbam vloxv )- A 40 0/2 J r pcoxciii.o 20 0/2 TL-1112 Benzene, 1,3-6is(dimct hylcar- X/ OCOXTCIIj), Sc.W. Mice so 0/2 bamyloxy)- A — 40 0/2 1 20 0/2 1 1 l0C0X(CHj)j TL-1114 Benzene, 1,4-6£s(dimet hylcar- OCON(CHi)j Sc.P Mice SO 0/2 bamyloxy)- A 40 0/2 o 20 0/2 V OCOX(CTL)i TL 1348 Benzene, 1,4-6/s(met hylcar- OCOXHCIlj Sc.P. Mice SO 0/2 bamyloxy)- A 40 0/2 o 20 0/2 v OCONHCH, — III. Benzene compiunds with two carbamate groups and other groups. TL 1117 Benzaldehydc, 3,4-Wa(dimethyl- OCOX(CHs)j Sc.P. Mice - so 0/2 carbamyloxy)- A 40 0/2 r |ocox(cHi)i 20 0/2 V CHO SECRET 213 CHEMICAL STRICTURE AND TOXICITY Code Xame Structure Route and solvent Species Dose mg Kffcct TL-1157 Benzyl alcohol, 3,4-6»s(di- OCOX(CIIa)I Sc. W. Mice 10 1/2 met 11 ylcarl >a myloxy)- /\ 5 0/2 [ jocoxccn.h i 0/2 X/ CHjOH TL 1160 Dimcthvlamine, X-[3,4-5is(di- OCOX(CHj)i Sc.W. Mice 10 2/2 me(hylcarbamyloxy)benzyl] /\ 5 2/2 — hydrochloride f jOCOX(CIIi), i 0/2 A J 0.5 0/2 — 9 CII2X(CHj)j-HC1 TL-981 Benzene, 1,2-bis( dimethyl- OCOX(CTI3)t Sc.lmp. Mice 80 0 2 carbarn yloxv)-4-ni tro- A Sc.O. SO 0/2 r pcoxxciuh 40 0/2 — NO, —- TL-I017 Benzene, 1,2-hi«( d i me t hy 1 ca r- OCOX(CIlj); Sc. N/10 M ice to 2/2 1 mmy loxy )-1-amino- /\ Ac. 20 _ - 2/2 — f pcox(cn5)s 10 2/2 I J 5 0/2 XH, TL 1155 Benzene, 4-(dirne(hylamino)- OCOX{CH,)i Sc.W. Mice 10 0/2 1,2-Ws{diincthylcarbamyl- A 5 0/2 oxy)-, methiodide f ]()COX(CHj)j 1 0/2 {J 0.5 0/2 - X(CH,),I TL-1150 Benzene, 1,2-W«(dimet hylcar- OCOX(CIIj)i Sc.O. Mice 10 0/2 bamyloxy)-3-allyl- /\OCOX(CH,), 5 1 0/2 0/2 X^JciIjCH—CHj 0.5 0/2 TL-1158 Benzene, 1,2-6/.<.{ dimethyl car- OCOX(CIL)j Sc.O. Mice 10 0/2 1 >a my 1< »xy )-3-pn tpy 1- |/\oc 'OX (CII a) j 5 1 0/2 0/2 I JtTLCTLCH, 0.5 0/2 TL-1102 Benzene, 1,2-/>w(dimetliylcar- OCOX(GH,)i Sc.O. Mice 10 0/2 bamyloxy)-4-allyl- A 5 0/2 i 0/2 - Kj 0.5 0/2 CHjCH=CH, __ TL-1156 Benzene, 1,2-his(dimel hylcar- OCON(CH,), Sc.M. Mice 10 0/2 bamyloxy)-4-propyl- A 5 0/2 f |OCOX(CH,)t i 0/2 f. 0.5 0/2 CHtCHiCHi TL-1086 Benzene, 1,3-b/»(X-met hylcar- OCOXIICH, Sc.P. Mice 40 0/2 bamyloxy )-2-ni t ro- Ano, 20 0/2 I Jocoxiich, — Table 2, Section III {Continued) SECRET 214 VROM A TIC CAR RAM AXES Tahle 2, Section III (Continued) Route and Dose Code Name Structure solvent Species mg kg Effect TL-1120 Benzene, 1,3-b/x( X-met hylear- OCOXIICIl, Sc.W. M ice SO 0/2 bainylo\y)-2-amino hydro- chloride /\xh2iici 10 20 0,2 0/2 1 sJdcoxiicii, ti, 1310 Renzene, 1,4 -bis( met hylearba- OCOXIICIl, Sc.P. Mice . SO 0 2 mvloxy )-2,G-dimet hyl- A 40 0 2 CHI 0". OCOXIICIl, - ' 20 0 2 TI, 1350 Ben zone, 1,4-his( met hylcarba- myloxy )-2-isopropy 1-5- OCOXIICIl, A Sc.P. Mice so 10 2 2 12 rnctlivl- ( VlKCH.I, 20 1 2 nij I J 10 0 2 v 5 0/2 • . Ot ONIRIC IV. Benzene compounds with three carbamate groups and no other group. TI 1115 Renzene, 1,2,3-//ix( dimethyl- OCOXfCHOs Sc.W. Mice 40 2/2 carbamvloxv)- A 20 2/2 ■ - 1 f KX'OX(CTI,)s 1 k)cox(cii3), 10 it 0/2 0/2 V. Benzene compounds with one carbamate group and one quaternary ammonium group in the ortho position. TL-903 Carbamie acid, N-mcthyl-2- aminophcnyl ester hydro- OCOXIICIl, A Sc.W. Mice so 40 0/2 0/2 chloride [ jXTIj-IlCl -- 20 0/2 T-{ ?) Carbamie acid, N-methyl-2- dimethylaminoplicnyl ester \/ OCOXIICIl, A Sc. Mice 430 ldm methiodidc r |X(cn,)ji VI. Benzene coin|)oimds with one carbamate group i ami one quaternary ammonium group in the ortho position ami alkyl groujis. TL-1488 Carbamie acid, X-methyl-2- di met hyla mi no-4-isopropyl- OCOXIICIl, A Sc.W. Mice SO 40 0/2 0/2 phenyl ester methiodidc r ]x(cu,),i 20 0/2 V CIKC1I,), SR-13 Carbamie acid, X.X-dimethyl- 2-dimethvlamino-4-methvl- OCOX(CHj)- A Sc. Mice Approx. 200 LD,„ phenyl ester hydrochloride f CH, SECRET CIIE.MKUL STRUCTURE AND TOXICITY 215 Tabi.k 2, Section VI (Coulin iied) Route and Dose Code Name Structure solvent Species n>R kf; K fleet SB-14 Carbamic acid, N,N-dimethyl- OCOX(CH,)j Sc. Mice 2.0 I.D,„ 2-dimet hvlamino-4-methyl- /\ phenyl osier mclhiodidc | |N(CHj)jl V CH, SB-15 .Carbamicacid, \,\-dimethyl- OC()N(CIIi)t 8c. Mice 27 2-dimet hvlamino-4-ethyl- A _ phenyl ester hydrochloride r jN(CHj)jll(T "'“I V c3h. SB-10 ('arhamic acid, X,X-dimethyl- OCON(CHj)j Sc. Mice 1.25 LDia 2-di met hvlamino-4-ethyl- A phenyl ester mclhiodidc r |N(cn»)»i V c,h5 - SB-17 Carhalnic acid, N,N-dimethyl- OCX)N(CIIi)* Sc, Mice >400 UK, _ 2-dimet h vlamino-4-isopro- A ■ _ - ” - pylphenyl ester hydrochlo- 1 |N(Cii;)-nci “ ride IJ — • CH(CH»)* SB-18 Carhamic, acid, N,N-dimcthyl- ()CON(CIIi)j 8c. Mice 4.8 LDU 2-dimet hylamino-4-isopro- A pylphenyl ester nicthiodide r ]N(cn,).i -— — — ■- — ■ V ciuciia). SB-10 Carhamic acid, X,X-dimcthyl- OCOX(CIIj), 8c. Mice > 500 UK„ 2-dimethylamino4-/frl hutyl- A phenyl ester hydrochloride f jN(CHi)jliCI -- V C(CHj)i SB 20 Carbamic acid, N,\-diinethyl- OCOX(CH,h 8c. Mice 13.5 LDn, 2-dimet hylamino-4-fcr< butyl- A ptienyl ester methiodide f |X(CHj)jI V C(CHj)j SB-21 Carhamic acid, N.N-dimethyl- OCOX(CHj)j Sc. Mice > 500 LDM 2-dimet hylnmino-4-fcrl A • . amylphenyl fitter hydro- f ]Nr(cir3h-nci chloride v — CVCCXCII.h SB-22 Carbamic acid, N,\-diinrthyl- OCOX((dlj)j Sc. Mice 12 ldm 2-dimct hylamino-4-/crf A amylphenyl ester met hi- (slide V1 CtlUC(CHa)k SECRET 216 UlOMATIC CARBAMATES Table 2 (Continued) VII. Benzene compounds with one carbamate group and one quaternary ammonium group in the meta position. Code Name Structure Route and solvent Dose Species mg/kg Kffect TL 1309 Phenol, 3-(dielhylamino)- nicthochloride Oil A Sc.W. Mice 80 40 20 22 2/2 0/2 - 1 1X(CoH:,)2CH.C1 10 0/2 T-1122 Carhamie acid, 3-dimethyl* aminophenyl ester methio- dide — OCOXIIj Sc. Mice 37 U). o AH 11 Carhamie acid, 3-dimethyI- aminophenyl ester met ho* sulfate OCOXII* (^X(CH,),Sp4CH, lv. Mice 0.7 /.a. TL 94ti Carhamie acid, X-methyl-3- aminophenyl ester hydro- chloride OCOXIICH, 1 lxH,HCI Sc.W. Mice — SO 40 20 — 0/2 0/2 0/2 ATC12 Carhamie acid, X-methyl-3- dimet hylaminophcnyl ester hydrocliloride OCOXIICH, (^X(CII,),HC1 Iv. Mice 15 T-1152_ Carhamie acid, X-incthyl-3- dimethylaminophcnyl ester mcthiodhle OCOXIICH, (^)nt(cii,),i ! I 1 | ■i-i-i Mice 0.44 Rabbit 0,26 Mice 30 LDi o TL 1178 Carhamie acid, X-methyl-3- dimethylaminophenyl ester methiodide. OCOXIICH, [^)x{CH,),l Sc.W. Iv.W. Mice 0.27 Mice 0.115 (Sec p. 219) UK, Llh , TL-1226 Carhamie acid, X-inctliyl-3- OCOXIICH, Sc.W. Mice 0.140 LDtt dimet hylaminophcnyl ester methochloridc ,)3('i Iv.W. Mice 0.070 LDu T-1090 Carhamie acid, X-met hyl-3- dimclhylaininophenyl ester OCOXIICH, A Sc. Mice 0.37 Uho met liochloride Mx(CII,),CI All 13 Carhamie acid, X-met hyl-3- dimet hylaminophcnyl ester met hosul fate OCOXIICH, A l\(CH,),SOtCH, Iv. Mice 0.1 UK.. TL 1323 Carhamie acid, X-melhvl-3- OCOXIICH, Sc.W. Mice 0.135 Uho T-T194 dimethylaminophenyl ester cthiodide ( A Sc. Sc. Mice 0.38 Rabbit 0.13 Uho ldm 1 ]X(CH,),C,H5I SECRET CHEMICAL STUUCTLRE AND TOXICITY 217 Code Name Structure Route and solvent Species Dose Effect AH 11 Carbamic arid, X-methyl-3- rlhylmcthylaininophetiyl ester met hobromide OCOXIIGlij s Jx(CIIi)jC-jIIiHr Iv. M ice 0.15 UK* TI. 1134 Carbamic arid, X-methyl-3- diniel hylaminophenyl ester propyl bromide ( OCOXHCII, Sc.W. IX(CIFj)iCMjCHjCIIaBr Mice (Sec p. 210) 0.100 (78 K) UK* Tbi i:r. Carbamic acid, X-methyl-3- dimel hylaminophenyl ester ally! bromide OCOXHCII, Sc.W. *1 IX(CH,bCH,CH—Cll-llr Mice (See p. 210) 0.102 (82 K) UK, TL 1324 Carbamic acid, X-methyl-3- dibulylaminoplienyl ester met h iodide OCOXHCII, Ix(C4H,WHJ Sc.W. Mice 0.48 UK* AR-15 Carbamic acid, X-mefhyl-3- diel by la minophenyl est er hydrochloride OCOXIICH, I.X(CjHt),HCI Iv. Mice 5.0 UK* Tb-1217 Carbamic arid, X-methyl-3- diet hylaminophenyl ester met h iodide OCOXHCH, lx(C.H„).CHd Sc.W. Sc.W. Sc.W. pH4 Sc W. Mice Mice Mice 0. pi>? (See p. 210) 0.122* 0.120 0.135 0.007 LDiu U),„ LD_, LDW T-1123 All-If. TE-1200 Prep. 1 Prep. 2 Prep. 3 Prep. 3 Carbamic acid, X-methyl-3- diet hylaminophenyl ester methochloride OCOXHCII, IX(C,Hi),CH,CI Sc. Tv. Sc. Sc.W. Sc.W. Sc.W. Sc.W. Mice Mice Mice — Mice Mice M ice Mice (See p. 220) 0.20 0.1 0.13 O.OOOf 0.007 0.105$ 0.005§ UK* UK, UKo UK,, ldm UK* UK* TI. 1317 Carbamic acid, X-methyl-3- diethylaminophenyl ester methosnlfate OCOXHCII, Qjxccai.MCH^SO, Sc.W. pi 14 Sc.W. Sc.W. Sc.W. Mice Mice Mice Mice (Sec p. 220) 0,100 0.114 0.107 0.102 IjDs t U)M UK, UK* TI-1250 Carbamic arid, X-methyl-3- diethylaminophenyl ester eth iodide ocoxncii, In(CiIU).1 Sc.W. Mice 0.23 UK, AR-31 Carbamic acid, X,X-dimcthyl- 3-dimet hylaminophenyl ester acid tartrate OC’ON(C’Hj), Iv. A |X(CH,>X—CHOHCOOH), Mice 00 UK* * At 8.'. F. t At 75 F. J At 77 F. s At 76 F. Table 2, Section VII (Continual) SFX’RET \ R OM AT 1C C A KB A M A TES Code Name Structure Route and solvent SjICCieS Dose mg/kg KITcct TL-1321 SH-24 Carbamic acid, X,N-dimethyl- 3-dimethylaminophenyl ester methiodidc OCOXfClb,). (^)x(CII,),I Sc.W. Sc. Mice Mice 0.475 0.55 Uho U),, AR-32 SB- 23 TL-1304 Carbamic acid, X.X-dimothyl- 3-dimethyIamitiopliciiyl ester methosulfatc (Prosl igmine) OCOX(CH3), (^X(CII,)1SO,CH3 Tv. Sc. Mice Mice (See p. 0.5 0.45 220) UK, LI),, TL-1238 Prep. 1 Prep. 2 Prep. 3 Prep. 3 Carbamic acid, X,X-dimethyl- 3-diethylaminophenyI ester melhiodide ocox(cir3h. (^)x(c.iii).cn3r Sc.W. Sc.W. Sc.W. Iv.W. Mice Mice Mice M ice (See p. 80 40 20 10 0.125* O.ITot 0.089f 220) 2/2 2 2 0 2 0 2 LDia Uho /.D,„ TL-1422 Carbamic acid, X,N-dimethyl- 3-diethylaminophenyl ester met bf (Chloride OCOX(CII3h [^)x(CsIi5)iCII3Cl Sc.W. Sc.W. Sc.W. Mice _ Mice Mice (See p. 0.058J 0.108$ 0 1 (HI 220) Uho W>». UK, TL-1481 Carbamic acid, X-elhyl-3-di- ethylaminophcnyl ester melhiodide OCOXHCjHs (^N(C;1W’IU Sc.W. Mice 10.0 5.0 2.5 1.0 2/2 2/2 0/2 0/2 AR-21 Carbamic acid, X-elhyl-3-di- met hylaminopheny! ester methosulfatc OCOX HCjHs 1 J\'(CHj)3SO,CHj Iv. Mice 1.0 LDoo A11-36 Carbamic acid, X-ethyl-X- incthyl-3-dimethylamino- phenyl ester methosulfatc cii, / OCOXCsHs A Iv. Mice 3.5 LJ)*o ;■ ' - ! 1 lx(CITJ)JS01CH1 AR-33 Carbamic acid, X,X-diethyl- 3-dimethylaminophenyl ester methosulfatc OCOX(CiHs)i (^N(CH.)3SO.CH3 Iv. Mice 8 /v/2%0 AH--19 Carbamic acid, X-allyl-3-di- methylaminophenyl ester hydrochloritle OCOXIICI!jCH—('Hj [^Jx(CII3):-HCI iv. Mice 150 ID,, AR-34 Carbamic acid, X,X-diallyl- 3-dime(hylaminophenyl ester methiodidc OC<)X(CH.CII-CH,)l Mx(CH3)3I Iv. Mice 10 LD,o • At S3 F t At 75 F. t At 80 K. 5 At 71 F. 1) At 7 3 F. Table 2, Section VII (Continued) SECRET CHEMICAL STRIXTLKE AND TOXICITY 219 Code Name St met ure Route and solvent SjM-cies Dose 111)!; kg Effect All-20 Carhamic acid, X-allyl-3-di- methylaminophenyl ester met la >sulfatc OCOX HCHjCll—Cll- 1 Ix(C1I,)jSG4CIIi Iv. Mice 0.75 L/Xo All 24 Carhamic acid, X-phenyl-3- dimethylaminophcnyl ester hydrochloride ‘ / OCOX lie, 11=, |^\x(CH,VIICl Iv. Mice 25 /■/),. AH 25 Carhamic acid, X-phenyl-3- dimethylaminophenyl ester met hosulfatc OCOXIlCslli 1 lx(CHj)jSOiCIIj Iv. Mice 2 /-/), 0 All 22 Carhamic acid, X-lx»iizy 1-3- dime thy la mi noplwnyl ester hydrochloride OCOXIlCHjtMIs 3),-IIC1 Iv. Mice 50 ADS„ AH-23 Carhamic acid, X-benzy)-3- dimcthylaminophenyl ester mcthosulfate OCOXTICHjC.Hs (^)x(CHJ)iSOtCH3 Iv. Mice 0.1 LDsa T-1125 Carhamic acid, N-benayl-3- (li met hylaminophenyl ester methiodide OCOXHCH.CdL (^)n(CIL),I Sc. Sc. Mice Rabbit 0.35 0,20 I.D.,, Lfho TL 1308 Carhamic acid, N,X-penta- melhylenc-3-dimethyl- aminophenyl ester methiodide OCOXCJI.o 1 Ix(CIIj)jI Sc.W. M ice 10 5 2.5 2/2 2/2 0/2 All-35 Carhamic acid, X,X-penta- met hylenc-3-di methyl- aminophenyl ester mcthosulfate OCONCsHi# (^)x(CII,)JSO,CHi Iv. Mice 0 TL 1340 Carhamic acid, X,X-|>enta- met hylene-3-diel hyl- aminophenyl ester methiodide OCOXCJI.o 1 jx(CJU, Sc.W. Mice 5 1 0.2 0.1 5/5 3/5 0/5 0/5 CH,I T 1207 Carhamic acid, X-(4-melhoxy- pheuyl)-3-dimcthylamino- phenyl ester methiixlide ocoxnocit3 A 1 IX(CHa) J Sc. Mice 0.24 LD* TL 1442 Carhamic acid, X-(4-methoxy- phenyl)-3-dielhylamino- phenyl ester methiodide N>OCII, A 1 IX(CjlTi)jCH,I Sc.F. Mice SO 40 20 0/2 0/2 0/2 Table 2, Section VII {Continued) SECRET 220 AROMATIC CARBAMATES Route and Code solvent Species (at Effects various doses) TE 1178 Sc.W. Rat Rabbit G. pig L>og Cat 0.10 0.125 0.25 0 .5 1.0 0/2 0/1 0/2 0/2 0/1 0/2 2/2 2/2 0/1 0/2 1/2 2/2 2/2 0/2 2 2 2 2 2/2 2/2 1/2 TL 1434 Sc.W. Rat Rabbit G. pig Cat Dog 0.025 0.0,50 0.100 0.200 0/2 0/2 0/2 0 2 2/2 1/2 0/2 1/2 1/2 2/2 1/2 2 2 2/2 2/2 2/2 — TL-1435 Sc.W. Rat Rabbit G. pig Dog 0.050 0.100 0,200 0 2 0/2 0/2 2/2 0/2 2 2 2/2 2/2 2/2 TL 1217 _ Sc.W. 0.0.5 0.1 0.2 0.3 0.4 Rat 0/2 0 2 0/7 6/7 Rabbit — 0/2 2/2 G. pig 0/2 2/2 Dog 1/2 22 2/2 Cat 0/2 2/2 2/2 -r— * Sheep 0/2 3/3 Goat 0/2 2 -'5 2/3 Monkey 0/2 2/3 1" Route and Effects Code solvent Species (at various doses) TL-1299 1in.Imp. 0.025 0.05 0.1 0.2 0.3 (2ml sample) Goat 0/1 0/1 l/l Monkey 0/2 0/4 1/1 1/1 Sc.W. I)o(J 0/3 1/3 8/10 TL-1317 Sc.WT. 0.05 0.1 0.2 0.3 Rat 0/2 3/5 2/2 J- P'K 0/2 1 /5 5/5 Rabbit 0/2 1/2 2/2 (*at 0/2 2/2 TL 1394 Sc.W. 0.2 0.5 1.0 1.5 - . Rat 0/2 1/2 2/2 Rabbit 0/2 2/2 2/2 > pig 0/2 2/2 2/2 2/2 Dog 0/2 1/2 1/2 — Cat 0/2 1/2 2/2 TL-1238 Sc.W. 0.05 0.1 0.15 0.2 0.3 0.4 1.0 Rat 0/2 0/2 0/2 1/2 2/2 Rabbit 0/2 1/2 2/2 L l>ig 0/2 3/0 2/2 Cat 0/2 2/2 Dog 0/2 /2 2/2 T LI 422 Sc.W. 0.05 0.10 Rabbit 0/2 2/2 Dog 0/2 2/2 Table 2, Section VII (Continued) SECRET CHEMICAL STKCCTl HE VXD TOXICITY 221 Table 2 (Continued) VIII. Benzene compound* with one carbamate group and one quaternary ammonium group in the met a position anti other .substituents. Code Name Structure Route and solvent Species Dose mg/kg Kffect TL-12r.fi Carbnmic acid, 2-me I hy 1-5- OCONH, Sc. Mice 10 2/2 dimethylainiiu.phenyl ester /\ 5 2/2 methiodide cn/ ] 1 0/2 0.5 0/2 TL-1184 Carbnmic acid, N-melhyl-2- OCONHCHj Sc.P. Mice 80 1/2 mcthyl-5-dimelhyltimiiio- A 40 0/2 phenyl ester C11/ ] 20 0/2 I jN(CIfj)i Sc.W. pH3 Mice SO 5/5 V 40 5/5 20 1/5 T 1708 Carbamie acid, N-methyl-2- OCONHCHj Sc. Mice 0.1-0.12 LDw TL-1071 me! lrvl-5-dimet h vlamino- /\ Sc.W. Mice 0.115 /.Djo phenyl ester methuxlide CIl/ ] Sc.W. pH4 Mice 0.1 as LD„ 0 1 tN'(CU,)J Sc.W. pH! Mice 0.107 Uh 0 V Sc.W. pH4 Mice 0.102 /.Dju TL-1236 Carbnmic acid, N-methyl-2- OCONHCHj Prep. 1 methvl-5-dimethylamino- /\ Sc.W. Mice 0,075 ID, 0 Prep. 2 phenyl ester methochloridc ch/ ] Sc.W. Mice 0.064 LD,a Prep. 2 — 1 In(CH,).C1 Ip.W. Mice 0.088 Uh. Prep. 2 V Sc.W. Rats 0.100 LDi0 Prep. 2 - Ip.W. Rats 0.078 LD,. — Prep. 2 Oral W. Rats 2.5 LD, 0 Prep. 2 - Sc.W. Dogs 2.0 4/10 1.0 0/3 Prep. 2 : Im.Imp. Monkeys 0,0.50 1/4 Prep. 3 : &.w. Mice 0.070 Uh0 Prep. 3 - —- Iv.W. Mice 0.035 TL-1185 Carbnmic acid, N-methvl-2- OCONHCHj Sc.W. Mice 0.110 LDia met hyl-5-dirnet hylarni no- /\ (See p. 224) phenyl ester methosulfate ch/ ] 1 InCCIDjSOjCH, TI,-1186 Carbamie acid, N-methyl-2- OCONHCHj Sc.W. Mice 0.103 metliyl-5-diinethylamino- /\ (See p. 224) phenyl ester methosulfuric CH/ ] acid 1 iN(CH,),HSO, TL-1340 Carbamie acid, N-methvl-2- OCONHCH, Sc.W. Mice 0.090 ID-,. methyl-5-dimet hyhimino- /\ — phenyl ester ethiodide CH/ ] ' t Jn(ch,), 1 - CjHjI TL-1339 Carbamie acid, N-methyl-2- OCONHCH, Sc.W. Mice ~ 0.075* LDw methyl-5-dimcthylamino- /\ phenvl ester elhochloridc ch/ ] TIn(CHj)» V I c2h*ci TL-1257 Carbnmic acid, N-methyl-2- OCONHCH, Sc.W. Mice 0.125 T-1739 met hvl-5-dietli vlamino- /\ 1 Mice 0.2 phenyl ester methiodide ch/ ] I ]N(C..Hs)jCH,I • At 8i F. SECRET 222 ARC) M A TIC C \ H B A M AXES Route and Dose (‘(Hie Xante Structure solvent Sjteeies ntR/kg Effect TL -1202 Carbamic acid, X-methyI-2- met hyl-5-( X-Ih'mzvI-X - OCOXHCllj /\ Sc.W. Mice 10 5 2 2 2 2 methylamino)phenyl CH/ ] 2.5 0 2 ester methochloride I Jx(CIIi)* 2 0, 2 X/ 1 CH.C,H»Cl T I,-1201 Carbamic acid, X-methyl-2- melhyl-5-(X-allyl-X-meth- vlamino)phcnyl ester me lb- OCOXHCH, C\l/ | (Cll.hd Se.W._ Mice 0.077 UK,, — ochioride \/ X C1 IjC 11 —CH, TL-1511 Carbamic acid, X-methyl-2- met hyl-5-dimel bylamino- phenyl ester 0-hydroxy- ethiodide OCOXHCH, CM A 1 Jx(CHi)iI Sc.W. Mice o.ns* v' 1 CHjCHiOIl __ TL 1512 Carbamic acid, X-methyl-2- OCOXHCH, Sc.W. Mice 0.050 f /./Co met hvl'5-dimethylamino- /\ ■ = , — phenyl ester aeetonylchlo- CH/ ] ride ... 1 IX(CHj)iCI CH-COCII, — TL 1513 Carbamic acid, X-methyl-2- ocoxiich. Sc.W. Mice 0.1051 U),a one t by 1-5-d i me t by 1 a mi no- /\ phenyl ester carbethuxy- CH/ ] methochloride 1 1x(CH,)2C1 CHCOGCjIIj T-1722 Carbamic acid, X-methyl-3- OCOXHCH, (In saline) Mice 0.75 dimethylamino-O-elhyl- phcnyl ester melhiodide C.«A 1 IX(CH,),I (In buffer solution) Mice 1.30 LDka T-1700 Carbamic acid, X-methyl-5- OCOXHCH, 9 Mice 250-300 TL 1501 dimethylainino-2-Lsopropyl- /\ (In buffer phenyl ester melhiodide (CII,)jCH[ | solution) Mice 125 LDu 1 IX(CH,),I Sc.W. Mice 80 0/2 - 10 20 0/2 0/2 T-T778 Carbamic acid, X-methyl-2- OCOXHCH, ? Mice 175 Uho cyclohexyl-o-dimet hylamino- /\ phenyl ester melhiodide CeHnf ] 1 JX(CH,),I • T-1842 Carbamic acid, X-methyl-2- OCOXHCH, ? Mice 45 LDi o cbloro-5-dimcthvlamino- A phenyl ester hydrochloride - 1 lx(CH,),-HC1 - T-1S00 Carbamic acid, X-methyl-2- chloro-5-diinethylaminO' phenyl ester melhiodide OCOXHCH, cA ? Mice 4 LDm 1 lx(CH,),I TT-1523 Carbamic acid, X-methyl-3- OCOXHCH, Sc.W. Mice 0.120 1st ipropy 1-5-d i me t hy huni no- A (78 F) phenyl ester melhiodide f 1 ~ (CHj).HCl JX(CIIj)jI * At: •e F. t At 73 F. t At 74 F. Tabi.k 2, Section \111 (Continued) SECRET CHEMICAL STRUCTURE VM) TOXICITY 223 Tab IE 2, Sectum VIII (Continued) Code Name Structure Houle and solvent Species Dose »"K kg Effect T ITtiS Carhamic acid, X-mclhyl-3- OCOXHCHj ? Mice 10-15 LD, o dimethvlamino-4-inethvl- /\ phcnvl ester hydrochloride I IX(CIIi)*- LIC1 \r CH, T I.-1187 Carhamic acid, X-methyl-l- OCOXHCHj Sc.P. Mice 80 2 /2 mcthyl-3-dimcthylamino- /\ 40 2/2 phenyl ester 20 2/2 — I JX(CII,), 10 0/2 V 5 0/2 cir. Sc.W. pl!3 Mice so 5/5 40 5/5 20 5/3 10 0/3 5 0/5 TIM216 Carhamic acid, X-methvl-4- OCOXHCHj Sc.W. Mice 0.170 LD,„ iuethvl-3-dimcthvlamino- /\ phenyl ester methiodidc fi 1 lX(CIIj)aI “ - V CHS — Tl. I t53 Carhamic acid, X-mcthyl-4- OCOXHCH, Sc.W. Mice 0.130 LDi o mcthyl-3-dimctliylamino- A — (75 F) phenyl ester mcthochloride 1 lx(cn,)jCi \r CH, TL U20 Carhamic acid, X-methyl-4- OCOXHCH, Sc.W. Mice 0.155 hO.» met h vl-3-dimel hylami no- /\ (72 F) phenyl ester ethiodide ( ] • . —'* I IxXCHjJjCjHJ — CU9 TIM 188 Carhamic acid, X-methyl-4- OCOXHCH, Sc.W. Mice 0.200 U) ss methyl-3-dimethylamino- /\ — (See p. 224) phenyl ester methosulfatc 1 Ix(CHj)jSOjCH, CH, TL-1354 Carhamic acid, X-methyl-l- OCOXHCH, Sc.W. Mice 0.005 />D« met hvl-3-dimet hylamino- /\ — ]>henvl ester allvl bromide ( IX(CH,),CHjCH —CHJIr ' - V CH, TIM 338 Carhamic acid, X-methyl-4- OCOXHCH, Sc.W. Mitre 10 3/3 met hyl-3-met hylbenzyl- /\ 5 2/3 aminophcnyl ester met ho- | I i 0/3 bromide 1 JXCHjCfHs 0.5 0/3 \/ \ CH. (CH,),Br T-17(19 Carhamic acid, X-mcthvl-3- OCOX HCH, (In buffer Mice 70 LD„a dimethylamino-4-isopro- /\ solution) pylphenyl ester hydrochlo- f 1 ride I Jx(CH.hHCI CTI(CH,)» SECRET 224 AROMATIC CARB \MATES Code Name Structure Route and solvent S[H'oies Dose ntR/kK Kffect TL-1502 Carbamic acid, X-methvl-3- OCOXHCH, Sc.W Mice 0.51 CDi0 dimelhylamino-4-isoprt)- A (75 F) T-1721 pyipbcnyl ester methiodide ( Mice 1 1 lx(CH,),l ——==■" CII(CH,), T-1770 Carbamic acid, X-methvl-2,4- OCOXHCH, 9 Mice 10 LD,, diincthyi-3-dimelhylainino- A phenyl ester hydrochloride CHJ 1 JX(CHj)j*HC1 CH, T-1707 Carbamic acid, X-methvl-2,4- OCOXHCH, 9 Mice 0.1 U>.n dimcthvl-.Vdimethvlamino- A phenyl ester methiodide CHJ — 1 |X(CH,),I V CH|_ TM710 Carbamic acid, X-inethyl-3- OCOXHCH, 9 Mice 0.17 LD*, dimethylamino-5- methyl- A plienyl ester methiodide 1 1 - CHJ lX(CH,),I T 1741 Carbamic acid, X-methvl-3- OCOXHCH, ? Mice 0.4 dimethylamino-4-ethyl- A phenyl ester methiodide ( — — 1 Jx(CH,),I C,H, — TL 1237 Carbamic acid, X,\-dimethyl- OCOX(CH,V Sc.W. Mice 10 2/2 2-met hy 1-5-di met hylamino- A 5 2/2 phenyl ester methiodide CHJ 2.5 2/2 1 2.0 0/5 V 1.0 0/5 TL-1423 Carbamic acid, X, X-dimcthyl- OCOX(CHj), Sc.W. Mice 10 2/2 2-met hvl-5-diethvlami me A 5 2/4 phenyl ester methiodide CHJ 1 __ 0.5 0/5 1 lX(CjHi),CH,l 0.25 0/5 TL-1325 Carbamic acid, X,X-dimcthyl- OCOX(CH,h Sc.W. Mice 10 2/2 4-met hyl-3-dimct hylamino- A 5 2/2 phenyl ester methiodide 1 f 1 i 1/2 1 [ Jn(CH,),I _ 0.5 0/2 V 0.2 0/2 CH, — TI,-1487 Carbamic acid, X-methyl-X- OCOXCH;,OCH, Sc.W. Mice 2.5 5/7 mot hoxy-2-met hyI-5-di meth- A 1.0 0/7 ylaminophcnyl ester meth- CHJ ] 0.5 1/10 iodide 1 0.25 0/10 TL-1300 Carbamic acid, X.X-penta- OCOXCJI.o Sc.W. Mice SO 1/2 methvlene-2-methyl-5-dimeth- A 40 0/2 ylaminophcnyl ester meth- CHJ 1 i 20 0/2 iodide 1 1 IX(CII,),1 TL-1355 Carbamic acid, X,X-penta- OCOXCjH.o Sc.W. Mice 20 2/2 methvlene-4-metbvl-3-di- A 10 2/2 met hylaminophenyl ester 1 f I _ 5 0/2 met hiodide 1 1 IX(CH,),I 1 0/2 \/ CH, Table 2, Section VIII (Continued) SECRET CHEMICAL STRUCTURE AND TOXICITV 225 Table 2, Section VIII (Continued) Route and Dose Code Name Structure solvent Species mg. kg Effect TL-1239 Carbarn! biolic acid, N-metby - SCOXIICHj Sc.W. Mice 80 0/2 3-diinctliylamino-4-mcth- / \ 40 0/2 ylphenyl ester mcthiodidc f — - 20 0/2 I |X(C11,),I / \ CM, Route ami Effects Code solvent Species (at various doses) _ ;TL 1185 Sc.W. 0.1 0.2 0.3 0.4 1.0 / Rat 0/2 1 /2 2/2 Rabbit 0 2 1/2 1 2 • 2/2 G. pig 0/2 2/2 — Dor 0 2 1/2 1/2 Cat 0/2 2,2 2/2 Sbeep 0/1 0/2 0/2 0 2 i/r _ Goat 0/2 2/5 0/2 0 2 Monkey 0/2 TE-11 Sti Sc.W. 0.05 0.1 0.2 0.3 Rat 0/2 2/2 Rabbit 0/2 2/2 — G. piR 0/2 2/2 2/2 Dor 0/2 1/2 0/2 TI.-1188 Sc.W. 0.1 0.2 0.3 0.4 Rat 0/2 1/2 0/2 0/2 — - Rabbit 0/2 1/2 1/2 G. piR 0/2 2/2 Dor 0/2 2/2 1/2 ... IX. Benzene compounds with one carbamate group and one quaternary ammonium group in the para [>osition (including thiocarbamates). Code Name Structure Route and solvent Species Dose mg/kg Effect TL-913 Carhamic acid, N-methyl-1- OCONIICHj Sc.W. Mice 80 2/2 aniinophenyl ester hydro- A 40 0/2 chloride o __ 20 0/2 ■ NHj-IICl T 1088 Carhamic acid, X-methvl-1- OCONIICHj Sc. Mice 50 I-tDt, o AH-17 dimethylaminophcnyl ester A Iv. Mice 2 LDa o TL-101)7 methiodidc ( i Sc.W. M ice 80 2/2 I J 10 2/2 V 20 2/2 N(CIIj)jl 10 0/2 5 0/2 TL-M69 Carhamic acid, X-methyl-4- OCONHCH, Sc.W. Mice 10 2/2 dimethylaminophenyl ester A 20 2/2 ethiodide f ] 10 0/2 . v 5 0/2 N(CH,)jC}H J secret 226 AROM ATIC C ARR A MATES Code Name Structure Route and solvent Species Dose mg kg Effect. TL 1150 Carhamie acid, X-methyl-4- OCOXTICHj Se.W. Mice 10 2 2 di met hy la minopheny 1 ester /\ 5 2 2 allyliodide () 2.5 0/2 \/ (ClLi'ijXCTLCU—CTljI TL-1431 Carhamie acid, X-methyl-4- OCOXI1CH j Sc.W. Mice 40 9/9 diethylaminophcnyl ester A 20 1 2 methiodide [ ] 10 0/2 v 5 0/2 X(C;lL’,):CHc4 . - . TL 1132 Carhamie acid, X-methyl-4- ocox iic Hi Sc.W. Mice 10 2/2 -- diethylaminophcnyl ester A 20 1/2 allyliodide f 1 10 0/2 v I) 0/2 X(CdL)iI CH;CH—CH* TL 1430 Carhamie acid, X-methyl-4- OCOXIIC1L Sc.W. Mice SO 2 2 - diethylaminophenyl ester A 40 1 /2_ ethiodide f i 20 0/2 v 10 0/2 ' X(C-lL).d TL-1437 Carhamie acid, X,X-diinetliyl- OCOX(C!L>. Sc.W. Mice SO 1/2 4-dimet hylaminophenyl A 40 0/2 ester ethiodide o 20 0/2 _ V XCCHahCsHJ TL-1486 Carhamie acid, X,X-dimethyl- OCOX(CH3)2 Sc.W, Mice SO 2/2 4-dimethylarninophenyl ester A 40 1/2 /3-hydroxyet hobromidc f 1 20 0/2 '\J 10 0/2 X(CIIj)2Br - — | CILCILOII TL-1470 Carhamie acid, X,X-dimethyl- OCOX(CIL). Sc.W. Mice 80 2/2 4-dimet hylaminophenyl A 40 1/2 ester allyliodide f I 20 0/2 v 10 0/2 N(CHi)jI • — '■ CH*CH-=CH, TL 1438 Carhamie acid, X,X-dime(hyI- OCOX(CTL), Sc.W. Mice 80 0/2 4-diet hylaminophenyl ester A —: ■ _ 40 0/2 met hiodide o 20 0/2 V X(CjHs)iCHjI TL-1472 Carhamie acid, X,X-dimethyl- OCOX{(TL,)j Sc.W. Mice SO 0/2 4-diel hylaminophenyl ester A 40 0/2 allyliodide () 20 0/2 V N(CjH»)»l - (’IIjCII—CHj Table 2, Section IX {Continued) SECRET CHEMICAL STRUCT I.'RE AND TOXICITY 227 Code Name Structure Route and solvent S|iecies Dose mg kg Kffect TL-1471 Carbamic acid, N,X-dinielhyl- OCOXtCH.h Sc.VV. Mice SO 0/2 4-diet hylaminophenyl ester /\ 40 0/2 ethaxlidc || 20 0/2 . v —' X(C.IU)3I TL 1220 Carbamlhiolic acid, X-methyl- SCOXHCH, Sc. I*. Mice 80 1/2 4-nit rophenyl ester A 40 — 1/2 ■— I 1 20 0/2 u 10 0/2 - V NO. TL-1258 Carbamthiolicacid, X-methyl- SCOXHCH, Sc.W. Mice SO 0/2 4-dimet hylaminophenyl A - 10 0/2 ester methiodide 0 20 0/2 V X(CII3),1 — TL-1054 Carbamthiolthionic acid, X,X- SCSX(CH,), Se.P. Mice 40 0/2 dimethyl-4-nit rophenyl A 20 0/2 ester o T V xo. — TL-1128 Carbamthiolthionic acid. X,X- SCSX(CII3)s Sc.W. Mice SO 0/2 dimethyl-l-aminoj)henyl A 10 0/2 ester hydrochloride () 20 0/2 V NHj-HCl TT-1179 Cnrhamtbiolthionie acid, X,X- SCSX(CIIj), Sc.W. Mice SO 1/2 dimethyl-4-dimet hylamino- A (suspension) 40 0/2 phenyl ester metliiodidc () — 20 0/2 v X(CH,),I — X. Benzene compounds with one carbamate group and one quaternary ammonium group in the — para position and other substituents. TL-1478 Phenol, 3-isopropyl-i-dimetli- OH Sc.W. Mice 80 2/2 ylamino-, methiodide A 40 2/2 — 20 0/2 McHfCIh), • 10 0/2 X(CH,),I TL-1322 Carbamic acid, X-methyl-2- OCOXHCII, Sc.W. Mice 0.51 LDia isopropyl-4-dimet hylamino- A phenyl ester methiodide r jcii(cii,)i — v X(CII,),T TL 1446 Carbamic acid, X-methvl-3- OCOXHCH, Sc.W. Mice 10 2/2 methvl-4-ecics Dose mg kg Effect TL1147 Carbamic aeid, X-niclliyl-3- OCONHCH, Sc.W. Mice It) 2 2 incthyl-1-diinct hylamino- A 5 2 2 phenyl ester cthiodide 1 2/2 1 Jen, 0.5 2/2 V 0.25 0/5 N(CHi)jCj!Isl TL-1448 Carbamic acid, N-methvl-3- OCONHCHj Sc.W. Mice 0.24 /,/>.„ met hyl-4-dimet hylamino- — A (73 F) phenyl ester allyliodide ( | vrH‘ (CTOjNCHjCII—CHS1 TL-1434 Carbamic acid, N-methyl-3- OCONHCHj Sc.W. Mice JO 2/2 mcthyl-4-diel hylamino- A 5 2 2 phenyl ester methiodide I I i 1 2 I Jell. 0.5 0/2 V 0.25 0/2 N(C*Hi)iCIIiI TL 1407 Carbamic aeid, N-methyl-3- OCONHCHj Sc.W. Mice 0.145 LD> o ethyl-l-dimethylamino- A (74 F) phenyl ester methiodide | 1 _ - — \ A'11' N(CH,),I ~ TI/-1468 Carbamic aeid, N-methvl-3- OCONHCHj Sc.W. Mice 0.39 LD, „ propyl-4-di met hytami no- A (78 F) phenyl ester methiodide ( | — I ICHjCH-CHj N(CH,),I TL-1381 Carbamic acid, N-methyl-3- OCONHCH, Sc.W. Mice 10 2/2 isopropyl-4-dimet hylamino- A 5 2/2 phenyl ester hydrochloride 2.5 2/2 1 JCH(CHj)j 1.0 1/2 — V 0.5 0/2 — N(CH,VHC1 _ _ TL-1327 Carbamic acid, N-mcthvl-3- OCONHCH, Prep. I isopropyl-4-dimet hylamino- A Sc.W. Mire 0.067 Prep, 2 phenyl ester methiodide— Mice 0.070 LD„ 1 JCH{CH,)j (79 F) Prep. 2 V pH 4 Mice 0.064 LD, „ N(CH,),I . — TL 1345 Carbamic acid, N-methyl-3- OCONHCH, Prep. 1 isopropyl-4-dimcthylamino- A Sc.W. Mice 0.045* LD„n Prep. 2 phcnvl ester methochloridc I | Sc.W. Mice 0.0474 LD:,n Prep. 2 1 JCH(CHj),* Sc.W. pH 4 Mice 0.050} Prep. 2 V Sc.W. Rats 0.1035 U)M N(CH,),C1 (See p. 234) TL-1522 Carbamic acid, N-methyl-3- OCONHCH, Sc.W. Mice 0.057} LDia isopropyl-4-dimethylamino- A — phenvl ester alls ! bromide 1 k'lKCH,), (CH,hNCH,CII -CH,Br TL-1475 Carbamic aeid, N-methyl-3- OCONHCH, Sc.W. Mice 10 2/2 but vM-dimet hvlamino- A 5 2/2 phenyl ester methiodide i 0/2 1 ICILCH-CTLCH, 0.5 0/2 N(CH,),I « At 00 F. t At 87 F. 4 At 78 F. i At 81 F. SECRET CHEMICAL STRl’CTUKE AND TOXICITY 229 Route — anil Dose Code \ ante Structure solvent Species niK/kK Effect TL 1476 CarLamic arid, X-mcthyl-3- OCOXHCII, Sc.W. Mice 10 2/2 am vl- 1-dimet hylamino- /\ 5 2/2 phenyl ester methiodide 1 i 0/2 1 ICILCILCILCII.CIL 0.5 0/2 X(CH,),I TL-1416 Carhamic arid, X-mclhvI-3- OCOXIICH, Sc.W. Mice 10 2/2 cyclopentyl-4-dimethyl- /\ CIL -CM, 5 2/2 aininophcnyl ester meth- i 0/2 iodide Jen 0.5 0 2 V V ciL-cn, X(CH,),I TL 141K) Carhamic acid, X-melhyl-3- ocoxncit, Sc.W. Mice 10 2/2 hexyl-4-dimethylamino- /\ 5 2/2 phenyl ester methiodide 1 2.5 2/2 - v JcJL, 1.0 0/2 V 0.5 0/5 X(CH,),I Tl, 14 SO Carhamic acid, X-metlivl-2,5- OCOXIICH, Sc.W. Mice 0.325* hDi o dimelhvl-4-dimethvlamino- /\ phenyl ester elhiodide chj I \JVlh - : ■ XiCFLWdU - TL-1254 Carhamic acid, X-methvl-3,5- OCOXHCII, Sc.W. Mice 80 2/2 dime) hyl-4-dimcthyla mino- A 40 2/2 phenvl ester hvdroiodidc 1 20 0/2 chJ ijc". 10 0/2 — X(CH,)jHI — TL 1482 Carhamic acid, X-methvl-4- ocoxiich. Sc.W. Mice 0.145f LD.0 dimet h vhuninocarvacrvl A ester ethiodide CHJ 1 - 1 lcH(CH,), X(CH^C,HSI SB-26 Carhamic acid, X-mcthvl-4- OCOXHCII, Sc. Mice 23 LD* dimet hylamiiiothymyl A ester hydrochloride {Clique 1 _ nJch, X(CH,),-HO SB-27 Carhamic acid, X-melhvI-4- OCOXHCII, Sc. Mice 0.22 LDi0 dimet hylamiiiothymyl A ester methiodide (Client 1 — iyH' - X(CH,),I TL 1451 Carhamic acid, X-methvl-2,6- OCOXIICH, Sc.W. Mice 80 2/2 dils< ipropvl- 4-dimet hyl- A 40 0/2 aininophcnyl ester met h- (Clique ( |CH(CII,), 20 0/2 iodide KJ X(CH,),I SB-2 Carhamic acid, X,X-dimethvl- OCOX(CH,h Sc. Mice >400 LDtri 4-dimolhvlamino-2-mcthyl- A phenyl ester hydrochloride l)"‘ V X(CIL), TIC1 * At 75 F. t At 73 F. Table 2, Section X (Continued) SECRET AROMATIC CARBAMATES Table 2, Section X (Continued) Code Name St met tire Route and solvent Species Dose mg ku KlTcct SB 3 Carhamic acid, X,X-diniethvl- OCOX(CHi). »Sc. Mice 6.5 4-dimelhvlamino-2-methyl- /\ phenyl ester methiodide ()"■ — . v N(C1I,),I TL-I313 Carhamic acid, X,X-dimethvl- OCOX(CHj)j Sc.W. Mice 0.17 4-dimethylamino-2-isopropyl- A phenyl ester methiodide [ jcii(cih)j V SB 1 Carlmmicacid, X,X-dimethvl- OCX)X(CII,)* Sc. Mice 105 t-diinet hvlainino-3-rnel hvl- /\ -- phenvl ester hydrochloride f 1 U1"' -- — X(CHj)i-IICl SB 5 Carhamic acid, X,X-dimethvl- OCOX(CHi)j Sc. Mice 13.0 LD,„ 4-diiiiclhvlamino-3-methvl- /\ phenvl ester methiodide — I ] - X(CH,)»1 TL 11 10 Carhamic acid, X,X-dimethvl- cuj SB-6 ('arbainic acid, X,X-dime(hy 1- OCOX(CH,)» 8c. Mice 45 4-dimethylamino-3-ethyl- /\ phenyl ester hydrochloride ( ] — 1 ICjII* _* . X(CHj)»-IK1 SB 7 Carhamic acid, X.X-dimethyl- OCOX(CH.h 8c. Mice 1.15 LD it, TL-1412 4-dirnet hvlamino-3-elhyl- /\ Sc.W. Mice 10 2/2 j>henvl ester met hiodide f 1 5 2/2 I kyi, i 1/2 V 0.5 0/2 X(CHj)iI SB-8 Carhamic acid, X,X-dimcthyl- OCOX(CHi)j Sc. Mice 0.075 LDio TIXiOy l-dimcthylainim>-5-isopro- A Sc.W. Mice 0.080 Uh 0 pvlphenvl ester methiodide l i Sc.W. Mice 0.080 l JcM(CHj)j Ip.W. Mice 0.108 LDi 0 - V Ip.w. Mice 0.220 U)i* X{CIIj)jI Ip.w. Mice 0.265 I'D it. (See p. 234) TL-1460 (. 'arhamic acid, X, X-dimet hyi- OCOX(CH3)j Sc.W. Mice 10 2/2 3-propvl-4-dimet hylamino- A 5 2/2 phenyl ester methiodide 1 1 i 2/2 1 JCIIiCHjCHi 0.50 0/2 V 0.25 0/2 X(CHj)iI SECRET CHEMICAL STRCCTl RE VM) TOXICITY 231 Table 2, Section X (Continued) ('ode Name Structure Route and solvent S|>eeies I lose niK k* Kffect, TL 1143 Carlmmic acid, X,X-dime!hyl- OCOX(CHah Sc.W. Mice 0.065 ldm 3-isi >i >n >py l-4-di met hyl- /\ (73 F) arninophenyl osier met ho- I I chloride 1 IcirtcH,), - X(CHj)jCl - TL-1521 Carbamic acid, X,N-dimcthyl- OCOX(CIL)- Se.W. Mice 0.182 3-isopropyI-4-dimethyl- /\ (71 F) aminoplienyl os tor elhio- f 1 dide 1 lcH(CHj)« XXClLbCdU TL-1461 Carbamic acid, X,\-dimethyl- OC()X(CHj)i Sc.W. Mice 10 2 2 3-hut vl-4-dimet hvlamino- /\ 5 2 2 phenyl ester met hiodide \ | i 0 2 - 1 kcn,),cH, 0.5 0 2 x(ciis)a TI/-14152 ('arbamie acid, X,X-dimclhyl- ocoxxcn,). Se.W. Mice 10 2 2 3-umyl-4-dimelhvlamino- /\ 5 22 phenvl ester incthiodidc ( 1 i 0 2 • 1 I(CHj)iC11j 0.5 0/2 Tl. 1163 Carbamic acid, X,X-dimel hyl- ()COX(Clh): Sc.W. Mice 10 2/2 3-hexyl-l-dimet hvlamino- A 5 2/2 phenvl ester mcthia mic acid, X, X -diuicthyl- OCOX(ClUh Sc.W. Mice 10 2/2 3-cyclopentvl-4-diinethvl- /\ CHj —CH; 5 2/2 arninophenyl ester 1 2/2 methiodide 1 Jem 0.5 0/2 — V \ CH,—t H, - TL-1166 Carbamic acid, X,X-dimethyl- OCOX(CH3)i Sc.W. M ice 80 0/2 3-phenvl-l-dimethylamino- /\ 10 0/2 phenyl ester methiodide 1 1 20 0/2 VCcH' X(CIT,)al SB-28 Carbamic acid, X-methyl-4- OCOXIICHj Sc. Mice 2.1 LD„ dimethylarainocarvacryl /\ ester hydrochloride 1 P1* (CHahlld \ X(('lf»)** 1IC1 ' SR-29 Carbamic acid, X-methvl-4- OCOXIICHj Sc. Mice 0.09 LDm dime! hylaminocarvacryl /\ ester methiodide I FH» {cii.wid 1 X(CHj)jl SIX’HIT 232 AROMATIC CARBAMATES Taiu-E 2, Section X (Continued) Code Name Structure Route and solvent Species Dose mg kg K fleet SHI 1 Carbamicaeid, X.X-dimcthvl- OC()X(CHj)j Sc. Mice 20 LD> o 4-dimct hvlaminocarvacrvl /\ ester hydrochloride 1 Flh (C1I3).1IC1 1 x(cii»)*-nca SIM 2 Carbamic aeid, X,X-dimethyI- 0(ox(cir,)- Sc. Mice 0.24 U)» 4-dimct hvlaminocarvacrvl /\ ester methiodide (cn,)*nd 1 X(Clh),I — SIM) Carbamic acid. X.X-dimcthvl- OCOX(CHj)- Sc. Mice ICO Uh 0 l-dimet hylaminot hymyl (CH,yiic/\ ester hydrochloride It ijcu. — X(CHj)i- IIC1 ■ - SU-10 Carbamic acid, X,X-dimethyl- OCOX(Clf3)j Sc. Mice 0.72 LDt o •1 -dime! hylaminot hymyl /\ ester methiodide (CH3)2U('| ] [JVH. _ . X(CHa)»I TL Hilo Carbamic acid, X,X-dimethvl- OCOX(CIL)i Sc.P. Mice SO — 0/2 3,o-di me t hy 1-4-nit r< rphenyl /\ 40 0/2 ester f 1 20 0/2 chJ Jciu XOj - — TL-1233 Carbamic acid, X,X-dimetliyl- OCOX(CHj)i Sc.W. Mice SO 0/2 3,5-dimcthyl-4-di methyl- /\ 40 0/2 aminophenyl ester hydro- 20 0/2 iodide cnJ leu, X(CHj)ilII TL-1377 Carbamic acid, X-cthvl-3,5- OCOXIICjH, Sc.W. Mice SO 0/2 diisopropyl-4-di methyl- /\ — 40 0/2 aminophenyl ester [ I 20 0/2 (CH.hCllI IcH(CII»)j - X((’H,)j TL 1077 Carbamic acid, X,X-dicthyl- OCOX(CjH5), Sc.O. Mice 80 0/2 3,5-dimethyl-4-nil roso- /\ 40 0/2 phenyl ester I I 20 0/2 CllJ IcH, - ■— xo TL-1197 Carbamic acid, X,X-diethvl- OC()X(Cdl„)5 Sc.P. Mice 80 0/2 3,.5-dimethyl-t-nit rophenyl /\ 40 0/2 ester f 1 20 0/2 CllJ ICH, s/ NO, TL -907 Carbamic acid, X',X-diethvl- OCOX(CjHi)j Sc.W. Mice 80 2/2 4-dimet hylaminot Itymyl /\ 40 1/2 ester methiodide ] 20 0/2 K/"' 10 0/2 X(CHj)J SECRET CHEMICAL STRUCTURE CM) TOXICITY 233 Code Name Structure Route and solvent Species Dose tng kj? Effect TL 778 Carbamie acid, X.X-dielhyl- OCOXCJLO Sc.W. Mice SO 0 2 eneoxy-4-dimelhylamino- /\ 40 0/2 carvacrvl ester met ho- 20 0 2 cldoride \ X(CH,),C1 TL 770 Carbamie acid, N,\-dielliyl- OCOXC.IbO Se.W. Mice 80 1/2 eneoxy-4-dimet hyiamino- A 40 0/2 carvacrvl ester methiodide — 10 0/2 (CH.JjIIcI 1 — X(GHa).r TL 1073 Carbamie acid, X,X-6(S(2- ocoxcciun.cib Sc.W. Mice SO 1/2 chloroethyl )-4-dimet hyl- A ' 40 1/2 luninothymyl ester metho- (CHiVt ’1I| ] * 20 0 2 cldoride . V"' 10 0/2 X(CIIj)>CI TL-1079 Carbamie acid, X-(2-chloro- C’jHs Sc.O. Mice SO 0/2 ct h vl )-X -et h vl-4-uit roso- 40 0/2_ thvmyl ester ocox 20 0/2 A x — (CIIihCHj 1 CILCILC1 u™- — X/ xo TL-1080 Carbamie acid, N, X - fciX 2- OCOX(C 11 jC 1 IjCl ), Sc.O. M ice 80 0/2 chloroethyl )-4-»itro- A 40 0/2 thvmyl ester 20 0/2 — - l/H- X/ xo, TL 900 Carbamie acid, X-(2-chloro- CjHs Sc.W. Mice 80 2/2 e I hyl )-X-ethyl-4-dimet h- / 40 0/2 ylaminothymyl ester ocox 20 0/2 — methiodidc A x (ClL)JICf 1 CILCH..C1 1 Jch8 v X(CIIi)jI TL-1074 Carbamie acid, X-(2-chloro- C.H, Sc.W. Mice SO 2/2 ct hy 1)-X -ct hy)-4-dimet h- / 40 2/2 vlaminothymvl ester ocox 20 0/2 methochloride A \ 10 0/2 (cir,)2cnf ] cilcilci A- - X(CHj)jCl — - TL-104S Carbamie acid, X,X-6m(2- ocox(c,n4ci). Sc.W. Mice so 0/2 chloroethyl M-dimelhyl- A 40 0/2 amiiiolhymyl ester meth- (C1I,)2IIC| 1 20 0/2 iodide V"‘ X(C!la)jI TL-119S Carbamie acid, X-(2-ehloro- CjHi Sc.P. Mice 80 0/2 — ethyl)-X-ethyl-3,5-di- / 40 0/2 inethyl-4-nil rophcnyl OCOX—C H,C I I, Cl - 20 0/2 ester A — chJ Ich, — X/ xo. Tabi.k 2, Section X (Continued) SECRET 234 A ROMATIC CARRAMAXES Code Name Structure Houle and Dose solvent Species mg kg F fleet TL 1075 Carhamic aeid, X,X-6/s(2- ehloroet hyl )-3,5-dimet hyl- 4-nilrosophenyl ester t’llj DC'O\(C1 l.-C 11..Cl)j Set). Mice 80 I 2 /\ 40 0 2 | 20 0 2 K/'"‘ xo TL-1255 Carhamic acid, N',N-l»i*(2- chlorocthyl V-3,5-dimethy 1-4- _ nitrophcnyl ester trihy- drate CH; OCOXTCIM'IIiCDj 3H;0 Sc.P Mice 80 0 2 /\ — 40 0,2 20 0 2 A xo. TL-1413 Carhainie acid, X,X-pcnta- niet h vlene-3-ot hyl-4-di- ocoxcdf,. A Sc.W. Mice 80 2,2 40 2 2 methylaminoplienyl ester metliiodide A"' X(CHj)jI . 20 2 2 It) 0 2 5 0.2 TL 1414 Carhainie acid, N,X-penta- rne t hylcne-3-isopn ipyl-4 - dimcthylnminophenyl ester methiodide OCOXCill,. X(C».)3l Sc.W. Mice 0.51 /jDju (78 F) TL-1418 Carhamic acid, X.X-penla- methyleno-3-cyclo|>entyl- 4-dimethylnminophenyl ester methiodide OCOXCJLo or CUi-t X(CH»)J Sc.W. Mice 80 2/2 ’ll. 40 2/2 20 1/2 10.J 0/2 •II, TL 1049 Carhamic acid, X.X-penla- inethyleiic-4-diinelhyl- aminothymyl ester mctho- (CI1,)2C11 chloride OCOXCill,,. Qr". x(cn,)3a Sc.P, Mice 0.36 LDio TL-968 Carhamic acid, X,K-penta- met hylene -4-di met hyl - aminothyniyl ester meth- (CIIs)»HC iodide (K'OXCiH,,, 0™- X(CHj)il Sc.W. Mice 0.44 LI)ia TL-777 Carhamic acid, X,X-|ienta- methylcne-4-dimet hyl- aminocarvacryl ester methiodide (CH»)*IIC OCOXCill,.. 0~ X(C1I3),I Sc.W. Mice 3 2/3 2 0/3 1 0/3 Iv.W. Mice 3 3/5 2 0/5 TL-1106 Carhamic acid, X,X-pen(a- mct hy lene-3,5-d i rne 1 hyl- 4-nitrophenyI ester CII, OCOXCill, „ 6"- xo. Sc.P. Mice 80 1/2 40 1/2 20 0/2 10 0/2 Tahi.k 2, Section \ (Continual) SECRET CHEMICAL STRUCTURE \ X l> TOXICITY 235 Table 2, Section X (Continued) Route and Dose Code Name Structure solvent Species nig kg Kffect TL I2G0 Carlmmic acid, X,X-penta- OCOXC»H,. Sc.W. Mice 80 0 2 mcthvlene-3,5-din»clhvl- \ 40 0/2 4-dimcthylaniinophcnyl f ] 20 .02 ester hydroiodulc CIlJ X(ciij)j m TL 1465 Carlmmic acid, X-phenvl- OCOX HC.Hi Sc.W. Mice .40 2/2 3-i-sopropyI-4-dimethyl- / \ 20 2/2 atliinophcnyl ester meth- ( 1 10 •> o iodide I JcH{CIIi). — 5 1 /2 \ / 2, 5 0 2 X(C1I.)J Route and Kffccls ( ’ode solvent Species (at various doses) TL-114S Sc.W. 0.2 0.3 0.5 1.0 Hal 0/2 2/2 Rabbit 0/2 2/2 - 0. pig 0/2 1/2 1/2 2/2 T I.-1345 Sc.W. 0.025 0.05 0.1 0.15 0.2 tisi sample; G- pig 0/2 1/2 2/2 Rabbit 0/2 2/2 1/2 4/4 Dog 0/2 1/2 1/2 2/2 Cat 0/2 1 2 2,2 2/2 Monkey . . . “ 0/2 3/3 SB-8 Sc.W. 0.1 0.2 0.3 TL-500 Rat 3/6 6/6 „ . Rabbit 1/3 2 3 G. pig 1/4 4/5 5/5 Dog 0 2 2/3 3/5 Cat 0/3 2/2 XT. Benzene compounds with one carbamate group and an alkyl side chain having a quaternary ammonium group. Code Name Struct tire lloutc and solvent Sjiecies Dose mg/kK Kffcct Til HO Carliamic acid, X-methyl-2- dimctbylaniinomethyl- phenyl ester met hiodidc OCONHCH, [ |CII2X(CH,)»I Sc. Sc. Mice Rabbit 7.2 3.5 ID, o AK-3!» Carbamic acid, X,N-dimet hyl- 2-diethylaininomctbyl- phenyl ester hydrochlo- ride \/ OCOX(CH>)t Iv. Mice 1.5 /,D,. All-40 Carbamic acid, X,X-diiueth\T 2-diet hylaminomct hyl- phenyl ester inethiodide 0(‘0N(CHj)s [ VlI,X(C,II»W KJ \n. Iv. Mice 0.5 UKu SECRET 236 AROMATIC CARBAMATES Table 2, Section XI (Continued) Code Name Structure Route and solvent Sjieeies Hose mg /kg Effect T-(?) Carbainic acid, X-methyl-N- ( X'-me thylcarba myl )-2-di- methylaminomcthylphenyl ester methiodide CH, OCOX \ /\ COXHCHj r jCH*N(CHiM Sc. Mice 343 1.D, o T-20G5 Carbainic acid, X-mcthyl-2- (1-dimelhylamino-n-pro- — pyllphenyl ester methiodide v OCOXHCH, /XcilCIIiCH, \/X(CH,).I Sc. Mice 10 t T-2068 Cart Mimic acid, X-methyl-2- (1 -diliiethylamilio-n-pro- pyOphenyl ester hydro- chloride OCOXHCH, /\ciich3cii, • HC1 Sc. Mice 40 /.D„ T-1890 Carbainic acid, X-methyI-2- di met hytaminomet hyl-C- methylphenyl ester hydro- cliloride OCOXHCHi CH/ |CH,N(CH,),HCl ? Mice 350 I'Diu T-1S0I Carbainic acid, X-methyl-2- dimethylaminomcthyl-5- methylpheny! ester hydro- chloride _ OCOXHCH, ( ]CIliX(CH,),HCl c,v - ? Mice 150 LDt,o T-1802 Carbamic acid, X-melhyl-2- dimeltiylaminomet hyl-4- methylphenyl ester hydro* - chloride OCOXHCH, j^CHjXtCH.VHCI CH, ? Mice 140 LDio T-1893 Carbainic acid, X-methyl-2- dimcthylaminomethyI-4- methylphenyl ester melh- iodide OCOXHCH. J^jC'H.X(CH,),T CH, ? Mice 75 LDi (, T-1847 Carbamic acid, X-methyl-2- (o-dimethylaminoethyl)-4- methylphenyl ester hydro- ctiloride OCOXHCH, /\ciIN(CH,)i • HCI O. CH, ? Mice 70 IjD$ 0 T-184C Carbamic acid, X-mcthyl-2- (o-dimet hylaminoel hyl )-4- methylpheayl ester meth- iodide OCOXHCH, /\CHX(C11,),I Oh. CH, ? Mice 12 LD.u T-1821 Carbamic acid, X-methyI-3- (dimet hylaminomet hyl)- phenyl ester hydrochloride OCOXHCH, (^)t'HIX(CH.)lIICI Mice 10 LDi0 SECRET CHEMICAL STRUCTURE AND TOXICITY 237 Table 2, Section XI (Continued) Code Xante Structure Route and solvent Species Dose mg/kg Kffecl T-1825 Carbamic acid, X-methyl-3- (dimethylaminomethyl)- phenyl ester methiodide OCOXHCHi (^)cll,X(CUJ)J ? M ice 7 IDi0 T 1887 ? T-1939 ? Carbamic acid, X-methyl-3- (/3-dimet by laminoet hylV pbeuyl ester methiodide 1 OCOXHCHi QcH,CH,X(CH.),I 9 ? Mice - Mice ca. 100 7.5-10 LD, o I.D,„ A R 28 Carbamic acid, X-methvl-3- OCOXHCHi Iv, Mice 1.0 T 18-13 (o-diinet hy laminoet hyl)> phenyl ester hydrochloride (miotine) 1 V CH, Iv. Mice Rabbit G. pig Rat Mice 0.5 1.0 ± 0.5 1.0 ± 0.5 1.0 ± 0.5 1.0 ± 0.5 Lf}$v LD* o T-1894 Carbumic acid, X-methyl-3- (o-dimethylaminopropyl)- phenyl ester hydrochloride 1 OCOXHCHi ? Mice 3.0 LDu T 1895 Carbamic acid, X-methyl-3- (o-dimethylaininopropyl)- phenyl ester methiodide 1 OCOXHCHi c,h6 ? Mice 5.0 LDm AR 29 Carbamic acid, X-methyl-3- (a-dimethylaininoethyl)- 6-meihoxyphcnyl ester hydrochloride CHaOj OCOXHCH, [^CilXiCHihHCl CH, Iv. Mice 0 h Dsn AR30 Carbamic acid, X-melhyl-3- (o-dimethyl:iiiiinoethyl)-6- mcthoxyphenyl ester methiodide CII30| OCOXHCHi CH, Iv. Mice 5 Uhi T-1886 ? T 1938 ? Carbamic acid, X-methyl-3- (d-dimet hylaminoel hyl )- phenyl ester hydrochloride 1 OCOXHCHi ? f) 1 tcH,CHiX(CHi)j • HCl Mice Mice 35 Approx. 3.0 LD$q LD$u T-2040 Carbamic acid, X-methyl-3- (2-dimcthylamino-n-]»ropyl>- phenyl ester hydrochloride 1 OCOXITCH, [ | X(CH,),-HCI N/CHiCHCH, Sc. Mice Approx. 16 Uhl T-2064 Carbamic acid, X-inethyl-3- (2-dimclhylamino-n-prcpyl)- phenyl ester methiodide i OCOXHCH, 2CHX(CH.),l CH, Sc. Mice 0.6 LDjri SECRET 238 \KOM VTIC CARBAM VTES Route and Hose Code Name Structure solvent Sjiecies mg kn KfTect T 2038 Carbamic add, X-methyl-3- (3-dimet hy la mino-n-but yl y plienvl ester hydrochloride OCOXHCH, ■A, X(CH3)j - IIC1 So. Mice 9 Uhu 1 1 JcHiCll.CU CH, - - T '2039 Carbamic acid, N-methyl-3- OCOXHCH, Sc. Mice 1(1 U):, o (3-dimet hylamino-n-butyl)- /\ X(CH,),1 phenyl ester methiodide 1 JcHjCHjCH \/ CH, — T-1845 Carbamic acid, N-methyl-4- OCOXHCH, 7 Mice 00 diniel hvlrminomelhyl- /\ phenyl ester hydrochloride o V CHiX(CHa)j. HC1 T-1844 Carbamic acid,X-methyl-4- (a-dimethylaminoct hyl )- phenyl ester hydrochloride 1 OCOXHCH, o f Mice 25 LD„ o CH,CHX(CH,), HC1 AR-28a Carbamic acid, X-me thy 1-4- (rt-dimethylaminoethyl )-2- methoxypheuyl ester hydro- OCOXHCH, [ pCH, Iv. “ Mice 1-1.5 Uho chloride 1/ CH,CHX(CH,V HC1 . T-1S96 Carbamic acid, X-methyl-4- («-dimct hyl ami nopropyl)- OCOXHCH, A ? Mice 300 LDta phenyl ester methiodide I 1 — CH,CH3CHX(CH,),I T 1834 Carbamic acid, X-methyl-4- (d-riimet hylaminoct hyl )- plienyl ester hydrochloride OCOXHCH, 0 CHiCHiX(CH,)2-HCl 7 Mice 10 LDbn AR 41 Carbamic acid, X,X-dimethyl- 4- (/3-dimct hylaminoet hyl)- OCOX(CH,)i A Iv. Mice 15 TJKo phenyl ester hydrochloride f 1 C11.CH2X(CH,)jHC1 AR 42 Carbamic acid, X,X-dimethyl- 4-(/J-dimct hylaminoct hyl> phenyl ester methiodide OCOX(CH,)i n Iv. Mice 55 UK* CH3CH3X(CH,),I T-1935 Carbamic acid, X-methyl-4- (y-dimet hylaminopropyl y phenyl ester hydrochloride OCOXHCH, 0 7 Mice 5-7.5 Uh0 V CHiCH,CH,X(C H,), • IIC1 Tabi.k 2, Section \I (Continued) SECRET CHEMICAL STRUCTURE AM) TOXICITY 239 Code Name Structure Route and solvent Species Dose mg kg KtTeet T-1936 Carbamic and, X-meihyl-4- (y-dimet hylaminopropyl )- phenyl ester methiodide OCOXHCHj 0 9 Mice Approx. 50 Llh o T-15)81 Carbamic acid, X-methyl-4- (7-dirnethylamino-n-butyl)- phcnyl ester hydriK'hloride CH,CHiCHsX(CHj),I OCOXHCHj n Sc. Mice 1(H) LDu CHiCIIjCHN(CHi)j- HCl T-1U82 t 'arbamic acid, X-met by 1-4- (■y-diinetliylamino-n-lHityl)- phenyl ester methiodide CTIi OCOXHCHj o Sc. Mice 40 LDt0 \/ CI I..CH.(' H X (C H ijal - TT.-H15 Carbamicacid, X,X-dimethyl- 3d/3-2-pyndy lothyl Iphcnyl ester methiodide CHj OCOX(CIIj)i 0-0 - Sc.W. Mice 0.33 (78 F)_ LD, o — i CHd T 1827 XII. Benzene compounds Carbamic acid, X-mcthyl-2,4- &i«(dimethylamino)phenyl ester dihydrochloride with one carbamate group ami two OCOXHCHj | |X(CHj)»-HCl quaternary ammonium groups. ? M ice tit) LD„ T-1826 Carbamic acid, X-methyl-2,4- 6i«(dimethylamino)phenyl ester dimethiodide V x(cn,VHa OCOXIICII, 9 Mice 7 LDuo T 1800 T-1S11 X(CITj)31 Carbamic acid, X-methyl-2,5- OCOXHCIIj bis( ilimet hylamino)phenyl /\ ester dihydrochloride | |X(CHj),-HCI (CIIi)>X • IICll 1 9 Mice 50-75 /.Dio T-1810 Carbamic acid, X-methyl-2,5- his{ di mcthylam ino)phcnyl ester dimethiodide OCOXHCH, ( ]X(CH,).I (CHj)jXll \ 9 Mice 500-1,000 /./Co A11-27 Carbamic acid, X-methyl-3- £methyl-(d-dielhylam!iio- ct hyl )-aminojphenyl ester hydrochloride OCOXIICII, Iv. ! In cHiCH;x(c,h,), • iici Mice 0.1 /.Dso CH, - . “ .* T\bi.e 2, .Section XI (Continued) SECRET 240 AROMATIC C VR BA MATES Code Name Structure Route and solvent Species Dose mg kg IcfTeet T 1780 Carbamic acid, X-methvl-4- OCOXHCH, ? Mice 16 LLK, [met hyl-{ d-diel hylamino- /\ ethyl)-amino1phenyl ester ( ] dihydrobromide U CHi—X—CHiCIIjX(CiIIi)j-llllr X-1779 Carbamic acid, X-incthyl-4- OCOXHCH, 9 Mice 100 LDm [methyKd-dicthylammo- /\ * et hyl)-amino]phenyl ester f ] monomet h iodide v rij cifj—x circifxtc.ihh T-1S33 Carbamic acid, X-methvl-5- OCOXHCH, 9 Mice 500 2,500 /XL.. dimet hylamino-2-di met hvl- /\ aminomethylphenyl ester ( VlLXCClhh 1IC1 dihydrochloride - \ XITI. Benzene compound with one carbamate group and one sidfonium or arsonium group. XL-1306 Carbamic acid, X-methyl-3- OCOXHCH, Sc.W. Mice 0.370 LDia methylthiophenyl ester A — — met humiliate ( i 1 kCH.^SO.CH, . XL 1452 Carbamic acid, X-rnethvl-2- OCOXHCH, Sc.W. Mice so 1/2 dime! hylarsinopheny! ester A 40 0/2 methiodide r Wh,),i 20 0/2 XL 1479 Carbamic acid, X,X-dimeth\l- V OCOX(CH,)> Se.W. Mice 10 2/2 3-diinelhylarsinophenyl A 5 2/2 ester methiodide l 1 1 2/2 1 lAs(CH,),I — 0.5 0/2 XL-1504 Carbamic acid, X-methvl-3- ocoxiich j Sc.W. Mice 1.0 2/2 diet hylarsinopheny 1 ester A 0.5 2/2 methiodide \ 1 0.25 0/2 I lAs(CjH5),CH,T 0.125 0/2 XL-1459 Carbamic acid, X,X-dimethyl- OCX)X(CH,), Sc.W. Mice 80 0/2 4-dimct hylarsinophenyl A 40 0/2 ester methiodide o 20 0/2 \/ As(CH,),I XTV ’. Carbamates of naphthalene derivatives. XT-1096 Carbamic acid, X-methyl-2,4- OCOXHCH, Se.B. Mice SO 2/2 dinitro-l-naphthyl ester AA 40 0/2 (Tr 20 0/2 W xo. XL-1053 Carbamic acid, X-methvl-1,6- xo. Se.P. Mice 40 1/2 dinit ro-2-naphthvl ester AA 20 0/2 ( Y pcoxcn, A 10 0/2 HA/ Table 2, Section XII (Continued) SECRET CHEMICAL STRUCTURE VXD TOXICITY Code Route and Name Structure solvent Species Dost* mg kg Effect T-1889 Carhamic acid, X-melhyl- ? 5,6,7,8-tctrahydro-5-di- /\/\ methylamino2-naphthyl f s f jOCOXIICHj ester methiodide I I 1 Mice 20 LDbo X (CH.).l T-iaS8 Carhamic acid, N-methyl- /\/\ 7 5,B,7,8-tetrahydro5-di- \ * \ pCOXHClh melhyhunino2-naphthyl 1 ' J 1 ester hvdrohromide \/ \y X (CHjVHUr— Mice 4.0 /.Dm TL 110*'. Carhamic acid, X-melhyl- OCOXIlCIIj Sc.W. o,fi,7,S-tetrahydro4-di- methylamino-l-naphthylester I si methiodide 1 J I Mice 0.31 (74 F) ID. o X(CHS)3I XV. Carbamates of quinoline and isoquinoline derivatives. T-1934 Carhamic acid, X-methyl-8- /X/\ 7 quinolinyl ester hydro 1 T 1 chloride III CIIjXIlCO -HC1 II 0 Mice Approx. .*>00 LD. o All-37 Carhamic acid, X,X-dimelhyl- /\/\ lv. 8-quinolinyl ester hydro chloride 1 1 J (CIIi)-XCO 1ICI II () Mice 150 LD,o A H 1S Carhamic acid, X-rhelhyl-8- /\/\ Tv. Mice 0.1 LDu T-(7) quinolinvl ester methiodide ( T 1 lv. Mice 10 LDt o 1 1 J Sc. Mice 90 \y^s/ sc. CHjXHCO dial (In buffer || solution) O Mice 31 uh„ AH 3S Carhamic acid, X,X-dimcthyl- /X /\ lv. 8-quinolinyl ester metho sulfate Mice 0.5 ID SO (CHahXCO ‘| 11 CHaSO.CITa — 0 — T 1072 Carhamic acid, X-methyl-1- /X/\ ? met hyl-1,2,3,4-tet ra hydro 7-quinolinyl ester hydro CHjXHCOl 1 SJ cldoride || \/'X o I CHj-UCl Mice 30 LD,» T-11)73 Carhamic acid, X-mclhyl-1- /\/\ 7 methyl-1 ,2,3,4-tet rahydro 7-quinolinyI ester meth- CllaXIlCOl Is! iodide 11 X/'X o I (CH.hl M ice 0.33 Table 2, Section XIV (Continued) secret 242 AROMATIC C ARB AM AXES Houle ami Dose Code Name Structure solvent Species mi! kg Kffect T 1937 Carbamic acid, \-methvl-l- ? M ice Approx. 45 /./> „ met hyl-1,2,3,4-tel rahydro- 1 S I 8-quinulinyl ester meth- iodide CII, XI ICO 11 o Ax^ 1 (CHS)2I T-1970 Isoquinolinc, 2-methvi- CILXIICO, V\ s XCHj llCl V •} Mice 20 1,2,3,4-tetiahvdro-5,tt-5(.s- A (X-methvlearbamyloxy) (11, XI ICC)/ J hydrochloride I - / T-1071 Isoquinoline, 2-met hyl- 1,2,3,4-tel ra h v d ro-5, G-6is- ClliXHCXL /\ 9 M ice GO /,/),„ f X-met hvlearba in vloxy) CM XHCo/ J" s X(CIL)j 1 melhiodide T 1968 Isoquinolinc, 2-methyl- Cl LX 1 ICO- /\ Mice Approx. 400— /,/).,0 1,2,3,4-tct ra h vdro-tLZ- 800 bis( X-met hvlearhamyl- ClLXHCoJ 1 s XCII, I1C1 oxy) hydrochloride \/ — T 1909 Isoquinolinc, 2-niethyl- 1,2,3,4-tel rahydro-6,7- C1LXHCO, /X 9 Mice >800 /./),„ j * X(CILM bis( X - met h v Icarha my 1- ClLXHCoJ oxy) methiodidc 'v XVI. Carbamates of aliphatic alcohol derivatives. TL 1251 Carbatnic acid, 2-(dibutyl- I(C4IL)5XCILC1LOCOXIL Sc.W. Mice 80 0/2 _ amino)-cthyl ester bulo- 40 0/2 iodide 20 0/2 TL 1224 Carbamic acid, X-met hvl-2- KC.lDACHjCI LOCOX IIC ’1L Sc.W. Mice 80 0/2 (dibutylamino)-cthyl ester 40 0/2 butoiodide 20 0/2 TL-1234 Carbaraic acid, 2-(dictfiyl- 1(C-I LLXCH .C1LOCOX1L Sc.W, Mice SO 0/2 amino)-ethyl ester ethio- 40 0/2 dide 20 0/2 TL-1152 Carbamic acid, X,X-di- 1( Cjl DjXCIIjC 1 LOCO X (CH 3 >2 Sc.W. Mice 80 0/2 met hvl-2-diet hvlamino- 40 0/2 ethyl ester ethiodide 20 0/2 TL 1151 Carbamic acid, X-mct hvl-2- I(Cj 1LLXCILC1LOCOX I1CH, Sc.W, Mice 80 0/2 diethylaminocthyl ester 40 0/2 ethiodide 20 0/2 CIIjC I LOCOX HC11 j Sc.W. Mice 80 0/2 TL 1154 Carbamic acid, X-methy 1-2- xy )-, met hiodide I Sc W. CIL(C2IU=XCI1.(IICJLOCOXHCI1j 1 ocoxiicn. Mice SO 40 20 0/2 0 2 . 0 2 TL 1514 I lexync, 2,5-6i«(X-me( hyl- carbamyloxy)- high melting form ('—CII(Cllj)0( OXIIClIi Sc. P. I C (IRCIIdOCOXHCH, Mice SO ~ 10 20 0/2 0/2 0 2 TL 1515 Ilexyne, 2,5-bis( X-methyl- ea rba n ty luxy)-lo\v melt- ing form C—CIKCliriOCOXllClU Sc.P. i - <' -cii(cn5)Ocoxiicn, Mice SO 40 20 0/2 0/2 0, 2 T-{?) Carlmmic add, X-benzyl- 2-dimetliylaminoetliyl ester met hiodide I(CII1)1XCil.( 1 Sc. Mice 6.25 /-/)» 0 T-(?) Carbamie aeid, X,X-di- 1 ienzy 1-2-dimet hyl- aminoethyl ester met hiodide >"<0 RCIIjIjXCILCILOCOX Mice 75 Lt>i„ T (?) Carbamie add, 3-di- methylami nopropyl ester methiodide l(CHi),XClI;( 'IIjCITjOCOX Hi Se. M ice 37.5 LDa T-(?) Carbamie aeid, 4-di- met hy laminobutyl ester methochloride CKCTDiXTCIDjCHjOCOXTIi Sc. Mice 12.5 LD&a T-(?) Carbamie aeid, 10-di- mcthylaminodecyl ester methoehloride C ’l(CH3)JX(CH,)9CH,0( OX 1 r2 Se. Mice 75 LD„ T-1090 Carbamie acid, 5-di- methylaminoamyl ester methoehloride C1(CI I,i)5X(CI DiCH/KOX 11, Se. Mice 20 LDiB T 1124 Carbamie acid, X-methyl- 4-dimetliylaminol)eiizyl ester methoehloride ('1( CI I,),X ■ I I.OCOX HCHj Sc. Mice 79 LDsa T (?) Carbamie acid, X-methyl- 2-dimef hylaminoel hyl ester methoehloride C1(CH,)3XCH2CHi(XOXIICHj Sc. M icc 15 T (?) Carbamie aeid, X,X-di- met hy 1-2-dimet hyl- aminoethyl ester methiodide KCID.XClIiCHiOCOXtCIU), Sc. Mice 20 LDin T-(?) Carbamie acid, X-ethyl- 2-di met hylaminoel hyl ester methoehloride Cl(CH,)jXCI l.CHOCOXIICdL Sc. Mice 60 LDb o T- (?) Carbamie add, X,X-di- elhyl-2-rlimelhyl- aminoethyl ester methiodide I(('II,),NCI12CHiOCOX(CjI lib Sc. Mice 42.5 LDi o SECRET 244 AROMATIC CARBAMATES Code Xame Structure Route and solvent Sjiecies Dose mg kg Effect H» H, I I T (?) Carbnmic acid, X,X-penta- mcthylene-2-dimet hyl- aminoethyl ester mcthiodide - ■ — 1 1 C—C / \ I(CHj),XCH;CIIiOCOX CH, \ / C—C 1 1 — Sc. _ Mice 1 LUW T (?) T-(?) T 1003 Carbamic acid, X-allyl- 2-dimethylaminoethyl ester mcthochloride Carbamic acid, X-phenyl- 2-dimcthylaminoethyl ester mcthiodide Morpholine, X-(d-earba- myloxyet hyl)-, methcx'hloride H, H, C1(CHi)»XCIIjCHsOCOXIICIIjC1I—■ CH, Sc. l(CH,)iXCH,CIW)COXIIC»Hi Se, Cl CHr X—CH,CH,OCOXH, Sc. / \ CH, CH, i i Mice Mice Mice 37.5 450 175 LDJV Uhn * ‘ . it CH, CH, \ / - _ O — TL-1380 AR-II All 45 TI.-HOO XVII. Miscellaneous Carbamates, Physost igmine salicylate CHa Sc.W. CHjXIICOOp \ | j| V'n V ch, ch, c,h,o, Physostigmine salicylate Iv. Physost igmine mcthiodide Tv. Ammonium comjmund, snbsti- CHj Sc.P. tuted di mcthyl-[/J-(X- /\ methylcarbamyloxy)-y-(3,4- 0 j inethylenedioxyphenyl)pro- / 0 pyl] (3,4-methylcnedioxy- /\S benzyl) iodide M i«!C Hats G. pigs Rabbits Cats Dogs M ice M ice Mice 0.370 1.500 1.500 1.500 1.2Q0_ 1.000 0.800 1.400 1.200 1.000 0.5 0.75-1.0 80 40 20 __ J.l)W 0/2 0/2 0/2 2/2 2/2 2/2 1/2 1/2 0/2 I. IK, /,/),„ 0/2 0/2 0/2 - •— CII, ---- 1 CHjNHCOCH ii i ■ it i 0 CH, 1 X(CH,)J CH, ■ 0. \ O 0 \l CH, Table 2, Section XVI {Continual) SECRET CHEMICAL STKI CTI RR AMD TOXICiTV 245 Code Name Structure Route and solvent S|>ccies Dose mR/kg Kffect TL-1411 Carhamic acid, X-methyl- II Sc.W. Mice 80 0/2 3-di met h via in ino-d-born vl 40 0 2 ester mcthiodide /VOCOXHCH, 20 0/2 — 1 lx{CIIj)jl N/ 11 - — AR-43 Carhamic acid, X-melhyl ester A A Tv. Mice GO Uh„ of Harmol hydrochlorkle ( i (i - 1 L 1 Jocoxiicii cn. SB 2o Carhamic acid, X,X-dimcthvl- 0(’()X(CHj)i Sc. Mice 120 LD /t-pyridyl ester hydrochlo- /\ ride (1 V VXHCI XVIII. Carbamide* and carbazates. TL-1517 Carbazic acid, 2,2-dimethvl-o- OC*OXHX(CIIj)i Sc.W. Mice 80 0 2 dimethylamino-2-methyl- A 1 40 0/2 phenyl ester dimethiodide cu/ ] 20 0/2 1 Jn(CH,)3 Vi TL-1516 Carbazic acid, 2,2-dimethyl- OCOXHXfCIIsb Sc.W. Mice 80 0/2 5-dimethvlamino-2-methvl- 40 0/2 phenyl ester dihydrochloride ch/ | 20 0/2 VyxfCH,), AR 2fi Carbazic acid, 2-phenyl-3-di- OCONHXHCtlU lv. Mice 0.25 LD.„ methylaminophenyl ester /\ met hiodidc [ 1 _ ' - TT.-1402 U rea, 1 -(4-hy drox v-2,3,o-t ri- 11 X—COXIICH, Sc.P. M ice 80 0/2 me thylphenyl)-;4-methyl- Acn. - ■ 40 0/2 1 1 - 20 0/2 CII\ X'H, - on TL-1401 Benzene, 1,4-bis CITj—X —COXIICHj Sc.W. Mice SO 0/2 (1,3-di met hyhireido)- A 40 0/2 () 20 0/2 V CHj—N—CONHCH* Tabus 2, Section XVII (Continued) SECRET Chapter 1I MISCELLANEOUS COMPOUNDS PREPARED OK EXAMINED AS CANDIDATE CHEMICAL WARFARE AGENTS Marshall dates ill INTKODUCTION 1.\ table 1 of this chapter m grouped all those compounds which for one reason or another have not. l«>en subjectod to detailed toxicological examina- tion. Wit.li the average example, these substances showed insufficient toxicity to he seriously considered as chemical warfare agents, although other consider- ations, such as limited availability, or lack of means for tactical employment have influ- enced decisions to abandon exploration of some com- pounds or the classes to_\vhieh they belong. Several of the compounds included or classes cov- ered have I>een treated in other chapters of this vol- ume. For example, cadmium, cadmium oxide, other cadmium compounds, some selenium derivatives, and several metallic carbonyls form the subject of Chap- ter 11. The tabulation of this chapter is intended to supplement such chapters by including references to the preparation and screening of the less promising members of such classes for the sake of completeness. Although a number of compounds examined by the British have been included in the tabulation, no at- tempt has been made to give comprehensive coverage to British screening tests, since such systematic lists are provided elsewhere.** Perhaps worthy of mention in passing is the .sub- stance diehloroformoximo (“phosgene oxime”). It was examined in this country ljecau.se intelligence reports and published literature indicated that some attention had been paid it, by the Germans and per- haps by the Russians. Diehloroformoxime possesses marked irritating action against skin which is mani- fested by an immediate burning sensation and the production of blisters. For this reason, the substance has Ik'Cii proposed as a “nettle" gas, but its limited stability, relatively low toxicity, and difficult prepa- ration preclude serious consideration of it as a chem- ical warfare agent. Diehloroformoxime exists w hen pure as a colorless solid of nip 39 40 C. It boils at 129 C w ithout decom- position at atmospheric pressure*3 and at 47 49 C at 23 nun 42:1 and is soluble in water and in organic solvents. It is rapidly destroyed by alkalies and is slowly hydrolyzed by water.®* It possesses a pene- trating and unpleasant odor and attacks (be mucous membranes and the eves severely.** The substance appears to lie reasonably stable, when pure and kept from contact with moisture ■’*'■** or when stored in anhydrous ether solution 60 but crude material rap- idly decomposes on standing.44* Three distinct methods of preparation are de- scribed in the open literature: 1. The reduction of triebloionitmsomethane by hydrogen sulfide or aluminum amalgam,64 2. The action of chlorine on fulmintc acid *' or on mercury fulminate,*2 3. The chlorination of chloroisonitrosoaeetone.** The first anti third of these methods have been briefly examined by investigators under Division 9 of (lie National Defense Research Committee £NDRC] with disappointing results.42l 44b The first gave rise to unspecified yields of material of poor quality which decomposed in less than a day; the second gave only 30-40 per cent yields of crude ma- terial. The material which was examined physiologi- cally by the University of Chicago Toxicity Labora- tory melted below 35 C, and it is doubtful whether a pure sample of diehloroformoxime has been pre- pared or exanrned in this country. The chlorination of fulminic acid salts has been in- vestigated briefly in England.*** The yields obtained (24 45 per cent) did not approach those claimed by Birckonbach and Sennewald.61 It was found that twice reerystallized material is considerably more stable than distilled material and can l»e stored for several weeks without undergoing appreciable de- composit ion. The related dibromoformoxirne has also Ijcen pre- pared and screened for toxicity.511’ It is less toxic than the prototype. SECRET MISCELLANEOUS COM POL NDS AS CHEMICAL AVAR FAKE AGENTS Tahlk I. Miscellaneous compounds prepared or examined as candidate chemical warfare agents The compounds in Table 1 arc arranged in two large groups; (1) derivatives of heavy metals; and (2) miscellaneous organic compounds Within the heavy metals group, the compounds are classified according to the periodic group of the mi tal, and, among each group of the periodic tahlc, according to increasing atomic number. Tite miscellaneous organic compounds have liccn arranged according to the Heilstein system. The following abbreviations are used: refractive index at1 C; sjiccific gravity at t, C in reference to water at hi nip, melting point in (4; hpJ', boiling jxdnt in C at p mm lig; vp', vapor pressure in mm Ilg at 1 C; vol', saturation concentration (volatility) in mg 1 at I C; and dec. p., dcconi|M)sition point. llrilish reports concerned with those compounds marked hv at asterisk are not all available in this country. Centigrade scale is used throughout the table. Reference Refer, to to Physical projicrties toxicity Compound synthesis Property Reference data 1. Cupric (luoroaeelate 52 51 2. Cupric 2,4-dinitrols‘n/eiiearsonale 40a 3. Cupric 2,4,fi-(riiiitrobenzenearsonatc 40a ... .... —. . , t; Silver nitRite Commercial 24 5. Zinc fluoborate 40r 24 fi. Zinc fluosilicate 40i| 24 7. Strontium fluolroratc 40r 24 8. Strontium fluosilicate 40r 24 it. Cadmiumt Commercial ... - .... See ('hap. 11 10. Cadmium fluoridet 40o — 21 11. Cadmium chloridet Commercial 24 12: Cadmium uitratef Commercial 24 13. Cadmium osidef 24 14. Cadmitiih sulfidef Conimercial 24 15. Cadmium sclenide 22 24 Hi. Cadmium selenite 40q 24 17. Cadmium selenatet 22 ... .... ... * 24 18. Cadmium phosphite 24 19. Cadmium phosphate! 24 20. Cadmium fhiol>ora(et lOp 24 21. Cadmium fluosilieatcf 40q dcc.p. Approx. 100“ 40q 24 22. Cadmium lactate 6 23. Cadmium butyrate .5. 24. Cadmium caproatc 6 25. Cadmium palmitate fi . . _ 20. Cadmium oleale fi 27. Cadmium stearate (j ... 28. Cadmium naphthenate 6 29. Cadmium oxalate 6 30. Cadmium malonate fi 31. Cadmium maleate fi . ; . 32. Cadmium fumaratc 6 33. Cadmium succinate 6 34. Cadmium malate fi 35. ('admium tartrate - - - 6 30. Cadmium glularale 6 ... .... 37. Cadmium adipate 6 ... 38. Cadmium mucatc fi 39. Cadmium citrate 6 40. Cadmium chelate of acetylacctonc 0 dcc.p. 280-285° 6 41. Cadmium enolate of ethvl nitromalonate 0 42. Cadmium salt of nitrated oxidized starch Commercial 24 13. Cadmium salt of 2,4-din it rophenol — 6 ... .... 44. Cadmium picrule 0 dec.p. 250° fi 45. Cadmium chelate of dinitroresorcinol fi 40. Cadmium styphnate fi 47. Cadmium m-nilrobonzcncsiilfonate fi .... 48. Cadmium 2,4-diiiitrohenzencsuifonatc 0 Cadmium p-nitrolicnzoate fi 49. 50. Cadmium 2,4-dinitrolienzoate 6 ... 51. Cadmium 3,5-dinitrobenzoate 6 t These compounds are discussed more fully in Chapter 11. SECRET 248 MlSGEULANEOl S COMPOUNDS \S CHEMICAL WARFARE AGENTS Compound Before ncc to synlliesis Physical projH'rties Property Hcferencc Refer, to toxicity data 52. Cadmium 2,4,6-trinitrolicnzoatc 6 53. Cadmium chelate of salicvlaldchvdc 6 mp >300 0 54. Cadmium chelate of salicylaldoxime 6 mp >300° 6 55. Cailinium salicylate 6 dcc.p. 280° 290° 6 56. Cadmium 3-nil rosalicylate 6 57. Cadmium 5-nil msalicvlate 6 58. Cadmium pbthalate 40k 50. Cadmium o-nitrocinnamate 6 (iO. Cadmium /«-nitrocinnamalc j « 61. Cadmium p-nitrocinnatnale / 8 62. Cadmium s;ilt of hcxanitrodiphcnylamine f ti (13. Cadmium o-nitrolieiueneareunate 6 64. Cadmium 2,4-dmitrol)cnzencarsonatc 0 65. Cadmium 2,4,6-lrinitrobeii7.cnearsonate 6 66. ('admium 3,5-dinitro-4-bvdroxybcnzcncarsonatc 6 1)7. Cadmium 3,5-dinil ro-2,4-325° 6 73. Dimethvleadmium 6 i>p - 08-99° 6 74. Diclhvlcadmium 6 bp5" 62 64° 6 75. Dipropylcadmium 6 I'P" 67° ti 76. Barium fluoboratc 4 Op mp >200° 40p 24 77- Barium fluoeilicate 40p mp >200° lOp 78, Barium succinate .... 70. Barium salt of 2,4-dinilrophenol 40c <80. Barium 3,5-dmitrobenzoate 40e 81. Barium 2,4,6-trinil robenzoate 40c 82. Barium salt of dipicrvlamine lOc 83. Barium 2,4-dinitrobenzcnearsonate 40a 84, Barium 2,4,6-trinilrobcnzcncarsonatc 40a 85. Barium 5-nitro-2-furoate 10c 24 86. Barium 5-nit ro-2-furvlacrvlale 40e 87. Mercuric chloride Commercial 24 88. Mercuric fluoroacelatc 52 51 80. Mercury salt of nitrated oxidized starch Commercial 24 00. Mercuric 2,4-dinitrohenzcnearsonate 40a 01. Mercuric 2,4,6-trinil robcnzcncarsonatc 40a 02. Chlorovinylmercuric chloride 40j 24, 33 03. Butvlmercuric iodide 40e 04. Butvlmercuric hydroxide 40c 05. 2-( ’hloromercurifuran 28 mp 151-152.5° 28 24, 33 06. 2,5-( IJicliloromercuri)furan 28 24 07. 2-Chloromcrcurithiophenc* 28 mp 183-184° 28 24, 33 08. 2,5-f>is(Chloromercuri)thiophene* 00. Difurylmercury 40c IO CO CO 100. Thallous fluoride 15 bp 298° 15 24 ■ _ — mp 288“ 15 101. Thallous fluoborate 15 i»p8 300“ 15 24 102. Thallous selenite 4 Or 103. Thallous fluosilicate 15 bp* 340° 15 24 104. Thallous ethoxide 15 , , . 105. Thallous /3-chIoroethylmcrcaplide 40p mp >300° 4 Op 106, Thallous formate 15 107. Thallous acetate 15 24 108. Thallous fluoroacelatc 52 51 100. Thallous trifluoroacetale 40p mp 116-110° lOp 24 110. Thallous salt of ethyl nitromalonale 40c Table 1 (Continued) SECRET MISCELLANEOUS COMPOUNDS AS CHEMICAL WARFARE AGENTS 249 Table 1 (Continued) Compound Hefercnee to synthesis Physical pro|H‘rlies Property Reference Refer, to toxicity data' 111. Thallium sail of nitrated oxidized starch Commercial 24 112. Thullous lienzoate 15 113. Thallous /j-nilrolx'iizoate 15 114. Thallous 2, 1-dinit robenzoate 15 115. Thallous 3,5-dinitrobenzoate 15 110. Thallous /H-trifluorometbvlbenzoatc 40p 24 117. Thallous salt of 2f4,6,2',4',6'-hexaaitrotiiphenyl- amine 15 118. Thallous furoate 15 1 111, Thallous o-nitro-2-furoate 15 120. Thallous 5-nil ro-2-fnrylacry late 15 ... . r. 121. Thallous N - me t h v Id i t h iocarba mate 15 122, Thallous X,X-dimelhvldi(hiocarbamale 15 mp 121-125° 15 123. Thallous X-ethvldithiocarbamatc 15 121, Thallous X-isopropyldit hiocarhama te 15 125. Thallous X,X-diethy!di(hiiK'arl>amate 15 bp""1 190° 15 120. Thallous X-butvldithiocarbamate 15 'HP 110 111° 15 137, Thallous X,X'-diisoj>ropyldilhioearl>amate 15 .... 128. Thallous X-cvcIohewlilittiioearliamate 15 -v . . • 129. Thallous X, X-dibit tyldilhiocarba male 15 bpo.oi-ae 230-235° 15 130. Thallous X,X-diisobutvldithiocarbamato 15 nip mp 75 77° 165-105.5* 15 15 131. Dimcthylthallium fluoride 15 .... 132. Dimethylthallium iodide 15 — .... 133. Dimethylthallium hydroxide 15 131. Dimethylthallium fluoborate 15 mp 303° 15 135. Dimethyllhalliuni fluosiheate 15 nip >300° 15 130. Dimethylthallium cthoxidc 15 24 137. Dimcthylthallium ethylmercaptide 24, 33 138. X-Dimethyllhallium dimethylaminc 40i ... 139. X-Dimelhylthallium diethylamine 40i 110. X-Dimethylthallium dibutylamine 40! 141. Dimelhvlthallium acetvlacetone 15 mp 214 215° 15 142. Dimethylthallium cthvl acctoacetate 15 nip 128-130° 15 143. Dimcthylthallium trifluorohexoylacetone 2 4, 33 I It, X-Dirnethvlthallium mellivlaniline 4 Of .... 145. Diniethyllh.allium salieylaldchyde 15 mp 200°d 15 140. Dimethylthallium X,X-dicthyldithiocarbamate 15 bp1 130° 15 24, 33 117. Dimethylthallium X,X-diLsopropyIdithioear- 15 bp4 bp* 138° 130° 15 15 24, 33 bamate bp55 115° 15 .. . ’ • mp 150° 15 148. Dimcthylthallium X.X-dihutyldilhiocarhanmte 15 bp"-* 147 148° 15 149. Dimelhvlthallium X,X-diisobutyldithioearbamatc 15 bp»» 104-105° 15 24 150. Dicthylthallium bromide 15 mp 73-74 15 151. Diethylthallium ethoxide 15 152. Dietlivltballiuin Irifluoroaeetate 15 mp 233-235° 15 153. Diethylthallium aectvlacctone 15 dcc.p. 240° 15 154. Diethylthallium benzoylacetone 40*1 155. Diethylthallium thioacetale 15 mp isi-i83° 15 150. Dipropvllhalliuin ethoxide 15 157- Dipropyllhallium-d-camphor-l 0-sulfonate 15 15 158. Diisopropylthallium chloride 15 mp 150°d ■ 15 15 159. Dibutvlthallium fluoride 15 mp 22t>-230° 15 15 100. Dibulvllhallium chloride 15 mp 240-245° 15 15 161. Dibutvlthallium bromide 15 mp 245-250° 15 15 102. Dibutvlthallium iodide 15 mp 220-225° 15 15 103. Diisoamylthallium acet vlacetone 40d 104, Diphenylthalliuin chloride 40(1 — , . . SECRET 250 MISCELLANEOUS COMPOUNDS AS CHEMICAL WARFARE AGENTS Table 1 (Continued) Reference Refer, to to Physical projwri ics toxicity Conqmuml synthesis Property Reference data 165. Diphenylthalliuin iodide 15 mp >300° 15 166. Diphcnylthallium hydroxide 40.1 167. Difuryllballium fluoride 15 mp 235 210.1 15 . , . 168. Difurvlthallium iodide 15 — mp 238-240° 15 166. TctrainethylKermaniimi 24 170. Stannic 2,4-dinit rolicnzenearsoiinte 40a 171. Stannic 2,4,6-trinitrobenzcncarsonate 40a 172. Rutvltin triiodide 2 bp‘ 154° 2 2 173. Dipropyltin dibromide 2 bp"1 112° 2 2 « mp 49-50° 2 2 ~ 174. Dibutyltin diiodide 2 bp55 1572 2 2 175. Di-terl-butvltin dibromiile 2 bpu 128° — 2 2 176. 6is(2-Pyridyl)tin bromide 10f ... - 177. Trimethyltin bromide 2 bp* 46-47 2 — 2 17S. Trimethyltin hydroxide 2 sublim.p. 105-108° 2 179. Triethvllin hydride 2 bp*- 36° 2 180. Triethyltin bromide 2 1'P 216-217° 2 2 24 181, Tripropyltin hydride 2 bP* t>5‘ 2 21 dt* 1.1452 2 182. Tripropyltin bromide _ 2 bp* 123° 2 2, 24 183. Triisopropyltin bromide 2 bp1 79° 2 184. Triisopropyltin iodide 2 bp4 108 no 2 2 185. Tributyltin hydride 2 bp* 115° 2 24 d,n— 1,108 2 186. Tributyltin chloride 2 bp1 5 119° 2 2 200° 40q 24 209. Ix'ad salt of nitromethane 1 210. Ixad salt of nitroaminogiianidinc 1 211. la-ad salt of dinitrotartaric acid 1 212. Ix-ad-w-nilrolxuizenesulfonate 1 213. Ix>ad 2,4-dinitrolx-nzenesnlfonate 1 214. Lead lienzoate I 215. Leaad m-nitrolienzoatc 1 217. Ixad p-ni(robenzoate 1 218. I>ead 2,4-dinitrobenzoate 1 219. Ijead 3,5-dinitrolienzoate 1 .... ... SECRET MISCEI.I. VN KOI S COMPOUNDS VS CHEMICAL WARFARE AGENTS 251 Tabi.E I (Continued) Hefereiex1 Refer, to tft Physical properties toxicity Compound synthesis Property Reference data 220. I/oad 2,4,6-trinitrohenzoate 1 221. I*'ad sail of p-nitrophcnylhydroxamic acid 1 222. Load salt of m-phcnylenedinitroamine 1 • ... 223. I.ead «-nit rolrenzenearsoimle 1 224. Ijcad m-nit rolienzenearsonatc 1 225, Ix-ad 2,4-dinit rolrcnzenearsonatc . . * 220. Iz>ad 2,4,6-trinitrohenzcncarsonnte I. 227. Ix'ad 3-nit ro-4-hvdroxvbenzcnoarsoimlc 1 228. Iz*ad 3,5-dinitro-l-hydro.\vhen2encarsonate 1 .... 220. 1 .cad 3,5-dmil ro-1-aminobcnzenearsonate 1 ..:— 230. Iz\id 5-nitrofuroate I 231. Iz-ad 5-nit rofurvlacrvlate 1 .... 232. Diethvllead dinitnUe 1 233. Diethvllead scionito 16 mp >286" 16 234. Dicthylh'ad fcis(p-chlorolx'nzoale) 16 mi) 18.551 16 235. Dicthvllcad hix(»«-hromohenzoate) 16 nip 178 179 d 16 —.,. 230. Dietlivllcad b/s(m-nilrolxMizoate) 16 mp 179 18051 16 237. 1 )ict livllcad b/s(/>-tohiate) 16 mp 18651 16 238. Diet livllcad hiM N -lint vlant hranilatc) 16 mp 169 109.551 16 230. Dicthvllcad dinicotinatc 16 mp 14351 16 240. Dicthvllcad dithioacetatc* 56a mp 84.5-85° 56a 241. Dihutvllcad dinitratc 1 242. Diphcnyllead dinitratc 1 243. b/«(»»-Nitropheiiyl)lead dichloridc 1 211. bis{ m-N ilrophcnyl)lcad dibromidc 1 215. hix( i/i-Xil rophenvl )lcad diiodide 1 246. 6i*(m-Nitrophenvl)lead dinitratc 1 .... 217. his(m-Nitrophcnyl)lcad oxide 1 .... 248. 'Primetlivllcad p-tolucncsulfonalc* — . 249. Trictlivllcad thiocyanate* 16 mp 26.5 27° 16 24, 33 250. Triethvllead sclcnocyanate* 16 mp 33-34° 16 251. Trict livllcad nitrate I 252. 6/s(Tricthv!lcud) fluosilicate * 24 253. Triet hyl-p-chlorot hiocthoxylead 40m 24 254. Triethvllead fluoroacetate 56e mp 180.5° 56e 55c 255. Triethvllead a-chlorocrotonale 16 mp 153 155° 16 256. Triethvllead acid oxalate 16 mp >300° 16 257. bi,s(Trietlivllcad) oxalate 16 mp >300° 16 258. hiM Trict livllcad) fumarate 16 dcc.p. 165° 10 259. b(s(Trictlivllcad) adipate 16 mp >360° 16 260, f>is(Ti iothyllcad) d-eamphoratc 16 mp >310° 16 16 261. (rt.s(Triclhyllcad) citrate 16 nip >350° 16 262. Triethvllead m-chlorol>cnzoatc 40d 263. Triethyllcad p-chlorobenzoate 16 mp 123 124° 16 264. Triethvllead o-hromolienzoatc 16 mp 134-135° 16 265. Triethvllead w-bromol>enzoate 16 mp 113-11-1° 16 16 266. Triethvllead p-bromobenzoate 16 mp 127-128°- 16 267. Triethvllead o-iodolrenzoatc 16 mp 138.5 139" 10 268. Triethvllead m-iodolienzoate 16 mp 135-1.36° 16 269. Triethvllead /Hodolienzoatc 16 nip 129.5-130.5° 16 270. Triethvllead <>-nitrot>enzoate 16 mp 142-14351 16 16 271. Triethyllcad w-nitrobenzoate 16 mp 1 72 I73°d 16 272. Triethvllead p-nitrolienzoate 16 mp 1 67-168.551 16 273. Triethvlleadsalicvlatc 16 mp 75-76° 16 274. Triethvllead p-anisate 16 mp 97 98° 16 . . . 275. Triet livllcad /eaniinobenzoatc 16 dcc.p. 265° 16 276. Triethvllead p-aminolienzoate monohydralc 16 mp 84-86° 16 277. Triethvllead X-inethvlanlhranilatc 16 mp 132.751 10 278. Triethvllead N’-phcnvlanfhranilatc 16 mp 124.5-125° 16 . 279. Triethvllead phcnylaectatc 16 nip 96-97° 16 280. Triethyllcad /r-aminophcnylaeetatc 40d 281. Triethyllcad phcnylpropiolate 16 mp 149-15051 16 16 SECRET 252 MISCELLANEOUS COMPOUNDS AS CHEMICAL WARFARE AGENTS Compound Reference to synthesis Physical properl Projierty ics Reference Refer, to toxicity data 282. Tricthvllead cinnamate 10 mp 122 123°d 16 283. Triethvllcad /4-lie nzoylacry late 16 mp 139 110(1 16 284. Triethylload 9-fluorcnccarl>o.\ylate 10 dcc.p. 208° 16 285. Trie thy Head /3{3-naj»hthoyl (propionate 16 mp 134 135° 16 16 280. Tricthvllead diphcnylacetale 16 mp 164-165“ 16 287, Trielhyllead triphcnylacetatc 16 mp I34-136°d 16 288. Triethvllcadsulfanilamidc 56b mp 171° 56b 56b 289. Triethvllcad furcate 16 mp 156-157°d 16 290. Tnethylleail furylacrylate 16. nip 132133 d 16 291. Tricthyllcad lepidino2-eai lioxylale 16 mp 153 155“ 16 16 dcc.p. 197-199' 16 292, Triethvllcad X -c t h vlcarba zi)le-3-earlx>.\yla 1 e 16 mp 19551 16 16 293. Triethvllcad thioacctatc* 56a mp 44° 56a 294. Tricthyllcad cvclohexvlsulfinatc 16 mp 132-134’ 16 295. Triethvllcad p-tcluencsultiimlc 16 mp 86 88° 16 296. Triethvllcad o-tohienesulfonate* 50a mp 87“ 56a 297. Tricthvllead p-toluencsulfonate* 298. Triethvllcad 2-amino-5-tohicncsuJfonate 16 mp 21051 16 299. Triethvllcad imphthalciie-2-sulfonate* 300. Tricthyllcad d-camphor-l 0-sulfonate 16 mp - 172° 16 16 301. Tricthvllead p-tolylthiosulfonatc 16 mp 109° 16 302. Tricthvllead methanesulfoliamidc* 66b mp 97“ 56b 56b 303. Tricthvllead mcthanesulfonanilidc* 56h mp 115.5° 56b 56b 304. hi.s(Triethvllead) niethaliedisulfonate 50b 56b — 305. hits(Triethvllcad) methanedisnlfonanilidc 561) 56b 300. Tricthvllead cthancsulfonanilidc 56b mp 110° 56b 56b 307. Triethvllcad benxenesulfonaraide 55a 308. Triethvllcad p-aminolxaizenesulfonamidc 16, 56h mp 173 174° 10 56b 309. Triethvllcad o-tolucncsulfonamidc* 56b mp 133° 56b 310. Tricthyllcad p-toluenesulfonamidc* . . . ~ 56a 311. Tricthvllead p-toluciicsulfoiianilide* 56b mp 134° 56b 312. Tricthvllead p-tolucncsmlfon-p-ehlonuiilhle 56b mp 111.5° 56b 56b 313. Triethvllcad p-toluenesulfon-p-bromanilidc 56b mp 117° 56b 50b 314. Trielhvllead (7carl»oxvl)enzenesulfoiimiide* 561) mp 135° 56b 315. Tripropyllcad o-tohicnesu donate 56a mp 87° 56a 310. Tripropyllcad p-tolucncsulfonatc 50a mp 7:4-74.5“ 56a 317. Tricthvllead l-amino-d-naphthalcncsulfonatc 16 mp 238 240° 16 318. Tripropyllcad mclhanesulfonamide* 56b mp 67° 56b 56b 319. Tripropyllcad hcnzcncsulfonamidc 55a 320. TripropyHead p-aminobenzcncsulfonainide 50b mp 101° 56b 56b 321. Tripropyllcad p-lohiciicstilfotiainlide 56b mp 104° 56h 55a 322. Tripropyllead /Mohicncsulfon-p-chloranilide* 5Hh mp 123° 56b 56b 323. Tripropyllcad o-carboxyhcnzcncsulfoninikle 56b mp 130° 56b 56b 324. Tributvllcad p-tolucncsulfonate 56a mp 81-82° 56a 325. Trihutvllead naphthaleiie-2-sulfonatc 56a mp 08° 56a 320. Triphcnyllead nitrate 1 mp (sinter) 220-225° 1 327 Tri(»«-nitrophcnyl)lcad chloride i .77” 328. Tri(m-nilroplicnyl)lcad nitrate i .... — 329. Tel ramet hyllcad" 24 330. Triethvlallvllcad dimer 10h 331. Antimony trifluoridc Commercial 24 332. Ethyldichlornsl ihine 13 bp' 62-83° 13 24 — A 2.182 13 333. p-Thiocyanophcnyldichlorost ihine* 334. p-r.lhvlthiophenyldicliloroKt ihine* 335. p-(3-Chlorocthvllhio>phcnvl dichlorostibine* 330. p-Phcnylcncarsincst ihine tetrachloride* 337. l>is(«i-Aminophcnyl)chloroMtihine dihydrochloride 24 338. his( m-Aminophcnvl thydroxystihine 24 339. 5,10-1 lichloro-5,10-dihydrost ibarsan t hrcnc* 340. Diphenyl-o-l hknylst ihine * Table 1 (Continued) SECRET MISCELLANEOUS COMPOUNDS AS CHEMICAL WARFARE AGENTS 253 TabI-K J (Continued) Compound Reference to synthesis Physical projjerties Property Reference Refer, to toxicity data 341. Phenyhlit hienylst ihim* 342. Trifury (antimony lOe • .V. — 24, 33 343. /r/*( 5-iert-B u ly 1-2-f ury 1 )anl i mon y 40f 24, 33 344. (m(2-Pyridyl)antimony 40g 24, 33 345. Trimelhvlstihine sulfide* 346. fci«(Trimelhylstil>o)t risulfide* ... 347. bis( Diphenylstibine)sulfide* 34.8. Sulfate of 6i.s(m-aminophenyl)hydroxystibine* 349. 5,10-Dihydro-5,10-di< >xys t il >a rsanl hrene-5,10- monoxide 330. 1 )iphenylbismuth thiocyanate* 56d 331. //■/*( 2-lurvl )bismuth 4 Of 352. Chromyl chloride 28 bp 114° 28 24 353. ('hromium hoxacarbonyl 40g Jc5 1.912 28 354, ('hromium 5-nilro-2-furoate 40c 355. Tungsten carlxmyl 36 mp 125° 36 35 yp6T 1.2 36 350. Manganous 2, l-dinitrol)enzenearsonatc 40a 357. Iron jxmtacarbonyl* Commercial See Chap. 11 35.8. Ferric 2,4-dinitrobenzenearsonatc 40a .77 359. Cohaltous fluohorate 24 300. Salcominc Commercial 35 361. Cobalt 2,4-dinitrobcnzcncarsonate 40a 362. Nickel carbonyl* ~ . . — See Chap. 11 303. Nickel fluoborate 40q 24 364. Nickel fluosilicate 4 Or mp >275° 4 Or 24 305. Nickel 2,4-dinitrolienzenearsonate 40a 300. Chlorine Commercial 24, 59 307. Bromine Commercial 24, 59 368. Nitrogen fluoride — ... 24 369. Ammonium fluoride Commercial 24 370. Lithium hypochlorite 24 371. Hydrogen sulfide . . . mp -85.5° 59 59 — bp -60.3° 59 372. Sulfur monofluoride* 54, 57 bp -35° 54 54, 57 _ -99° mp . -105.5° 54 ... ,u" 1.5 54 373. Sulfur tetrafluoride* 54, 57 bp -40° 54 54, 57 374. Sulfur hexafluoride ... mp -124° 54 24 375. 1 hsulfurdeeafluoride See (’hap. 4 See Chap. 4 376. Thionvl fluoride* 54, 57 bp -43.8° 54 24, 57 - mp -129.5° 54 377. Sulfuryl fluoride* 54, 57 bp -52° 54 24, 57 mp -120° 54 378. Salfuryl cblorofluoride . . . 24 379. Pvrosulfurvl chloride ... 24 380. Hydrogen selenjde bP -41.5° 59 59 381. Sodium selenitic Commercial 24 382. Selenium monochloridef 59 bp;“ 127° 59 59 2.7741 59 nun 1.5962 59 383. Selenium monobromidet - 384. Selenium hexafluoridet 57 sublim.p. -46.6° 54 57 385. Carbon snlfidcsclenidcf . . . 386. Carlm>ii diselenidet 22 bp 117 118° 22 24 387. Selenium oxychloridef 23 bp21 84 85° 23 24, 33,59 nip 10.9° 59 nil5” 1.6516 59 t These compound* and other selenium compounds are discussed more fully in Chapter 11. SECRET 254 MISGEI.I.ANEOl S COMPOUNDS AS CHEMICAL WARFARE AGENTS t'oni|>ound Reference to synthesis Physical properties Property Reference Refer, to toxicity data 388. Selenium ox v bromide! 38!). Selenium oxide Commercial 24 390. Selenium dioxide! 391. Chloroselenious acidf 392. Sodium selenite __ 24 393. Hydrazine hydrate Commercial 24 391. Ammonium fluosilicate Commercial 24 393. T riehloronit rosomethanc 7 24 3s< ipn >pa ne 49a bpioo 40 49a 405. 2-Nitrobulene-I vol3" 32.36 26 24 40(4. 1,4-Dil>romo-2-l mtene 7 nip 54° 7 24, 33 407. /n‘s( Chloromel hyl )uit romel hane 47a 24, 33 408. 3-Chlon>-3-nit rosopentane 7 bp1' bo35 44° 1.4190 7 7 24 . d,3i 1.016 7 409. Methyl sulfite ... 24 410. Methyl silicate 7 hpiw 75“ 7 24 411. Dimethyl sclenide 22 bp 56-58° 22 412. Trirncthylselenonium fluoride 20 rf® 1.378 20 24 «ir" 1.4600 20 413. Dimethyl tclluride* 414. Monoehloromet hyl sulfate* 415. f>/.t(Chloromet hvl) sulfate* 416. his( Chloromel by I) et her* 417. 6/s(Bromomcthyl) etlier 59, 401 mp bp d3" -34° 154 155° 2.2013 59 59 59 24, 59 418. fl-ChlorovinylsoIenium chloride* 23 nip 86° 23 24 419. Ethyl sulfite 24 420. Ethyl fluorosulfoiiate 24 421. Ethyl chloroselenite* 422. Ethyl selenomcrcaptan* ... . 423. Elhoxysclenyi chloride* i>p,s 81.5-82.5° 55e 55c 424. Diethyl sclenide* 22 bpls 79 82° 22 24 425. Diethyl diselenide* 426. Diethyl tclluride 427. (r/jf(/J-ChIoroethyn liorate 40s lip1 97-99° 40s 24 428. ltlrnkis(J}-( 'hloroelhyl) orl hosilicatc 21 bp1 142 113° 21 24, 33 429. /3-Chloroelhvl nitrite 7 l.p’" 33° 7 24. 33 Till30 ,P" 1.4115 1.212 7 7 430. Methyl 5-ebloroethvl sulfite* ... — 24 431. hi${(}-{'hloroelhyl) sulfite* ... 24, 33 432. 6(x(d-Chlomethvl) selenite* nip 44-45° 55e 55c 433. d-Chloroet hylsulfnryl chloride* 12 bp° s 60-64° 12 24 t These compound* and other selenium compounds are dis ■usseti more fully in Chapter 11* Tabi.k t (Conlinutd) SECRET MISCELLANEOUS COMPOUNDS AS CHEMICAL AY A K FAKE AGENTS 255 Tabus 1 (Continued) Reference Refer, to to Physical properties toxicity Compound synthesis Property Reference data 134. f»(s($-Chloroelhyl) sulfate 12 bpos 117-133° 12 24 435. W.P 113-117° 27 24 n ir" 1.4330 27 430. 3-Rrorao-2-propyn-l-ol 27 bp= 49-53° 27 24 MU*" 1.5140 27 440. 3 Iodo-2-pmpvn-l-ol 27 bp5 82-85° 27 24, 33 mp 40-43° 27 44 U Methyl 2-propynyI ether 27 bp 01 65° 27 24 /(u'4 1.4052 27 4 12. Methyl 3-bromopropynyl-2 ether 27 bp1* 34-38° 27 21 air* 1.4703 27 443. 3-Chloroallvl alcohol 24, 33 114. Allyl methyl ether . . . 24 445. si/m-Dichloroisopropyl eblorosnlfinatc — 32 l>l>15 108 110° 32 24, 33 «w*° 1.5130 32 - —. . . J--a 1.432 32 446. 2-Xilro-l-butanol silicate 40b .... 4 17. Ethinyldimcthylvinyl carbinol Commercial 24, 33 448. 2-Butyne-l ,4-diol Commercial 24 440. bi.x-0-ChloructhyI formal 12 bp1- 02-04° 12 24 450. Mcthylformylchloride oxime 19 bp 63-66° 19 24 mp -64 to —60° 19 »Da 1.4193 19 — — — d* 1.135 19 451. Acetaldehyde a zinc 7 bp 96 08° 7 3, 24 «ua 1.4370 7 452. i lemiaoetal of chloral and chlorctonc 471. mp 68-69° 47b 453. Chloral oxime 7 bp*9 69 70° 7 24, 33 7lu“ 1.4905 7 • d,a 1.571 7 454, Acrolein Commercial 24 455. Propionaldehyde azine 7 bp 139-141° 7 3, 24 n ir5 1.4497 7 456. Acetone azine 7 bp 120-133° 7 3, 24 nu4 1.4511 7 457. 6jx(Selenoacelone)* .... 458. Chloroacetone oxime 7 bp* 70-71° 7 24 . , . n 1.4777 7 1.221 7 459. Bromoaectonc 21 bp13 35.5-36.5° 21 24 400, Butyraldehyde azine 7 bp17 77-78° 7 3. 24 «na 1.4504 7 461. Methylethylketone azine 7 bp-» 71 72° 7 3, 24 n u’5 1.4517 7 462. l-Bromobutanonc-2 21 bp30 62-66° 21 24 «n15 1.4700 21 . . . 463. 3-Bromobutanone-2 21 bp- 49 53° 21 24 H,,11 1.4595 21 464. Selenovaleraldehyde * . . , 465. Diethylkelone azine 7 bp“ 94-96° 7 3, 24 Bn3 1.4539 7 406. 1 - LI romopen t a n one-2 21 467. 3-Bromopent anonc-2 21, 58 bp15 75-76° 21 24 «u!1 1.4576 21 vol-" - 21.59 26 468. a-Chloromesitvl oxide 30 bp31 66-69° 39 24 400. 1-Hy droxy-2-pcntync-4-one 27 bp3 79-83° 27 21 «ir° 1.4587 27 vol” 0.111 26 SECRET 256 MISCELLANEOUS COMPOUNDS AS CHEMICAL W Alt FA HE AGENTS C oin pound Reference to S) nthesis Physical pr< >i>ert ies Property Hi ■ferenec Refer, to toxicity data 470. l-Mcthoxy-2-pcntync-4-one 27 bp3 47-50° 27 24. 33 471. Carbon sulioxide 38 n n!H 1.4462 27 24 472. 1 ,l,4,4-Tetraethoxy-2-butyne 27 »>P: 97-102° 27 24 «iru 1.4346 27 473. Diketone *»pm 43° 38 24 474. Hydrocyanic acid See Chap. 2 Sec Chap. 2 47.5. Sodium cyanide Commercial ... 24 476. Triallvl orlhoformate 24 477. 2-Propynyl formate 27 bP 105-109° 27 24, 33 478. Allvl formate a,,1'1 1.4203 27 24 479. a, d-Dicblorovinyl acetate 49c bp11 41-13° 49c 24 480. (3-Triazoethyl acetate 21 bp'-“ 74° 21 21 «ie" 1.4345 21 1.123 21 481. Acetyl fluoride 12 bp 20-22° 12 24 482. Acetyl azide 2L ... 483. Acetonitrile-boron trifluoride addition product 39 mp 118-120° 39 24 484. Methyl selenolaeetale 22 iip” 29 31° 22 485. Sodium chloroacetatc Commercial bP 112-114° 22 21 486. Chloroacetyl fluoride 49n bp™ 366.2 26 24 - vol-° 74-76° 49u 487. Sodium bromoacctatc 24 4SS. Itromoa cetyl bromide Commercial 24. 33 489. Sodium iodoacetate 24 490. Ethyl iodoacetate 40k — 24, 33 491. Propiolic acid 27 bpa 73-77° 27 24, 33 492. Methyl propiolate - — 27 bp 100-102° 27 24, 33 493. Ethyl propiolate 27 »>P 119-120° 27 2 4, 33 494. p-Chloroethyl propiolate 27 bp‘3 n ir“ —79-82° 1.4588 27 27 24, 33 49.5. Allyl propiolate 27 hpeo 70-73° 27 24 — . «i.lss 1.4378 27 496. Hromopropiolic acid 27 mp 85.5-87° 27 24, 33 497. Methyl bromopropiolate 27 bp“ 40 45° 27 24 . tiua 1.48S4 27 498. Acrylonitrile Commercial bp 75.5-76° 24 499. Methyl a-cliloroacrylatc 7 bpin 51-55* 7 500. 0-Chloroacrylonitrilc »u!0 dt'° 1.4400 1.201 7 7 24 501. o,0-Dichloroacrylonitrile Commercial bp®" 58-59° 24, 33 .502. o,d,/3-Trichloniacrylonit rile 48 mp 17 19° 48 24 — bp710 141-142° 48 503. Ethyl /3-chloropropioniminocstcr hydrochloride 12 rnp 96°d 12 504. o,o,d-Trichloropropionitrile 505. Mcthoxytetrolic acid 24 27 bp’ 114-118° 27 24, 33 506. Methyl methoxytetrolatc 27 nij!t lip’ 1.4669 56-58° 27 27 21, 33 507. Crotonyl fluoride 19e n n5" bp 1.4438 88° 27 49e 24 508. Ethyl vinylacctifhinoester hydrochloride 12 nip 90 100°d 12 509. Allyl cyanide 24 510. y-Chlorocrotononitrile 49m bp10 60-62° 49m 24 .511. /8-Chlorocrolononitrile 48 bp7* 125.5 126.5° 48 24 512. Butyryl fluoride 49c bp 6.5-67" 49c 24 513. Methyl a-chloroisobutyratc . . . 24 514. a-Triazobutyric acid 21 bp"7 80° 21 24, 33 515. Methyl o-nitro-0-methylerotonatc 49p nir4 bp5< 1.4536 129-125° 21 49p Table I (Continued) SECRET MISCELLANEOUS COM POINDS AS CHEMICAL WARFARE AGENTS 257 Tahi.e 1 (Continued) Compound Reference to synthesis Physical proper! Property ies Reference Refer, to toxicity dat a 516. Methyl melhoxyacetate 24 517. Dimethyl diglycolatc Commercial ...— 24 518. hi*{(}-{ 'hloroelhyl) diglycolatc 7 1>PS 195-199° 7 51ft. Formaldehyde cyanohydrin 24 520. Methyl 2,2,2-trieblorolaetate Iftq i.p* 92-94° 4ttq 24 521. Chloralcyanoliydrin 4!th mp 59-60° 49h 522, d-Cvanoelhyl nitrite Iftd cannot be distilled 49.1 523. l-Chloni-l-isoiiitrosoacctone 1ft mp 108-109° 19 24, 33 524. l-Cbloro-2-methylglyoximc 1!) mp 183-IS4°d 19 2 4, 33 525. Triehloronectylcyanide. 49g bp 118-121° 49g 24 526. Vinyl mncocblurate Commercial vol20 0.289 26 2 4. 33 527. Hexaehlorodimethyl oxalate 4fth mp 79-80° 4ftb 24 528. Oxalvl fluoride 10 i>p approx. 2 3° 10 24 52ft. Oxalvl chloride 12 bP 64-65° 12 24. 33 530. Methyl cyanoformate 42c bp 98 9ft 42e 24 531. Chlorocyanofomuddosimc 42.1 bp5 53-549 42d 24, 33 mp 54 56 4 2d 532. Cyanogen .... 7T. 24 533. Diethyl dichloromalonale 17 i»pu 115-116° 17 24 no54 1.4386 17 534. 1 Wet hv! ethoxvmethylenem.-donate 47c 1.4600-1.4620 47c 24 535. Ktlioxymethyleneinjilononitrile 4ftj mp 60-63° 49j 24 536. Diethyl diethoxymcthvlmalonatc 47d bp1* 133° 47d 24 n u2« 1.4220 47d 537. Dimethyl acetylene* 1 icarlsixylate 4ftc bp2" 1.17 26 24. 33 vol29 98-99° 49o 538. Diethyl acetylenedicarboxylate 27 bp3 84-88° 27 24, 33 -- — . . . nr.2" 1.4435 27 53ft. h/xf $-(' h loroe t hy 1) acetylenedicarboxylate 27 bp* 175 21551 27 24 nu1'5 I..5004 27 540. Diallyl acetylenedicarboxylate 27 bp* 112-118° 27 24 ■ - tin" ‘ 1.4718 27 541. Diisopropyl acetylenedicarboxylate 27 bp* 103-107° 27 24 * ... nDlil 1.4408 27 542. f)/s(2-Ethylhexyl) acetylenedicarboxylate .... "" 24 543. Dimelliyl maleate 27 bp 199-204° 27 24, 33 544. Dimethyl fumaratc bp 189-192° 27 24, 33 545. Diallyl fumaratc 27 bp2" 137-140° 27 24 546. Fumaryl chloride 48 bp'3 62 63° 48 24 517. Dimethyl chloroinaleale 24, 33 548. Diethyl bromomalcate 49g i.P°‘ 85-86° 49g 24 54ft. Diethyl chlorofnmaratc • 27 i>p5u 137 139° 27 24. 33 550. Chlorofumaronitrile Commercial 24. 33 551. Diethyl bromofnmarale 27 bp8 120-123° 27 24 552. Chlorofnmaryl chloride 27 bp21" 140-143° 27 24 553. Dimethyl dibromomaleate 17 bp" 134-136° 17 24 554. Dicthvl «,a'-diehlorosuccinate 17 bp* 106 108° 17 24 555. Dimethyl «,o'-dibroniosiiccinate . 17 mp 00-61° 17 24 556. Dimethyl tctrachlorosuccinatc 24 557. Dimethyl «,«'-dicbloroglntarate 17 bp1 95-96° 17 24 558. Dimethyl or.o'-dichlorondipate 17 bp2 126 128° 17 24 «rr* 1.4660 17 55ft. fusCTriehloromethyl) carbonate 24 560. Methyl ft-chloroelliyl carlxmate 24, 33 561. 6i«(£-CbIor.s‘thyl) carbonate 12 bp11 119 122° 12 24 562. Methyl fluorocarlsinale 4ftg bp 43-15° 49g 24 503. Methyl chlorocarbonatc 24 564. Trichloromethyl cblorocarlsmate (diphosgene) See Chap. 3 See Chap. 3 .565. Ethyl chlorocarbonatc ... 24, 33 566. 0-Chloroethyl cblorocarlsmate 7 bp™ 152.5° 7 24 »i>t0 1.4465 7 d.20 1.3825 7 SECRET 258 MISCELLANEOUS COMPOUNDS AS CHEMICAL WARFARE AGENTS Table I (Continued) Compound Reference to synthesis Physical pro|>erl ies Property Reference Refer, to toxicity data Allvl cbloroearbonate 40f bp 107-111° 40f 24 .'>68. Methyl triazoformate 4!li bp 97-101° 40i \ 24 560. Carlsmvl chlon iflnoride See Chap. 3 See Chap, 3 570. Carbonv] chloride (phosgene) 8ee Chap. 3 See Chap. 3 571. X.X-Dichlorourelhanc 40d 24, 33 572. Dimethyl azoformate 7 bp“ 08° 7 24 bpe 104“ 7 «u:* 1.4180 7 t/.,rn 1.222 7 573. 6is(/3-Chlorocthyl) azoformate 7 bp' 140-143° .-z- 7 33 (Id" 1.4752 7 •IF 1.300 7 574. Cyanogen chloride See ('hap. 2 See Chap. 2 575, Diehloroformoxime (phosgene oxime) 42a, 56c, 62, bp-1 47MO° 42a 21, 55b 64, 66 1V1|) 30 10 63 — - - bp 129° 03 576. Cyanogen bromide Commercial 24 .577. 1 >ibromoformoxime 55b .... 55b 578. Methyl chlorothiolformatc ■12c b|> Ill 112° 42c 21 ' - — mr" 1.4001 42c . . . - if-'" 1.200 42c 570. Tridden .met hyl chlorothjolformate- 42c lip-4 153 162° 42c 24 > 1.52 42e ,po 1.654 42c 580. Thiophosgene 42c bp 73-76° 42c 24 581. Thiocarlsmyl chloride polymer 42c 7-.— 24 582. Acetyl thiocyanate 40h bp" 60.5 61.0° 40h 24 583. CarlMWiielhoxy isothiocvanate 40h bp14 58-61° 49h 24 584. Methyl thiocyanate 28 bpJ« 128-129° 28 24 «ua 1.4081 28 •hr' 1.0732 28 585. 2-Chloroethvl thiocyanate 5 — ... 24, 33 586. Hexyl thiocyanate 28 bp1 5 84 87° 28 24 ttu 1.5650 28 . •hr* 0.041 28 587. Dodecyl thiocyanate 28 bp14 177 170° 28 24 «i>“ 1.460 28 — mct hyl) et her* . ,T~ 24 501. Aceloliyl thiocyanate 40e .... 24 502. Cyanogen sulfide* , . . 503. Methvl chlon>dithkiformafe 42c bp13 47 10 42c 24 504. Allvl sclcnourea* 505. 1,3- Diseleiiocyanopropane* 506. Cyanogen diselenide* 5!>7. (3-Chloroethencselcnjnvl chloride* 28 24 508. Elhaneseleninie acid 23 500. Elhaneseleniiivl chloride hydrate 23 mp 72-75° 23 24 GOO. N, \-Dimef hvlformamule Commercial 24 601. 2,5-6/s(N-Methylearbarnyloxy)-3-he.vyne (two forms) 35 602. X,X-DimethvIcarl>.imvl fluoride 49k bp** 05" 19k 24 603. Ihmcthvlc.irbamyl chloride 11 bp 166 168° II 24 604. Methvl isocyanate 37 bp 37-30° 37 35 605. Dimethylsulfamyl fluoride* 4 On bp14 48.5° 4 On 24, 55d 606. Dimethvlsulfamvl chloride 11 bp* * 34 II 24, 33, 55d 607. X, X-T )ict h vlchloroacet amide Commercial 24, 33 608. X,X'-Dicthyloxamidc n nip 175° 11 24 600. Ethyl X,X-6/s(j3-chlorocthyl) carbamate 39 bp" 131-132° 39 24 dif5 1.4688 39 ,if 1.214 39 SECRET MISCELL VXEOCS COMPOl M)S AS CHEMICAL WARFARE AGENTS Compound Reference to synthesis Physical pro [Kit Pro(>erty It'S Reference Refer, to toxicity data 010. \-But vlmaleimide 24 Oil. Dibutvlearbamyl chloride 11 l',p* 108-109° ii 24,33 012. 4-Chlon>|ieii( vldielhylamine hydrochloride 34 mp 99.5 100° 34 24 013. Kthvlenediamine thiosulfate 24 014. 1,0-llexanedianiine Commercial 24 015, 1,0-Hexanediol diisocvanate Commercial 21, 33 010, /1-DimethvlaminoelbvI formate 14 bp7* 125-130' 14 24, 33 n if0 1.4262 14 “ ■ 0.905 II 017. d-Dimethvlaniinoellivl acetate 14 bp1*' 148-151° 14 24, 33 — a u10 1.4178 14 d„r" 0.928 11 618. fJ-MetKvIelhvlaminoethyl formate 14 bp711 117-150° 14 24, 33 . _ «..»* 1.4287 14 0.919 11 610. /J-Methvletlivlamiinielhvl acetate 14 bp711 162 163° 14 24. 33 «!!*" 1.4226 14 dio50 0.918 14 020. Formylcholine chloride 14 mp 144 146° 14 24 021. Acetylcholine chloride Commercial 24 022. ('arhaminoylcholine chloride (Doryl) 35 023. ti-( N-Met bvlcarbamvloxv )ei hvlt rimet hylain- mcmium chloride 49b 24.33 024. 0-( X-Pro|*vlcarl*iimyl)ch(>line iodide 24 025. (i-( N-HutvlcarhamyDcholine iodide 24 026. /3-Diet hylaminoethyl formate 14 bp7* 157-160° 14 2 4, 33 n i>so 1.4358 14 0.900 14 027. (J-DiethylaminoethvI acetate 14 bp61 101-103° 14 24, 33 W|.5“ 1.4259 14 djo™ 0.911 14 028. /S-Diethvlaminocthvl carbamate elhiodidc 29 mp 150-150.5° 29 24 629. 0-Diethylaminoelbyl N-methylcarbamate eth- iodide 29 nip 90-92° 29 24 030. /l-l)ihul vlarniniK-thyl carbamate but oh slide 29 nip 99.5-100.5° 29 24 631. /J-Dibutylaminoethyl N-mcthylearbamate bulo- iodide 29 mp 100 101.5° 29 24 032. vdroxycthvl )methylamine ('ommercial 24 033. hylaminoethyl formate 14 i,p7 126-127° 14 24, 33 — n if" 1.4698 14 1.045 14 034. Mcthyl-l(i*{d-fornio.\yethylfamine 14 bp7 110-111° 14 24, 33 «u20 1.4501 14 -l ti.amy la mi nopropyl X-methylcarbamate amvliodide 29 nip 78-83° 29 24 611. I-Diethylamino-2,3-6«f(X-mcthylcarbaiiiyloxy) melhiodide 29 mp 122-123.3° 29 24 642. Ethyl diazoacelatc 21 bp1- 42 43° 21 21 nip" 1.1592 21 043. 1 )imel h vlaminoacet onit rile 49h bp11 55-56° 49h 24 644. /r/xfd-Thioevanoel hyl famine ... 51 — <>45. Trifluoromel hvlsilieane 24 610. T rich Ion >mel hvlsilieane 24 647. Dichlorodiraethylsilicane 24 Tabi.k I (Cimtinueil) SECRET 260 MISCELLANEOUS COMPOUNDS VS CHEMICAL WARFARE AGENTS Compound Reference to synthesis Physical projicrties Pro|*crty Reference Refer, to toxicity data HIS. Chlorotrimcthylsilicane 24 649. Ethvltrifluorosilicane . . . 24 650. Kthyltrichloroeilicuno 4 On bp7*" 96-98° 40n 24 651. Tetracthylsilicanc 40f 24, 33 652. Trifluoropropylsilicanc 24 653. Trichloropropylsilicane 24 654. Trichloroisopropylsilicane 24 655. ButyK rifluorosilicane ... .... ... 24 656. Buty 11rieldorosil icane . _♦ . . 21 657. Tributvlboron 40a 65S. 1 l/xachlorncvc!ohexane (impure) Commercial 24 659. rt-Broino-2-chloro-6-uiirotoluenc Commercial 24,33 600. 2-N'ilro-l-phcnylpropene 18 bp'° 64.5 65.5“ 18 mp 139° IS 24. 33 66!. 1,2-fus(fl-Chlorocthyl)bcnzenc 32 i>p 120-122° 32 24 602, Phenvl chlorocaHamate 24, 33 663. 2,4,6-TrichIorophcnyl chlorocarbonatc 24, 33 664. Picrvl silicate 40n 065. Cvclohoxvl dithiocyanate* 666. o-C’hlorophcnyl thiocyanate* ~ . . 667. m-Chlorophenyl thiocyanate* 668, p-('lilorophcnyl thiocyanate* 669. roacel<»|>lu-ii<>nc 24 702. tt.o-Dichloruacetophcnone 24 703. «-ChIi>n>-f>-nitrosoaci‘(ophcnone 47e mp 138,1 17c * - . 704. a-('hh.r(..p-phcnylacctophen(.nc 24 705. Phcnylpropargyl aldehyde 27 i»p” 114 117° 27 24 — n i>'5 1.0029 27 700. Phcnylpropargyl acetal 27 l.p*3 153 150° 27 24 n [i3 1.5100 27 707. n-Bromopropiophem>nc __ 24 70S. Selemicyanoacetophenone* 709. S-Isonitrosocamphor (d) 44e mp 154-155° 14c 24 — - bp1" 179° 44e 710. 1,3,5-/ r /.t( C h 1 oroa ce t y 1) lie ll ze nc 32 mp 148 1.50° 32 24, 33 711. 2-MethvH,4-naph(li1 K'nzoylazide 21 mp 07 08° 21 24 713. rt-Hromolien/oylcyanide 59 mp 29° 59 24, 69 in." 132-134° 59 714. Methyl phcnylpropiolate 27 i»p! 04 99° 27 24 715. n-Amyl-X-(-/>-phonylencdi- lla ... 24 amine 34 mp 178 180° 34 24, 33 SECRET MISCELLANEOUS COMPOUNDS AS CHEMICAL AVARFARE AGENTS Table 1 {Continued) Compound Reference to synthesis Physical proper! Property ies Reference Hefer. to toxicity data 743. X, N '-bi*{r}-i ’hloroc.trbethoxy V/i-phenvlenecii- aniine 34 mp 201° 31 24 74!. l,4-5(s(X,X'-Oimcthylureido’ibenzene 30 nip 229.5-230.5° 30 24 74">. '-Disulfinvl-p-phcuylenediarnine 40a nip 115-110° 40a 35 740. p-l)imcthvlaminoanilinc Commercial 24, 33 74 7, p-Di md hylaminophenyl isothioeyanate 34 bp5 148-150° 34 24 mp 09-70° 34 748, p-Dimcthylaminophenyl isothioeyanate hydro- chloride 34 mp 144-145° 34 24 741*. p-Dimct hylaminophenyl isothioeyanate moth- iodide 34 mp 171° 34 24 750. X-\lelhyl-X-(p-dimethylaminophenyl)thiourea hvdrochloride 24 751. X,S-I)iincthyl-N'-(p-dimethylaminophenyl)- thiuu rea 11 y droit slide 24 752. X,X '-Di met hvl-p-phenylenedian due 34 bp” 157 100’ 34 24, 33 ■ _ — mp 53-54° 34 753. X,X‘-Dinudhyl-p-phenylonediamilie dihydro- chloride 34 nip 224 (1 34 24 754. X,X'-Dirarbethoxy-X,.X'-dimethyl-p-phenyl- enediamine 34 nip 106-107° 34 24 755. X,X,X'.X '-Telrameth\T«-pheii vlenediamine 34 bp" 92 93° 34 24. 33 750. X, X,X ',X '-Tetramethyl-o-phenylenediamine —meihiodide 34 mp 19451 34 24 757 X,X, X ',X '-Telramethvl-m-phenvlenediamine 34 bp10 121-124° 34 24, 33 758. X,X,X',X'-Tctramrthyl-m-phenylencdiamine meihiodide 34 mp 18751 31 24 759. X,X,X',X '-Tetramethyl-p-pheiiylenediaminc Commercial 24, 33 700. X,X,X',X'-Tetramethyl-p-phenylenediamine di- hvdrochloride 34 mp 22251 34 24 701. X,X,X',X'-Tetrame4hyl-/)-phenyleiiediamine meihiodide 34 mp 20051 34 24 702. i>-Phenylene-f)ts(oxasMdidone-3) — 34 mp 253 254° 34 24 703. X ',X '-Diet hvl-X,X-di met hvl-u-phen vlenediamine 34 bp,u 137° 34 24, 33 mp 203-205° 34 764. X,X'-Dielhyi-X,X'-diincthyl-p-|i!icnylenedi- ainine 34 bp” 150-151° 34 24, 33 705. X,X',X '-Trielhyl-X'-methyl-p-phenylenediamine 34 bp"1 144° 34 21, 33 mp 22° 34 766. X,X,X'-Triethyl-X'-mcthyl-p-phenylencdiamine dihvdrochloridc 34 mp 22051 34 24 707. X,X,X'-Triel hyl-X'-methyl-p-phenylenediaminc meihiodide 34 mp 177° 34 24 70S. X,X,X ',X '-Tetradhvl-p-phenvlenediamine 34 bp” 155-156° 34 24, 33 mp 51-52° 34 709. X,X,X',X'-Tet raelhyl-p-phony lenediaminc meihiodide 34 mp 185° 34 24 770. X, X’ '-his(d-Hydroxyet hylVp-phenvIenediainine 34 mp 123 34 24 771. X,X'-6is(l-Methyl-l-diethylarninohiityl)-/>- phonylenediamine 34 bpio* 145° 34 24 772. X-Melhyl-X'-(p-dimei hylaminomet hvlphenyl)- ihiourea hvdrochloride - 24 773. Polymer of X,X '-decamet hylenc-X,X '-dime! hy 1- l,4'-diamiiiodiphenylmethanc ftis-metho- bromide 21 774. Phenylhydrazine Commercial 24 775. X-Carbomet hoxv-X '-phenvlhvdrazine 49f mp 114.5-110° 49f 24 770. p-Phciivlenedihydrazine dihvdrochloride - 34 dec.p. 200° 31 24 777. Tetra-m-nil rophenvlsilicon 40a 24 77S. Tel rahvdrofurfuryl alcohol Commercial 24 779. Tetrahvdrofurfurvl fluorocarbonate 49r bp-15 92-94° 49r 24 780. Tetrahydrofurfuryl chlorocarbonate 49o bp1" 81-83° 49o SECRET MISCELLANEOUS COMPOUNDS AS CHEMICAL WARFARE AGENTS 263 Tabi.k 1 (Continued) Compound Reference to synt hesi.s Physical propel ProjxTty 1 ies Reference Refer, to toxicity data 78!. 3,6-K|h rxvcvel, tbcxcnc 24, 33 782, Adduct of fumn and maleic anhydride 7 nip 110111° 7 24, 33 783. Furan Commercial 24 784. Met hylfuran Conunercial 24 7 So, l-(2-Furvl)-2-nilroethyIene IS bp"' 74 75° 18 24, 33 786. I 42- FuryI)-2-nit roprojiene IS inp hp10 110° 48.5-49.5° 18 IS 24, 33 787. Furfurvl alcohol Commercial mp 125° IS 24 788. 2-Furaldchvde Commercial 24 780. Furoic acid Commercial 24 790, 5-Hvdmxy-2-cbl«roinethyl-y-pyroBC 12 nip 162163° 12 791. 5-Meth<>xv-2-ehloromethvl-> -pvrone 12 mp 119 120° 12 24 7! >2. 5-llydroxy-2-henylethyl)-pyrro- colinium bromide 25 24 812. Oct ahydn>4-(d-hy dr, ixj'ct hyl)-2-met hylpy rm- colinium bromide 25 24 813, 4-( d-Aco t, ixyet hyl)-oc( ahy dro-2-met hylpyrro- colinium chloride 25 _ 3 24 814. 2-TriacetvlnoreholyIoetahvdropyrrocoline 25 mp 75-95° 25 24 815. N-ChlorocarhamyIpi|ieridine 24 816. 3-{ Pi)S‘ndvl-X-carliMiml (choline iodide 24 817. 1-Pipcridylsulfamyl chloride . ..v 24 818. X -<•)-11 vdroxyet hylpiireridine 12 bp™ 95-96° 12 819. 2-Pijieridyloethyl X-mel hylcarbamale methiodide 29 mp 103-105° 29 24 820. X-Cyanoinethylpiperidine 49h i.p" 83 84° 49h 24 821. 2-Vinvlpvridine 12 822. Coniine (o-propvlpi|)eridine) Commercial 24 823. 2-0-1 lydroxyel hyl (pyridine 12 bp"4 84 90° 12 824. 3-Ilromoacetylpyridine hvdrobromidc 24 825. Xicotfne Commercial . . 24 826. 2-( X-CarlK,mcthoxvamino)pyridine 49j mp • 122° 49j 24 827. 4-(/S-I)imcthvlaniinoethyl)pyriiline 30 bps" 135 145° 30 35 828. 4-(tf-l)imethviaininoot hyl)pvridine dime) hiodide 30 rnp 207-208° 30 35 829. 2-(/3-Hydroxyethylamino)pyridjne 12 bP” 180-185° 12 mp 109 110° 12 X These substances were obtained from natural sources. SECRET MISCELLANEOUS COMPOUNDS VS CHEMICAL WARFARE AGENTS Tabi.E 1 (Continued) Reference * Refer, to _ to Physical properties toxicity Compound synthesis Property Reference dat a 830. 2-Carbclh»xvoxv-4-carbcthoxyaininopyrindane 34 inp 139-141° 34 24 831. 4-Carbet hoxvamin<>-2-p-tosyloxy-4-pyriiidane 34 nip 132.5° 34 24 852. 4-Amim>-2-liydroxypyrindine nip 309“ 34 24 833. 4-Acetylamim>-2-liydroxypyrindinc 24 834, 2-Aectoxv-4-acelvlaminopyrindine X . . 24 835. 2-Methylpyrrocoline hydrochloride 25 nip 01 02° 25 24 836. 2-Plie n vlpyrn icolinc 25 mp 214-215° 25 24 837. 3-Acet vl-2-rnct hvlpyrroeolinc 25 nip 83-85° 25 24 838. 2-Triacol vlnorcholvlpyrrocolinc 25 nip 109-170.5° 25 24 839. 1,2,3,4-Tctracarbomethoxyquinolizinc 25 nip 180-188° 23 24 840. 4,7-Dichloroquinoline 47c mp 84-85° 17c 24 841. 2-p-N itrophenylqninolinc 401 842. 8-Methoxy-5-methylquinoline .... 24 843. 2-{»i-DimelhylamiiiophenyI)quinoline 40k . . X 844. 2-4 />-! )iincthylaminophenyl )qnin-1,3-isoquino- lincdione — Commercial — ■ - 24 847. 9-Vinvlcarbaxole 24. 33 SIS. Kthvhme-N-nilrofcourea 19 mp 102 I04d 19 24 849. 1,4-1 >itlhyl-l ,4J»?s{3-hyilnixvi'thyl)pi|MTazinium diehloride 24 850. 2-PhenyIimidazo-[l ,2-o] pyridine hydrobroniide 25 mp 122-124° 25 24 851. N-Morpholinoacetonitrile 49i nip 60’ 49i 852. 1,4-Selenoxan-4-dichloridc* . . . * 853. Phenoxtellurinc* — 854. 10,10-Dichlorophenoxtellurine* 855. Bisajiometliylbrucinc hydrochloride 24 850. Bisapomethylhrucine diacetate 24 857. Dimcthvlfuraxane ■ 12 bp760 153° 12 24 858. Dimethylfurazane oxide 12 bp5* 170-171° 12 859. Dicarbellmxyfurazane oxide 12 bp!s 173° 12 24 860. Methvl N-{5-totrazalvDcarbainate 49g nip >300° 49g 801. Product of thermal destruction of cyanogen chloride — 3.5 862. Veratrinc Commercial ... 24 803. Ricin* Sec Chap. 12 See Chap. 12 804. Ficin 24 865. Lubricating oil, S.A.E. No. 10 24 - 860. Fog oil, SGF No. 1 24 SECRET PART II SPECIAL PHYSIOLOGICAL AM) TOXICOLOGICAL STUDIES SECRET Chapter 15 THE ASSESSMENT OF PARTICULATES AS CHEMICAL WARFARE AGENTS William L. Doyle and l\. Keith Cannon I5.i INTRODUCTION During tiik vkars 1941 1915, more than 1,500 compounds were examined in the United States as potential chemical warfare agents. The volatilities of the majority were so low that they could have little offensive value if used in the form of vapors.*11 On the other hand, a few were intrinsically so much more toxic or more vesicant than were the standard chemical warfare agents61012 that the question of disjiersing them in particulate form commanded con- sideration. Ricin (\V), for example, was several score times as toxic as phosgene, whereas l,2-6ts(/3-chloro- ethylthio)ethane (Q), when applied in a solvent to the skin, was vesicant at one-tenth of the minimal blistering dose of mustard (II). Apart from observations incidental to the study of the screening power of smokes, little attention was pair! to the toxicological properties of particulate dis- persions until the decision was made to submit finely powdered ricin to field tests. As a result of this de- cision, an expanding program of work was under- taken on the physical and toxicological assessment of dispersions of this material. As a result of the experi- ence gained, the investigations were later extended to a study of the vesicant effects of aerosols of f».s(/3-chlo- roethylthioethyl) ether (T), Q, and fm(/3-chloro- ethyl)amine (HX3). The point of departure of all the work was an ap- preciation of the paramount importance of particle size in determining the effectiveness of a particulate cloud (see Table 1). In the first place, the particle size determines the stability of the cloud under given meteorological conditions. Secondly, it controls the fraction of the area dose which will impact upon an obstacle in the path of the cloud, and therefore de- termines the hazard to the unprotected skin and eyes of an individual in the cloud. Finally, the im- pacting characteristics of the particles also control the inhalation toxicity of the cloud, since they affect the fraction of the inhaled material that will pene- trate to and lie retained in the lungs. The problem of the assessment of particle size in fine liquid particulates had been greatly advanced by Table 1 warfare t . Relation of pari •haracteristics. tide diameter to chemical Pari icle diameter (microns) Type of cloud Characteristics of clouds 10 3 - 10 - Vapor (molecular) Airborne, non|»ersistent, sub- ject to laws of diffusion. Invades lungs, eyes, cloth- ing, and skin. 10 ' Aerosol Airborne and non|)ersistent. Invades lungs. Din’s not impact out of streamlines. I to 5 Fine particu- lates As for aerosol except that it is more readily filtered and the lung retention is more complete. Five a is close to up[»er limit of nasal penetration. 20 to 100 Particulates ('loud persists in mild lapse conditions. Does not reach lungs. Impacts on surfaces and should invade eyes and skin. 200 Sprays Sediment rapidly. Impact efficiently. Not dealt with in this chapter. the British in (lie invention of (he cascade impaetor.33 The attempt was made to adapt (his instrument to the assessment of clouds of solid particles. However, the irregular size, shape, and density of the particles raised a number of difficulties which have not yet boon satisfactorily resolved. Much fundamental work has, however, been carried out on the calibra- tion and use of the cascade impaetor with dusts.1318 The relation of particle size to the inhalation toxicity of toxic particulates was investigated by direct assays in animals of various species. Ricin aerosols of controlled ranges of particle size were utilized.lsi At the same time the filtering character- istics of the human nose were measured by observa- tions of the extent of penetration of a variety of nontoxic particulates.13'1 •,8ch The effectiveness of dis- persions of nonvolatile and of slightly volatile vesi- cants on human skin and on the eyes of animals were investigated as a function of particle size, wind speed, etc.,3k SF.CRFT ASSESSMENT OF PARTICULATES AS CHEMICAL WARFARE AGENTS The results obtained in this work have shown clearly that the significance of the size of the air- borne particles cannot be reduced to any simple formula. However, the following broad conclusions would appear to be justified and may serve to indi- cate the status of the problem. 1. The size, shape, and density of the particles, as well as the wind speed and other meteorological con- ditions, all contribute to the aerial behavior of a particulate cloud. Particles with effective diameters greater than alxmt. 100 n sediment rapidly in a stable atmosphere. They will remain airborne for significant periods only under conditions of considerable turbu- lence. On the other hand, clouds containing effective concentrations of particles smaller than 0.1 n in di- ameter are subject to rapid aggregation. For example, if a cloud with a concentration of I mg 1 were com- posed initially of very small particles, it would attain relative size stability only when the average particle diameter had grown to about 0.7 m- —These considerations lead to the conclusion that the problem of toxicological effectiveness may be restricted to a consideration of clouds whose parti- cles (if of unit density) fall within the size range of 0.1 to 100 n. 2. In general, toxic agents are much more effective if they enter the lungs than if they are retained in the nose. The probability that a particle will penetrate the nasal barrier increases as the size of the particle diminishes. Available evidence indicates that the optimum size for penetration to and retention in human lungs probably lies within the limits of 0.5 3 ft in diameter.l8* h The optimum size for lalx>ra- tory animals is appreciably lower.18* These values are for resting animals and are further lowered at the high ventilation rates associated with exercise. 3. The probability of the impaction of a particle on a surface in the path of a cloud increases with the size of the particle. At moderate wind sjieeds, the fraction of the area dose which may lie expected to impinge on the surface becomes significant if the particle size is above 10 m and becomes an important, fraction of the area dose above a size of about 70 a*-46 4r It would api>ear from items 2 and 3 that no single dispersion can exploit to the full the potentialities of an agent which, like Q, is both vesicant by contact and toxic by inhalation. This is a fundamental di- lemma which imposes serious limitations on the offensive potentialities of aerosols of this type. 4. If munitions were available which would dis- perse particulate material in either of the optimum size ranges indicated above, new orders of inhalation and of vesicant effectiveness in the field should he obtainable. Such munitions have not yet l>een ade- «iuately «leveloj)ed.3'J 15.2 TYPKS OF I’AKTTCUIATKS 15.2.1 Sternutators Classical sternutators such as diphenylamine- chlorarsine (adamsite), diphenylaminecyanoarsine (cyan DA), and toxic sternutators such as aconitine and nitrophenyldichlorarsine are primarily harassing agents. These agents act at concentration time prod- ucts (Ct's) considerably lxdow (hi mg min in*. At present there is little interest in these agents because they are stopped by available masks and because trained troops carry on effectively despite their pres- ence. It is possible, however, that the utility of sternutators has not been adequately considered. Larger particles than those that have been utilized may be more harassing. German interest in mixtures of sternutators with mustard 16 may indicate at- tempts to hide the presence of more toxic agents. 15.2.2 Toxic Particulates 1. Inorganic substances, e.g., cadmium selenium. 2. Synthetic organic compounds, e.g., aromatic carbamates. T Naturally occurring substances, e.g., ricin (W). The metals are thermally stable and may be in- corporated in standard smoke incendiary S!> or high- explosive weapons. As indicated in Chapter 11, these substances are not more toxic than standard chem- ical warfare agents, but they may be used without ready detection in various types of munitions. Although the aromatic carbamates are consider- ably more toxic than standard agents,10 they are un- stable in aqueous solution and to heat. For these reasons, little serious consideration has been given to their use as particulate clouds. Ricin (W) is intrinsically somewhat more toxic than the best of the carbamates. It is also thermo- labile. Its toxicity when dispersed as a cloud has been studied extensively in the laboratory and prelim- inary field trials, using special munitions, have been carried out. This interest in ricin was not entirely dependent on its own merits as a toxic agent. It was recognized as a prototype of toxic protein materials of bacterial origin which wore known to have even greater toxicity but which were less conveniently prepared and handled. SF.GRKT EFFECTIVENESS OF PAIIT1CULATE CLOL DS 15.2.3 Vesicants 1. Volatile vesicants, e.g., mustard (II), tris(0- chloroethyljamine (HN3). 2. Nonvolatile vesicants, e.g.,12 b is (/3-c Ido root hy I- thio)ethane (Q). All the members of this group are toxic, but not so toxic as (hose in Section 15.2.2, (1) and (2). They are, however, vesicant. The best nonvolatile vesi- cants are intrinsically more toxic and more vesicant than the volatile ones. Q is inherently 10 to 20 times as vesicant as II and at least 5 times as toxic.7,91*a l8h They should lx* more difficult to detect than the volatile agents. In the field they will not be expected to create a vapor hazard, but, by contamination of equipment, should establish a contact hazard for bare skin which it would be difficult to eliminate by decontamination. It is doubtful if nonvolatile vesi- cants can be effective through clothing.1*1 I*1’ The volatile members of this group can lid dispersed thermally, by means of airplane spray, or by high explosive-chemical shells. The particle size achieved will markedly influence the action of the agent. Thus, very small pat t ides (0.2 1.0 g in diameter) may Ik* non vesicant because of streamlining, but will be more toxic by inhalation and will also yield (he great- est and most rapid vapor return. Larger particles (5 to 25 g) will have greater vesicancy, but probably at the expense of toxicity. The largest particles (200 - 2,000 g) may lx* most effective for the penetration of clothing, particularly of the permeable protective type. The largest particles may also create a contact and traversal hazard. These statements are broad and tentative general- izations based upon contemporary views of the char- acteristic behavior of particles of different diameter. Many of these generalizations require further experi- mental investigation. 'Hie results of contemporary work are reviewed in Section 15.5.1. 15.3 TilK EFFECTIVENESS OF PAR- TICULATE CLOUDS 15.3.1 Stability Srdimcntation. The rate of sedimentation of particles in a static atmosphere increases with in- crease in particle size. Computations based upon kinetic considerations indicate that precipitation lx*- comes rather rapid when the effective diameters of the particles exceed about 100 g. Clouds of such large particles could Ik* maintained in the air for significant periods only under strongly turbulent conditions. They are sprays rather than clouds and find their natural use for contact and ground contamination — e.g., as airplane sprays. The opt imum part ide size for such sprays depends on a variety of factors — the speed of the plane, the turbulence, the vola- tility of the agent, etc. — which it is not the province of this chapter to discuss.|J Suffice it to say that for direct assault upon exposed personnel there is general agreement that the optimum range of particle size to obtain massive and diffuse con- tamination with a vesicant is about 0.3-2 mm in diameter. If ground contamination is the objective, the upper limit of size may be unimportant. Coagulation. Although the rate of sedimentation sets the upper limit of size in a jiersistent aerosol, the tendency of particles to coalesce upon collision estab- lishes a lower limit of particle size stability. On simple considerations of collision frequency, the half life of a particle should l>e roughly proportional to the con- centration of the aerosol. With increase in particle size, the concentration required to give a fixed half life increases with the mass, and therefore with the cube of the radius of the particles. It has been esti- mated47 that the half life of an aerosol containing 5 X 10* particles jw-r milliliter is 6 minutes at room temperature. For particles of 0.1 g in diameter and unit density, this corresponds with a concentration of 2.5 nff I. For particles of 1 g in diameter, the cor- responding concentration is 2.5 gg 1. If these clouds were initially established in higher concentrations, they would aggregate until the numbers of particles in unit volume had fallen to a relatively stable level. It should be noted that the tendency to coagulate does not lead directly to a reduction in mass concen- tration, but rather to an increase in average particle size. The phenomenon is important, therefore, only if clouds of small particle size are required. There is no purpose in attempting to disperse a particulate in a smaller particle size than can be sustained by the concentration that is to Ik* established. If a concen- tration of I yg I is accepted as the lowest which the toxicity of the material would justify, then the small- est particle size which it is worth while attempting to disperse is of the order of 1.0 g. In summary, therefore, considerations of rates of sedimentation and of coagulation suggest that we should concern ourselves with the behavior of air- borne particles in the size range of 0.1-100 g in diam- eter, corresponding to a HP-fold range of particle mass. SECRET 270 ASSESSMENT OF PARTICULATES AS CHEMICAL WARFARE AGENTS 15.3.2 The Significance of Particle Size The tactical use of a chemical warfare agent in the form of a cloud is, in general, to lie justified only when the conditions of the operation will l>e such that personnel exposed to the cloud will absorb casualty-producing doses through the lungs or through the skin. When the cloud is a true vapor, the actual dost* that is inhaled under standard con- ditions of respiration can be predicted from the prod- uct of the concentration and the time of exposure (Cl). The amount of vapor absorbed from the skin under standard conditions of temperature and hu- midity can, likewise, be predicted from the Cl, since the rate of diffusion of the vapor to, and (he rate of penetration of, the skin may generally lx* taken to lx* proportional lo the concentration. It follows that an effective dosage on the target can lx* assured if muni- tions expenditure is properly adjusted to the meteor- ological condit ions. The tactical requirements cannot lie formulated so simply when the cloud is composed of particles with colloidal or larger dimensions. In this situation, the concentration of the agent may actually be less im- portant than the sizes of the airborne particles. It has been noted that this is a factor in determining the stability of a particulate cloud. In the case of an agent which is toxic by inhalation the particle size also controls the proportion of the inhaled material which is filtered out of the inspired air in the respira- tory passages. Likewise, in the case of a vesicant, the amount of material deposited upon an exposed sur- face at a given Cl and wind speed is a function of the particle size. In brief, the effect ive dose of an inhalant and of a vesicant depends on the impinging char- acteristics of the particles which, in turn, depend upon the size and density of the particles. 15.3.3 The Impingement of Particles The amount of an airborne particulate which will deposit on an object in the path of a cloud w ill ho the sum of the amount which impinges upon the object and the amount which is deposited under gravity.15 Since this discussion has ix*en limited to clouds in which the rate of sedimentation is small, considera- tion may l« confined to the amoimt which impinges on the object. The probability that a spherical particle w ill im- pinge upon a cylindrical surface in its path is given 45 by /* = «( 1 + ■-) (1 - e~^/D) (1) In this equation, n is the velocity of the particle, <1 its diameter, and p its density. D is the diameter of the target, while a and n are constants. It will lie seen that the tendency to impinge increases with the size and with the density of the particle and also with the wind speed. It depends also on the size and shape of the target. According to Sell,15 when D » e largely determined by the rela- tively small number of the larger particles which are present. A heterogeneous cloud may, therefore, Ik* a much more effective vesicant than a homogeneous cloud having the same mass median diameter. Further complications are introduced if, as may occur with solid particulates, the particles vary in shape and density as well as in volume. A very im- portant example of such variations is (he formation SECRET PRODUCTION VND CONTKOT, OF PARTICl LATE CLOUDS 271 of loose irregular aggregates of low density from smaller primary particles of uniform density. This type of aggregation is prone to occur during the dis- persion of powdered materials, particularly if they are somewhat hygroscopic. The effects of these com- plicating factors on the impingement of solid parti- cles are elalxjrated in Section 15.4.3. When one is dealing with a particulate cloud of a slightly volatile agent such as HNS, consideration must lx? given to the toxic effectiveness of the vapor as well as to that of the dispersed phase. It must bo rememliered, also, that the characteristics of the cloud continually change with time. The particulate phase suffers progressive loss in concentration and size as volatilization pnx-eeds until a pure vapor cloud results. An analysis has been made of the fac- tors which determine the rate of evaporation of air- borne particles.” It is of interest to note that the wind speed has two opposed effects on the tact ical efficiency of a vesicant particulate cloud. The greater the velocity of the wind, the lower Is the concentration of an agent which is being generated at a fixed rate. On the other hand, the greater the wind speed, the gieater is (he impaction efficiency of a given concentration of the particles. Experimental studies of the relation of particle size to the vesieancy of aerosols of Q, T, and TINS arc reviewed in Section 15.5.1. 15.3.5 Effective Inhaled Dose It is generally acknowledged that toxic particles are less effectively absorbed from the nasal and respiratory passages than from the alveoli of the lungs. Considering the pulmonary toxicity alone, the effective dose of an inhalant may be given as the product of Cl, v, and (1-/) where v is the minute vol- ume of respiration, and/is the fraction of the inhaled material which is trapped in the respiratory passages. It will lx1 agreed that this fraction is determined in large measure l»y the amount of impaction in the nose. It may lx* expected to vary with the species of animal, and, to some extent, from animal to animal of the same species. It will also vary with the physi- ological state of a single animal. To the extent that impingement in the nose determines/, an increase in rate of respiration will, by increasing the velocity of the particles in the nasal passages, result in a greater nasal retention and a reduced effective dose. The question of (he extent to which particulate material which enters the alveoli is retained and al> sorlxxl has been investigated in a preliminary way. The results are summarized in Section 15.5.2. Experimental studies of the effects of particle size on the toxicity of ricin for animals and on the reten- tion of nontoxic particulates in the human nose are reviewed in Section 15,5 and in Chapter 12. 15.1 LABORATORY PRODUCTION AND CONTROL OF PARTICULATE CLOUDS 15.1.1 Dispersal Liquid* and Solutions. In a few speeial studios the Sinelair-LaMer homogeneous smoke generator has Ik'cii used.1 3 Thermogenerators may lx> employed for the dispersal of stable, slightly volatile agents. In most cases, however, various types of atomizer have-been used under conditions of operation which have been empirically determined to give clouds of the desired characteristics. Preliminary studies of the fundamental proi>erties of atomizers have been reported.Mb A useful method of producing clouds of varying particle size from a standard atomizer has been the following. A nonvolatile cosolvent is mixed in vary- ing proportions with a dilute solution of the agent in a volatile solvent. When these mixtures am atom- ized, the mass median diameters of the particles in the cloud vary with the proportion of nonvolatile solvent in t he original mixture. For example, glycerol has been found to bo a satisfactory cosolvent for aqueous solutions of ricin and dibutyl phthalate for solutions oLnonvolatile vesicants.13*-,8b Solids. Electric arcs employing the toxic agent as one component of the electrodes provide useful sources of finely divided metals and their oxides. Thermal generation of toxic clouds by the incorpora- tion of the agent in incendiary or fuel block mixes may also lx? used when the agent is thermostable. Sucli thermal generators tend, however, to give dis- persions which coagulate rapidly.**-40-41 The obvious alternatives in the ease of a thermo- labile solid such as ricin are to disperse by atomiza- tion of a solution or to generate a dust cloud from a finely comminuted powder. Most devices which have, been desenix'd for the dispersal of powders lead to a fractionation of the sample. In some it is the smaller particles, in others, the larger particles which tend to disperse the more rapidly. In most there occurs a considerable formation of loose aggregates in the cloud. Although some attempts have been fairly suc- cessful,nblsh no really satisfactory method for the SECRET 272 ASSESSMENT OF PAKTICl I VIES AS CHEMICAL WAKFAKE AGENTS uniform dispersal of a powder at a rate of a few milli- grams a minute has been described. The devices which lead to least aggregation in the cloud have the disadvantage of a variable rate of delivery. (See Section 15.(1 for dispersal in (he field.) 15.1.2 Measurement of Size The assessment of particle size in a cloud requires not only the observation of the range of sizes in the cloud, but also the amounts of material in the differ- ent size categories. The results are comprehensively expressed as curves in which the cumulative amount of material is shown as a function of the diameter of the particle. Three types of curves may lx- distin- guished, according to whether the particle diameter is plotted against (1) the number, (2) the volume, or (3) the mass of airborne particles. From these curves may be derived respectively a number median di- ameter [XM D], a volume median diameter [A MD], and a mass median diameter [MMD], The number distribution is appropriate if one is interested in ef- fects dependent on the numlter rather than on the mass of airborne particles — as, for example, in the knockdown of mosquitoes. The volume distribution has no particular practical significance, but is the form in which results must be cast if the densities of the particles are not known and the amount of ma- terial must be evaluated from microscopic observa- tions of the numbers and diameters of the particles in the sample. The mass distribution is the descrip- tion of particle size which is most significant to the problem of the vesicant and toxic effects of the cloud. The clouds from atomized liquids have fairly typi- cal distributions and the densities of the particles are uniform. In such conditions the MMD is sufficient to characterize the cloud satisfactorily. In dust clouds generated from powders, on the other hand, the distribution of sizes may be quite abnormal, the unitary particles may lx- far from spherical, and many aggregates of low density may I>e present. The MMD of such a cloud may be a quite misleading in- dex of the impaction efficiency of the cloud. The complete mass distribution is required for the char- acterization of such a cloud. Methods. When dealing with dusts it is desirable to make counts of the undispersed material for com- parison with the airborne cloud. The MM I) and the range of sizes in a given preparat ion are best deter- mined by direct microscopic examination It the M MD is below 10 g. The work is tedious and various meth- ods have been discussed to save labor but critical in- vestigatora agree with Fairs on the procedure to lx* followed. Hard and fast rules for (lie numlxr of parti- cles to bo counted cannot be stated.130 The statistical features of the problem are well presented by Dalla- valle.4- The suitability of a laboratory or field proce- dure for the measurement of the particle size in a cloud depends upon the size of the particles, whether they are liquid or solid, upon the time available for sampling, and upon the concentration. Optical meth- ods suited to the analysis of homogeneous smokes have been developed.* These methods are not readily applicable to heterogeneous clouds, but some at- tempts in this direction have been made.13 In general, the optical methods result in neglect of the relatively small numbers of coarse particles winch may carry an appreciable fraction of the mass. An instrument capable of photoelectric measurement of the surface area of individual particles is not theoretically impos- sible. In view of the labor required in available pro- cedures, some such device is highly desirable. Ultra microscopic and dark field observations of falling particles have frequently been employed.18 Such methods are limited to particles small enough to remain airborne prior to observation and to con- cent rations so low that coagulation is avoided. There is great danger that large particles will be lost in the sampling procedures^prior to observation. 'The thermal precipitator47 is very useful for parti- cles below 5 to 10 n in diameter, provided that the cloud is available for a sufficient period of time so that the necessarily slow sampling rate provides an adequate sample. For clouds ranging from 2.0 to 50 g in diameter, there is one instrument at present which avoids many of the difficulties inherent in other methods. This is the cascade impact or.2* •:w It merits more detailed consideration than those already referred to. ]5.t.3 'The Cascade Impactor This instrument consists of a series of four jets ar- ranged in series so that the sampled cloud impinges at four increasing velocities on to suitably prepared microscope slides (A,B,C, and D). In this way the particles are separated into four impacted groups. 'The size ranges trapped on successive slides overlap to some extent, but the MM 1> of the material on a " The British workers employed the effective drop size [EDS] m place of the MMD to characterize the slides. The EDS is approximately the size lielow which !)8 jjer cent of the number of particles on each slide is found and for most clouds is about 1 times the diameter of the mass median.ui SECRET PRODUCTION AND CONTROL OF PARTICULATE CLOUDS 273 particular slide is, under favorable conditions, char- acteiistic of that slide. Under such conditions, there- fore. it is necessary only to measure the amount of material on each slide in order to obtain a rather satisfactory assessment of the mass distribution. The amount of material on a slide may lie computed from microscopic counts or by chemical analysis. The cascade impactor has a number of obvious ad- vantages over single jet instruments such as koni- ometers, the Owen’s jet, etc. [t was originally de- vised and calibrated 23 30 for the assessment of liquid particulates. For nonvolatile liquids quite pre- cise data can lie obtained if proper consideration is given to the following variables. In the first place the MMD of the material im- pacted on any one slide depends to some, extent, on the MMD of the cloud as a whole. It deqiends also on the shape of the distribution curve of the cloud. Values of the MMD’s on the four slides have been determined experimentally for clouds of MMD 5, 10. 10. and 100 g.13' Impingement of a particle on a given slide is a question of statistical probability. The particles of a homogeneous cloud are distributed over more than one slider Calculations have been made of the mass distribution on the slides which should be obtained with strictly homogeneous clouds.Secondly, the MMD of the particles on a slide depends on the velocity of operation of the impactor. Experimental results indicate that the MMD is proportional to the reciprocal of the square root of the flow rate.iai lHh As the result of the analysis of the counts of a large number of slides a characteristic mass distri- bution curve has been constructed.181* By means of this curve it is possible, by chemical analysis of the amounts of material on the slides in a given experi- ment, to arrive at a fair estimate of the MMD’s on the four slides. When an instrument lias been cali- brated in this way, the use of chemical methods of analysis eliminates the very tedious process of micro- scopic assessment of the slides. Dust Clouds. The use of the, cascade impactor for the assessment of clouds of solid particles was first investigated in this country.1*"-' -,,h It will be evident from what lias already lieen said that its use for this purpose is complicated by a number of factors which arise from the diversity of the characteristics of solid particles. Solid particles may be highly irregular in both shape and density. It has lieen found, for ex- ample, that samples of ricin prepared by the spray drying of aqueous solutions consisted largely of hoi- low spheres. When this material was further de- graded by air grinding the product was chiefly in the form of thin disks. Again, the particle size distribu- tions in dusts may lie quite different from those char- acteristic of atomized sprays. The spray-dried ma- terial referred to was remarkably uniform in size, whereas ball-milled preparations of Hein contained a wide range of particle sizes with a large number of extreme fines. Finally, the adhesion of impinging solid particles may lx* incomplete and the degree of slippage may change progressively as the slide be- comes coated with the agent. These factors combine to give a wider distribution of particle sizes on a single slide than is obtained when an atomized liquid is assessed. When the parti- cles are not. spherical, the problem arises of the proper method of computing their volumes from observations of their dimensions under the micro- scope. Serious errors may arise if they arc1 treated as if they were spheres. The volume of a sphere is 0.52 UP. Hey wood 44 has listed some of the factors by which the cube of the observed “diameter” should be. multiplied when the particles depart from the spheri- cal. The factor fora rounded particle is given as 0.54, for a prismoidal object it is 0.47, and for a tetra- hedral particle it is 0.38. A mean value of 0.5 is sug- gested for a heterogeneous assembly of nonspherical part ides. Recent work has confirmed the validity of this factor for slides (’ and D, but it has not always been possible to apply it to slides A and B because it is on these slides that the large highly irregular and often disk-like particles are found. To measure the mean lateral dimensions of such particles and compute their volume as though they were spheres leads to an MM1) for the slide which is much greater than the true value. Some attempt should be made to measure the thickness of plate-like objects and to calculate their rectangular volumes. The frequent occurrence of aggregates in a solid particulate has proved to Ik1 particularly trouble- some.41 The MM I) of t he particles impacted on a particular slide varies with the square root of the density. Since the density of a loose aggregate may 1)0 less than one-tenth of that of the unitary particles of which it is composed, it is evident that the presence of many aggregates on a slide may profoundly change the MMD of that slide. The problem of the density to be assigned to an aggregate in order to compute its mass is also a difficult one. Microscopically the l)est that can be done is to take a few representative SECRET ASSESSMENT OF PARTICULATES VS CHEMICAL WARFARE AGENTS aggregates, count the numlier of unitary particles in them, and sum their volumes. The density may then be taken to be the ratio of this volume to the volume of the whole aggregate treated as a sphere. Many aggregates disintegrate when they impact on a slide. They will be assessed as though they corre- sponded in impinging properties with the unitary particles of which they were composed, although the latter would probably not have appeared on that slide had they not been aggregated. The result will be artificially to reduce the MMD below its real value. When unusually large (Kirticles are present in a cloud, losses may occur by impingement on the walls of the orifice of the instrument, particularly when the impactor is operated in a-static cloud. Under conditions of isokinetic sampling of clouds moving with average wind velocities it has boon calculated for liquid, droplets that orifice losses become appar- ent with droplets about 50 g in diameter and in- crease as the size further increases. Similar calcula- tions for aggregates with a density of 0.1 indicate that the upper limit for reliable sampling is about 100 g. The upjjer limits for static clouds are probably appreciably below these figures because of increased turbulence around the leading edge of the orifice. Summary. The emphasis which has been laid upon the evaluation of the sizes of particles on the impactor slides has tended to distract attention from the fact (hat the cascade impactor does not measure the size of a particle but rather its impingement tend- ency. Most of the difficulties in applying the instru- ment to the assessment of dusts have been in express- ing the distribution of impacted material in terms of volumes and masses computed from microscopic ob- servations of the dimensions of the particles. This has been a necessary preliminary to the calibration of more direct methods of interpreting the results obtained. In so far as the toxicity of particulate ma- terial is a function of the amount of material which will impact in the nose or on exposed surfaces the efficiency of a cloud should, most logically, be de- scribed in terms of its impaction factor under stand- ard conditions. The use of particle size to characterize the cloud is a convention which may, perhaps, be discarded when instruments which measure impinge- ment have been properly calibrated. The MMD of slide B of the cascade impactor is close to or slightly greater than the maximum size of particles which have been found to penetrate the nasal barrier in most animals. The fraction of air- home material which collects on slides B, (', and D under standard conditions of operation should, there- fore, be somewhat greater than the effective inhala- tion dose. Calibration of the instrument in such a way as to establish a relation between these two fractions should make possible an estimation of the inhalation toxicity of a cloud from a chemical analy- sis of the impact or slides alone. The effective dose of a vesicant is dependent on the fraction of airborne material which is large enough to impact efficiently. Most of this fraction should be captured by slide A. The analysis of im- pactor slides operated in clouds of nonvolatile liquid vesicants should lead without much difficulty to satisfactory estimates of the effective vesicant dose. 15.5 PARTICLE SIZE \ND TOXICITY 15.5.1 Vesicant Effects The dose of a particulate which is deposited on_an object, depends upon the amount settling out plus the amount impacting.45 The amount impacting will vary with the wind speed, density of the particle, area of the particle, diameter of the target, and nature of the surface of the target. A heterogeneous cloud of MM I) 2.0 g may have the same impactibility for a given surface as a homogeneous cloud of M MD t.O g. The impingement pattern on the object will vary with particle size from a diffuse pattern with vapors atid stpokes to a localized (upstream surface) mosaic with coarse sprays. The volatility of the agent and the rate of absorption of the material by the target will affect the physiologically effective dose. Preliminary indications of the order of magnitude of effect of particle size on vesicancy of a nitrogen mustard (HN3) and a nonvolatile vesicant (T) were obtained by exposures of forearms in a wind tun- nel.13k~m,l8“ At 5 mph wind speed and under condi- tions of temperature (about 80 F), relative humidity, and skin resistance (sweating index) such that a vapor of HN3 at a Ct of 1,200 mg min nr produces an erythema, the following tentative conclusions were reached. Smokes of MMT) below 2.0 g are less effective than vapor. A heterogeneous (atomized) cloud of MM D 2.0 g was equally as effective as vapor. A heterogeneous cloud of MMD 8.0 g was twice as effective but the erythema was more localized. HN3 is less volatile than mustard. T is practically non- volatile. The relation of volatility and particle size to vesicancy is illustrated by the following relation- ships. By topical application of single drops to fore- SECRET PARTICLE SIZE VND TOXICITY’ 275 arms it lakes 10 limes as much IIN3 to produce the same skin reactions as a given amount of T.7 As a 2. 0-/i (heterogeneous) particulate, a Cl of to of T (area dose = Cl X 5 mph) is the equivalent of a Cl of 1,200 mg min m* of 11N3; T is thus 27 1 hues as vesicant as TINS. When the particle size is raised to 8.0 g, a Cl of 0 to 10 of T is as effective as a Cl of 600 of HN3, thus demonstrating a factor of 100 or more in the vesieaney of these agents.18* These findings demonstrate the importance of designing munitions which will dis|>erse the chosen particulate in an opti- mum size range. Owing to the great effect of temperature and humidity 4 817 on skin reactions to given exposures, it is difficult to generalize from these data to other agents and conditions. By employing the appropriate factors for comparison of-vesicant power,71xh com- parisons may l>e made with the values given in Chapters 5 and 0. Under the conditions obtained in the exjKM'iments described in the preceding para- graphs, I] vapor is about one-half as effective as HN3 vapor. The effect of evaporation of the agent after deposition on the skin has hern found to bo approximately (he same for H and ITN3,,8a b despite differences in volatility. It will be indicated in the next section that of a heterogeneous cloud of T of MM I) 8.0 n. only 10 15 per cent will penetrate the human nose. The results on vesieaney in relation to particle size apply to exposed skin areas. The presence of clothing profoundly modifies the situation. A droplet of nonvolatile agent on the surface of clothing can under some circumstances !>e considered innocuous, whereas a volatile agent will generate vapor which may lie drawn over the underlying skin by the bel- lows effect of clothing. Numerous tests have been carried out on the droplet diameter required to pene- trate clothing by wetting the cloth. The sizes in- volved are well above the particulate range consid- ered here. Relatively few data on the penetration of clothing by small particles are available for chemical warfare agents. The amount of a particulate found on clothing is a function of filtration and impaction. A given expenditure of agent will with increasing wind speed deposit decreasing amounts on (and through) the cloth by filtration (bellows effect) but will deposit increasing amounts by impaction, espe- cially for coarser particulates. For nonvolatile sub- stances the amount penetrating cloth by impaction forces appears to l>e a small fraction of the amount penetrating by filtration.I8b For nonvolatile materials there is definite disad- vantage to the use of particulate clouds coarser than 1 to 2 n in diameter if penetration of clothing is to he achieved. Increased turbulence at higher wind speeds appears to reduce the percentage that penetrates by filtration.18'* 15.5.2 Penetration of the Nose , Initial experiments were designed to determine the particle diameter at which 50 per cent of the mass of a given cloud passes the nose.1*-'-*-2* Values obtained on four human subjects are given in Table 2. Tmilk 2. Penetration of the human nose by particulates. Agent Diameter for 50 per cent Density Flow rate penetration (g-ml) (1pm) (n) Corn oil Dry NalK'Oj 0.93 17 5.0 00 IS 2.2* 17 2.1 00 0.8 * Actual density in tides. i nose somewhat lower Ijecause of hydration of par- These experiments wen* extended in an attempt to determine the percentage penetration of the nose at. various sizes for materials of differing physical char- acteristics, e.g., liquid corn oil of density 0.9 and dry NaHCOs of density 2.0, and at various rates of breathing. The results are presented in Figures 1- and 2. It is of interest that there is little difference in rir.rRE 1. Nasal retention of particulates in man. nasal penetration Ik*tween flow rates of 17 and 29 1pm (unpublished data). A change from 17 1pm to 60 1pm changes the value for 50 per cent penetration from 2.1 to 0.8 n. Regardless of flow rate or density, parti- SECRET 276 ASSESSMENT OF PARTICULATES \S CHE.MIL VL WARFARE AGENTS eles 10 g in diameter have approximately a 10 per cent chance of penetrating the nose. Over the size range shown in Figures 1 and 2 it would ap|icar that for a given particle diameter the MASS MEDIAN DIAMETER IN MlC»0NS FiiiCRE 2. T.ung retention of particulates in man. lung retention is about 20 per cent more efficient than the nose. From another viewpoint, the same efficiency in retention obtains for particles in the nose which are 2.5 times the diameter of those in the lung. When molecular dimensions arc reached the nasal and lung retentions increase al>ove those found at 0.2 g.18'-29 15.5.3 Inhalation Toxicity in Animals Mice, rats, and rabbits were exposed, while at rest, to particulate clouds of W in glycerol at controlled HMD’s. 'Phe relation between particle size and L{Ct)ho is shown in Figure 3. For the size range 0.5 to 7.0 g the effect is much more pronounced in rats and mice than in rabbits. These data, which are reviewed in more detail in Chapter 12, should be compared with those of British authors29 using other tech- niques. 13.6 DISPERSIBILITY OF PARTICULATES In the laboratory it is relatively simple to prepare clouds of unitary particles by atomization, thermal generation, or in electric ares. Previously comminu- ted powders may also Ik- disperses! largely as unitary particles in special apparatus. Munitions capable of dispersing previously comminuted powders in the unitary state have yet to be developed. Powders differ in ease of dispersibility, as shown in various Fioitre 3. -Inhalation toxicity of ricin in relation to particle size. laboratory tests. Sueli tests tire, however, generally meaningless in terms of dispersibility by field muni- tions. The factors involved in field munitions which are difficult to scale up from laboratory tests include aggregation phenomena occurring prior to, at. the time of, and immediately subsequent to dispersal. These aggregation phenomena are influenced by ge- ometry, strength of materials, brisance of explosives, and scale of munition.** •** To date field experiments 39 have, however, almost universally confirmed the finding 15 that suspensions in organic nonsolvent media result in much higher dispersion efficiencies than can l>e obtained by use of gas ejection munitions or standard munitions with dry fillings. Owing to low bulk density of dry fillings, suspensions permit a higher ratio of active filling to munition weight. 15.7 FIELD ASPECTS OF PARTICULATE ASSESS M ENT The outstanding observation resulting from fit !d experiments on dispersion of previously comminuted powders by field munitions is the fact that the frac- tion of material airborne in the size range of the original filling is generally insignificant. Most of the mass of the filling appears in a highly aggregated SECRET FIELD ASPECTS OF PARTICULATE ASSESSMENT 277 state. Field sampling must not only evaluate the gross clumping and spillage but also account for the low toxicity (in terms of chemical Cl or area dose) of the more lastingly airlxxrne clouds. The frequently occurring light fluffy (snowflake) aggregates are gen- erally not encountered in laboratory investigations. They are particularly deceptive since they may be readily disrupted in the sample and thus appear as a group of component unitary particles. The orifice velocity of field sampling equipment should not deviate markedly from the wind speed if particles above 50 g in diameter are to lx* readily sampled. Where power-operated devices ait* em- ployed, the requirements of pump capacity for ap- propriate sampling of particulates, which are higher than for vapors, may cause some embarrassment. In early exjx*riments fine wires were tested as sampling devices but discarded because of (he differ- ing impaction efficiencies for small and large parti- cles.31’ By employing wires or tubes of three different diameters, however, the relation of collection effi- ciency to wire diameter can be utilized to calculate particle size and area dose from the mass of material collected on each size of wire."**r d 44 This device dispenses with power requirements when sampling in wind speeds above 3 mph. At I mph corrections for settling are required. There are marked difficulties in the assessment of initial clouds containing vapor and particulate con- centrations. The chemical drop trap 24 and the chem- ical selector*4 indicate possible methods to be de- veloped. The use of impingers M or filters is recom- mended when numerical values of (he MMD or impactibility are not required, 'fixe total chemical Ct, without regard to size, can be measured for dry par- ticulates with filters. Hayon-aslx’.stos, esparto-as- bestos, and gas mask filter papers may lx- used. For use with \V those papers are unsuitable owing to the strong adsorption of the protein on the paper. Cellu- lose acetate filter batts 12 do not absorb proteins ami in addition are soluble in appropriate organic liquids. Another qualitative device for evaluation of aggre- gates is the “sticky finger.” Ha Methods have been developed and results obtained during the period 1911 to 1945 which indicate the desired particle size for various purposes. In the same jxeriod, however, no adequate munition capable either of producing such sizes or dispersing materials already prepared at those sizes has been developed. Thus, at the date of writing, W (which has in the laboratory several score times the toxicity of phos- gene) has (in the field) been found to be only seven times as toxic as phosgene in the best munitions available.** SECRET Chapter 16 APPARATUS AND TECHNIQUES UTILIZED IN TOXICOLOGICAL STUDIES ON CHEMICAL WARFARE AGENTS By //. .1. Wooster and W. L. Doyle 16.1 INTRODUCTION In this chaptku are summarized methods de- veloped and utilized for toxicological studies at the University of Chicago Toxicity Laboratory [UCTL]. Pertinent contributions of other NDRC Division 9 contractors arc included, but no attempt is made to review systematically developments made by other agencies. The apparatus and methods are described under the following major headings: (1) gassing chandlers, (2) methods of dispersing agents into chambers, (3) sampling equipment, (4) precision methods of testing inhalation toxicity, and (5) methods of test- ing vesicants. Each section starts with a discussion of the relevant principles and is followed by a brief description of specific items of equipment and pro- cedure, together with an evaluation of the merits and limitations of each. Descriptions and construction details for the more important items of equipment will l)e found in the reports listed in the Bibliography and referred to in the text. The work leading to the development of appara- tus included in this report was initiated prior to March 15, 1945, at which time the contract with the University of Chicago was assumed by the Chemical Warfare Service. Subsequent work has Den reported where it was in logical extension of apparatus initi- ated under the prior contract. J6.2 GASSING CHAMBERS 16.2.1 General Description of Design The earliest form of gassing chamlier was a closed container in which the animals were placed and the agent dispersed. Despite the simplicity of such an apparatus, its use introduces many complexities. The actual concentration of agent in it at any one time is a result of the action of at least two variables — the rate at which the agent is sprayed into (he chamber and the decrease of the concentration. The latter is influenced in several ways — absorption on the chamber walls, chemical changes of the agent (the hydrolysis of dichlordialkyl arsines, for exam- ple), and, in the ease of particulates, aggregation of the smaller particles. Animals kept, in a closed eham- Ikm for any period of time may /hange the carl ton dioxide content of the air sufficiently to distort their respiratory patterns. Nominal concentrations in such chambers are almost meaningless, and analytical concentrations are difficult to interpret. Lehmann, in Germany, in a long series of investi- gations (1884 1918) st udied the effects on animals of various toxic vapors used in industry, llis method was to expose animals in a modified Pettenkofer respiration apparatus to a continuous flow of air con- taining a constant and known concentration of the agent being studied. Almost all the gassing chambers used in this country since 1918 are based on this constant Row, or “dynamic” principle. (It should lie noted that English workers, in many of their screen- ing runs, employed “static” chambers during World War II.) The ratio of chamber volume to air flow is critical in the design of such chandlers. Silver23 derives the basic equation covering chamber equilibration t imes: tv = 4.(5 X I 0 where = time for the chamber concentration to attain 99 per cent of the theoretical nominal concen trat ion. a = volume of the chamber in liters. h = (he rate of air flow in 1pm. It will Ixi seen from this that a chamber having an air flow of 1 chamber volume per minute will come to equilibrium in about 5 minutes; with 10 chamber vol- umes per minute equilibrium is attained in 0.5 min- ute. A (puck equilibration time has several advan- tages— momentary changes in concentration, such as ( hose produced by the introduction of animals, arc quickly rectified, and unstable materials have less time in which to decompose. The saving in material by the use of a shorter equilibration time is overbalanced by the larger amount of material necessary to set up SECRET GASSING CHAMBERS 279 a given concentration, but a 5-g sample is generally adequate for a single test of a substance toxic at 0.3 mg 1. When slowly volatile materials, which exist as both vapors and aerosols, are dispersed in cham- bers of very high How rates, such as the auxiliary chamlier for the 200-1 medium How chamber (see below),-effects of the flow rate on toxicity may lx1 encountered. The flow of air through the chamlier may Iic pro- duced by either positive or negative pressure. In most chamliers negative pressure is used, to mini- mize the tendency for toxic materials to escape into the laboratory. Standard equipment for this is a gear-type (Roots) blower V-belted to an electric motor. An air ejector is used on the chamlier for the large Benesh atomizer, and water aspirators have been used on some small smoke chamliers. Positive pressure has been used on three chambers — a large screening smoke chamlier, a small chamber used for testing the toxicity of gasoline, and the microline. In these chamliers the air How is controlled by the volume of air blown into the chamlier. Standard equipment for measuring air flow through the larger chamlx'rs has lieen an orifice in a Monel plate in the effluent, line between (he chamber and the filters. A differential manometer, filled with butyl phthalate, is connected to each side of the orifice. To calibrate such a flowmeter a large dry- type gas meter is connected to the chamber and all other openings are sealed. A working calibration chart is prepared from these readings. The dry meter is calibrated by positive displacement of a measured volume of air. The standard orifices are about 0.8 inch in diam- eter. Because of their location they are subject to contamination and corrosion. When aerosols are used in the chamber they tend to clog up the hole and make it smaller. It is advisable to recalibrate such orifice flowmeters at least once a year. A much larger orifice (about 2.5 inch) has been used in the chamber for the large Benesh atomizer, which was designed specifically for use with smokes. An inclined differ- ential manometer is necessary to read accurately the small pressure gradient resulting from the use of such a large orifice. The effluent from these chambers contains a large proportion of the original toxic material. It is passed through replaceable charcoal filters. When nonvola- tile vesicants have lx*en used, the filters become heavily contaminated, and their removal and re- charging is a hazardous procedure. The effluent from all chambers plus (he effluent from all rooms is drawn olT by ml ary bh>wers and discharged into a large incinerator stack. The dilution afforded by the stack provides a larger margin of safety and in some cases permits dispensing with charcoal filters. The UCTL stack has an average inside diameter of I6J2 feet and is 100 feet high. Under normal conditions the stack discharges 750,000 cfm. The chamlx'rs are of metal and or glass construc- tion. The all-metal chaml>ers are constructed of welded sj'fi-inch mild steel plate, which is protected on the inside with a baked-on vitreous or bakelite resin (l.ithcole) enamel. Connections to these cham- lx'rs are made with standard plumbing pipe fittings. Ten-liter wide-month glass bottles with holes drilled in them have been used for several small chambers. The chamber on the small Benesh machine is made entirely of triplex safety glass, cemented together. Composite structure is represented by the 400-1 chamlier. made from a length of Pyrex industrial pipe 12 inches in diameter, fitted with brass ends, and the 488-1 chamber, made of metal lined with plate glass. One of the more important procedures in gassing animals is the method of introduction of animals into the chamber. The simplest method, which is entirely feasible with mice, is to open a port and insert the caged animals. This is routinely done with the small smoke chambers and with some of the larger cham- bers which have small auxiliary ports on their larger doors. With larger animals, some sort of sliding car- riage for the cages must lie provided. This is, in essence, a three-sided box, the ends of which are plates, and the bottom an open structure. The side view can be represented by | J. Either end may serve as a closure for the opening in the chamber side. When such a carriage is rapidly pushed into a cham- ber, a certain piston action is exerted. The carriage on the big Benesh machine was designed to avoid (his. When the carriage is out of the chamber, closure is provided by a vertically sliding glass panel. Thus (he carriage needs only the form ). In high-flow chambers the animals may be placed in the chamber before the agent is put in. This is practicable because of the short equilibration time of such chambers. However, it should not be used for short exposures to substances which deviate markedly from Haber’s law. One difficult problem in the design of chambers is the position of the port through which the agent is to lie introduced. This may be at either the top or SECRET 280 VI’P.AHATL'S \M> TKCHMQLES IN TOXICOLOGICAL STI'DIES the side of the chambers. The top is a somewhat more convenient, location, inasmuch as all the parapher- nalia connected with dispersal may be placed on top of the chamber out of the way. When dealing with gases or with aerosols set up by a baffled atomizer, the position is not so important as with other de- grees of dispersion because the materials enter the chamlier at a low velocity and loss by impaction on inlet tubes is negligible. With concentric atomizers dispersing semi volatile materials, introduction from the top means that the spray must undergo a right- angle bend to get. into the chamlier, with consequent loss on the mixing bowl. A jet fed in from the side of the chamber must lie aimed with care to clear the animal cages. It would seem advisable to design future chandlers with provision for the optional use of either route of entry. Little attempt has been made so far to control the temperature and humidity of air entering the cham- lier. In most cases the chambers withdraw air from the lalioratorv and operate at the ambient temper- ature and humidity. In the microline provisions were made to humidify the entering air. Some small cham- bers have lieen operated in a thermos fated water bath. The most elaborate regulation is in the man- chamlier, which has automatically controlled equip- ment for heating or cooling, and varying the water content of the entering air, as well as temperature control of the room surrounding the chamlier. 16.2.2 Description of Specific Chambers Rectangular Chambers Larger than 200 Liters .QUO-Liter Standard Chamber.g -3 The first large chamber used at UCTL was built from a design standard at Ed go wood Arsenal. This chamlier is fitted with a sliding carriage 8 inches high and 15 inches wide. This, at most, can hold 4 cats or rabbits, or 20 guinea pigs or rats. The chamber air flow can be regulated between 50 to 90 per cent of the cham- ber volume per minute. A wooden sliding carriage with stocks for surrounding the necks of exposed animals was made to study body and head exposures. In use, this chamber was found to have several limitations. Animals larger than cats could not be exposed routinely (singledogs were used in body ex- posures). Appreciable difficulty was encountered in working with lewisite, owing to wall loss at the low How rates --e.g., the nominal LC:,o of lewisite for mice was approximately three times as high with the standard chamber as with the Benesh machine. SSO-Liter Standard Chamber.* This chamlier is identical in principle and operation with the 100-1 chain her. The sliding carriage is somewhat higher in relation to the height of the chamlier. Its cross- sectional dimensions are 23x55 Inches. This gives it a maximum animal capacity of I small dogs, or 2 large dogs, or 1 or 2 goats, or 6 monkeys. A mixed group of 1 small dog, 1 rabbits, 1 cats, 10 guinea pigs, 10 rats, and 20 mice can Ik? exposed at the same t ime. At a later period a small door was built into the outside plate of the sliding carriage, making it possi- ble to put small animals into the chamber without pulling out the carriage. This chamber has been calibrated with mustard gas (11), using (hi1 Northrop 1 itrimeter.3*1 The air flow was 700 1pm. and the nominal concentration may be expected to be in error by about + 5 percent- The concentration built up is the same in all parts of the chamber within 1 per cent and the drop in con- centration on moving the carriage in or out is prob- ably not more than 5 per cent. This chamlier has, perhaps, Ik'om the most con- sistently useful for general work. 300-Liter Medium Flow Chamber,u This chamlier was designed to provide a chamber in which dogs and other large animals could be exposed to agents at rates of chamber exchange comparable to those at which mice had been exposed in smaller chambers. By interchanging a glass door on (he side of the chamber for one which is provided with platforms and head stocks, mice, rats, or guinea pigs may I>e exposed to gases either by inhalation or by body ex- posure alone. A similar arrangement can be attached to the front carriage for similar exposures of cats, rabbits, or dogs. Some time after the chamber was built an auxiliary high-flow chamlier was added.14 The new chamber was built onto a removable side plate which could be substituted for the side door. The cross-sectional diameter of the high-flow chamber is about one-ninth that of the main chamber. Air is drawn from the main chamber into the auxiliary chamlier and thence into the exhaust line. When the chamber is operated at 500 1pm, the velocity is increased to 3 mph just before the toxic agent reaches the animals, with a minimum velocity of 0.5 mph in the center of the compartment in which the animals are exposed. These velocities may be increased or decreased by varying the air flow. The incident velocity may lx; changed by changing the size of the slits through which (he air stream enters. The high-flow chamlier is 18x7x3 inches. It. SECRET GASSING CHAMBERS is divided into three compartments by two longi- tudinal walls, each of which contains 10 slits, 2x Yi incl u*s. the slit size may be varied. The compart- ment in which the animals are exposed is 18x3x3 inches, and is located lx-tween the other two com- partments. the long, slender compartments on each side have openings in the floor through which ana- lytical samples may lx- drawn. The inner of the small compartments is o|x*n on the side communicating with the main chamber, and on the distal side has the slotted wall openings. It serves as a mixing chamber to insure that all the animals are exposed to the same concentration. The outer of the two compartments has a slotted inner wall through which the air stream leaves. The effluent is carried away through an open- ing in the end of this chamber. Analytical samples can lx* drawn Ix-fore and after the agent passes the animals. Animals may be exposed by total exposure, or by body or head exposure atone. A special manifold is provided for the last two types. The lower portion of the side plate to which the high-flow chamber is attached can also be used for either Ixx.lv or inhala- tion exposures at low flow rates. Total exposures for low flow rates can be carried out by placing animals in the main chamber. Animals may, therefore, lx- exposed simultaneously to high and low flow rates either by total exposure, body exposure, or inhalation exposure. The 200-1 chamber differs in several design details from the standard chambers. The carriage is provided with castors, making it more convenient to slide it in and out. In the standard chambers the bare metal of the door seats against the bare metal of the chain) tor. In this chamber sponge rubber gaskets are pro- vider!. The toxic agent is usually admitted at the top of this chamber instead of at the side. In the use of this chamber a good agreement has lx‘on obtained between analytical and nominal con- centrations. L{CI):M’s obtained by this chamber cor- respond with those* obtained in the small high-flow chamlxws. This is not the case with values obtained from the 100-1 standard chamber. 429-Lttrr (Ham-Lined Chamber. This chamlx*r was designed specifically for use with aerosols. It is lined with plate glass. The sliding stainless-steel animal carriage is attached to a glass panel which forms the front wall of the chamber when the carriage is in place. When this is not used a counterweighted glass panel drawn down from the top seals the chamber. Interlocks are provided so that the carriage cannot be pushed in until the sliding panel is fully raised. This scheme is a trifle complicated and requires two oper- ators for rapid action, but has the advantage that it does not exert the plunger effect of the usual cham- ber carriage. A small circular auxiliary port in the panel on the carriage permits caged mice to be placed in the chamber without opening the main door. A large air injector is used as a pump to exhaust air from the chamber. This gives a maximum air flow of 900 1pm at 35 lb air pressure. An inclined differen- tial manometer, reading across a large orifice, gives t he chamber air flow. Such a large orifice is less sensi- tive to fouling than those commonly used, flic air injector is also less subject to fouling with aerosols than gear-tyjx' blowers. Variations in the pressure of the air running the Venturi are corrected by a diaphragm-actuated regulator. The glass lining makes this chandler particularly easy to clean. It is much.quieter in operation than the mechanically driven chambers. This chamber has lieen calibrated with 11 by means of the Northrup titrimeter, at a nominal concentra- tion of 38.8 gg 1 and an air flow of 1.5 chamber vol- umes per minute.21® The following conclusions were drawn: 1. The Ct calculated from the nominal concentra- tion will be in error by about -flG |x*r cent. 2. The 10-minute Ct calculated from an analytical concentration measured at about the mid-point of a 10-minute exposure will be in error by about +3 per cent. 3. The Ct calculated from an analytical concen- tration based on a sample drawn over the entire period should be in error by less than 3 per cent. 4. In general, errors caused by the fall in concen- tration that occurs upon pushing in the animal cages may Ik; neglected for 10-minute exposures and can lx* corrected by analytical samples drawn at intervals during the entire exposure period. Screening Smoke Chamber.316 This chamber was designed for the repeated exposures of animals to low concentrations of agents employed as screening smokes. It was made large enough for monkeys to live in and was fitted with automatic controls. The chamber is 4 feet square and 7 feet high. Its volume is 3,078 I. The base is a concrete block fitted with a drain and lined with sheet metal. The top is wooden, as are the comer posts. The sides are of glass. A com- mon wooden door with a glass panel is let into one side, this door is weafherstripped. The whole struc- ture is lined with a very heavy wire mesh. SECRET 282 APPARATUS AM) TECHNIQUES IN TOXICOLOGICAL STUDIES Two fans are used with this ehamlier. A continu- ously running exhaust fan provides ventilation. An intermittently operating centrifugal blower giving 2,180 1pm is mounted on the top of the ehamlier. Just Inflow the ceiling outlet is a suspended baffle plate. A six-jet atomizer (DeVilbiss experimental model No. 7030 1), connected to the compressed air line, discharges into the inlet of the centrifugal blower. A General Electric time switch controls the solenoid valve, feeding compressed air to the atomizer and the relay actuating the inlet fan. These go on and off to- gether, in a cycle of 30 minutes on and 30 minutes off. The chamber was found to come to equilibrium in 10 (±2) minutes. 'This is somewhat longer than the theoretical time. Forty-five |>er cent of the equi- librium concentration is reached after I minute, and eighty per cent at 5 minutes. Lnrrimotor Chamber"'* This chamber is essentially a 400-1 standard ehamlier with a maximum air flow of 1,0001pm. The adaptation for use with lacrimators consists of three ports projecting from the center of the ehamlier walls on three sides. Eye pieces, which fit the ports snugly, consist of rubber diaphragms edged with rubber tubing. Swimming goggle frames are cemented around holes cut in the diaphragm. The sternutator provision consists of industrial-type, nose and mouth respirator masks connected to the ehamlier with lengths of gas mask host*. There are six of these. The subjects signal their response by tapping keys, located under the ports, which cause signal magnets to mark an automatically timed rotating kymo- graph. The subject taps the key when irritation is first experienced, and again when he feels tears start- ing to form. Thereafter he depresses the key each time he is forced to close his eyelids and releases it when the lids are once again open. At the end of the run there is a graphic record of the onset of irritation and of lamination, as well as of (he periods during which the eyes were open or closed. Owing to the low priority assigned to lacrimators and sternutators, this chamber was never extensively used or completely calibrated. Great Lukes Man-Chamber’""*"r" * This cham- ber was designed for the exposure of human subjects under conditions of temperature and humidity con- trollable by the investigator and independent of ambient conditions. The chamber is made of :,s-inch boiler plate, lined with i6-inch sheet lead. Its volume, exclusive of Ilie air lock, is about 17.300 I. Tlic maximum How rate through the ehamlier is about 5,100 1pm. All control of concentration (H has l>eon (he only agent used) is dune with the Northrup titrimeter, so that exact values for this flow are not so necessary as when an attempt is made to estimate the nominal con- centration. This chamber is equipped with automatic pneu- matic controls for temperature, relative humidity, rate of flow, and pressure. They function as follows; 1. All air coming into the chamber passes through a eommeicial air-conditioning unit. It emerges from this into the chandler at 26 F, saturated with water vapor. When warmed to 70 F, this air is at about 35 per cent relative humidity. The temperature and relative humidity of this air represent the lowest levels at which the ehainber can be operated, 2. The desired wet bulb and dry bulb tempera- tures are set on the controlling-recording apparatus and the steam lines are opened. Heating is cont rolled by a steam coil controlled by the dry bulb tempera- tures. Lowered wet bulb temperatures cause the automatic humidity valve to open, injecting steam into the ehamlier. When the wet and dry bulb tem- peratures reach the desired values, the humidity valve closes and the by-pass dampers open; thus the incoming air is conducted underneath the heating coil rather than through it. 3. When the air is pulled through the heating coil, there is more resistance in the system than when the air is by-passing the coil, so that adjustments of the flow are necessary. This regulation is controlled by dampers on the discharge side of the exhaust fan. When the flow rate drops below 5,600 Ipm, these dampers open and permit more air to lie drawn out of the ehamlier; as the flow rate rises, the dampers close and cut down the flow. The flow rate usually oscillates between 5.300 and 5,900 1pm. 4. Ordinarily the fluctuations in the amount of air discharged would produce variations in the pres- sure inside the chamber. Such variations are elimi- nated by automatic control of the dampers on the discharge side of the supply fan. The pressure con- troller is set for a differential of 0.1 inch of water; when the pressure in the chamber increases, the con- trol damper effects an opening of the dampers to the room, so that less air is passed into the chamber. Similarly, when the inside pressure falls to a value lower than 0.1 inch of water Inflow the outside pres- sure, the dampers close* to jiermit a larger volume of air to enter the ehamlier. SECRET GASSING CHAMBERS 283 An air lock is equipped with motor-driven ports by means of which fresh air may be diverted through the air lock when men wearing contaminated clothing are leaving the chamber. Measurements of the wind speeds in the chamber showed that the velocities vary from less than 0.4 mph in the corners to over 8 mph in front of the fans, with an average of 2.5 mph for 32 positions. Constant-Flow Uhamokbs Smallkk than 100 I, The Microline.1 The 100- and 800-1 standard cham- bers were found to be unsuited for “screening” new agents of which only small amounts were available, and for working with unstable substances such as the arsenical*. The mierolmo together with its ancillary chambers was designed to provide a small chamber through which a relatively high How of air at con- trollable humidity could be sent. The influent air is delivered via two parallel series of bubblers and absorbers. One of these delivers dry air, the other saturated. These arc mixed in the de- sired proportions and passed through a dispersing bubbler or an impinging atomizer containing the agent and thence into the chamber. The first chamber used with this mieroliue was a 10-1 screw-capped wide-mouthed bottle. A cylindrical cage fastened to the bakelito screw cap contained six mice. A U-shaped manifold, both ends of which passed through the screw cap, was user! for body ex- posures. The heads of mice were stuck through holes in the manifold while fresh air was circulated through it, and a concentration of toxic agent was set up in the chamber. A branched manifold for testing toxic- ity by inhalation could be substituted for the cham- ber. This enabled 8 mice to inhale the agent while their bodies were exposed to room air. These chambers and manifolds had several draw- backs. Only mice could lx? used in the chamber, and not more than six of these. The agent flowed linearly through the chamber, so that if the first animal af- fected the composition of the agent the last might, get a lowered dose. The inhalation and body expo- sure manifolds could hold only 8 and 6 mice, re- speetively, and were difficult to manipulate. Later a commercial Lectrodryer unit8 was in- stalled to supply adequate amounts of dry com- pressed air. The size of the water-saturators was in- creased proportionately. An 11.5-1 chamber was con- structed of plate glass cemented together and sup- ported inside of an angle iron framework. This was designed to assure equal distribution of the toxic-air mixture directly to each animal. To do this the material is conducted into an H-sha|ied channel, each arm of which has an opening connected with a slit in the glass side of (he chamber, 0.1 rum wide and extending from front to back. The channel is de- signed to give uniform flow through the whole length of !>olh slits, which form two horizontal lines on each side of the chamber and are so centered that, when the mouse cage is placed in the chandler, the animals am directly opposite the slits in line with the flow of the material. This permits a high degree of uniform- ity in exposure of the mice. The effluent is carried off by an identical arrangement on the other side of the ehaml)er. This chamber has a capacity of 20 mice, 3 rats, or 3 guinea pigs. A body exposure manifold which holds 20 mice and fits into the chamber and a separate inhalation mani- fold holding 16 mice have also boen-construct<*d. Using an aerosol of HN3, recoveries of about. 65 per • cent were obtained from the chamber in the absence of animals and of about 75 per cent from the inhala- tion manifold. Recoveries of more volatile materials are well above 90 j>er cent. Small Smoke Chamber. This was essentially a vastly simplified mieroliue. Dry air was passed through a dispersing bubbler or impinging atomizer. The re- ducing valve on the compressed air tank, with the atomizer connected, was calibrated in liters per min- ute versus pressure. Auxiliary air could he bled in through a Y tube to bring the flow to the desired value. The air flow was then led through a water or steam jacketed condenser into the wide-mouthed bottle used as a gassing chamber. The toxic material was blown out of the chamber into the air of the hood in which the whole setup was placed. This chamber has been used in a thermostated water hath for exposures alxjve or Ixdow room temperatures. This setup, which required a minimum of appara- tus, proved to bo quite useful for screening materials of low vapor pressure. Its use; was limited to materi- als which would melt without decomposing, so that they could lx> dispersed with an impinging atomizer. Mice, guinea pigs, and rats were (he only animals that could Ixs fitted into the chamber. A mollification of (his chamber was used to set up very high concentrations of gasoline vapor.*’* All air entering the chamber was blown through a concen- tric atomizer, and then passed through a steam- heat ed Friederieh’s condenser. A trap in the line re- moved non volatilized material Indore it entered the chamber. SECRET APPARATUS AND TECHNIQUES IN TOXICOLOGICAL STUDIES The Wind Tunnel*1' The UCTL became inter- ested in the relation of particle size to vesication on bare skin and through clothing and in the relative efficiencies of the vapor and aerosols of the same ma- terial as vesicants. It was necessary to construct a wind tunnel in which the arms of human subjects could be exposed to airborne agents moving at vari- ous and variable velocities. The tunnel, circular in cross section, is 11 feet long and 2J2 feet in diameter in (he largest places. It is fashioned after certain Port on models30 designed to give an even distribution of droplets across the work- ing sect hip. It differs from st reamline tunnels, in which markedly higher velocities exist at the center of the stream than at the edges. The wind tunnel proper (Figure 1) is of cylindrical cross section. Two truncated cones (li and C) are placed base to base with a short base diameter cylin- der in between. This assembly precedes a longer cylindrical section (£)), IS inches in diameter and 3 feet long. The source of vapor or particulate spray is an atomizer or bubbler orifice located at (he mouth of the tunnel. In order to mix the narrow plume of agent with the main air stream, the diameter of the tunnel is increased (li) to produce turbulence. The expanding cone is followed by a reducing cone (C) to give approximately constant velocity across the stream in the cylindrical working section (D). The flow through the working section is somewhat turbu- lent at 7 mph. This turbulence can lie decreased by placing a hardware cloth screen in the reducing cone, l»ut such a screen causes an increase in concentrat ion of the larger particulate droplets in the center of the stream. Without the screen the droplet distribution is quite homogeneous across the tunnel. Turbulence creates vortexes in the working section, producing differences of about 10 per cent (at 7 mph) in the velocities at opposite sides. This difference could probably lie decreased by increasing the length of (he cylindrical section between the two cones B and C. With particulate clouds of nonvolatile droplets in which 30 |xu' cent of the mass is in the size range 10-30 fx> there is a just perceptible loss on the walls; the loss is negligible wit h smaller droplets. With drop- lets of 150 + 50 fi in diameter there is a slightly greater loss on the bottom of the tunnel than on the top. Most of the loss occurs in the reducing cone (C). The source of suction is the room ventilation which leads via filters to the incinerator stack. The flow is regulated by adjustable louvers. To obtain velocities above 25 mph a tul>e with its own reducing cone and Specialized Chambers The Explosion Chambers. Chambers were required to assay the action of high explosives on the toxicity of certain chemical warfare agents. It was necessary to construct a rugged chamber in which small amounts of explosives could be set off and the toxicity of the resulting airborne material assayed. The first of these was a 1-ton shipping container for war gases such as mustard, A port (12-inch di- ameter) was welded on (his. It was fitted w ith a steel cover 2 inches thick, bolted down with 1-inch bolts. T1 iis chamber was used while a specially designed -chamber w as being built.21* The latter was constructed from 18-8 stainless steel. The interior is polished to a No. 4 finish. The vessel is 48 inches in outside diameter and approxi- mately 8 h'et high. The volume is 2 cu m. It is mounted over a concrete pit in a specially const meted laboratory and is shielded by heavy concrete walls. The dome-shaped top of the chamber is held down by SO bolts under spring tension to act as a safety valve yielding at SO psi. Easy access to the interior is provided by an 18-inch manhole with single-screw closure. There are eight 4-inch ports which can lie closed with :t4-inch Pyrex or stainless-steel plates; additional ports are provided for valves and electri- cal leads. The chamber is equipped with a shower for Hushing. The walls and interior fittings are designed to permit complete drainage to the valve at the bot- tom. This permits maximum recovery of the prod- ucts. Steam lines lead to the chamber for decontamina- tion. The residual gases in the chamber may be drawn through a 200 cfm collective protective canister. When metal bombs are exploded they are sur- rounded by stainless-steel baffles to protect the walls of the chamber. This is not necessary for glass or plastic bombs. The resultant gas-smoke mixture is drawn through Pyrex glass piping to a small glass constant-How exposure chamber. The effluent from t he exposure chamber is filtered and absorbed in the usual fashion. This chamlier is somewhat small for testing the effects of high explosives on chemical warfare agents. Twenty-five grams of explosive is the maximum that can lie detonated. It would lie desirable to have means of heating and cooling the chamber walls. Other than this, the chamlier has proved quite satis- factory. It is the only known explosion chamber per- mitting recovery and analysis of the entire residue. SECRET METHODS OF DISPERSING AGENTS INTO CHAMBERS 285 A Entry port equipped with ancinosiat. H Expansion cone. C Reducing cone. I) Exposure chamber (working section). K Removable, Inch vi‘locityf reducing cone. F Velocity control damper. Cl Kjfil to stack. Figure 1. Wind tunnel, elevation. smaller working section is available. Fairing of the incoming air stream is accomplished with a commer- cial “auemostat” with the three central vanes re- moved. The working section is provided with a door, windows, and sampling ports for introduction of animals, arms, and instruments. Air speeds are meas- ured with a commercial Velometer. The wind tunnel has been employed in studies on vesication by particulates and in the development of methods of assessment of particulates (see Chap- ter In). 16.3 METHODS OF DISPERSING AGENTS INTO CHAM HERS 16.3.1 General Liquids may be dispersed as vapors or as aerosols. Solids may have been previously comminuted or it may he required to subdivide them in the process of dispersal. The choice of method to be employed should Ixi based on the following criteria. 1. The dispersing technique must not produce am chemical change in the material. 2. The delivery rate must be constant during the experiment. 3. The rate of delivery should be readily measur- able in onler to provide a measure of the nominal concentration. 4. The rate of delivery should be readily adjust- able to provide an adequately wide range of con- cent ration. 5. The material must he dispersed in particles of desired size. 16.;l.2 Techniques of Dispersal The UCTL has had to test (fie toxicity of materi- als ranging in volatility from gases to metals. The physical state of a substance determines the method of dispersal to ho used. Compounds Boiling Below 0 C.# Those substances are usually available compressed in small steel or copper cylinders. A pressure-reducing valve is at- tached. A capillary or orifice flowmeter is then cali- brated for (lie rate of flow of the gas by the liquid displacement method. A liquid in which the gas is insoluble is used in the flowmeter as well as in the pneumatic trough. The gas is delivered at the desired rate through the flowmeter directly into the gassing chamber. A nominal concentration, as a cheek on that derived from the rate of flow, is obtained by weighing the cylinder before and after each run. Un- stable gases (e.g., ketone21*) have been generated directly into the chamber. Liquids Boiling Between 0 C and Room Temper- ature. These compounds may be dispersed as gases or, w ith proper cooling, as liquids. If they are to be treated as gases they are distilled into a glass am- poule. A calibrated flowmeter and a reducing valve, are attached as described. The nominal concentra- tion is obtained by determining the volume dis- placed during a run or by weighing. It is usually more convenient to treat such com- pounds as liquids. With proper cooling, a solution may be made up. Any dispersing device for liquids which ran Ixi adequately chilled can then be list'd. Such devices are concentric and impinging atom- SECRET APPARATUS AM) TECHNIQUES IN TOXICOLOGICAL ST I DIES izers, bubblers, and the small Benesh and constant- delivery atomizers. Liquids Boiling Above Boom Temperature. These may lx* dispersed by vaporizing or spraying. They are vaporized by passing nitrogen through a bubbler. The volatility of the material determines the size of bubbler and the degree of heating or cooling required. In general it is desirable to use as little heating as possible. Heat is supplied by a water bath at a tem- perature somewhat higher than that desired for the liquid in the bubbler. Liquids may lx* atomized either undiluted or in solution. Solutions should not lx* dispersed from im-_ pinging atomizers since the solute and solvent are usually refluxed to different degrees with correspond- ing changes in concentration of the solution in the atomizer. Solids Which Can Be Dissolved or Which Melt with- out Decomposition. A solution of a solid can be sprayed in the usual fashion. The volatility of the solvent is important. If too volatile it may evaporate sufficiently rapidly at the atomizer tip to produce clogging. Agents which melt without decomposition can l>e dispersed from a direct or impinging atomizer im- mersed in a water or oil bath. Solids Which Cannot Be Dissolved and Which De- compose When Melted. In most eases these materials must Ik* ground to the desired particle size before dispersal. They can lx* dis|x*rsed from the dry duster (see Section 1(1.3.3 under “The Dispersal of Par- ticulates”). Very fine aerosols of metals have been produced by means of an high-tension arc, using the metal as one of the electrodes. 16..1.3 Apparatus for Dispersal Dispersing Bubblers 6 i3 A method of dispersing liquids with appreciable vapor pressures is to bubble a non reactive gas through them. The output is controlled by varying the flow of the gas and the temperature of the water bath in which the bubbler is immersed. The gas pass- ing through (he liquid is broken up into small bubbles by passage through a sintered glass disk (coarse porosity) or a Folia bulb. The type of bubbler used depends on the volatility of the toxic agent. Agents boiling lx*low 50 C are kept in bubblers with stopcocks at both inlet and outlet to minimize the possibility of leakage when the bubbler and contents are being weighed at room tempera- turc. Compounds with high boiling points arc kept in bubblers with outlets large enough that the rapid How of the gas mixture does not blow out material condensing in the outlet nozzle. When small amounts of agent are to lx* dispersed the bubbler should be kept small and light enough to I>e weighed on an analytical balance. (This also applies to atomizers.) hen substances are vaporized from bubblers it is desirable to keep the bath temperature as low as practical. This minimizes the decomposition of ther- molabile agents. To get high concentrations in such cases increased gas flows ate employed in large bubblers through which as much as 12 1pm of gas can lx.- passed. * The use of bubblers in toxicity determinations is limited by the purity of the substance available and the amount of air (or nitrogen) which can l>c passed through them. If the toxic material is quite pure and stable the amount of substance volatilized per vol- ume of gas passing through is quite constant . A very slight degree of impurity will, if the impurity is volatile, result in a changing-output from the bubbler. As a result it. is always necessary to make a series of preliminary runs to bring the output down to “con- stant volatility.” Bubblers designed to hold 10 to 50 ml of toxic agent usually permit the passage of gas at a maximum flow rate of 2 1pm. The most con- slant operating conditions are obtained when the rale of gas flow through the bubbler is sufficiently slow to permit at least 95 per cent saturation of the gas with the vapor. Atomizers An atomizer functions on the Bernoulli principle. A tube is positioned in the center of a jet of air. This tnl)e is immersed in the liquid to Ik* dispersed. The liquid is aspirated up the tube and sheared off the end. The size of droplets produced depends on the diameter of the tubing at its orifice, the viscosity of the liquids, and the rate of flow of air. The tulx; sup- plying liquid may be concentric with the jet of air or at right angles to it. Concentric Atomizer*." These are commonly made of glass, A capillary tube {A, Figure 2) is drawn down to a tip and bent at right angles and sealed into a bulb B. The tip of the capillary is adjusted so that it is precisely centered in the orifice 0 of the bulb. The annular space, between the orifice O and the tip of the capillary is drawn to such dimensions that the desired delivery is obtained at air pressures of 5 to 20 psi. SECRET METHODS OF DISPERSING AG ENTS INTO CHAMBERS 287 the whole chamber is raised by a rack and pinion. When the chamber is lowered, a gaslight seal is main- tained by rubber gaskets. A constant air flow of ISO 1pm is maintained by a combination pump and meter driven by a synchronous motor. This is also geared to the mechanism for delivery of the toxic liquid, so that even if the motor should vary the same proportion of toxic agent to air would be maintained. The agent is displaced from a buret by a rising column of mercury. It flows through stainless-steel tubing to a small stainless-steel concentric atomizer. The mercury column is connected through a U tnl>e, omitted in Figure 3, to a brass cylinder filled with oil. A stainless-steel piston, 0.250 inch in diameter, is driven into the cylinder at a known constant rate. This drives oil into one leg of the U tube, and mercury out of the other. The piston is driven by a lead screw, connected through a change gear box to the synchronous motor. Three hundred and eighty-five gear changes are provided.-A high-s|>eed motor is belted to the lead screw for rapid return of the piston. The main air stream is divided so that 158 !pm goes directly into the chamber while 22 1pm enters a compressor which feeds the atomizer. The spray from the atomizer enters a spiral ev aporator which is provided with a flow of hot water of controlled tem- perature; the air stream of the atomizer may also be heated. Less volatile materials arc condensed on and evaporated from the spiral coils. An inverted mercury-water U tube provides an estimate of the nominal concentration and a check on the accuracy of displacement. In use, the mercury level is set at the zero mark in the fust leg of the U- tube. During the run, mercury is driven into this bulb, displacing water, which displaces mercury from the next bulb into the third bulb containing the solu- tion to be displaced. At the end of the run the mer- cury in the first bulb is drained off back to the zero mark, and weighed. When the machine is free from leaks, letter than 99 per cent recovery is obtained. The Benesh machine is used as follows. Either the density of the agent is determined, or a solution of known density and concentration is made up. From this is calculated the revolutions per minute needed to give the desired .concent ration. The change gears are then set to give the correct rpm. It. is usually possible to select a gear setting such that the rate of delivery is within 2 per cent of the amount desired. Revolution counters on the carriage are set to give the number of revolutions needed for a run of the A Capillary. C Air inlrt. II Hulb. H Rinjt mil. O Orifice. FmviiE 2. Concentric atomizer. Maximum efficiency is obtained when the tip of the capillary either extends slightly beyond or is w ithdrawn slightly into the outer orifice. The adjust- ment is most readily made by heating the atomizer in the region of II (Figure 2). The delivery rate and particle size are determined by the dimensions of the t ip and the orifice. A constant head device can be applied to the flask so that the delivery does not vary with the level of the fluid. Concentric Atomizers — Constant Delivery Type. Concentric atomizers supplied with liquid solely by the Bernoulli effect are subject to variations in their delivery rate. The delivery rate decreases as the liquid level falls. Furthermore, the delivery rate is influenced by fluctuations in pressure of the gas driving the atomizer. At the UCTI. certain atomizing units have been constructed which are provided with liquid by a constant-flow motor-driven pump. Two of these- are called Benesh machines, after M. E. Benesh, Chief Engineer in charge of Research and Testing of the People’s Gas, Light and Coke Com- pany, who designed them. 1. The small Benesh machine (Figure 3).l-* This machine consists of a chamber and an atomizer built into one compact unit. The all glass chamber has a volume of IS I. It can hold 10 mice, 7 rats, or 7 guinea pigs. It is built with double walls between which a suction of IJ4 inches of water is maintained. This prevents leakage of toxic material. To insert animals, SECRET 288 \PPAKATUS AND TECHNIQUES IN TOXICOLOGICAL STUDIES Figure 3. Small Benesh machine, diagrammatic. desired time. The animals are placed on the chamber floor and the chamber lowered. The toxic agent is placed in the buret. The machine is placed in gear and started. The exposure does not start until the first revolu- tion counter is tripped. Prior to this, the agent is dis- persed into the system, but exhausted before reach- ing the ehamljer. When the first revolut ion counter is tripped solenoids are actuated which turn off the ex- haust valve and open a stopcock to admit mercury into the measuring buret. The exposure continues until the second revolution counter is reached and thrown. This automat ically disengages t he motor and turns on the exhaust valve. The animals are then re- SFX’RRT METHODS OF DISPERSING AGENTS INTO CHAMBERS 289 moved. If a different concentration of the same, agent is to be tested, it is only necessary to change the gear and revolution counter settings. It is possible to make as many as five 10-minute inns on the same solution within an hour. The maximum concentration that can theoreti- cally be attained with any substance is one-eighth of its equilibrium volatility at the temperature of the heating coils. Since the air flow through the coils is too rapid for saturation to take place, the actual con- centration obtainable is somewhat loss. The machine should not lx- used with substances which attack mercury and stainless steel. In practice, materials which react slowly with mercury can be used. 2. The large Benesh atomizer. The atomizer on the small Benesh machine was permanently connected to one small chamber. It could not be used with aero- sols, which came to form an increasingly important part of the work. The large Benesh machine was built to retain the advantages of the smaller machine in a somewhat more flexible form. The essential de- sign was retained, but the following changes were made. a. The volume of the buret containing the toxic liquid was increased from 25 to 130 ml. This permitted longer runs or higher'con- centrations to lx- used. 1). The oil-mercury system was somewhat cumbersome and prone to leakage. It was made necessary by the use of a brass cylin- der. Changing the size of piston used was a major operation. In the large machine direct displacement of mercury was made possible by the use of an all-steel system. Three concentric pistons, 1 inch, inch, and ’4 inch in diameter are used. The two larger pistons can be quickly locked down and used as cylinders for the next smaller size. The pistons and lead screw are mounted vertically on a section of channel which can lx- inverted for removing air bubbles from (he cylinder. The largest piston displaces mercury at the rate of 0.0435 ml per revolution of the lead screw; the other pistons displace in proportion to their areas. The stroke is about 10 inches, the maximum displacement about 130 ml. c. In place of the change-gear box on the small machine, three pairs of standard loose change gears connected l»v idlers are used. These are changed by hand, with a wrench. Twenty sizes of change gears an- available. By using the several gears and pistons available several thousand rates of displace- ment are theoretically possible, ranging from 0.008 to 7.000 nil min. In practice both extremes are avoided, because of the inaccniacy of the first and the high pres- sures produced by the second. The ease of changing geais in the small Benesh machine made it convenient to make up one solution and change concentrations by changing the rate of displacement. With the large Benesh atomizer it was frequently more convenient to leave the gears set at a certain ratio and make up different solu- tions for the desired concentrations, d. The atomizer, driver by a refrigerator com- pressor operating at 15 to 70 psi, sprays directly into the chamber rather than into a condenser. This makes it possibleTo use aerosols. Solut ions of agents of low vola) ility, such as glycerine, may be used to produce aerosols of a size determined by the concen- tration of the solution. Thus 0.1 per cent solution gives clouds of mass median di- ameter jTM MD] 0.3 n and 5 per cent gives an MMD of 4.0 n from droplets initially 7 p. o. This atomizer has none of (he automatic controls used on the small Benesh machine. Return of the pistons is made by a hand crank geared to the lead screw. This atomizer cannot be used with ma- terials with low boiling points, inasmuch as no provisions were made for cooling the storage buret. It should not lx- used with materials which react with mercury or stainless steel. 3. The small, constant-floic atomizer.2,: Many of the features of the Benesh atomizers can be retained in a very simple apparatus. Standard, all-glass syringes are used as pistons and cylinders. These an- connected to a small glass eon- centric- atomizer. Connection to the atomizer may be made by an all-glass system, but a short piece of rub- ber tubing is preferred to prevent breakage. Syringes ranging from 1 to 100 ml may Ik? used. They are held in two metal brackets by rubber collars. The lead screw is geared by bevel gears to a 1/150 hp Bodine synchronous motor geared down to SECRET APPAKATUS AND TECHNIQUES IN TOXICOLOGICAL STUDIES 3 rpm. No provisions for changing the gear ratio are made, and changes in concentration must lx* made by changing the syringe size and the concentration of solution. The machine is made to give IS minutes of running, which allows for equilibration time and a 10-minutc exposure. The machine will deliver from 0.011 to 1.71 ml min with the various syringes. The piston is returned by reversing the mot or and running the machine I tack wants. The syringes can lie .cooled by laying rubber bags filled with ice water across them. This cooling is adequate for a 75 (ter cent solu- tion of hydrogen cyanide in ethanol. This machine is quite satisfactory. Nominal concentrations can i>e estimated from the change in reading on thcAvringe. It can Ihj set up on almost any chamber and used with almost any agent. Slightly mom complex design would provide changeable gear ratios and a quick return device. 4. Modified Sinks atomizer.2" For work with the wind tunnel an atomizer was needed that would give an estimate of the amount of agent delivered at any time during a run. A commercial spray nozzle was modified for this purpose. The nozzle was a Binks f 174 humidifier nozzle obtained from the Binks Mfg. Company of Chicago. The body of this is a bronze casting which contains a needle valve concentric to a conical air passage. An indicator arm fitted with a hairline was attached to the handle of the needle valve. A 360-degree protractor dial was fitted to the body of the nozzle. With these, precise and repro- ducible settings of the needle valve could be made. The fluid feed inlet was fitted with a 25-ml burnt ce- mented into a brass sleeve threaded to fit the laxly of the valve. Compressed air at. 10-25 psi was supplied through a corrugated host* fitting threaded into the lower inlet. At 15 psi and with a fluid feed of 2.0 ml/ min. a cloud of MMD 8.0 ju was obtained. Finer sprays were obtained with higher air pressures and slower feeds. The principal advantage of this atomizer is the direct reading of the amount of solution delivered during a run. The output tends to vary somewhat with the hydrostatic head in. the system. The atomizer cannot lie used with substances which attack brass. Impinging Atomizers.* In these atomizers the jet of spray from the atomizer strikes a baffle plate. Larger particles stick to the wall and run back to the liquid reservoir, whereas smaller ones remain air- borne and are swept out of the chamber, either by the air blast from the atomizer itself or by an auxiliary air supply. Theoretically those devices could use either a con- cent ric or a right-angle atomizing unit. To save space the right-angle unit is commonly used. This unit must bo adjusted before sealing into its container. The size of particles obtained (and inversely the out- put of the atomizer) is largely determined by the distance of the, jet from the baffle plate. The wall of the container may l>e used as a baffle, or a small plate may be fitted in front of the orifice. 1. impinging atomizers.-1' These im- pinging atomizers work at a very low efficiency. Per- haps 5 per cent of the output of the atomizing unit passes out of the nozzle as aerosol. This results in a quite low delivery. It was necessary to develop an atomizer which would set up larger amounts of ma- terial as an aerosol for use in the wind tunnel. Im- pinging atomizers of partially metal construction were developed. The atomizing unit consists essen- tially of a hollow brass tube, about an inch in diam- eter, with the lower end plugged and the up|>er end connected to the air line. Near the lower end #55 holes are spaced equally around the circumference. A small brass tube, with the upper end machined, is soldered to the body of the cylinder at right angles to the axis of each hole. The lower end of t his tube dips into (he liquid to l>e dispersed; the upper end is centered in the jet of air from the hole. The most successful of the atomizers has eight of these jets. A shield in the form of a truncated cone is soldered base down around the units to form a baffle. This does not greatly affect particle size but it facilitates return of fluid to the bottom of the bowl. The vessel in which these are placed consists of a 1-1 Florence flask to the neck of which is sealed a side arm of the same diameter. The shape and diameter of (he side arm determines the particle size. 'Phis side aim is fitted with a trap which returns liquid to the reservoir, A bulge on the bottom of the flask provides for efficient scavenging of small amounts of liquid. The diameter of all tubing through which the aerosol passes is kept as large as possible. The eight-jet atomizer, operated with 5 cfm of air at 20 lb pressure delivers from 1.5 to 2.3 g 'min with agents of low volatility. These atomizers can produce clouds of MM I) from 2.0 to 3.5 m- These atomizers overcome the main drawback of impinging atomizers, i.e., low delivery. Impinging atomizers cannot be used with binary systems, as they fractionate them, the more volatile component distilling over. Impinging atomizers tend to give a flat and fairly linear curve for output versus SF.CRKT METHODS OF DISPERSING AGENTS INTO CHAMBERS 291 pressure, which makes fine adjustments in output practical. The Dispersal of Par tic elates Certain of the atomizers used above may be used for the dispersal of particulates, as solutions or molten solids, as well as for vapors. There are in addi- tion several methods peculiarly adapted to the dis- jiersal of particulates. The Dry Dusting Atomizer.'* '11' *1'' It became neces- sary. to test the toxicity of dry dusts in comparison with that of atomized droplets of solutions. The “dry-duster” was developed for this purpose. It is essentially an atomizer for dispersing dry powders. The body of this atomizer is a straight tube, 25 mm in diameter. It is separable in the middle by a ground- glass joint, for ease in filling, A glass nipple is sealed to the lower end. A sintered glass disk (40 60 mesh) is sealed across the bottom of (he lower member, just al>ovo the constriction. The powder is placed on this sintered disk. A side arm, constricted distally, is sealed to the upper member. A tube side of smaller diameter is ring-sealed through the, opposite wall to extend concentrically into the side arm. In operation this device is charged with powder, assembled, and placed in a flexibly mounted clamp. A clamp attache's the assembly to an eccentric mounted on the shaft of a small electric motor. The vigorous agitation so provided tends to prevent channeling, and to ensure a uniform rate of disper- sion. The two concentric tubes sealed to the upper portion constitute an atomizer. The Venturi vacuum produced by passage of compressed air through the inner member draws a current of room air through the sintered disk. This current draws the particles up to (he atomizer and into the chamber. Much closer regulation of the output is possible if instead of re- lying on the v acuum, a slow current (1 1pm) of dry nitrogen is passed through the lower inlet. A good estimate of the nominal concentration is provided by the weight loss of this duster. The ap- paratus disperses particles at. approximately their original size. The shearing action of the air blast shatters aggregates to a certain extent. Fractionating Devices. As it is difficult to obtain clouds of a desired particle size, a fractionating de- vice is sometimes introduced between the atomizer and the exposure chamber. Two forms of fraction- ators have been used. 1. Fractionating lower. This device makes use of the fact that the mass and volume of a particle de- termine its rale of settling. This relation is formu- lated in Stokes’ law. Hy passing a current of air up a vertically mounted tube those particles which fall at a velocity greater than that of the air current will settle out; smaller particles with slower rates of fall will lie swept up the tube. Such a tower may be used to reject either small or large particles, depending on whether the outlet for desired particles is placed at the top or the bottom of the tube. A tower of this sort was used for work with one particulateS1h to exclude all particles al>ove 5 y in diameter. One has been used for work with another agent dispersed from an impinging atomizer in the molten state.2"1 In this tower particles below 75 y were sucked upward and rejected, while the larger particles wore allowed to fall downward into a small wind tunnel. 2. Rotating macro-impingcr.2" One of the princi- ples widely used in analytical instruments for aero- sols has been that of impingement. A jet of aerosol- laden air is driven at high velocity against a surface. The larger the particle, the better its chance of sticking. An attempt was made to use this method on a larger scale to reduce the mass median particle di- ameter of a dry aerosol (see Chapter 12). This was done by impacting the dispersed agent at a high velocity against a moving kymograph drum which had been coated with vaseline. A moving surface was used for impaction to prevent overloading. The smaller particles which did not stick to the drum were passed into an exposure chamber. This equipment was able to reduce the MMD only from 0.5 to 3.8 y. It was somewhat bulky and its use was abandoned. However, this method of reducing the MMD has certain inherent possibilities. 3. Serial mac ro-impi tigers.-'1 Large impingers (see Figure 4) operated in series have been successfully used to reduce the MMD of a NallCO* cloud below 1.0 y. Filtration flasks lined with vaseline are used, with a central tube extending down from the top. The size fraction taken out is regulated by adjusting the position of this tube and varying the air flow. This method is less cumbersome and more effective than the rotating impinger. Electrical Atomization.-1* This method of dispersal is peculiarly well adapted to the dispersal as aerosols of metals and other conductive, heat-stable materials which are not obtainable in a finely powdered form. The material to be dispersed is used as an electrode for a high-voltage arc. If the material is to Im» dis- SECRET 292 APPARATUS VXD TECHNIQUES IX TOXICOLOGICAL STUDIES A Critical pressure orifice from rompressed air line at l.'i fisj (de- livers 38 1pm). B Manometer (maintained at 3 psi). 1 Flask boldine powder to be dispersed. 2 Air jet (directed to side to facilitate mixing). 3 Settling column (130 rm high, 5 cm in diameter). 4 Vaseline-coated impinger. 5 Mncro-imping«>r (FliliT filter fla>k runt ui nine Vasrlino and oil mixture). tl Maera-impinger (500-ral flo.sk as above). _ 7 Mixing flask (2-15torK 8 Manifold. *.♦ Stationary fan blades of o|)|>osing rotation. 10 Cotton plug (permits cmcapc of exeess air). II;-12 Exits to mask and to sampler. Figure 4. Dry cloud apparatus. persed as an oxide, the arcing is carried on in an at- mosphere of oxygen; otherwise helium or hydrogen may be used. The aerosol is mixed with dry air and led into a small chamber; regulation of the concen- tration is achieved by varying the amount of diluent air. Aerosols obtained by this method are extremely fine, somewhat less than 0.3 m in diameter. The out- put of the are is quite constant, and regulation of the concentration is made by regulating the air flow. 16.t SAMPLING EQUIPMENT It is usually necessary to know not only the con- centration of agent put up in a chamber, referred to as the “nominal” concentration, but also to know the concentration actually existing in (he chamber. This concentration is determined by chemical or physical methods anil referred to as the “analytical” concentration. With vapors, it is only necessary to know how much of the material is dispersed in a cer- tain volume of air. With particulates, in addition to this information, it is necessary to know something about the size of the individual particles. 16.1.1 Equipment for the Sampling of Vapors Most of the apparatus for determining the concen- tration of vapors in gassing chambers is of common use iii the study of air pollution.34 UCTL practices are as follows. 1. Withdrawing of the gas sample is effected by either a water aspirator or an electrically driven pump. 2. Measurement of the-vohttne- withdrawn is usu- ally made by a wet test meter. This meter is cali- brated by the positive, displacement of a known vol- ume of air through it. Familiarity with methods used in field trials led to the use of critical pressure orifices for regulating sampling rates, 3. The type of absorbing bubbler most commonly used in this laboratory is made of glass. A coarse sintered disk is used to break up the gas into bubbles. An investigation was made of.the efficiency of three types of bubblers,2,i the sintered disk type,6 the Bushnell type, which has a plain inlet tube ex- tending about 5 cm into the absorbing liquid, and the Kdgewood type, which is filled with glass beads. The absorption of H was studied, and a Northrup titrimeter used to measure the slippage. The sintered disk type was found to be (he most efficient. The fol- lowing conclusions governing the ust* of bubblers were drawn from this study and coincide with others independently obtained.'5 1. The absorbing solvent should have a low vapor pressure. 2. If possible the solvent should react with the absorbate to give a nonvolatile compound. SECRET suipum; equipment 293 3. The absorbent should dissolve water vapor if the air has an appreciable humidity. 1. A solvent which foams considerably is to lx* preferred to a nonfoaming solvent, other factors lx*- ing equal. 5. Low tem|x*ratures are conducive to lx*tter ab- sorption. 6. The flow rate of the gas should lx* kept as low as possible. 7. The kind of bubbler is of less importance than has usually been assumed. Two devices for the analysis of vapors have been developed at the UCTL. They are described below. f.ow-1{(.Asia nee Absorber.27* Investigations of the hydrogen cyanide content of expired air (“precision” gassing) required a bubbler that would combine low resistance, small volume of absorbent, and small deail space with high efficiency at intermittently high flow rates. It was necessary to design a new type of bubbler to meet these specifications. This absorber consists essentially of a pair of con- centric glass tubes. The outer is 37 cm long and 2.2 cm inside diameter. Inside, it is a tulx* with both ends closed, 33 cm long and of such a size as t o leave a 1-mm annular space between it and the outer tulx* as an air passage. The outer tulx- is fitted at its ends with male ball joints so (hat it can l>e freely and con- tinuously rotated about its long axis (Figure 5). It is cent, when tin.' (low into is 30 Ipiii. Only 30 nil of wash liquid arc needed, as compared to 500 ml needed for a bead bubbler of somewhat lower efficiency. The absorber and its motive power form a fairly compact unit. An Electronic Interval Timer for the Northrup Ti~ trimeter.'2 The Northrup titrimeter is an electro- chemical analytical instrument* for the quantitative determination of the airborne concentrations of chemical warfare agents (sec Chapter 30). A sample of contaminated air is drawn in at a constant rate. At intervals it is titrated with a dilute bromine solu- tion. The titration is carried on in one half of an Ag AgNOj Br2 Br cell, with a platinum indiffer- ent elect rislc. When oxidation is complete, an excess of bromine creates an electrical potential, which is recorded on a galvanometer. The amount of bromine solution needl'd is determined by the time required for it to flow from a constant-head burnt. This instrument is made in two forms. In the sim- pler field model the bromine solution is run in by the operator, who shuts off the flow when the galva- nometer shows a positive reading. In the automatic model the galvanometer mirror reflects a beam of light on a photocell when the titration is complete. The photocell actuates relays which shut off the buret and start another sampling period. The length of sampling period is controlled by fixed cams, which give a choice of four periodicities; from I minute sampling and 2 titrating to 50 minutes sampling and 10 titrating. Bet ween the end of one titration and the start of the next sampling period (he cell is kept in equilibrium — the agent sampled during this period is balanced by intermittent addition of bromine. The owning and closing of the bromine buret is recorded by a relay-actuated marking pen writing on a paper record wound around a kymograph drum. In this form the automatic model was incapable of accomplishing some of the determinations that were desired at L’C'TL. In particular, the shortest time interval available (cycle repeated every 3 minutes) was too long for showing variations in concentration occurring at a frequency greater than that, whereas the provision for 1-hour sampling periods was un- necessary. The cyclic rate could have been increased by cutting another cam. However, it was desirable to eliminate the time lost between the end of.one titra- tion period and the start of the next sampling period which results from the use of the cam timing mech- anism. An electronic method was adopted. The sampling Fin cue 5. Low-rcsistancc absorber. rotated by two micarta pulleys, 2 inches in diameter, boitil out to fit the outer tube, and cemented to it. These pulleys rest on t he rollers of a small ball mill (Fisher Minimill). The whole assembly is mounted on two rods attached to the sides of the ball mill. Two brackets hold the corresponding female ball joints flexibly. The absorber is held down against the rollers by helical springs attached to slip rings. In use S ml of absorbent are poured into the ab- sorber. This is more than enough to wet all exposed surfaces when the absorber is rotated. Thereby the absorbing surface is continually being renewed. Tests have shown resistance to lx* very low, about 1 cm of water at an air flow of 30 1pm. When 3 per cent NaOH in ethanol is used as an absorbent, the absorp- tion of hydrogen cyanide is 100 per cent from air con- taining 2. t mg 1 and flowing at 7 1pm. It is 05 per SECRET \PPAR ATI'S AND TECIIMQL'ES IN TOXICOLOGICAL STID1ES time interval is governed by the time required to dis- charge a condenser of high capacity through a high resistance. The resistance was controlled by a po- tentiometer; changing this set ting varied the time of discharge, ami hence the sampling rate. The sizes of the elements used were such as to give continuous variation between 0.25 and 5.8 minutes; a longer sampling jx'riod proved unnecessary but use of larger condensers would provide it. At the end of the sam- pling period titration starts and continues until all the agent collected during the pretit ration period plus, that collected during the titration period is titrated. Thereupon the titration stops and essen- tially instantaneously a new cycle begins. This addition to the laboratory model Northrop titrimeter has the following advantages. 1. Continuous variation in sampling times is avail- able merely by turning a knob. 2. As soon as one titration-period has been com- pleted, a new sampling period begins. 8. By using short time intervals the concentration of agent in the absorption cell is kept very low at all times, thus reducing the loss of material by slippage. 4. The original instrument is now adapted for use in determining concentration changes in gassing chambers over short periods of time. Ih. 1.2 Equipment for the Sampling of [’articulatesfi Filters. One of the simplest ways to determine the concentration of a smoke is to draw a measured sam- ple of air t hrough an efficient filter and determine its gain in weight. At UCTL much early work with smokes was done with cotton-asbestos mats (40 per cent cotton — 00 per cent asbestos) I to 2 mm thick, pressed into perforated or sintered glass filtering fun- nels (25 mm in diameter). Suction of from 0.0 to 2.0 inches of mercury was needed to pull 3 to I 1pm through these. Work on certain types of aerosols (see Chapter 12) introduced several new requirements for a filter.11'1 Since the determinations involved a micro procedure, it was necessary that the filter material have a low blank (less than 20 /ig). The filter chosen should not lie clogged by as much as 10 mg of a standard prepa- ration and should offer low resistance to air flow. Several filter papers were tried Ix-fore one made from cellulose acetate was found to l>e satisfactory. This paper contained no material simulating the material determined in the analytical procedures. Insoluble material could lie completely floated off, ami (he bait could lx1 completely dissolved in a suitable organic solvent, leaving the particles unaffected and ready for counting. In use. both in the laboratory and in the field disks of the paper are stamped out. They are held in brass holders in which they are backed with a wire screen. Precipitators. Particles have been removed from the air by direct precipitation. A Watson 40 thermal precipitator has been constructed and used. In this instrument the air passes over a Niehrome win- kept at 100 C. Smokes are precipitated on cover slips backed by brass blocks. Electrostatic precipitation has also been used in a small Cottrel-type precipitator.33 This is essentially a long glass cylinder. Copper screening is wrap|xnl around the outside, and attached to one terminal of a I 5,000-volt transformer. A wire in the tube at its long axis forms the other pole. Impinging Devices, An impinging device is one in which the smoke-laden air is drawn or driven at high velocity against a prepared surface. The particles may be trapped on the baffle plate or absorbed by some liquid medium. Impinging devices may housed either to collect all of the particles in one stage or to fractionate the particles by using jots of various speeds. 1. The atomizing imping/r.21' This unit (see Fig- ure 6) consists of a concentric atomizer mounted in- A Inlet f uix- B Battle (supported by ted from .W not shown). C Capillary. r> Distanrr- between cajiillary and battle. K K\it to vacuum. F Flui.l to be atomized. Fiiiimt; R. Atomizing irnpinger. side of a glass vessel in such fashion that its jot strikes a baffle plate and drains down to a sump from which it is re-atomized. Dust particles in the incom- ing air ring strike the baffle plate and are trapped. A straight tube is ring-sealed concentrically through tho top of a side arm test tube. The inner tube is constricted at its inner end to give a jet. A secret SAMPLING EQUIPMENT 295 smaller tube is ring-sealed through the wall of this inner tube in such a fashion that its outer end ex- tends at right angles, and its inner end is concentric to and extends through the constricted portion, forming a concentric atomizer. Tins outer end dips down into a bulge* on the outer tube which acts as a sump. The apparatus is mounted horizontally. A glass arm supports a baffle plate which is carefully positioned in front of (he jet. If this plate is too close to the orifice the atomizer will not function; if too far away the unit will act as an atomizer and not as an impinger. A source of vacuum is attached to the side aruuand the inlet tube connected to the charal>er. This pattern has proved 1)0 per cent efficient in the collection of clouds which had 1 »eon allowed to settle for 30 40 minutes and contained particles with an MMD id 6 ju. A practical advantage of the apparatus is that the collecting volume is small and, conse- quently, small amounts of toxic agent are not diluted too much for injection into animals. The Cascade Impactor. The construction, method of use*, experimental results, and theoretical principles of the cascade impactor are fully described by K. it. May.’*' Experiments at the UCTL have emphasized the desirability of the instrument with dry particles and have employed slightly different, calculation pro- cedures, such as (hi* substitution of the MAID on each plate for the effective drop size [EDSj used by Port on. With dry clouds a total sample of about 0.350 mg represents the maximum which can be ob- tained without overloading if the cloud is distributed on all four slides. W ith dry powders the presence of aggregates in the airborne cloud complicates the calculation of the MAID. These aggregates fre- quently break up upon impaction so that they can- not be measured microscopically. Since the density of an aggregate is lower than that for unitary parti- cles, the aggregate is impacted along with unitary particles of smaller size. 'Phis property leads to the recommendation (Chapter 15) that particulates should be assessed in terms of impactibility rather than in terms of diameter. 1. .4 device for increasing the load on cascade im- pactor slides.-'11 The amount of a particulate impacted upon slides No. 3 and No. 4 of the cascade impactor must l>e kept very small to prevent overloading. The quantity obtained on one streak is barely within the limits of the available analytical methods. To allow collection of a larger sample, a method of moving tin; slides at intervals during sampling was devised so that it is now possible to obtain eight streaks on slide No. 4 and four streaks on slide No. 3 during one sampling period. A heavier cap is screwed on to the appropriate tubes of the cascade. In the center of (his cap a hole is drilled and tapped for a '.pinch bolt. The Iwdt is passed through this cap. The inner end bears on the slide. This bolt is turned by band at in- tervals during the run to move the slide. The distance it is moved .is determined by the numlxuof rotations of the screw. 2. .1 modified cascade impactor for use with small particulates.-1'' The cascade impact or was originally designed to handle the range of drop sizes set up by munitions in (he field. In work with nasal filtration of small droplets it was necessary to work in a range of much smaller sizes. The larger drops in this range- were of about the smallest size that the standard cascade impact or would handle at 17.5 1pm. The standard cascade with critical orifice impinger and filter )tacking would trap the particles in this range but would not fractionate them. A modified cascade was constructed which frac- tionated the drops which slipped past slide No. I and were caught by the impinger. The standard cascade lavs a rate of flow through its jets of 5, 30, 50, and SO mph, with the impinger giving 700 mph. The mod- ified cascade has jet velocities of 56, 80, 177, and 700 mph, the last slit being a critical orifice. (A criti- cal orifice gives a speed of How equal to the speed of sound — approximately 700 mph.) ft will lie noted that the first two jets of this cascade correspond, roughly, to the last two jets of the standard cascade, while the last two correspond to the impinger, as used with the standard instrument, and an intermediate value. This modified impactor has proved capable of efficiently fractionating a cloud which slips past the standard instrument. This modified instrument re- quires a backing filter to collect all material. The present experimental model is blown of glass. The four separate sections fit together with rubber stoppers. The slides arc held in place between in- dentations in the walls. Wall losses can be easily detected by inspection. Particle Fractionators. Drop traps, chemical noses. In an attempt to simulate the characteristics of nasal passages with respect to particulates, various devices have been made to fractionate the cloud into a lung fraction and a nose fraction. Glass tubing in the form of Z’s and S’s such as were used by British workers for liquid droplets were coated with a sticky film of alkyd resin in an effort to fractionate dry particulates.-^30 Better results SECRET VI’IVVR \Tl S VM) TECIIMQI KS tX TO \ I COM )<; ICA L STUDIES were obtained with selectors which were essentially the first stage of a cascade impactor backed by a filter.2" Wires fur Sampling Particulates. The use of slides, tubes, and wires of different dimensions for the de- termination of particle size and cloud concentration has been described in detail.27“ b e 16.5 MKT]lODS FOR "PRECISION GASSING’’ In the usual gassing procedure no account is taken of the effect of the toxic agent on the respiratory volume or rate. Consequently, there is no means of determining the inhaled LIh» from the LC:,„. Some species, especially rabbits, hold their breath to agents which are apparently undetected by other species. Methods which take account of the actual respira- tion during exposure have been termed “precision gassing” methods. A tracheal cannula and Douglas bag were employed in studying the effects of phos- gene on the respiratory pattern of dogs.211' In some instances the respiration was modified by the use of CO;.-,f For the early investigations on the effects of hy- drogen cyanide, the animal was mounted in a body plethysmograph attached to a Rrodie bellows. More precise methods were later developed 27b r-r in which a mask was fitted to the animal. A valve with minimal dead space was used and a special low- resistance absorber const ructed. 16.5.1 Equipment The Mask. The first mask tried was made of vinylite sheeting, shaped in a truncated cone. The ai>ex of this was cemented to (he male half of a 16 22 standard taper joint. This was used with dogs. The animal’s snout was taped shut, and the cone bound over it so that the apex of the cone was in contact with the nostrils. When this facepiece was applied in the usual man- ner, an average leakage of 17.5 j>er cent was found. When it was applied very tightly, the leak was re- duced to an average of 1.4 per cent. In order to achieve this low leakage the facepiece had to be ap- plied with sufficient pressure to embarrass respira- tion seriously. This facepiece subsequently was replaced by a pair of nasal tubes leveled at one end to facilitate in- sertion. These tubes (1 '4 inch long, and 4 mm inside diameter) were attached by paragum rubber tubing to the diverging arms of a glass V tube scaled onto tiio end of a 15 20 male standard taper glass joint. The tube ends were closely approximated to the Y arms so as to expose as little rubber surface as possible to the agent. After gassing no adsorbed agent could be detected on the inside of the nosepiece. It was possible to handle animals so intubated with local anesthesia alone (cocaine or butyn) but this was not entirely satisfactory. Therefore intubation was carried out on lightly anesthetized animals (for dogs, 20 mg kg of Nembutal intravenously) after a swab of 1 per cent cocaine or butyn had been applied to the nostrils to prevent sneezing. The month was closed with elastic bandage and the tubes were then inserted and fixed with additional taj>e. No leakage could be detected in 11 of 12 animals tested, and the other had only 1.9 per cent leakage. The Valve System. The first valve used was a copy of the all glass valve designed by Weston and Tobias.'5 The laxly of this valve was made from two female and one male 16 22 joints sealed together in a 1'. The long arm was made from a male and female joint, and the side arm from a female joint. In use the long arm is mounted vertically with the male joint at top. The valves proper are composed of glass disks ground flat, of such diameter as to fit inside the female joint. The end of the male joint is ground flat and serves as a valve seat. The disks are held down in place by their own weight. The assembly of male joint and disk is inserted in a female joint which keeps the disk in line. Such a valve will pass air in the direction away from the male joint. In use, the dog’s snout is connected to the female joint on the side arm. The lower valve is connected to a gassing chamber, and the upper, through a suit- able absorber, to a spirometer. On inspiration the lower valve opens and permits contaminated air to enter. On expiration, the lower valve closes, and the upper valve opens and passes air to the absorber. It is desirable to minimize the dead space as much as possible. The use of glass imposes limits on the re- duction which can be made. In addition the convul- sions of exposed animals place great strain on the valve. It was possible to machine a valve from brass which would have less dead space. The valve is of the same general design, except that both the laxly of the valve and the flaps arc made from brass. The valve lias only 13 ml of dead space as compared to 25 ml in the glass valve, and is practically unbreak- able. It was found that while untreated brass ab- SECRET METHODS FOll "PRECISION GASSIXo” 297 sorlied appreciable quantities of cyanide, brass “blued” by immersion in a hot solution of As.()3 in HC1 did not react with cyanide. The Absorber. The use of an ahsorlier to collect gases from air, as expired, imposes certain peculiar requirements. It must be efficient at intermittently higli flows (as much as 30 1pm), it mus‘ have low re- sistance (conventional absorliers have a resistance of several inches of mercury under these conditions), and it must lie possible to rinse it out with a small volume of liquid, since very small quantities of agent are present in the expired air. The first absorber used was the Edge wood low- resistance, glass-bead type.25 This absorber is filled with fluid and drained just before use. Thus, the gas passes over the surface film and not through liquid. However about 500 ml of wash liquid was needed to transfer all the ahsorlied agent to the titration vessel. With the very small amounts of material present, this large volume of solution led to appreciable titra- tion error. The UCTL low-resistance absorber-7'1 described (Section 16.1.1) proved more satisfactory. 16.5.2 Determination of Inhalation Toxicity of Particulates As descrilied in Chapter 15, the effect of particle size on physiological action is appreciable. In order to determine, whether an inhaled particle was trapped in the nose or in the lung or whether it was exhaled again, various methods were devised similar in prin- ciple to those employed in “precision gassing” experi- ments with vapors but moilifieri for the assessment of particles. 1. One procedure for use with animals involved exposure to toxic clouds of controlled particle size. It was used with agents which are highly toxic in the lung but of negligible toxicity in the nose. From these experiments the relation of particle size to toxicity in mice, rats, and rabbits was determined (Chapter 15). 2. Correlated with these experiments were meth- ods for the determination of toxicity by intrapul- monary instillation of solutions of the toxic agent.276 In general the trachea of an anesthetized animal is canmilatcd, and the solution instilled into the trachea through a tube which fits inside the cannula. The method has previously been used for rats.35 A small catheter is used. The neck is transilluminated with a Spencer microscope lamp to aid obscuration of the trachea. For mice this method was modified as follows. A cannula whic h fits snugly into (lie trachea of a 20-g mouse is made by rounding off the beveled (ip of a 1 j-2-ineli 18-gauge needle. A lJ4-ineh handle is at- tached to the hub. The solution to be instilled is con- tained in a '4-1111 tuberculin syringe tipped with a 25-gauge needle. The mouse is etherized and tied on its back. The mouth is held open and the tongue held against the mandible with a pair of blunt forceps. The throat is transilluminated and the larynx is visible as a bright spot opening and closing with the respiratory movements. The cannula is inserted into the trachea through the larynx. When the cannula is situated correctly, it is possible to cause a pulsation of the chest, by blow ing gently into the cannula. 'The 25-gauge needle is then inserted into the cannula, and the solution instilled. When in position, the tip of the 25-gauge needle should ice Hush with the (ip of (lie 18-gauge cannula. If the cannula is in the right position, the breathing becomes labored upon in- sertion of (lie 25-gauge needle, and returns to normal when the needle is removed. 16.5..1 Nasal Filtration and Lung Retention 21fc 1 The mensuration of these factors involves setting up a particulate cloud and determining its particle size with cascade impactors before and after passage through portions of the human respiratory system. Obviously these tests could be done only with non- toxic materials. Either non hygroscopic or hydrophi- lic aerosols may be used; dyed corn oil and calcium phosphate were used for the former, and NaHCO* for the latter (see Chapter 15). The basic techniques involved in these determinations are (lie same; differ- ences will be discussed under the subheadings. Selling up the Particulate Cloud. In the first work com oil (Mazda) dyed with Sudan Red was sprayed from the large Benesh atomizer. When NaHCO;) was used, it was dispersed from a 250-rnl Erlenmeyer flask with a two-hole stopper. A glass tube drawn out to a fine tip extended through the stopper to near the bottom of the Ha.sk. When compressed air was forced through this jet, a cloud of NaHCOa dust emerged from a tube in the other hole of the stopper. This cloud was directed into a 12-1 bottle, and the large particles allowed to settle out for 5-10 seconds. The particles remaining airborne were then drawn into the common entrance of the Y tube. It was found early in the experiments that the cloud dis- persed as described was quite heterogeneous. Though SECRET VPl'AK ATU S AND TECHNIQUES IN TOXICOLOGICAL STUDIES passing through a common tube, samples drawn si- multaneously from the two arms of the Y tube showed markedly different distributions on the im- paclor slides. This was remedied by placing two small, oppositely oriented, metal propellers in the common tube. This stirred the passing air sufficiently to make the cloud uniform. The same setup was used for calcium phosphate smokes. Full her fractionation was provided by the use of serial Tnaero-impingers, lined with Vaseline (see Figure 4). 1. Dry rlnini apparatus. The dry cloud of NallCOj was set up by the apparatus shown in Figure 4. Fif- teen pounds pressure in excess of atmospheric applied to a critical pressure orifice for IS 1pm (at atmos- pheric pressure) gives a flow of about 38 Ipm, suffi- cient to supply the lung and the control without di- lution by unfiltered room air. The material enters the settling column. The agitation here is adequate to maintain the cloud at-nearly the same concentration. The pressure in the manometer w as about 3 psi, practically all of it living due to impinger No. 6. The jet in impinger No. 4 was simply a large glass tube. In No. 5, the end of the tube was somewhat flattened-, whereas in No. 6 the orifice in the end of the tube was about 1x5 min. For smaller clouds a still smaller jet may be used, the pressure in the column-being" larger. To maintain the same flow rate only a slight shift in the initial pressure is required. If one starts with considerably higher initial pressures with a proportionately smaller orifice in No, 1, changes in pressure in the column may l>e ignored. Between runs the impingers were warmed to re- surface the bottoms of the flasks and the mixing flask 7 and tube 8 were blown clean. At the end of a run the last flask should not be too heavily coated. Passage of the Cloud through a Portion of the Human Respiratory System .The expo rimen t s conduc ted were; 1. Nasal filtration with corn oil. The oil was sprayed from the Benesh atomizer into the 700-1 chamlier. This was operated at a flow of 300 Ipm and acted as a settling chamber. A glass Y tube. 22 mm in diameter. led from the chamber. One branch of (his Y led to a mask which fit ted tight ly over the nose and mouth of the subject. This mask was from a com- mercial dust respirator; an inflatable rubber tube formed a tight gasket with the subject’s face. Pro- truding into the mask was a second exit tulx* about which the subject closed his mouth. The exit tube led through a cascade impaetor, backed by an impinger, to the pump. The second branch of the Y tube led to another impaetor, also hacked by an impinger and the pump. This impaetor sam- pled the incoming cloud. The tubing between it and the fork of the V tube was comparable in length and shape to the tubing which led to and from the mask. As far as could be controlled, the only difference between the two air streams was that one passed through the subject’s nose and the other did not. The impingers backing the impactors were of such size as to be critical orifices, with a flow of 17 Ipm. Ivtch experiment was done in duplicate with the positions of (he control cascade with its corre- sponding impinger, and the mask exit cascade with its impinger, reversed during the second experiment. Th is canceled out instrumental errors. Sealed to the mask exit tube-and preceding the cascade impaetor was a small glass tulie through which air could be drawn from or added to the sys- tem. This could lx1 used to increase or decrease How rate through the mask while maintaining the same flow through the cascade impaetor. A similar tul>e preceded the control cascade. A second small opening in the tube which connected the mask to the cascade impaetor led to a manometer which indicated the reduction in pressure caused by the resistance of the nose when air was flowing through it. During each experiment, which lasted 30 seconds, the subject held his breath so that there was no ap- preciable passage of the aerosol into and out of his lungs. For the flow rate of 10 1pm, the 30-second rim was repeated immediately in order to obtain a larger sample. The nasal resistance was found to be very low excel)! in individuals whose nasal passages were congested or who were not sufficiently relaxed during the experiment, [f a person is tense, his posterior nares may become constricted, with a marked in- crease in resistance. Subjects were used in the experi- ments only after they had had sufficient practice on t he apparatus to allow a 10 1pm flow with a resistance of 0.3 inch waf er, 17 Ipm with 0.5 inch, and 2!) 1pm with 1.0 inch or less. In the case of some subjects this low resistance could often be achieved only after in- halation of benzedrine, or the nasal instillation of neosynephrine solid ion. 2. Nasal filtration with NnllCO* particles. Com- mercial powdered NaHCOj was chosen as a nonirri- tating, non aggregating powder containing particles of a size range from 1 to 15 a (microns). The same assembly of mask, Y tube, impactors, critical pres- sure impingers, and pump was used as with the corn oil work. The impaetor slides were covered with SKCKET METHODS FOK TESTING VESICANTS 299 alkyd resin, and the NaTICOj on them analyzed elect romet rically. The hygroscopic!tv of XallC’Oa introduced several difficulties In-cause of the moisture picked up by passage through the nose. It was necessary to oven- warm the impactor Indore use, and to warm the cloud from the nose by passing it through an 8-inch length of 15-mm tubing, electrically heated to give an emergent air stream of approximately 90 C. A dupli- cate heating device was used in the cent ml air stream. The wetting and subsequent drying of the cloud passing through the nose compacted and rounded the particles in it. It was necessary to humidify the control cloud also. The humidifier was a metal tube (1 foot long, 1 inch inside diameter) lined with a water-soaked blotter, placed in a thermostated water bath. This humidifier was also an inefficient im- pactor, taking out about 20 per cent of the airlmrnc material in the cloud going through it, A similar tula-, lined with Vaseline, had to be placed in the path of the cloud to be transmitted through the nose. After these modifications quantitative agreement lx-tween the NallCOj contents of the dry and humidified cloud could l>e obtained. With the use of cellulose acetate filters instead of the cascade impactors it was unnecessary to have the various driers. 3. Retention of particles in human Jungs. In these experiments the subject’s nose was plugged. Stop- cocks were placed in each of the two sampling tubes between the critical orifice and the filter. During an experiment the subject inhaled for a fixed period, the beginning and end of w hich were in- dicated to him by an operator. During the inhalation period a sample of the incident cloud was drawn through filter A, by opening the corresponding stop- cock (the vacuum pump operated continuously). During exhalation, which was also for a fixed number of seconds, the exhaled cloud was drawn through filter H. The cycle was repeated 10-15 times,depend- ing on the length of inhabit inn and exhalation periods. Since the incident and exhaled clouds were sampled at the same rate and for equal periods, the material found in the exhaled cloud represented the unre- tained fraction of an inhaled quantity equal to that on the other filter. The total volume breathed during an experiment was governed by the rate of withdrawal of the ex- haled cloud from the mouth. The volume withdrawn during each period was considered to be the tidal air. The exhaled tidal air was, of course, constant from period to period since it was controlled by the sam- pling instrument. The inhalotl tidal air, however, varied from period to period depending on whether or not the subject inhaled a volume which exactly compensated for the amount withdrawn during the previous exhalation. Over a number of cycles, of course, the average volume inhaled had to be equal to the volume exhaled. This method has been studied with smokes of NatiCOj and calcium phosphate. Since calcium phosphate is nonhydroscopic it is possible to dis- pense with the humidifying and drying sections of t he apparatus, 16,6 METHODS FOR TEST I ISO VESICANTS The usual method for testing the vesicancy of an agent is to put a known amount of it on the skin of the forearm and to observe the results at a later time. The agent may be put on as either a liquid or a vapor; it may be either still or flowing. 16.6.1 Testing Vesicants as Liquids The Edgcwood Rods* n One of the simplest meth- ods for testing compounds for vesicant action in- volves the use of a series of stainless-steel rods of standard weight with tips varying from 0.0 to 2.OS mm in diameter.” With the exception of the smallest rod, all of them deliver 0.022 to 0.029 mg of H per square millimeter. These rods, usually known as “Edgewood rods,” are touched to the surface of a pad saturated with the vesicant anti then applied to the skin. Liquids were used either undiluted or dis- solved in diphenyl ether. Solids were also dissolved in diphenyl ether. The method is simple and although all the material on the surface of the rod is not delhered to the skin, easily reproducible burns result from the iise of a given rod with a given compound. In general the rods have not proved satisfactory for comparison of vesicants since a separate calibration is required for each compound tested. It is not possible to test oint- ments with these rods, since the droplet cannot satis- factorily 1m‘ delivered to the surface of ointment- covered skin without breaking the covering. Further, the method is not desirable for compounds that react with steel, although it has been "used with lewisite. Nonstandard sets of glass rods have also been made. ”Drod" It was necessary to devise some form of micropqx't which would deliver small, known vol- umes of vesicant. Trevan v> used a standard microm- eter caliper to drive a 1-cc syringe. A modification 2 SECT! ET 300 APPARATUS VXD TECHNIQUES IN TOXICOEOC.fCAL STUDIES called the Drod used a specially constructed microm- eter head to drive the plunger of a 1 j-ee tuberculin syringe. A spring click bears on 12 longitudinal grooves on the barrel of the head, each click corre- sponding to 31) degrees of rotation and the delivery of about 0.2 cu mm of liquid. (The amount would Ik* constant for each syringe, but commercial syringes are not interchangeable, being individually ground to fit. The diameters of the pistons, and hence the volume delivered, vary between syringes.) A 27- gauge needle, w ith the tip ground flat and square, is used to deliver the liquid. The instrument is sturdy and portable. It delivers an accurately measured small dose, which is not so dependent on the physical properties of the agent as is the case with the Edge wood rods. This apparatus requires considerable time to fill, and the change from one vesicant to the other n*qwires decontamina- tion of the syringe and tip, making it unsuitable for use when many different liquids are to be handled in one day. The amount of liquid delivered per click is large, with (he result that dilutions must frequently be used. The Modified Drod/ An attempt was made to modify the original Drod to make it. more suitable. Several modifications were made on the driving head. It was redivided, so that a click occurred for each 7.5 degrees of revolution (48 clicks per revolution). The instrument then delivered 0,065 mg of H per click, instead of the original 0.2 mg. A 6-inch indi- cator disk, with 192 divisions, and a pointer arm at- tached to the head made it possible to split- the clicks in half, and possibly into four. These are equivalent to 0.032 mg and 0.016 mg of mustard. The 1 pee tuberculin syringe was retained. It is filled w ith mercury, which is used to expel the agent from a removable delivery tip. The syringe and screw are attached by a ground joint to a three-way stop- cock. With the sto(>eock in one position, the agent in the tip may lie expelled by turning the micrometer. With the stopcock in the other position, the lip may be filler! or washed by liquid which enters from a side arm. A platinum or graphite surfaced stopcock is iisihI to avoid fouling the agent. It is possible with this modification to remove one vesicant, decontaminate the apparatus, and load an- other vesicant in less than I minute. It was found that dividing the clicks into four did not give repro- ducible lesions, but 0.032 mg of mustard, correspond- ing to half clicks, can he delivered quite accurately. This amount, although small, was not small enough for some purposes. The increments were too coarse to discriminate I>etween vesicants of nearly similar potencies. Other Pipits. Capillary tubes have been used for the application of measured amounts of vesicant.24 The capillaries were however rather fragile and the method is not adapted to testing large numbers of men. A device for blowing drops of measured size off a microburet tip was developed at Port on.3' 32 The Be tush Micropipet.1* In the I) rod type of micrometer syringe the piston was of a diameter equal to the'bore of the cylinder. To achieve a smaller displacement with the same pitch lead screw, it was necessary to reduce both bore and piston diameters. The 1 j-cc syringe already in use was the smallest available size. Micropipets capable of delivering smaller quantities of liquid have previously been de- scril »ed.3r*8 Various features in their design were not, however, suited to vesicant testing. The lienesh micropipet was based on a some- what different displacement principle. The piston was a steel wire 0.0122 inch in diameter. This entered a mercury chamberthnaigh a Neoprene gasket, The volume of mercury displaced was equal to the volume of wire which entered the chamber, but. since the piston worked by displacement it was unnecessary for it to be tightly fitted to a cylinder. This scheme avoided the difficulties of accurately machining such a small size hole. The wire piston is driven by a micrometer head, somew hat larger t han usual, but of standard design. Twenty-five grooves are cut on the thimble actuating a spring click. The lead screw has the standard micrometer pitch of 40 threads to the inch. The. dimensions of the wire and (he pitch of the lead screw are such that each click (1 25th revolu- tion) advances 0.002 cu mm (2.5 gamma) of II. The mercury chamber communicates with a re- movable tip, made out of capillary tubing. The end of the tip is optically polished. The instrument is mounted to move up and down on a rod screwed to a wooden base. The base forms the bottom of the carrying ease, with the rod serving as a tie rod to hold the top and bottom of the ease together. The instillment can bo transported as easily as a com- pound microscope. The apparatus is durable and simple. It lues been found to give reproducible lesions. The principal de- fect is incomplete delivery of all the material ad- vanced to the capillary tip. It is necessary that each subject’s arm come in contact with the tip with the same pressure. The extent of loss duo to the evaj>ora- SECRET METHODS FOR TESTING VESICANTS 301 tion of the compound between the time it gets to the tip and the time that it is applied to the subject is unknown. It is minimized by maintaining a regular, rapid rate of application. The instrument can best be list'd by a trained pair of operators, one operating the micropipet, the other holding the men’s arms against the tip. A regular rhythm soon leads to both speed and accuracy. It was found that the necessity for counting a number of clicks repeatedly let! to personal error. An attachment was made for the pipet that made it possible to advance the desired amount in a single motion, rather than by counting a number of clicks. A brass plate with 25 holes equally spaced around the periphery was attached to the instrument. An index arm was attached by a ratchet to the lead screw. By placing taper_pins in the appropriate holes any number up to 25 clicks can lie delivered without counting. Liquid Vesicant Cup.'11 Occasion arose to compare the action of 11N3 as a liquid with saturated 1IN3 vapor. The vapor concentrations were set up in vapor cups (set' Section 16.6.2). The apparatus employed for the application of liquid consisted of a small cup, 12 mm outside diameter and 8 mm inside diameter, with two capillaries leading from it. One capillary leading directly upwards from the cup, was attached to a safety flask and a charcoal column aspirator; the other tube, coming from near the base of the cup at a 45-degree angle, is connected with a three-way stopcock. A pear-shaped bulb with a small hole in the side is sealed to the vertical arm of this stopcock. The liquid vesicant is placed within the bulb, and the stopcock is turned so that the vertical arm is con- nected with the cup. The cup is placed on the sub- ject’s arm, and the vesicant is drawn by suction out of the bulb, through the capillary, and into the cup until the area on the arm is covered with a continu- ous layer of vesicant. At the end of the exposure period 5 per cent hydrochloric acid is sucked through the instrument, followed by water; by applying the suction intermittently, the surface, of the arm is flushed and decontaminated. In control tests with fat-soluble dyes all visible dye Was removed within 5 seconds. 16.6.2 Testing Vesicants as V apors For proper evaluation of the vesicancy of a com- pound the vapor hazard must also be determined. Edgewood Vapor Cups.'0 One of the simplest ways of producing vapor burns is by the use of small glass cups with a Hat rim. A pad of filter paper or some other absorlient material is placed in the bot tom and moistened with the liquid vesicant. The cups are then taped on to the arm of the subject for the de- sired length of time. The amount of vapor (and its effectiveness) in the cups will vary as a result of the interplay of outside temperature, skin temperature, amount of moisture under the cup, and (he presence or absence of sun- light on it. The actual concentration in the cup is un- determinable and may be changed by cooling or warming the cups. In addition to vapor burns, “rim burns” sometimes occur. These are the result of con- densation of liquid agent on the lip of the cup. The use of these cups permits the application of approxi- mately saturated concent rations of vapor. Modified cups have been devised which permitted the application of subsaturation concentrations, pro- vided for circulation of the vapor, and eliminated rim burns.17 IS — The Vapor Train.11 Some of the objections raised to the use of the Edgewood cups are similar to those raised against the use of “static” chambers. A dy- namic method of exposure was devised to overcome some of these difficulties. This apparatus consists of the following essential parts. (1) A bubbler from which the agent is vaporized. (2) A serum I bubbler in which water is vaporized, (it) A )’ tube that unites the streams of vapor-laden air from the two bubblers. (4) Glass tubing which is branched and rebranched to divide the vapor-air stream into four identical streams. This tubing, 20 mm inside diameter, is in several sections that are joined by 29 12 standard taper joints. (5) Four appli- cator orifices. Each may be described as an open cup with a delivery tube for conducting the vapor-laden air to it and a side arm that serves as an outlet. The cup is formed from a 24/40 male standard taper joint. The delivery tube, 8 mm inside diameter, enters the cup at the bottom through a ring seal and pro- trudes to within 3 mm of the upper, open end. The vapor-air stream, therefore, flows upward through the delivery tube, impinges upon the skin of the arm which a subject holds over the opening of the cup, and out through a side arm. The velocity of this jet is about 5 mph. (6) Four slain less-steel adapters for the applicators, each with an 8-mm hole in the cen- ter. Use of these; adapters reduces the area of skin exposed anti thus minimizes the severity of the re- sultant lesion. Each cap has a small ridge at its outer edge (1 32-inch deep) to prevent an arm from mov- 8ECRET APPARATUS AM) TECHNIQUES IN TOXICOLOGICAL STUDIES ing around during exposure. The caps are held in place by rubber bands. (7) A branched and rebranched glass tube, identical with (4) but used for uniting the effluent streams from the applicators. (8) A tube to conduct the combined effluent into a suitably venti- lated duct. (9) A sampling apparatus to draw a meas- ured volume of the effluent through a suitable ab- sorber for determination of the analytical concentra- tion of the vapor (see above). (10) Platforms upon which subjects rest their arms while holding them over the applicators. These are small tables of ap- propriate height with holes through which the appli- cators protrude alwait 14 inch. The skin is thus held firmly against the cap of the applicator without any possibility of excessive pressure, and the arm rests comfortably during the exposure. This apparatus, with a volume of 2 1 and an air flow of 20 1pm, can be classified as a small, high-flow chamlter. Concentrations of agent can l>e used up to saturation; the humidity of the air can be varied from dryness to saturation, (loud analytical-nominal ratios are obtained. The apparatus is rapid and con- venient to use. i’se of Dynamic Chambers for Body Exposures. Almost all of the standard type chambers in this lab- oratory have, at one time or another, been equipped for body exposures. The bodies of the animals are exposed to contaminated air, while their heads are in fresh air. A gasket around their necks prevents leak- age of the noxious air and its inhalation. In one of the earliest methods1 for use with a small smoke chamber the animals were placed in the chamber and provided with a manifold through which pure un- circulated. It has been more common practice to place the bodies of the animals in the chamber and let their heads protrude. The first chamlxT to have built-in provisions for body exposures was the 200-1 chamber.6 I sc of Wind Tunnel for Testing Vesicants on Man. ''1 The wind tunnel (p. 285) is equipped with ports through which arms can be inserted perpendicular to the air stream. Since turbulent flow occurs, it was necessary to expose an annular space around the arm. The arm was prepared for exposure by wrapping (he hand and wrist to a point 5 cm above the distal end of the ulna with oilcloth sealed with adhesive tape. A piece of adhesive tape 2 inches wide was placed around the forearm leaving an exposed an- nulus of skin 1 cm wide between the wrist covering and the adhesive tape. Another piece of oilcloth cov- ered the. remainder of the forearm and elbow, leaving a second (proximal) annulus Indween the 2-inch (ape and the elbow covering. To deliver two doses to the same arm the distal (wrist) annulus was left exposed for the whole exposure period; the proximal (elbow) annulus was kept covered with oilcloth except for the appropriate terminal fraction of the exposure period. At the end of the exposure the coverings were re- moved and discarded. The use of the wind tunnel permits testing the relative efficiencies of aerosols and s apors at various wind speeds. Temperature and humidity of the air stream can bo controlled only by controlling the temperature and humidity of the laboratory. The (ircal Lakes Man-ChamberThis apparatus for test ing effects of vesicant vapor on masked men has been described in Section 16.2.2. SECRET Chapter 17 PHYSIOLOGICAL MECHANISMS CONCERNED IN THE PRODUCTION OF CASUALTIES BY EXPOSURE TO HEAT Alan it. Moritz 17.1 INTKODL CTION At a MKKTixti called at the instigation of the Technical Division of (he Chemical Warfare Service on March 22, 1944. certain deficiencies in the existing state of knowledge concerning the casualty- producing effectiveness of the flame thrower were discussed. Attention was called to the fact that, al- lhough both heat and the inhalation of irrespirable or poisonous gases probably contribute in varying degrees to these effects, little was known regarding their relative importance. It was recommended that the physiological section of Division 9 of the National Defense Research Com- mittee [NDRC] investigate the various mechan- isms by which flame thrower action may cause dis- ability and death. In this chapter are reviewed the studies that were made of the mechanisms by which excessive environmental heat may lead to early dis- ability and death. 17.2 PILOT EXPERIMENTS TO EXPLORE CASL ALTY-PRODUCING ATTRIBUTES OF GASOLINE CONFL VGR VITONS A certain amount of general information concern- ing the thermal and chemical attributes of gasoline conflagrations was prerequisite to the planning of an experimental program. For the purpose of orienta- tion, certain exploratory investigations were made of the rate, magnitude, and duration of the changes that occur in the temperature as well as of those that occur in the atmospheric concentrations of oxygen, carbon dioxide, and carbon monoxide incident to the tanning of measured quantities of flame thrower fuel in both closed and ventilated spaces. 17.2.1 Experimental Procedure A series of experiments 1 were accordingly under- taken in which gasoline was burned in a fireproof room having a capacity of 14.4 cu m. The construc- tion of the room was such that it could be cither closed or ventilated at will. The fuel was poured into shallow metal pans which completely covered the floor, which measured 1.6x3 m. Approximately 1 li- ters were burned during each conflagration. To measure the changes in temperature, 10 gauge iron-const an tan spot-welded thermocouples were suspended in the center of the chamber. The thermo- electric potentials provided by the thermocouples were amplified by means of an electronic optical bridge circuit.1- It was found that the use of a split circuit is capable of amplifying a 1-rnv input poten- tiometrieally to a 5-ma output in less than 0.2 sec- ond. Since this amplifier was a null-point, instru- ment, it was independent of all the electronic tube characteristics, of the intensity of the light beam focused on the photocell, and of the input resistance of the thermocouple leads. Two such amplifiers were const ructod. Two recorders were used. One was an Esterlinc- Angus recording milliampere meter (5 mil, full scale) with a response time of 0.5 second. The other was a General Electric photoelectronic recording milli- ampere meter with a response time of 0.2 second. Both recorders had 12 inch per minute chart drives. By means of a selector switch the sensitivities of the amplifiers were usually set so that a 40-mv input produced full-scale deflections of the recording pen. Method of obtaining samples of atmosphere for gas analysis: Three long tubes, each having an in- ternal diameter of 2 mm, extended from the outside to the center of the conflagration chamber. These tubes passed through the wall at, the bottom, middle, and top of the room. Samples of 300 ml were with- drawn as desired by attaching evacuated flasks with ground joints to the ends of these tubes. The gas samples obtained in this manner were analyzed for ()2. CC2, and CO by means of a standard Orsat ap- paratus. SECRET 304 ST I DIES OF THERMAL INJURY— CUTANEOUS AM) SYSTEMIC IT.2.2 Temperatures Developed during (.asoline Conflagrations Unventilatod conflagrations: In these experiments the door was kept closed during the fire. Oxygen de- pletion resulted in extinction of the conflagration in about 30 seconds after ignition. Approximately’ half of the gasoline contained in each pan remained un- burned. When the door was opened following the premature extinction of the file, the room was found to lie filled with dense black smoke and there was a strong odor of gasoline. the lower. The sharp peaks in the temperature curve of (he upper thermocouple are also due to convert ion currents. The average temperatures recorded by the two thermocouples over a 30-second period were ap- proximately the same, namely, about 500 C. At the termination of the combustion, the ambient temper- atures fell rapidly and uniformly. The curves shown in Figure IA are typical of all experiments in which the conflagration was unventilated. Ventilated conflagrations: Figure IB shows a con- tinuous temperature recording of a thermocouple which was situated about 1.5 m above the floor dur- ing a conflagration in which ventilation sufficient to maintain complete combustion was provided. The temperatures obtained were about the same as those recorded during unventilated conflagrations. The duration of the high-temperature plateau de- pended on the length of time that the door was left open. In (lie experiment in which the record shown in Figure. IB was made, the door was left open for 50 seconds, IT.2.3 Extrapolation of Experimental Temperature Changes to Conditions Likely to Prevail in Bunkers and Pillboxes Incident to Flame Thrower Attack It was judged that the circumstances which pre- vailed in the experiments just described probably predisposed to the development of maximal temper- ature elevations. It is regarded as unlikely that higher temperatures would be developed in hunkers or pillboxes incident to flame thrower attacks in which gasoline was used as fuel. Due allowance should lx> made for the toleraneo of commercial re- cording instruments in the interpretation of data pertaining to temperature changes in bunkers and pillboxes incident to field tests of the effectiveness of flame thrower equipment. Thermocouples of the usual size and potentiometers or millivolt, meters of the usual period are not capable of following the rapid temperature fluctuations that occur in nn- ventilated or incompletely ventilated gasoline con- flagrations. Furthermore, temperature observations made by such apparatus may be lower than the actual temperatures obtained by as much as 500 C. I7.2.t Exposures of \nimals to Burning Gasoline Adult dogs (6-8 kg) and young pigs (7 12 kg) wore exposed in various ways to burning gasoline. Figckb 1. Continuous temporal hit recording dining burning of gasoline in rectangular (fixlOx 10ft)combus- tion chamber. (A) No ventilation. Two thermocouples, one 5 ft and the other 3 ft above floor level. Distance from floor to ceiling was 10 ft. (B) Room ventilated for 50 seconds. One thermo- couple 5 ft alxivc floor. Figure 1A shows continuous temperature records provided by the two thermocouples, one of which was hung midway between the floor and ceiling in the center of t he 3 m high conflagration chamber, and the other approximately 0.9 in above die floor. Because of rapid convection currents, the upper thermocouple reached higher temperatures than did SECRET \TTRIBLTKS OK GASOLINE CONFLAGR VTIONS 305 Tabi.r 1. Effects of temperature and com Oust ion products resulting from gasoline conflagrations on animals. Conflagration thermal exposure combustion Comp. (* f ) of air r ate of animal Blood Kef. Avg Temp C Body Body only With After after fire Dead Survival expt CO sat sec face only fire fire (): COj CO min or days SC 1 30 320 + 4" 5 ruin 14.0 4.0 o.s + 30 2 40 600 ' + + Xo 16.2 4.0 0.8 + 7 3 30 400 4- No No + . . 4 30 370 + No Xo + 5 73 700 + Nil Xo + k 30 350 Xo 5 min 14.7 4.6 1.0 + 6 7 30 350 + + 4 min 15.7 3.5 0.3 4~ T race 8 30 450 + —4" 4 min 16.1 3.6 0,7 4- 37 9 30 500 + + 2 min + 32 The animals were anesthetized by the intraix'iitonoal injoetion of sodium pentobarbital ami fastened by asbestos tape to an iron frame situated in the center of the conflagration room 54 inches above the floor. The principal data pertaining to these experiments arc included in Table 1. COMBINED CTtaNEOES AND RESPIRATORY EXPOSURE Animals 1 and 2 were exposed to the full effects (cutaneous and respiratory) of the burning gasoline. Throughout the entire exposure of animal 1, the door of the conflagration chamber .remained closed. The fire burned out in about 30 seconds because of in- sufficient oxygen. The average temperature of the air surrounding the animal during tins period was 320 C. The animal was allowed to breath the at- mosphere of the unveatilated room for 5 minutes after the fire was extinguished. Samples of the atmosphere were taken for gas analyses as soon as the fire had burned out. The mean concentration of CO in the atmosphere was 0.8 per cent, and the oxygen concentration was 14.6 per rent. The CO saturation of a .sample of the animal’s blood taken 5 minutes later was 30 per cent . Although there was no indication that the fire had resulted in a dangerously low oxygen or a danger- ously high CO. concentration, it did appear likely that, the animal would have died of CO poisoning if it had remained much longer in the unventilated room. Although animal 1 had been severely burned, it did not develop early shock, required several post- exposure injections of Nembutal to keep it quiet, and was beginning to become restless with returning con- sciousness when sacrificed 6 hours later. Its air pas- sages contained an excessive amount of mucus but them was neither clinical nor pathological evidence of significant thermal or chemical injury of the larynx, air passages, or lungs. In the case of animal 2, the door remained open during the first 10 seconds of the conflagration, with (he result that a larger amount of gasoline burned and a higher tem|H‘iature was achieved and was main- tained for a longer period of time than was (he case in the first experiment. At the end of 10 seconds, (he door was closed with the result that the file was ex- tinguished very soon thereafter. Samples of the at- mosphere were then taken for gas analyses and the animal removed. This dog was moribund when re- moved to (he o[X‘ii air. In view of the fact (hat the atmospheric concentration of CO was similar to that observed in the preceding experiment, it was sur- prising to find that (lie CO saturation of the blood was only 7.0 per cent. The explanation of this dis- parity probably lies in the fact that animal I breathed the atmosphere of the conflagration chamber for a total of 6-7 minutes, whereas animal 2 was moribund at the end of 2 minutes. Two factors may have contributed to the ex- tremely rapid death of dog 2. One is systemic hyper- thermia caused by overheating of the blood as it cir- culated through the extensive sii|x*rficial network of subcutaneous vessels. The. other is respiratory ob- struction due to pharyngeal edema. That a signifi- cant degree of hyperthermia had occurred was indi- cated by the finding of a rectal temperature of 41.2 C when the animal was autopsied 5 hours after the ex- posure. That obstruction to respiration may have contributed was indicated by the presence of severe burning of the mouth and pharynx with what ap- peared to he obstructive edema of the latter. The trachea and bronchi contained abundant mucus SECRET STL DIES OF THERMAL INJURY—CUTANEOUS AND SYSTEMIC mixed with carbon particles. The lungs were hy- peremie. The results ol the first two exposures dealing with the effects on animals of burning gasoline indicated that, even in circumstances considered to be particu- larly favorable to the production of CO and to the exhaustion of oxygen, the concentration of these gases was not sufficiently altered to cause uncon- sciousness or death within 5-6 minutes. Although the. results of the two experiments were not construed as proof that neither fatal anoxia noi fatal CO poisoning could result from a gasoline fire, they did indicate that such an exposure can cause rapid death from thermal injury alone. ( Vtax Kors Exposehe The next three experiments shown In Table I were undertaken to ascertain the effect of protecting the respiratory tract against heat and combustion prod- ucts during the time thaf~l.be body was being ex- pose* I. To investigate this question, animals 3, 4, and 5 wore light-fitting asliestos-eovereil rubber masks through which a continuous stream of un- heated air was circulated during their exposure to heat. The first two animals of this series (3 and 4) were exposed to an un vent dated conflagration of about 30 seconds duration and average atmospheric temperatures of 400 and 370 C, respectively. Al- though both animals showed extensive burning of the skin, they survived the immediate effects of heat and were in reasonably good condition when killed 6 hours Inter. In the ease of animal 5. the door of the room was left open for the firsl minute of the fire and for 65 seconds the temperature of the room was in ex- cess of KK) C. Within 15 seconds after the door was closed the fire went out and the animal was removed. This animal died immediately on reaching the ojicn air and showed severe burning of all the body surface except where the skin had been protected by the mask. 'These experiments provided evidence that a rela- tively brief (75 seconds) exposure of the skin to a sufficiently high temperature could cause almost im- mediate death independently of other factors. Kespiratokv Kxposvhe 'The last four experiments shown in 'Table 1 were undertaken in an attempt to investigate further the effects on animals produced by the breathing of I lie combustion products of a gasoline conflagration. In each experiment the door was kept closed throughout the entire conflagration. By this procedure post con- flagration mixing of outside air with the combustion products was reduced to a minimum. The skin of the body was protected against excessive overheating by enclosing the animals to the neck in a heavy asbestos sack. With the exception of No, 6 the animals were free to breath the burning gases and hot air during the fire as well as the smoke which remained in the chamber after the fire. Dog No. (i breathed outside air circulated through the mask during the fire; as soon as the temperature in the room had dropped to 200 (' the mask was detached by remote control and for the next 5 minutes only the hot smoke and air of the combustion chamber were available for respira- tion. None of these four animals showed either clinical or pathological ev idence of thermal injury of the air passages or lungs. Two of them (6 and 7) may have held their breath throughout most oral! the exposure period. That animals No. 7 and 8 breathed during some of the lime that they were in the combustion chandler is indicated by their carboxyhemoglobin concentrations of 37 and 32 j)er cent, respectively. It is possible, of course, that even these two animals held their breath during the conflagration and ac- quired their carlnin monoxide by breathing during the interval between the time that the fire went out and the time that they were removed from the chamber. 17.2.5 Summary The ignition within a simulated pillbox or bunker of a well-spread layer of gasoline leads within 10 sec- onds to a temperature rise of be)ween 800 ami 1000 The duration of such a conflagration and t he temperature increase caused by it varied according to the oxygen supply and the fuel. In a closed room having a capacity of 14.4 cu m and measuring l.Gx 3x3 m, the fire was extinguished within 20 seconds and considerably less than 250 ml of gasoline was consumed for each cubic meter of air space. For ap- proximately 10 seconds of the burning time the tem- perature fluctuated between 500 and 900 C. If such a room is ventilated, the initial temperature rise is similar to that which occurs in a closed room, but the fire continues to bum until the fuel is exhausted, re- sulting in a temperature fluctuation of between 500 and 1000 C. In both instances, convection currents established by the conflagration resulted in marked fluctuations in the temperature at any given place within the room. During the period of rapid com- SEC11ET BASIC CHARACTERISTICS OF HEAT AND HEAT TRANSFER 307 bust ion the tem|x*ratures were highest near the ceil- ing and lowest near the floor. In none of the experiments conducted in this par- ticular tyj)e of conflagration chandler did the oxygen content drop below 11 per cent. The carbon dioxide level did not rise higher than 5 per cent nor the carbon monoxide level above 1 per rent. 'Pbe most important information gained from these exploratory experiments was the observation that animals as large* as dogs and pigs when exposed to tire kind of a conflagration for more than 30 seconds may receive injuries that are almost immediately fatal. Such fatalities were not necessarily contributed to by asphyxia, carbon monoxide poisoning, or in- halation of flame. It apjx’ared that the rapid death may result from systemic disturbances caused by the impact of heat energy on the surface of the laxly. It was obviously in order to conduct additional and better controlled ex|X'iiment.s to investigate the physiological mechanisms concerned in the produc- tion of casualties through the thermal effects of flame thrower attack. IT.3 It \SIC CHARACTERISTICS OF IIKVP \M) HEAT TRANSFER* It could lie inferred from the results of the pilot experiments reported in the preceding section that heat independently of other factors was an impor- tant, if not the most important, casualty-producing attribute of flame thrower action. This being., the case, consideration should be given to the nature of beat and to the factors which determine the transfer of heat from one medium to another and from one place to another within the same medium. IT.3.1 Theoretical Considerations The Nature of Heat The concept of temperature rises from the sensa- tions of hotness and coldness. Experience has shown that when two or more substances of different tem- perature are kept free of all outside disturbances, the hotter bodies will get colder and the colder bodies hotter; and that ultimately these substances will reach a state of complete thermal equilibrium (ident i- cal temperature). The hotter bodies are said to have lost heat, and the colder bodies are said to have gained heat. This concept of heat becomes quantita- tive by defining a unit of heat, the calorie, as the amount of heat gained by a 1 g of liquid water under atmospheric pressure when the temperature increases from I t o (' to 15.5 C. This gain in heat, which is discernible through a rise in temperature, is associated with an increase in the infra- and intermodular motion. Thus heat can be considered as the energy stored in a substance by virtue of the state of its molecular motion. Certain manifestations of this increase in energy are readily observable, for example, melting, vaporization, de- composition, alteration in rate of diffusion and in chemical reaction. Reside the definition of a calorie, there are other physical concepts pertaining to heat which are requisite to an understanding of the.general problem of thermal injury. Heat Capacity Heat capacity or specific heat of a substance is the amount of heat which is .required to raise the tem- perature of the substance 1C. The importance of heat capacity (Cp) in thermal injury is readily seen by considering the respective injury propensity of 1 gof water (CP — 1.00) and 1 g of silver (C,, — ().()(») both at 100 C placed in contact with 1 g of thermally insulated skin (Cp ~ 0.7) at 35 C. After equilibrium is reached In the former ease, the tem|)c.rature of the skin is increased to 73 C, whereas in the latter case it is increased only to 42 C. It is apparent, that, if the skin were to equilibrate rapidly enough when placed in contact with a hot body, there is insufficient heat in 1 g of silver at 100 C to produce injury to 1 g of skin. Actually, of course, the skin, because of its thermal insulating properties, does not equilibrate rapidly enough and the portion of skin nearest (he silver docs reach a sufficiently high temperature to produce injury be- fore thermal equilibrium is reached. Hence another physical property of importance is heat transfer. 11eatTransfer In the experiments to lx* described beat was trans- ported to the skin by three mechanisms: namely, convection, radiation, and conduction. In the ease of convection and radiation, heat reaches the skin under such circumstances that the heat uptake is primarily determined by the heat source. In the east; of con- duction, the amount of heat absorbed by the skin is primarily determined by the properties of the heat absorber, namely, the skin itself. * By F. C. Hcnriques, Jr. SECRET 308 STUDIES OF THERMAL INJURY— CUTANEOUS AND SYSTEMIC ('ONVKCTION Convection is the mechanism by which hot air transports heat to a cooler surface because of the eddying currents that arise. The air velocities of the eddy currents are about 1.6 km per hour. An equa- tion has been developed for the transfer of ambient heat by natural convect ion from a large envelope of hot air surrounding cylindrical objects about ;{f) cm in diameter.29 34 This equation shows that 7, (he caloric uptake per minute |>er square centimeter of surface, can l>e expressed as follows: 7 = 0.0026(7’,, - 7\)J “ (I) where T„ is the air temperature in C and 7’, is the surface temperature in (’. Thus, with a skin temperature of 40 C, air at 100( ' and 400 C will transport to the skin about 0.4 and 4 cal cm2 min. It is also apparent that as this heat is absorbed by the skin the surface temper- ature of the skin will rise and the caloric uptake of the animal will decrease with time. It is of interest to compare with this the caloric uptake rate of skin at 40 (’ when an atmosphere of steam maintained at 100 C is substituted for the air. Under these conditions, about 300 cal enr min would be absorbed by the skin 36 if the surface tem- perature could be maintained at 40 C. This 800-fold increase in caloric bombardment as compared with that produced by air is due to the latent heat of con- densation of steam. This, of course, is why steam is an enormously greater hazard than hot air in the production of heat injury. Radiation All substances give off heat in the form of radiant energy in amounts that are predetermined by the surface temperature of the substance. When this radiation impinges upon another body, a certain fraction is absorbed and changed into heat. Thus, if two substances at different temperatures are placed in an enclosure, there is a continual exchange of energy, the hotter body radiating more energy than it absorbs and the colder body absorbing more heat than it radiates. In the special case of an animal completely en- closed in a large box of source temperature 7%, the caloric uptake rate 7 of the animal, due to this inter- change of radiant energy between the skin and the wall of the box, is expressed by the following equa- tion.30» 7 = scJUT, + 273)4 - (T, + 273)4] (2) where s is the radiation constant and is equal to 8.2 X 10~u calorie, per square centimeter per Tx per minute, e is the effective emissivity of the hot walls of the box, and / is the absorptivity of the skin, to radiation emitted at Tr. Under experimental con- ditions to l»c described, the product rf can be taken as about 0.8. Thus, when the skin temperature is 40 C, the hot walls at 100 (' or 100 C will radiate to the skin about 0.7 or 13 cal cm- min, respectively. Conduction Conduction is defined as the transfer of heat from the hotter portion of a substance to a colder portion of the same substance, or from a hot laxly in physical contact with a cold body, where in each case there is no appreciable displacement of any of the molecules comprising these substances. It is the latter restric- tion that differentiates conduction from convection. In certain experiments to be described heat was conducted from either a hot solid or a hot liquid to the skin. In these experiments, the purpose of both the solid and liquid heat source was to maintain the tenqierature of the skin surface at a predetermined constant value and hence the conduction of heat through the heat source need not lx* considered. In the hot air experiments, thermal conduction through air is small as compared with convection, and this small contribution is included in equation (1). Thus it is only necessary to consider conduction of heat through the skin. In all cases of heat flow by conduction, a temper- ature gradient must exist within the substance. If this temperature gradient varies with time, the rate of heat flow will also vary with time. The type of heat flow where temperature is a function of both position within the body and time is called heat con- duction in the unsteady state. Heat conduction in the steady state refers to all cases where the temper- ature at any point within a substance does not de- pend upon time. Under these conditions the amount of heat flow through the medium is determined by this tenqierature gradient and the ability of the body to conduct heat (thermal conductivity). The latter case will be considered first . The equation for steady-state heat conduction inside a rectangular homogeneous body is based upon Fourier’s law111 and is as follows: 7 = f (T, - T„) (3) where K, the thermal conductivity, is expressed in SECRET BASIC CHARACTERISTICS OF MEAT AMI HEAT TRANSFER 309 calories jxm- minute, per square centimeter perpen- dicular to (lie direction of heat flow per unit temper- attire gradient in (' per centimeter length of path. L is the path length through which the heat flows and T, and T„ are the temperatures in C at the be- ginning and end of the path, respectively; q has lieen previously descrilied. This equation permits the ex|>erimental determina- tion of the in vitro thermal conductivity of the four respective sections of tissue, namely epidermis, der- mis, fat. and muscle, and also of any combination thereof, Glnehal Tiikouv ok Heat Flow thkovgii Skix By making use* of the preceding brief definitions of the various physical factors involved in the transport of heat to and through the skin it is possible to con- sider how the application of heat affects the time- temperature relationship within a given skin site. It is-apparent that in order to make heat flow inward from the skin surface it is necessary to raise the tem- perature of the skin surface to an extent that over- comes the normal existing gradients. This can be ac- complished by means of an external source, of heat through conduction, convection, or radiation. Once the skin surface temperature is sufficiently high, the heat will start to flow inward, resulting in a general rise in tenqierature within the skin site. din's initial heat flow inward (and thus the rate of temperature rise within) will depend primarily upon two physical factors; namely (1) the heat capacity of the skin or the ability of the skin to absorb the beat, and (2) the thermal conductivity of the skin or the ability of the skin to transport the heat. After a cer- tain interval of time the amount of heal entering the skin site will be balanced by the amount of heat leaving the skin site, and the skin will l>e ‘‘heat- saturated.’’ In this state the new temperature dis- tribution within the skin site will become invariant with time and the amount of heat flowing through the skin will depend only upon (2) and the skin sur- face temperature. It is to be recognized that this picture involves not only the solution of the steady state of heat conduc- tion but also the solution of the initial unsteady state of heat flow. In order to solve even the “idealized” picture, it would lx? necessary to know the initial temperature gradients within the tissue, the thick- nesses. densities, thermal conductivities, and heat capacities of the various layers, and the skin surface temperature as a functiyp of time. The solution of such a problem involves the follow- ing Fourier heat equation:" (4) pC„iA ); with experi- ments (b), the heat transfer coefficient is finite and the numerical value readily obtained by combining the radiant and ambient contributions to heat trans- fer coefficient as computed by equations (1) and (2), respectively. In order to solve equation (1) under the boundary condition expressed by equation (5), it is necessary to assume that the ratio of the total tissue thickness to the epidermal thickness (approximately 80u) is infinite rather than finite. This assumption will lead to slightly longer time intervals for “heat saturation” of (he epidermis than are to be experi- mentally expected. The integration * of equation (1) under the above conditions results in equation (<»). j> —Tt f >1 hl:ki\+(hua^kt\~ T. ~ r„ = Ly/tJ !1-#[^( (,i) where r 0(Y) = f e~'dx (0a) V JT •' 0 and y is computed by means of equation (0b). y~2 ” (0b) 1 pCp - Ti is the temperature of the basal epidermal cells at the time I in seconds. T, is the temperature of the heat source. T„ is the temperature of the skin surface previous to the exposure to heat. 1. is the distance of the basal cells from the skin surface, p is the density of the basal epidermal layer. The other symbols lane been previously defined and are experimentally de- terminable. The integral that defines 0(1) (equa- tion Oa) is respectively equal to y/V 2 and zero when T is infinite (t ~ 0) and )' is zero (/ = <»). For other values of V, the numerical value of the integral is tabulated.59 The time-temperature relationships at the basal epidermal layer during an exposure of the animal to a source of constant ambient and radiant heat are evaluated by means of these equations in Sec- tion 17.3.2 (see also Section 17.9.2 under “Measure- ment of Heat Transfer”). In the experiments in which the skin surface was brought immediately to and maintained at a pre- determined constant temperature, //, the heat trans- fer coefficient, is nearly infinite, and equation (0) reduces to m-a.) where, as before, 0 is given by equation (Oa). It is to be noted that in this case T, can be taken as the skin surface temperature during the entire heat exposure, since tlie temperature of the heat source is identical with the surface temperature once heat exposure begins. It is to be noted that equation (tic) results in a basal layer temperature which becomes, after a cer- tain time interval, essentially identical with the skin surface temperature. Actually, a small but finite temperature gradient will always exist between the surface and the basal cell layer. This steady-state gradient can be experimentally determined by means of equation (3), and the true temperature of the basal layer can be quite accurately computed for any time t by using equation (tic) until the steady-state temperature obtained through equation (3) is reached. Computations using equation (tic) to ascer- tain basal epidermal temperature are given in Sec- tion 17.3.2, and the experimental justification for this theory will be considered in Section 17.0.5 (see also I7.fi.6). 17.3.2 An Kxperiinenlal Investigation of Quantities Involved in Botli Steady and Unsteady State of Heat Con- duction through Skin 3" It is apparent that certain types of special appa- ratus were necessary for the evaluation and assess- ment of the various physical factors involved in the time-temperature relationship to thermal injury. The description of these apparatuses will now follow in detail. Heat Capacity Apparatus The apparatus used for the determination of the heat capacity of the various skin layers need not lie described in detail since these specific heals were de- termined by the well-known method of mixtures.4* Briefly, this procedure consists of heating a known weight (about 10 g) of tissue in a brass container to 100 C and rapidly dropping it into a water calorim- eter. The heat capacity of the tissue was readily computed from the temperature rise of the water as measured with a Beckmann thermometer. SECRET BASIC CHARACTERISTICS OF HEAT AND HEAT TRANSFER 311 Automatic Exkrgv Recohdintj Applicator In order to measure the rate at which heat energy was taken up by the skin during the entire exposure period at any predetermined skin surface tempera- ture, the following apparatus was constructed to simulate an infinite source of heat at any given temperature. The effect of bringing the skin in contact with a source of heat having infinite capacity and constant temperature is shown schematically in Figure 2. The temperature of the surface of the skin immediately reaches and is maintained at the temperature of (he heat source. The rate of caloric uptake by the skin at the time of the initial contact is essentially infinite ami as the skin approaches its new state of temjier- ature equilibrium the rate of energy transfer dimin- ishes and finally- reaches a nearly constant value. Thus the curve representing rate of energy transfer is similar to that shown in Figure 2. gain as much heat from the surrounding area as it would lose to it. Thus the calorie uptake from the central area of the source (Figure 2) would he a meas- ure of the per|>endicular flow of energy through the directly subjacent skin if a sufficiently large sur- rounding area were in contact with the same or a similar source of energy. A scale drawing of the caloric applicator is shown in Figure 3A. It consisted primarily of three separate parts a crown, a brim, and an applicator disk. The crown and brim were brass, whereas the applicator disk was copper. The three units wen' maintained at (he same constant temperature by independent elec- trical heating units. The temperature of the crown and brim were controlled manually by means of Gen- eral Radio Variac transformers. The purpose of the crown was to prevent any leakage of heat from the applicator disk except via the exposed face. The brim compensated for the lateral spread of the heat from the surface of skin directly underneath the ap- plicator. The applicator was heated by means of an auxiliary' electronic apparatus which automatically recorded the wattage required for continuous main- tenance of the face of the applicator at a specified temperat ure T. The temperatures of the crown, brim, and disk were measured by means of three calibrated 10-mil iron-constant an Fiberglas duplex (Leeds «fe Northrup) thermocouple wires. The wire heating- units were of single, silk-insulated No. 40 manganin wire (negligible temperature coefficient, of resistance). This wire was held in the indicated spiral grooves with a thin coat of glyptal. (’upper le:ul wires were soldered to the ends of the manganin and the joints were electrically insulated from the metal parts with fine glass bushings. The electrical resistances of (he brim, crown, and applicator were .390, 277, and 71.75 ohms respectively. Three fine phonograph needles rigidly located the applicator disk inside the crown. The disk was held firmly against the needles by a rubber bushing under compression. A steel spring, (he tension of which could be regulated by a hard rubber screw, controlled the pressure of the applicator against the skin. This pressure could be set between 5 and 50 g/cm2. Guides served to keep the numerous lead and ther- mocouple wires apart, so that the pressure regulation was reproducible. The two lead wires to the heating unit of the applicator were held fast against the sides of the Fiberglas thermocouple wire by wrapping with thread for the first 10 cm and then with scotch tape. Fuu-re 2. Diagrammatic represent.'ition of rate of ealoric uptake of skin from heat source at constant tem- |>e rat ore, of infinite thermal conductivity and of in- finite heat capacity. The steady state of caloric uptake is a measure of the thermal conductance of the skin. The unsteady state is a measure of the ratio of the thermal capacity to the thermal conductivity of the skin. Unless the animal were completely enclosed by an infinite source of heat, there would be considerable lateral spread of energy from the application area (see Figure 2). It was apparent, however, (hat be- cause of lateral spread the skin in the center of the application area would, under certain conditions. SECRET 312 STUDIES OF THERMAL INJURY — CUTANEOUS VM) SYSTEMIC Floras 3A. Cross section of automatic energy re- corder applicator— A AplinUor disk (copper). B Brim (brass). — C Crown (brass). D Fiber washer. F Heater lead wire*. F Heater wires (3-mil silicic silk mancaiiini. (J Fine phonograph needles (three), I! Brass spider for boldine needlea. I Stainless steel screw. 4 Hard rubber dow'el. K Fiber handle. L Iron-constantan Fiherglaa duplex thermocouple wire. M Threaded hard rubber eup (Jot adjust inc spring pressure). N Rubber collar (for holding applicator disk tight against needles.) O Hraa« cup for rubber collar. P Thin stainless steel tube. Q Steel spring Figure 3TF Diagram of electronic apparatus that con- trols and measures wattage input into disk of automatic energy recording applicator; plate potential; thus, when the plate was positive the grid was always sufficiently negative to pre/ent the thyratron tube from firing. When light struck the photocell, the resistance of this part of the grid cir- cuit decreased sufficiently to alter the phase rela- tionship of the grid and plate circuit and the grid was not sufficiently negative to prevent the tube from firing during a portion of the cycle when the plate was positive. Once the tube fired, the grid lost control (gas-filled tube) and the tube conducted dur- ing the remainder of the positive plate cycle. When the plate became negative, the plate cur- rent became zero and the grid again gained control of the thyratron. Thus the amount of current which flowed during the positive plate cycle depended upon the phase angle between the grid and plate voltage. This phase relationship was a fimetion of the re- sistance of the photocell, which in turn depended upon the amount of light striking the photocell. Hence, the amount of light striking the photocell The electronic apparatus which controlled and measured the wattage necessary to maintain the face of the applicator at a constant temperature T is shown schematically in Figure 3B. The basic principle of the circuit was phase con- trol of the four-element (GE FG95) thyratron tube.40 In order to obtain sufficient filtered power at the moment that the applicator first touched the skin, it was necessary to operate the plate circuit with the 220-v alternating current that was available from a commercial power line. The grid circuit operated on 220-v alternating current from a radio transformer. This transformer was connected to produce a J 10-v potential between point .1 and point B. When no light was striking the photocell there was nearly a 180-degree phase difference between the grid and SECRET BASIC CIIAll ACTER1STICS OF HEAT AM) MEAT TRANSFER 313 gave a continuously variable control of the power output of the plate circuit. The 50-megohm re- sistance shunting the photocell added stabilization to the circuit. The 250-aaf variable condenser “tuned" the phase angle of the grid circuit to the l>est operating conditions. These conditions were that a 1-nun deflection of the light beam reflected from the galvanometer would give full control of the plate wattage. The purpose' of the capacities and chokes in the plate circuit was to filter the pulsating thyratron output into steady direct current. The values of the condensers and chokes were necessarily large lie- cause of the high current requirements of the appli- cator heater. Oscillograph Tests showed no appre- ciable ripple current in the filtered output. The wat- tage or caloric input rate into the applicator heater was measured with an appropriately shunted Ester- line-Angus recording milliammeter (5 mil, full scale and :i4 to 12 inch per minute chart drive). Six scales were provided by a selector switch with full-scale deflections of I, 2, 5, 10, 20, and 50 cal min cm2 of applicator surface area respectively. The highest value corresponded to a filtered output of 30-v across the applicator heater terminals. Operation. In use the galvanometer zero was set to provide sufficient illumination on the photocell to generate about 1 eaL min inside the applicator. The potentiometer was now set to the predeter- mined millivoltage (temperature). By turning off and on the low-sensitivity shunt button of the type K potentiometer the photocell was kept fully illumi- nated until the galvanometer started to deflect in the opposite direction. The high-sensitivity button was now locked down and the instrument was on auto- matic control. Thus, if the temperature of the appli- cator face as measured by the applicator thermo- couple tended to gel either hotter or colder, the gal- vanometer mirror moved in a direction that either decreased or increased the illumination on the photo- cell, which in turn either decreased or increased the wattage through the applicator. Thus by means of the thermocouple in the face of the applicator the output of the thyratron tube was thermally “locked” to a predetermined temperature of the applicator face. The sensitivity of the galvanometer was set to give a deflection of 4 mm for an 0.1 C change in tempera- ture. This deflection was sufficient to produce the maximum available power of 50 cal cm2/min. This was the maximum sensitivity that could be obtained without producing periodic heating and cooling of the applicator face (slow oscillations of the recorder tracings of caloric uptake rate). These oscillations in the power output were due to the short but finite time for the heat generated in the heater wire to affect the thermocouple. The heat losses of the applicator disk under the conditions of usage were determined by placing the disk and brim on a “perfect” insulator. The perfect insulat or consisted of a flat-bottomed, thin glass cone which was silvered on the inside, pumped out to I0~7 mm of mercury while being heated to 450 C for 8 hours, and then sealed off. All heat losses from the inside surface of the glass were prevented by the bright silver surface (no radiant loss) and t he vacuum (no molecular heat conduction). Lateral heat loss through the glass was prevented by maintaining the brim at the same temperature as the applicator disk. Heat losses from the applicator disk were deter- mined at two temperatures, namely 45 and 00 C. The results are given in Table 2. Table 2 . — Applicator 'I’eroperaluir C disk heat loss in Exp Crown Brim Disk cal/em!/'nin a 15 •15 45 0.020 b 45 Not heated 45 0.45 <* tiO 60 00 0.035 d 61 00 00 0.000 e 59 00 (iO 0.0H0 r 00 - Not heated 60 II . These data showed that when all three units wore heated to the same temperature the heat loss of the disk was trivial as compared with the caloric uptake of the skin at similar temperatures (see Table 5). The slow rate of heat transfer from the crown to the applicator disk was indicated by comparison of ex- periments c, SYSTEMIC constructed by threading a single silk-insulated 3-mil constantan wire through 4 feet of a No. 27 gauge trochar. The bimetallic junction was then made by honing down the end of the trochar and wire to a 45-degree angle; this removed the silk in- sulation from the constantan wire and permitted it to be surface soldered to the steel hypodermic needle. Through experimentation it was found possible to insert laterally a No. 22 gauge trochar along the natural cleavage plane of the dermis-fat interface, until a point directly underneath the surface area to be exposed was reached. Then the No. 27 gauge thermocouple needle was inserted into the No 22 gauge trochar until skin resistance could be per- ceived, and the No. 27 gauge couple was withdrawn about 1 cm. After the heat exposure was terminated, the skin was cut to the needle depth and the distance from the muscle-corium interface to the skin surface was ascertained with a depth gauge. This depth be- fore the application of heat was ascertained by a con- trol experiment on a neighboring site. The thermal emf of the steel-const ant an couple was read on a Leeds & Northrop Type K2 potentiom- eter and high-sensitivity galvanometer. The steel- const ant an emf seemed to be very reproducible in this temperature range 0 to 80 (.'. It was about 30 per cent lower than the iron-const antan emf. Tempera- ture differences of 0.1 C were readily determined. Thermocouple foh Measuring Surface Temperature of Skin The surface temperature of skin exposed to air de- pends upon two factors, namely, the rate at which heat reaches the skin surface from the underlying tissue and the rate at which (he skin surface loses heat to the atmosphere. When the surface tempera- ture of the skin reaches a steady state, these two rates must be identical. The use of the usual insulated thermocouples 19 for the measurement of skin temperature necessarily alters these conditions. Upon first applying an in- sulated thermocouple, no matter how perfect the insulation, the temperature measured will be con- siderably lower than the true surface temperature because of the relatively high heat capacity of the insulator. When a steady state of temperature is finally reached (in some cases a matter of hours), the temperature recorded by the insulated couple must be greater than the true skin temperature, since the skin site is no longer losing heat directly to the air. Thus an accurate measurement of surface tempera- tore by any apparatus similar to that just described would be fortuitous. A thermocouple for measuring the surface temper- ature of the skin in this investigation consisted of a bare 2 mil iron-constantan junction. The 2-mil wires were prepared by dissolving (by nitric acid) the ends of 15-mil iron and constantan thermocouple wires (Leeds A Northrop) for a distance of 5 mm. The re- duced ends of the two wires were then soldered end to end and stretched tightly by means of a bow made of brass tubing (see Figure 4). The heat capacity of the junction was trivial as compared with that of the skin. Fioube 4. Thermocouple for measuring skin surface temperature. In use the junction was placed on the skin for lateral contact and after It) seconds a reading was made. The couple was then completely surrounded by skin by pinching the neighboring epidermis and a second reading was taken within 5 seconds. Numer- ous such pairs of readings were recorded and in no case has there been any significant difference between the temperature of lateral and that of circumferential contact. Thus a bare fine wire rapidly reaches skin temperature (10 seconds) when it is in contact with the skin.1* The sensitivity of an iron-const an tan thermo- couple is such that with a Leeds A Northrop Type K2 potentiometer and high-sensitivity galvanometer '• A theoretical objection to the unprotected or bare wire junction has been that it is partially exposed to the air and thus will reach a temjieralure somewhere intermediate bet ween the air and the skin temperature. It should l>e kept in mind that the skin is also exposed to air. At normal air tempera- tures, the heat transfer coefficient for both wire and skin to air is quite small. Since the heat capacity of a fine wire is small and its thermal conductivity high, one would expect the wire rapidly to attain true skin temperature. SECRET BASIC CHARACTERISTICS OF HEAT AND MEAT TRANSFER temperature differences of 0.05 C were readily meas- ured. Determination of Heat Capacity of Four Pertinent Tissues (in vitro) The heat capacity of pig epidermis, dermis, fat, and muscle were determined on approximately 10-g samples of each tissue by the procedure given in Section 17.3.2 under “Heat Capacity Apparatus.” Determinations were made on each tissue of two 10-kg pigs. In order to obtain pure epidermis for these determinations the following method was used. After the hair was shaved as closely as possible, the pig was immersed in water at 55 C for about I min- ute, then removed, and the skin was carefully dried. It was then possible to remove strips of pure epi- dermis by scraping with a knife. The remaining tis- sues were readily obtained in a relatively pure state hy dissection. The values of the heat capacities of these tissues are given in Table 3. the thermal conductivity. The temperature of the tissue-cylinder interface was measured by means of an iron-const an tan thermocouple soldered into the face of the copper cylinder. The average t issue thick- ness was determined by measuring (he distance of the face of the applicator from the face of the cylinder. The thermal conductivities of all the tissues except epidermis were obtained by this procedure, since in view of the epidermal thinness the above method was not adaptable. The method of difference was used with epidermis. A section of well-shaved skin tissue consisting of dermis and epidermis was rigidly clamped to the copper cylinder, water at 55C was poured over the skin, and the excess water was removed by blotting. The clamps prevented lateral contraction of the heated tissue and the hot water facilitated subse- quent removal of the epidermis. The conductivity determination was now made, the epidermis was then scraped off, and the determination repeated. As a further check, in certain experiments, a strip of in- tact epidermis was placed over the denuded dermis and the measurement repeated. The thickness of numerous pig epidermal strips was determined with a micrometer. Tire thickness was about 80 + 10 g. At least triplicate determinations were made on each of the four tissues of three different pigs (ap- proximately 10 kg). The average values of the thermal conductivities obtained on each of these tissues are given in Table 4. Table 3. Heat capacity of pig tissue gram |ier C. in calories per Epidermis Dermis Fat Muscle Heat capacity 0.887 0.785 0.538 0.890 0.845 0.753 0.573 0.926 Average value 0.86 0.77 0.55 0.91 In view of the similar heat capacities of dry tissue, the above variations of the different tissues are prob- ably due to water content of tissue. In this respect the high value for pig epidermis (0.80) is understand- able since it was found experimentally that the water content, in spite of the presence of the cornified layer, averaged about 70 per cent. Determination ok Thermal Conductivities of Tissues (in vitro) The experimental determinations of thermal con- ductivities of pig epidermis, corium, fat, and muscle, were based on equation (3) of Section 17.3.1. The respective tissues were placed on a copper cylinder 2 inches in diameter and 4 inches high. The auto- matic energy recording applicator was now placed over and in contact with the tissue. Thus when the tissue became “heat-saturated,” the knowledge of the calorie input into the tissue, the temperatures of the tissue-applicator (approximately 48 C) and tissue- cylinder (approximately 30 C) interfaces, and the thickness of the tissue permitted the computation of Table 4. K given In vitro thermal conductivities K of pig tissue, in (cal — cm)/(cm* — min — degrees C) units. Kpidermis Dermis Fat Muscle K 0036 0.051 0.021 0.064 0.023 0.053 0.024 0.062 0.032 0.051 0.023 0.073 K 0.03 0.053 0.023 O.OCG In view of the thinness and uncertainty in the thickness of the pig epidermis, the wide variation in the epidermal thermal conductivity was to be ex- pected. The data pertaining to the other tissues were considerably more reproducible. It is of interest to compare some of these data with those of Brener,8 who determined the respective thermal conductivities of both muscle and fat of cow, horse, pig. and dog. This investigator found that the conductivities of pig muscle and fat, expressed in the above units, were 0.000 and 0.021 respectively; fur- thermore essentially the same values were found for SECRET STUDIES OF THERMAL INJURY — CUTANEOUS \MJ SYSTEMIC the muscle and fat of the other three animals. In view of the excellent agreement between Brener’s value and the present one for pig muscle and fat, it is diffi- cult to understand the value, 0.03, that Hardy and Soderstrom 15,22 report for both cow muscle and fat. Unfortunately no description of their experimental method was given. In order to investigate this dis- crepancy, the thermal conductivity of beef muscle was redetermined and an average value of 0.057, which checks Brener, was obtained. In view of the numerous indeterminate factors (Section 17.3.1) which enter into the in vivo conduc- tion of heat through pig skin, the in vitro thermal conductivities of these four tissues are not of them- selves too useful. They do however serve as a base- line-in the interpretation of certain experiments to be described. Observations {in vivo) of Caloric Uptakk of Pm Skin and Rise in Temperature at Dermal-Fat Interface as a Function of Both Time and Skin Sir fac e Temperate re It was of interest to ascertain (he caloric uptake of the skin when the epidermal surface was maintained at various temperature levels Iretween 15 and 100 C. Numerous such experiments have been done and as was to lie expected (see Section 17.3.1) the data were subject to wide variations and are extremely difficult to interpret in detail. Thus only a small fraction of these data Mill be reported and the variations to be expected will be indicated. During these experiments the temperature at the dermal-fat interface was also ascertained. A pig under Nembutal anesthesia Mas clipped and shaved. The hypodermic needle thermocouple Mas introduced laterally into the dermal-fat interface. The skin temperature at the chosen site Mas deter- mined and the automatic energy recording appli- cator Mas applied. Thus a continuous record of the caloric uptake of the. skin at a predetermined epi- dermal surface temperature Mas obtained. The tem- perature at the dermal-fat interface Mas determined either intermittently vith a & Northrup Type K2 potentiometer or continually xx ith a Ceneral Electric photoelectric recording potentiometer. Caloric uptake rate of pig skin: Typical caloric up- take data as a function of time and epidermal surface temperature are presented in Table 5. The data given in Table 5 are a composite of at least three determinations on the lateral thoracic area of different pigs; five pigs in all Mere used. As Table A guide ( +30 |xt cent1 to the caloric uptake* of the skin as a function of time and surface temperature as determined l»v the automatic energy recori ling applicator. Time inter- Skin surface temperature val in min 45 (' 501’ 55 (' - dcrmal fat in lateral thoracic area of a 10-kg pig lies about 2 mm below the skin surface. Ten-minute ex- posures to surface temperatures of 50 to 70C in- creased significantly the thickness of the dermis. This increase in thickness was due to the accumulation of edema fluid in the dermis and the effect was maximal when the skin surface was maintained at about GO C. Skin surface temperatures of 45 C or below do not activate the mechanism which gave rise to edema. Skin surface temperatures equal to, or greater than, SO C denature the curium so rapidly that (he mechan- ism by which edema fluid accumulated in the curium was destroyed. 2. Although the continual caloric uptake by the skin (ended to increase the dermal temperature, the appearance of relatively cool edema fluid tended to decrease it. At skin surface temperatures of 50 C and TOC, these two effects nearly counterbalanced each other, and after the first minute of heat exposure the dermal-fat interface temperature remained essen- tially constant. With skin surface temperatures be- tween 55 and Go C t he rapid appearance of a large amount of edema fluid more than compensated for caloric uptake, and the temperature at the interface between dermis and fat was temporarily lowered. This effect was maximal when the skin surface was maintained at about GO C. 3. When the skin surface temperature was main- tained at 45 C, and probably at all other tempera- tures that fail to cause edema, the dermis becomes “heat-saturated” after about 5 minutes of exposure. When edema fluid w as produced, the time for dermal heat saturation was essentially indeterminate, but it apparently was greater than 10 minutes. 4. Histological examinations showed that com- plete primary injury to the dermis was obtained in all experiments where the skin surface temperature was maintained at 65° or higher. These limited (5) time-temperature-injury data at the dermal-fat in- terface tended to indicate a quantitative relation- ship very similar to that found for epidermal injury' (see Section 17.7). 5. By making t he reasonable assumption that the dermis is essentially “heat-saturated” at the end of a SECRET 318 STUDIES OF THERMAL INJURY CUTANEOU S \ M) SYSTEMIC 10-minute heat exposure, the in vitro thermal con- ductivities of dermis can be computed by substitut- ing the approximate caloric uptake (Table 5), dermal- fat interface and skin surface temperatures, and the final dermal thickness into equation (3) (Section 17.3.1); the neglect of the epidermal temperature drop introduced no appreciable error. Table 5 also shows the results of these calculations. A comparison of these values with the experi- mentally determined in vitro values (Table 4) for pig dermis indicated that the presence of edema fluid increased the thermal conductivity of dermis two- to threefold. This increase in conductivity, however, was slightly more than compensated by the swelling of the dermis; an edematous dermis is thus a some- what better heat barrier to the underlying tissues than normal dermis. A comparison of the in vivo thermal conductivity obtained at 45 C with the in vitro value of 0.053 (see Table 4) tends to indicate that intact circulation probably increased the effec- tive thermal conductivity of dermis by about 15 per cent. Estimation of Temperature Changes at Epidermal-Dermal Interface during Exposure of the Skin Surface to Heat In view of the thinness (~80g) of the pig’s epi- dermis, the experimental measurement of (he time- temperature relationships at the epidermal-corium junction was not feasible. There are certain facts, however, that allowed the estimation of this time-temperature relationship with a considerable degree of certainty. In view of the ex- treme thinness of epidermis, the temperature of the basal layer was largely determined by skin surface temperature, which was an accurately known quan- tity. This is most readily seen by solving heat con- duction equation (3) for steady-state temperature of the basal epidermal layer. Of the four necessary ex- perimental quantities, namely, skin surface tempera- ture, epidermal thickness (alxnit HO/d, epidermal thermal conductivity (Table 4), am 1 caloric uptake of the skin at the requisite skin surface temperature (Table 5), only the last two were subject to a con- siderable variation (±30 pier cent). Fortunately, even variations of this magnitude resulted in uncertain- ties of less than 0.2 C in the steady-state tempera- tun' of the basal epidermal layer. Basal Epidermal Temperatures When the. Skin Sur- face is Immediately Brought to amt Maintained at a Temperature between C and 100 C. Before the steady-stale temperature is attained, the time-tem- perature relationship at (his epidermal-dermal junc- tion is given under these conditions to a good approx- imation by equation (tie) of Section 17.3.1, where y has the following numerical value: y — 0.15 if the time I is expressed in second*. The numerical constant, 0.15, is not subject to the experimental uncertainties of the quantities requisite to computation by equation (0a), since it can la1 quite accurately determined empirically fiom the temperature-time-epidermal injury data (see Sec- tion 17.0.5 for details). An identical value for y can also he.directly computed by substituting into equa- tion (0c) the experimentally determined values for heat capacity, thermal conductivity, and thickness of epidermis, and by assuming an epidermal density of 0.8 g cc (a most reasonable value). In view of (he two completely independent, methods, one of which was in vivo and the other in vitro, considerable confi- dence could be placed in the -adaptation of the in- finite body picture (see Section 17.3.1) to the solution of the time-tomperature relationship at the epi- dermal-dermal junction during (he unsteady state jjeriod of heat How. The computation of the temperature of the basal cell layer of the epidermis as a function of both time and skin surface temperature is given in Table 7A. Table 7A. The computed time-temperature relation- ships for (lie epidermal-dermal interface when the skin surface is immediately brought to and maintained at a specific temperature. Time in seconds Surface temperature, C 45 55 05 SO TemiMTature at basal epidermal 100 layer* 0 0 35.0 35.0 35.0 35.0 35.0 0.01 30.3 37.0 0.02 38.9 10.9 13.4 0.05 41.8 45.2 50.3 57.1 0.1 40.1 45.2 50.3 57.9 68.2 0.2 41.3 4 7.0 53.9 63.3 75.9 0.5 12.7 50.4 58.1 09.6 85.1 1.0 13.3 51.0 60.0 72.4 89.1 20 43. S 52.6 61.4 74.0 92.3 5 44.2 53.5 62.7 70.6 95.1 10 44.5 53.9 03.4 77.6 96.0 30 44.7 54.4 01.1 78.6 98.0 tiOCl min) 44.S 51.6 64.4 79.0 98.6 120(2 min) 44.9 54.9 64.5 79.4 99.2 300 (5 min) 44.9 54.9 64.7 79.5 99.3 000 (10 min) 44.9 54.9 64.8 79.7 99.6 Steady 44.8 54.5 61.2 — stale f * Computed by equation (t»c) and nrpprimpnt&I < lata of Section 3.2. t Computed by equation (3) and experimental < lata of Section 3.2. SECRET BASIC CHARACTERISTICS OF HEAT AM) HEAT TRANSFER 319 Table 7H. The computed time-temperature relation- ships for (lie epidermal-dermal interface when an entire animal {^- 30 cm in diameter) is surrounded by an on- velojie of i unbient and radiant heat that results from a constant (em|ierature source. ( 'ircumambient temperature, C 80 100 125 1.50 175 Time Heat transfer coefficient //* in in cal/cm*/tn in |ier f seconds 0.015 0.019 0.021 0.024 0.020 Temjierature at basal epidermal layer, ft,} 0 35 35 35 35 35 10 37 39 40.5 44 40 20 40.5 lit 30 38.5 41.5 44 49 52 40 51 54.6 50 30.5 43.5 40.5 53 57 70 to 44 48 56 00 100 41 45.5 50 59 04 130 42 47 52 01 100 42.5 48.5 54.5 03 200 43 50 50 05 ' 300 15 52.5 59 400 40 55 03 500 47 GOO 48 800 50 1,000 50.5 — 1,200 51 • In order U > make these data directly comparable to the. ex peri mental i(ivestiga lions t »f StTtion 17.0, the radiant contribution to // wa * computed by using a sour ce temperature 20 j»er cent in excess of the air temperature. t Computed by mean s of equations (o). (6), (fia), ! and (6b) and exjieri- mental data of Section 1 t Beeau.se of hoth the thinness of the epidermis and the slow rate of heat transport to the skin. there is no appreciable diffe renee between these tenijjeratures a nd 1 howe i r>f the skin surface after the first 20 sore »mls of heat ex|)0»urc. temperature and the skin surface temperature was trivial. It must he re-emphasized that these data apply only to situations in which the heat transfer coeffi- cient II from the temperature source to the skin sur- face is infinite.® In all cases where II is finite an analy- sis similar to that given below is required. liasnl Epidermal Temperatures When the Entire Animal Is Surrounded by an Envelope of Ambient and Radiant Ileal between SO and 175 In the previous section, the time-temperature relationships at the epidermal-dermal junction depended'only upon the rate of heat transfer through the skin and the con- stant temperature of the heat source. To this must now be added the slow rale at which heat is trans- ported from the heat source to the skin surface via air conduction, air convection, and infrared radi- ation. The mathematical solution of this problem is given by equation (0), where the only quantity that requires further consideration is //, the heat transfer coefficient from the heat source to the skin surface. This quantity is readily computed through the sub- stitution of equation (I), heat transfer by convection, and equation (2), heat transfer by radiation, into equation (5). The numerical values of the heat trans- fer coefficient which were obtained at certain source or air temperatures are shown in Table 7B. A com- parison of the numerical values of //, 0.015 to 0.020 calorie per square centimeter per minute per C, with epidermal thermal conductance K/L (Table 4) nu- merically equal to 4 in the same units, indicates the slow rate at which ambient and radiant heat is trans- ferred to the skin surface as compared with the rate this heat flows through the epidermis. Table 7B also gives tlie estimated temperature of the basal epidermal cell layer as function of source or air temperature as calculated by means of equation (6). These data show the ext reme slowness of tem- perature rise at this epidermal-dermal junction. In fact, under these conditions, the epidermal tempera- ture even after a heat exposure of 15 minutes is far lower than the temperature of the heat source, and one would expect an animal to succumb to hyper- thermia long before the temperature of the skin approached that of the air. Although the data for the time-temperature rela- tionship at the skin surface are not given, they can be The data given in Table 7A show that (here was a rapid rise in the temperature of the basal epidermal layer when the skin surface was immediately brought to and maintained at a specified constant tempera- ture. A comparison of the unsteady-state data com- puted from equation (0c) with the steady-state data obtained by means of equation (it) showed that the epidermis under the above conditions became essen- tially “heat-saturated” after a heat exposure of 0.5- to 1.0-minute duration. Actually, only the unsteady-state time-tempera- ture relationship as given by equation (6c) need be considered to elucidate the irreversible epidermal in- jury threshold data of Section 17.6.5; since these ex- perimental tinne-temperature-epidermal injury rela- tionships were such that for all skin surface tempera- tures above 50 C the epidermis never reached heat saturation, and for all temperatures below 50 (' the difference between the steady-state basal epidermal * Under (lie experimental conditions to be dcscrilicd in Section 17.6 (hot water experiments), II is not infinite 34 hut rather about 105 cal/eml/min j>er C. In these computations t tie sulwtitulion of for l(p is of no significance. SECRET’ 320 STUDIES OF THERMAL INJURY CUTANEOUS AND SYSTEMIC readily computed by putting L (the thickness of the epidermis) equal to zero in equation ((»). If this be done, it will be found that, except for the first, 20 seconds of heat exposure, the skin surface tempera- ture Is not significantly different from the values re- corded in Table 7B for the basal epidermal tempera- ture. This is due to the fact that heat transfer to the skin is the controlling factor. Thus, these data can also be taken as the temperature of the skin surface as a function of time. A comparison of Tables 7A and 7B indicates the importance to the epidermal time-temperature rela- tionships of the mode of imparting heat to skin sur- face. Thus,fora given source temperature, a mecha- nism that enables the surface temperature to be im- mediately brought to and maintained at the source temperature has, on a time basis, at least a thousand times greater injury propensity to epidermis than a heat source which raises the skin temperature by means of radiation, conduction, and convection of relatively immobile air. (See Section 17.9.2 under “Measurement of Heat Transfer ”) 17.3.3 Summary The various physical factors which determine the transfer of heat energy to and through (he skin and the temperatures attained thereby have been de- fined and discussed. A general theory of heat flow through the epi- dermis is developed. Experimental observations pertaining to the rate at which heat energy is taken up by the skin during surface exposures of varying intensity and the sub- surface thermal gradients established therein have been presented. The time-temperature relationship at the dermal- epidermal junction is computed under two greatly different experimental conditions: (1) when (he skin surface temperature is immediately brought to and maintained at the Temperature of the heat source, and (2) when the entire skin surface is exposed to a specified circumambient and eircumradiant temper- ature. These data indicate the extreme importance of the mode of applying heat to the skin surface to the time-temperature relationships within the epidermis. ITT EFFECTS OF INHALED HEAT It was inferred from the results of the pilot experi- ments (Section 17.2.5) that, so far as rapid neutral- ization of enemy personnel by flame thrower attack is concerned, the effects of heat on the surface of the laxly are probably of greater importance than are its effects on the air passages and lungs. The implication of this assumpt ion is too great to accept at face value the small amount of evidence provided by the pilot experiments. A search of the literature failed to disclose any re- liable information concerning the effects on the lungs and air passages of inhaled heat or the circumstances in which thermal injuries of the respiratory tract may be sustained. The following investigation was accordingly undertaken.*7 IT.1.1 Experimental Procedure In order to study the effects of heat on the respira- tory tract independently of the secondary changes that might result from concomitant burning of the skin, dogs were caused to breath hot air which was conducted directly to the trachea through an in- sulated transoral cannula. In some experiments heated air was pumped di- rectly into the air passages and in others it was in- haled by the respiratory efforts of the animal. The inner end of the cannula extended below the vocal folds of the larynx. Three types of inhalation experi- ments were performed. In the first the animals breathed room atmosphere heated to temperatures as high as 500 C in an oven. In the second, flame from a blast burner at temperatures estimated to be it! the vicinity of 1000 C was directed into the ex- ternal end of the cannula. In the third, a mixture of live steam and air was breathed from a generator (see Figure 5). All experiments were conducted un- der anesthesia induced by the intravenous or intra- peril oneal injection of sodium pentobarbital. The external temperature of the air available for respiration in each type of experiment was measured either by a thermometer or a platinum-rhodium thermocouple. Thermocouples (10 gauge eopper- constantan) were installed in the airway, one at the laryngeal end of the transoral cannula and the other at or near the bifurcation of the trachea, to measure the rate at which the inhaled air was cooled. I-eads from these thermocouples were connected with a Mold galvanometer having a period of 0.2 second. The excursions of the galvanometer wore observed directly and recorded manually. IT.T2 Kate of Cooling of Inhaled Air When the superheated air was inhaled, the tem- perature recorded by both the laryngeal and the SECRET EFFECTS OF INHALED HEAT 321 Fkii uf. 5. Experimental procedure used to investigate effects of inhaled heat on air passages and lungs. In all instances, insulated cannula conveyed hot air, flame, or steam from outside to animal’s larynx. Position of intra-laryngeal and deep tracheal thermocouples is shown. Top left view: Animal breathed room temperature heated in oven to 350 Top right view: Room temperature was pumped into animal’s lungs from combustion oven which was heated to 500 C, Bottom tefl view: Flame and combustion products of blast burner were projected into cannula during each inspiration. Bottom right view: A 400 ml blast of mixture of live steam and air was released into Iransoral cannula at the beginning of each inspiratory effort. Results of these exjjcrimonts arc shown in Table 8. Table 8. Results of experiments in breathing of hot air _ Kind of atmosphere breathed Xo, Animal Xo. Original pre- inspiratory temp of air (C) (approximate) No. of breaths Max temperature - recorded (C) Laryn- geal Lower cannula trachea Recovery period (hours) Site and severity of injury Upper Lower trachea trachea Tilings Air from dry- 1 423 350 46 182 19 Mild None None ing oven. 2 420 350 52 ISO 19 Mild None None See Fig. 5A 3 391 350 103 159 30 Mild None N one 4 390 350 lOti 175 Not (Complete clinical recovery —■ killed no autopsy) Air from com- 5 392 500 GO 267 4 Mild None None bust ion oven. G 432 .500 44 327 50 7 Moderate None None See Fig. 511 7 42G 500 22 291 24 Mild None None 8 431 17 135 7 Moderate Mild None Flame from 9 433 10 327 51 S Severe. Moderate Mild blast burner. 10 454 16 540 100 11 Severe Mild None Sec Fig. 5C 11 455 24 550 65 24 Moderate Mild None 12 405 14 510 64 Not (Complete clinical recovery - killed no autopsy) Steam from 13 456 Over 100 27 106 59 - 6 Moderate Mild None generator. 14 519 Over 100 18 98 79 7 Severe Moderate None See Fig. 5D 15 4S1 Over 100 20 94 53 10 Severe Severe Severe )G 475 Over 100 16 99 94 10 Severe Severe Severe 17 524 Over 100 10 90 24 Severe Severe Moderate IS 522 Over 100 12 75 18 Severe Severe Mild tracheal thermocouples rose throughout inspiration and fell during expiration. In each situation the high- est point in the temperature curve was reached at or near the end of inspiration. The inhaled gas lost most of its heat before reaching the lungs. When the in- haled gases were relatively dry, the intratracheal temperature rose to a sharp peak and fell away rapidly during expiration. When steam was inhaled, the curve described a plateau rather than a peak, probably because of the condensation of hot water on the thermocouple. The results of these experiments are shown in Table 8. When air heated to bet ween 350 and 500 C was in- haled, the temperature fell to about half of its ex- SECRET 322 STUDIES OF THERMAL INJURY — CUTANEOUS AND SYSTEMIC Finnic 7. Thermal tracheitis and pneumonitis. Photograph of respiratory tract of dog 10 hours after inhalation of steam, showing severe tracheobronchitis with dilatation of bronchi. There is central hemorrha- gic pneumonitis with generalized pulmonary edema and hyperemia. Finnic ft. Thermal laryngitis and tracheitis without pulmonary injury. Photograph of respiratory tract of dog 24 hours after inhalation of flame. Sufficient heal had licen conducted through wall of cannula to cause mild degree of laryngeal edema which may be recog- nized by bilateral olive-shaped mucosal protrusions from ventricular recesses. There was extensive destruc- tion of mucosa of upjKT trachea, diminishing rapidly to mild catarrhal inflammation in lower third Xo abnor- mality of bronchi or lungs of this animal was recog- nized. — of the trachea in such experiments was 135 C. When a mixture of live steam and air was inhaled, the in- spiratory peaks recorded at the laryngeal opening of the cannula ranged between 04 and 106 C and those by the deep tracheal thermocouple, between 53 and 94 C. 17.4.3 Effects on Animals The mildest thermal exposure used in the inhala- tion experiments was more than sufficient to cause severe injury to the skin. Every animal included in Table 8 would have sustained severe cutaneous in- jury if the skin had been exposed for more than a few ternal level by the time it reached the larynx, despite the fact (hat it was conducted through the mouth by means of an insulated cannula. By the time it, had reached the bifurcation of the trachea, the tempera- ture hail dropped to approximately 50 C. Flame and combustion products of a blast burner directed into the external end of the transoral cannula were de- livered to the larynx at temperatures between 300 and 550 C. The highest recording at the bifurcation SECRET 323 EFFECTS OF INHALED HEAT seconds to such temperatures. Circumambient air temperatures as low as 300 C produce severe injury of unprotected skin within a few seconds. Mixtures of steam and air at 100 C destroy epidermis even more quickly. Early in the investigation it was found that if ani- mals were to survive the inhalation experiments long enough to develop reactive changes in the lower air passages it was necessary to protect the larynx. Otherwise they died prematurely of asphyxia due to laryngeal edema. For this reason the transoral can- nula was inserted well lx*low the glottic folds. Primary thermal injury of the lungs occurred in none of the 7 animals that breathed hot air, in only 1 of the 5 animals that inhaled flame from a blast burner, and in 1 of the 0 animals that inhaled live steam. In the remaining animals thermal injury to the respiratory tract was confined to the upper air passages. In no instance did an animal die as a result of thermal injury of the lungs within the first 24 hours. All animals that sustained thermal injuries of the respiratory tract would, under nonexperimental conditions, have received severe cutaneous burns. Mucosal necrosis with desquamation of surface epithelium o< sirred in alt instances where the blast of hot atmosphere first struck the lower portion of the larynx and the upper portion of the trachea. In the case of hot air the injury was usually localized ami represented by shallow ulceration associated with catarrhal inflammation of the upper third of the trachea (Figure 6). Inhalation of flame or steam led to extensive destruction of the trachea with edema of the peritracheal areolar tissue of the neck and mediastinum and detachment of large casts ot ne- crotic mucous membrane, which were either expelled by coughing or subsequently inhaled into the lower portions of the respiratory tract (Figure 7). The portions of the lungs most vulnerable to in- jury' were the centrally located alveolar ducts and their communicating alveoli (Figure 8). Atmosphere not hot enough to damage the mucosa of the large bronchi or the alveoli of the more peripheral portions of the lungs was in some instances capable of causing central pulmonary edema and both intra-alveolar and interstitial hemorrhage. After more severe ex- posures the lungs became diffusely edematous and hemorrhagic. Focal patches of atelectasis and em- physema were observed and in some instances were obviously due to aspiration of mucus or mucosal debris. Bronchopneumonia was commonly' observed in animals that had received tracheal burns. It ap- pea red that, regardless of the mildness of the pri- mary thermal injury of the lungs, if the inhaled air was hot enough to damage the trachea it usually predisposed the animal to pneumonia. IT.t.l Discussion Tf was apparent, from the foregoing observations that air hot enough to burn the skin can be inhaled without causing damage to the trachea or lungs and that if the temperature of the air is high enough to damage the respiratory passages it will inevitably have caused burning of the surface of the body. This observation seemed paradoxical in view of the fact that the mucosa of the air passages is much thinner than the skin and should therefore be more vulnerable to thermal injury. The explanation of the experimental findings lies in the fact that the quan- tity of heat that can lie stored in the volume of gas that constitutes a breath is remarkably small. At any given air temperature the number of calories that can be transferred to the respiratory tract incident to the inhalation of a breath of hot air is limited by the vol- ume of that breath, whereas convection currents are capable of bringing a practically unlimited volume of hot air in contact with the skin. An infinitely greater caloric transfer can occur for each unit of surface exposed. Not only is the amount of heat energy available for transfer to the skin greater than that which is available for transfer to the respiratory membranes but also there are Important time differences be- tween cutaneous and respiratory exposures. In the case of the skin the exposure is virtually continuous, whereas the lining of the air passages is exposed in- termittently as each new breath is inhaled. An instructive illustration is provided by calcu- lating the potential heat trmisfer to the respiratory tract that might occur if air were inhaled at 142 C. Ix*t it be assumed that the amount inhaled with each breath would be sufficient to increase the pulmonary volume by 500 ml, that the air was dry when inhaled and saturated with moisture when exhaled, and that it was cooled to body temperature by the time it left the body. Approximately 13 cal of heat energy could be released within the hotly by cooling of one such breath from 142 to 38 C. Theoretically this amount of heat would be sufficient to raise the tem- perature of 1 g of tissue by approximately 13 degrees, providing none of it was carried away by the blood circulating in the subsurface capillaries. Actually no change in the net temperature of the respiratory SECRET 324 STUDIES OF THERMAL INJURY — CUTANEOUS UVD SYSTEMIC Fic.cue S, Primary thermal pneumonitis. Photomicrograph of lower lol>e of dog’s lung 24 hours after inhalation of steam. Although there was severe tracheitis, primary and secondary bronchi showed remarkably lit tie change. Evidence of pulmonary injury was confined largely to the central {tortious of lobes and consisted of hyperemia, edema, and partial atelectasis. tract would occur in such circumstances because the gain of 13 cal would be offset by a loss of 13 cal inci- dent to the evaporation of the 23 mg of water that would be required to saturate that amount of dry air. This is not to imply that the inhalation of air heated to 142 C would be necessarily harmless. Desic- cation would probably occur near t he portal of entry even though there were no net change in the temper- ature of the respiratory' tract as a whole. The calcu- lation serves to emphasize how important the heat capacity of the inhaled gas is in relation to the prob- lem of thermal injury of the lungs. A rise in tissue temperature is prerequisite to the occurrence of thermal injury and the amount (hat the tissue tem- poraturc is raised incident to any given exposure will depend in part on the magnitude of temperature dif- ferential and in part on the amount of heat energy that the inhaled gas is capable of storing. A more important attribute of an inhaled hot gas than its temperature in relation to its capacity to cause thermal injury is its water content. When steam or a mixture of steam and air comes in contact with a cool surface such as the skin or the lining of the respiratory tract, water is condensed on the sur- face with liberation of a relatively large amount of heat. Thus the cording of a 500-rnI mixture of equal parts of steam and air from 125 to 38 C would lead SECRET COMPAlii.SON OF PORCINE VM) III MAN SK.IN 325 to the condensation of about 800 mg of water. The heat energy liberated by this amount would lx* in the neighborhood of 175 cal. There is little doubt but that the sudden liberation of 175 cal to the lining of the air passages or on the surface of the skin would !x> capable of causing some injury. 17.1.5 Summary It was apparent from these experiments that ther- mal injury of the lungs is probably a negligible factor in the causation of disability or death incident to exposure trreonflagrations such as might result from Hame thrower action. A thermal exposure of suffi- cient, intensity to cause direct injury of the lungs was more than sufficient not only to cause extensive burn- ing of unprotected skin but also to result in rapidly fatal obstructive edema of the glottis. In the case of externally unburned or mildly burned casualties of a flame attack it can be assumed that no significant thermal injuries of the respiratory tract have been sustained. 17.5 COMPARISON OK PORCINE AND HUMAN SKIN The original choice of the pig as a suitable subject for this investigation was based on the fact that no other readily available animal has skin that bears so close an anatomical resemblance to that of man. A comparison of the structural characteristics of porcine and human skin at this point seems desirable in view of the extent to which the pig was used in ex- periments designed to provide information regarding ( I) the reciprocal relat ionship of time to temperature in the production of cutaneous injuries in man, and (2) the local and systemic disturbances in man which cutaneous hyperthermia may be capable of causing. Like that ol man the surface of the pig's body is covered by three layers of tissue. Progressing from outside in, these are the epidermis comprising strati- fied epithelial cells, the dermis comprising fibrous connective tissue, the hypodermis comprising fibrous connective tissue, and the hypodermis comprising fit>roadipose tissue (see Figures 9 and 10). 17.5. i Epidermis The epidermis of the pig varies in thickness, the average over the lateral body surface of immature animals (8 12 kg) Ix-ing approximately 0.1 mm, which is slightly less than that from corresponding areas of adult human subjects. As with man there are irregularities in contour of both the upper and lower surfaces of the epidermis, those on the upper surface being due to an intricate system of intercom- municating linear depressions and those on the lower surface corresponding to the dermal papillae over which it is moulded. The hairs penetrating the epi- dermis of the pig are thicker and more numerous than those of man. Microscopic appearance of epidermis: Like that of man the outermost zone of epidermis or stratum coraeu m of the pig consists of several loosely con- nected layers of the desiccated and intensely baso- philic remains of keratinized epithelial cells. The second or granular layer is thin and consists of several layers of dead or dying squamous cells, the acidophilic cytoplasm of which contains many fine, deeply basophilic kerato-hvaline granules. Many of these cells have lost their nucleuses. Others contain shrunkenTiyperchromatic nucleuses or Feulgen nega- tive nuclear ghosts... The third zone is comprised of several layers of aging squamous cells which nolonger have any direct cytoplasmic attachment to the dermis. The cyto- plasm is dense, deeply acidophilic, and ap|>ears des- iccated. The cells are so closely packed that neither intercellular bridges nor spaces can be recognized. Many of the nuclei are relatively small and more densely packed with chromatin granules than those of the deeper cells. The fourth zone consists of cells in transition lie- tween the squamous and the basal cell layer. The transitional cells are large and polyhedral and many of them still have an attenuated footlike cytoplasmic attachment to the dermis. It is in this zone that in- tercellular bridges of tonofibrils are most readily visualized. The cytoplasm is moderately basophilic. The cell outlines are distinct and the intercellular spaces are clearly defined. The nuclei arc larger and rounder than those of the more superficial cells and contain several coarse and many fine granules of chromatin. The fifth zone is comprised of the basal cells, which, except for their cuboidal or columnar shape and their palisadelike arrangement on the dermis, are essentially similar to the overlying transitional cells. Projecting from the inferior surface of the basal epidermal cells of the pig are many robust tono- fibrils which appear to be embedded in the dense felt work of fine collagen fibrils that comprise the super- ficial zone of dermis. No such fibrillar anchorage of epidermis to dermis can be seen in human skin (see Figures 19 and 20). SECRET 326 STUDIES OK THERMAL INJURY — CUTANEOUS AND SYSTEMIC Appearance of porcine (Figure 9) and human (Figure 10) skin under low magnification, stained with phloxinc-methylenc blue. Sections are representative of lateral thoracic region of pig and lateral abdominal region of man. Epidermis is slightly thicker in man, and dermal papillae are broader in pig. Collagenous bundles in dermis of pig are heavier than those in man. Glands shown in hypoderrnis of pig do not secrete sweat. Figure 9 Figure 10 Tlte microscopic appearance of ( he epidermis of hot it man and pig suggests that there is a progressive loss of intracellular water as the epithelial cells grow older anrcine skin which measured 2\2x2 mm. Series of thick (50m) benzidine-treated horizontal and vertical sections were mounted in such a way as to show distribution of veins, arteries, and capillaries at various levels beneath surface. No. I shows capillary plexus lying in most superficial (50m) portion of dermis. No. 6 shows vessels in most superficial layer of adi|«isc tissue of hypodermis. only slightly less compact than the reticular zone. The deeper portion of the reticular connective tissue sends trabecular extensions into the underlying adi- pose hypodermis. Blood Vessels of Porcine Skin It was observed in ordinary histological prepara- tions that the appearance of the capillaries in the dermal papillae of the body skin of the pig is similar to that in corresponding regions of man. In recog- nition of the fact that it is difficult or impossible to get an accurate impression of so complicated a structure as a capillary network by two-dimensional visualization, a modification of the Pick worth tech- nique 10 was employed in order that the dermal blood vessels could lie studied in three dimensions. Maximum cutaneous hyperemia was induced be- neath a circumscribed area of the lateral body sur- face of the pig by exposure to water at 50 C for 20 minutes. After such an exposure the erythrocytes were so densely parked in (he distended capillaries that there was practically no loss of blood from them when the skin was excised. Skin and subcutaneous tissue treated in this way was excised to a depth of 8 mrn, fixed in 10 per cent formalin, cut in thick sections, and treated with benzidine. The benzidine combined with the hemoglobin to impart a dark blue color to the contents of the en- gorged vessels. After skin treated in this manner was cleared, a three-dimensional study of its blood vessels could be made by use of a binocular microscope. The appearance of the dermal vessels of porcine skin at various levels below the surface is shown in Figure 11. To prepare this illustration a block of benzidine-treated skin was cut serially and parallel to the surface in sections measuring 50 p in thick- ness. Another block of the same skin was cut serially and at right angles to the surface. Photographs were made of both series and the prints were mounted in such a manner as to orient the horizontal sections in SECRET 328 STUDIES OF THERMAL INJURY CUTANEOUS AND SYSTEMIC relation to the depth below (he surface that each represented. The epidermis was removed from the surface of the block of skin shown in Figure 11. The excised skin was not clamped prior to fixation and postexcisional contraction resulted in an accentuation both in the height of the dermal papillae and also in the thick- ness of the dermis. It may be seen that (he fibrous dermis including the papillae measures approxi- mately 2 mm in thickness and that broad septa of fibrous connective tissue extend down from the der- mis at more or less regular intervals into the under- lying fat. In approaching the surface the blood vessels to the skin followed an oblique course through the hypo- dermis and after reaching the lower layer of the fibrous dermis branched horizontally with multiple intervenal and interarterial anastomoses. From these first approximately horizontal plexuses originated a series of broad vascular loops that penetrated to the mid-portion of the dermisT Interarterial and inter- venal anastomoses between these loops served to establish a mid-dermal plexus. From this mid-dermal plexus originated numerous hairpin-shaped capillary loops which extended upward into the dermal papil- lae. These capillary loops anastomosed freely with one another and constituted the most superficial or papillary plexus. It was apparent that capillary com- munications between the arterioles and venules oc- curred at different levels. Some followed a course that brought them to within a few microns of the basal epithelial cells over the tips of the papillae. Still others followed an almost horizontal course to establish communications between the arterioles and venules of the intermediate plexus. At all levels through the dermis there were numerous vascular communications with the mantlelike mesh work of capillaries that surrounded the hair follicles and dermal glands. As may he seen in Figure 11 the number, size, dis- tribution, and communications of the dermal blood vessels of the pig are remarkably similar to those de- scribed by both Lewis” and Spalteholz M in human skin. The similarity of blood vessels in human and porcine skin was found to be so great that it was with difficulty that one could be distinguished from the other in Pickworth preparations. It is not intended to imply that the anatomical re- semblance between t he vessels of human and porcine skin implies an equal degree of functional similarity. Certainly the vascularization of both indicates that ample and similar mechanical facilities exist either for the transfer of body heat to the surface to facili- tate its dissipation, or tor the conduct of surface heat to the interior to raise the internal temperature of the body. SweaTGlands and Sweating Several types of glands arc encountered in the dermis and hypodermis of the pig and, although one of them bears some resemblance to the sudoriferous glands of human skin, it does not secrete a significant amount of sweat. The fact that the pig does not sweat was verified by a series of experiments in which the water loss from the skin of living pigs was measured at various environmental temperatures, with and without the administration of pilocarpine (see Table 9). Tabi.K 9. Rate of water loss from surface of human and porcine "skin. Amount of water loss determined by weight of Mg (CICMj contained in base of weighing bottle during the time that the neck of the brittle was held in the skin. accretion in contact with Water uptake (mg/em’/min) during a period of 10 minutes Temp 21 C — Humidity 30-40% Temp 36 C -—Humidity 30—10% No, of No. of tests Min — Max Mean tests Min Max Mean Dead pig (lateral thoracic region) 4 Live pig (lateral thoracic region) 0.016 0.026 0.019 1 0.023 0.031 0.027 Without pilocarpine 5 Live pig (lateral thigh) 0.016 0.020 0.021 0 - 0.020 0.032 0.028 Without pilocarpine 1 0.018 0.028 0.024 With pilocarpine* (1 mg/kg bwt) Live man (forearm) 4 0.021 0.030 0.027 Subject #1 (A.R.) 1 • • • • . . . . 0.027 1 0.180 Without pilocarpine — Subject #2 (A M.) 2 Without pilocarpine 0.028 0.038 0.033 - 2 0.280 0.360 0.320 * Iodine color test negative. SECRET RECIPROCAL RELATIONSHIPS OF TIME ANI TEMPERATURE 329 It was found that the water loss from the skin of a live pig does not differ significantly from that of one that is dead. In a cool environment the water loss per square centimeter per minute is approximately the same in man and pig. At higher environmental temperatures, the rate of water loss from human skin is tremendously augmented, whereas the corre- sponding increase in water loss from the skin of a pig is relatively small and is due to more rapid evapora- tion of tissue water rather than to sweating. 17.5.3 Summary So far as can be judged by anatomic criteria the pig should be a suitable experimental subject from which to derive certain types of information regard- ing the effects of heat on human skin. Its various layers are of comparable thickness and structure. Its blood vessels are similar in size, number, and distri- bution. As will he show n later in Sections I7.(i and 17.7, its susceptibility and reactions to control epi- sodes of hyperthermia are remarkably similar. Since a pig does not sweat, allowance should be made for the inability of porcine skin to lose heat through the vaporization of moisture derived from sweating. The significance of heat loss through vapor- ization of moisture in respect to cutaneous burning will be discussed in greater detail in Section 17.9. 17.6 RECIPROCAL RELATIONSHIPS OF TIME AND TEMPERATURE* The most direct mechanism by which exposure of the body surface to excessive heat results in injury” is the transfer of heat energy to the skin at so rapid a rate that its temperature is raised to a level incom- patible with cellular survival. Such localized thermal injuries are commonly referred to as burns. Although it is common knowledge that there is an inverse re- lationship between temperature and the amount of time required to produce a burn, there is remarkably little precise information regarding the rate at which burning occurs at any given temperature. Because of the experimental difficulties inherent in the making of accurate measurements of either the time or the temperature characteristics of thermal exposures so intense that they are capable of burning the skin in a fraction of a second, it was decided to establish by experimentation the reciprocal relation- ships of time and temperature necessary to destroy cells at lower temperatures and to extrapolate from these data the time curve that should represent the minimum cell-destroying exposures for higher de- grees of temperature. 17.6.1 Method of Controlling Surface Temperature Direct exposure of the surface of the skin to a rapidly flowing stream of hot liquid was chosen as the method best adapted for the acquisition of these data. With this type of exposure, the surface of the skin could he maintained at the temperature desired without the establishment of an appreciable gradient between it and the source of heat. There was no in- sulation of the surface by a static layer of gas, liquid, or solid, no heat loss through vaporization of surface moisture, and no diminution of subsurface heat con- duction due to vascular occlusion by the application of pressure on the surface. The method was simple to .operate and led to remarkably reproducible, cut anc- ons effects. The applicator by which a running st ream of hot water was brought into direct contact with the skin consisted of a metal cup, the brim of which was cov- ered with a pad of closed-cell sponge rubber to insure a watertight contact. By means of an electric pump, water was circulated from a large constant-temper- ature reservoir through the cup, the open end of which was applied to the skin. The rate of flow was regulated by a screw clamp on the inlet, tube and by the height of t he outlet tube (see Figure 12). Fine he 12. Drawing of hot water applicator. Tangential flow of a liquid produced no vertical component of force and thus no vertical pressure. Vertical water pressure w ithin the cup could be var- ied between 70 and 8(i cm of mercury by suitable adjustments of the aperture of the inlet and the height of the outlet tubes. A copper-eonstantan ther- mocouple measured the temperature of the water flowing next to the skin. During any period of ex- posure the temperature of the water flowing over the skin could lie controlled to within 0.1 ('. SECRET 330 STUDIES OF TURK MAE INJURY — CUTANEOUS AND SYSTEMIC Two methods were used to equilibrate the appara- tus Ixffore applying it to the skin. In one, the appa- ratus was applied to a block of linoleum, adjusted to the desired pressure, and transferred to the skin site to be exposed as soon as the temperature equilibrium was reached. In the other, the applicator was allowed to remain immersed in the hot water reservoir with the pump turned on until thermal equilibrium was established. The cup was then transferred immedi- ately to the skin and adjusted to the desired water pressure. Provision was made in the construction of this ap- paratus for studying the relation of the size of the area of exposure to the intensity of the resultant in- jury, This was accomplished by making the brim of the cup removable so that the area of skin to be ex- postal could be varied according to the aperture size trf the brim selected for use. Thus, in the same region on the same animal and under identical conditions of time, temperature, and pressure, circular targets having a diameter of either 7 or 25 mm could be exposed. Individual bums in the animal experiments were 25 mm in diameter. This was larger than desirable for human subjects and the diameter of the aperture of the cup was accordingly reduced to 7 mm for the human experiments. Before this was done, however, it was established by animal experimentation that the reduction in the size of the exposure area did not make any appreciable difference in the effect of such exposures on the epidermis. Water was employed as the source of heat in all of the experiments summarized in Table 10. Because the question was raised as to whether or not a hypotonic fluid such as water might modify the effects of heat, a series of comparable exposures were made in which oil was substituted for water. There was no appre- ciable difference between the injury-producing po- tentiality of rapidly flowing streams of water and oil on either animal or human skin so long as the temper- ature and duration of exposure were the same. IT.6.2 Experiments on Pigs The primary purpose of this investigation was to obtain information relating to the tolerance of hu- man skin to episodes of hyperthermia of varying duration and of varying degrees of intensity, and the direct approach would have l>een to make all experi- ments on human subjects. For various reasons, this was not feasible, and it was decided to acquire the basic data from experiments on pigs. From an ox- tensive series of observations on pigs, it was thought that a relatively small number of critical exposures of human skin would establish the extent to which the more comprehensive animal data were applicable to man. Closely clipped young (8 to 12 kg) white pigs were used. It was found that different portions of the body surface of the pig vary slightly in respect to their susceptibility to thermal injury. The largest uni- formly reacting area was the lateral body surface be- ginning in front of the thighs and extending forward over the shoulders. The skin of the neck and for about 10 cm to either side of the spine had a slightly higher thermal tolerance than ( hat of the lateral body surface. The skin covering the thighs, the buttocks, the inguinal folds, and the mid-portion of the chest and abdomen had a slightly lower thermal tolerance. Results of experiments on pigs: The surface tem- pera! ure, duration, and results of 179 hot water appli- cations to the lateral body surface of young white pigs are summarized in Table 10. The surface temperatures at which these exposures were made ranged between 11 ami 100 C. The dura- tion of exposures varied between I second and 7 hours. The majority of the exposed sites were kept under observation until the reaction had subsided or the lesion had healed. In the ease of borderline re- actions duplicate exposures were made and excised at the end of 24 or 48 hours for microscopic study. As indicated in Table 10, a wide variety of reac- tions were observed. These ranged in severity from evanescent erythema to deep ulcers. In the beginning certain difficulties were encoun- tered in recognizing differences in the severity of cer- tain lesions. Although there was no difficulty in recog- nizing the difference between a reaction whose total effect was a mild and transient erythema and one that led to deep coagulative necrosis, it was not al- ways easy to recognize by clinical observations whether a given lesion represented a severe first- degree reaction with incomplete or focal epidermal destruction or a relatively mild second-degree re- action in which the epidermal destruction was com- plete. Apart from the microscopic appearance, the most reliable criteria by which to recognize (ransepidermal necrosis were (I) the ease with which dead but still intact epidermis could be displaced by friction on the second and third days after exposure, and (2) the de- velopment of complete encrustation of such a lesion within a week. SECRET RECIPROCAL RELATIONSHIPS OF TIME VM) TEMPER ATI’RE Taiii.k 10. Time-surface temjierature thresholds for thermal injury of porcine skin. Threshold Threshold Suhthreshold and supra- Subthresbold and supra- oxjH)sures t hreshold exposures threshold __ exposures exposures 1 reactions 2 and 3° 1" reactions 2"and 3' react ions reactions Focal Complete Focal Complete Hyperemia epidermal epidennal Hyperemia epidermal epidermal Xo. only necrosis necrosis Xo. only necrosis necrosis Trill) Time of Seal- Small Red Pale Temp Time of Seal- Small Red Pale (' Min See csp( Mild Severe ins ulcers burn burn C Min See expt Mild Severe ing ulcers bum burn -14 420 1 + j 52 30 1 + 45 130 1 + / 1 2 45 1 + ISO I 30 1 1 1 + 46 45 1 + 3 00 + '.<0 1 + 53 20 1 + 30 45 + , + 40.5 45 00 1 1 + + 1 2 2 + + + 47 35 45 1 1 + + 1 2 30 3 1 , . 50 fiO I + -f 54 15 1 + — 25 35 48 10 3 + 1 1 + 42 14 1 2 + 55 5 1 1 1 + 14 15 1 2 + + 10 15 + F 10 IS 1 1 + 20 25 1 I + + 20 _ 1 ' ' + 30 3 F 4!) 3 + 56 10 1 + + 4 5 + 15 1 5 2 • 20 1 + 0 5 + 58 5 1 + 0 2 + 10 1 + 0 2 + 60 2 1 + 7 7 7 8 8 8 0 2 1 1 4 1 2 11 + + + + + © + 10 2 3 5 7 7 10 1 1 1 1 2 1 F + + + + + + 10 5 + 65 1 + 50 1 2 i i + + 2 3 1 1 _ + + 4 i + + 10 1 + 5 i 70 1 2 + 5 3 + -—_ 2 1 + 5 5 2 2 + 3 2 + 75 0 2 0 30 2 + + 1 5 1 1 + 4- 51 4 1 » 2 2 + + ' 80 i 5 I 1 + + 1 30 2 + So 1 1 + 2 I + + 5 1 + 3 2 90 i 1 ■f 3 3 2 2 + + 5 1 + 4 5 2 I + + 95 i 3 1 1 + + 5 1 + 100 1 1 + 10 2 + 3 1 + SKCRET 332 STUDIES OF THERMAL INJURY—CUTANEOUS AND SYSTEMIC Figure 13. Photograph of right and left sides of pig with temperature and duration of each exposure indicated. Lesions on right side were 24 hours old and those on left side 7 days old. All exposures sufficient to cause vascular reaction but insufficient to destroy the full thickness of the epidermis throughout Ihe entire target area were designated as subthreshold. The entire range of cu- taneous responses to subthreshold exposures were characterized as first-degree reactions. The shortest time at any given temperature that was capable of causing transepidermal necrosis constituted a thresh- old exposure. The effect of a threshold exposure on the skin was characterized as a second-degree re- action. All exposures which were of longer duration or higher temperature than was necessary to cause complete epidermal destruction were designated as suprathreshold and their effects as third-degree re- act ions. The macroscopic appearance of different degrees of cutaneous reaction to hyperthermia may he seen in (lie photographs of the right and left sides of pig 924 shown in Figaro 13. At tbo time t ho photographs wore made, the lesions on (ho right sido wore 21 hours old and those on the loft were 7 days old. It is ap- parent from those photographs that the duration of exposure at any given temperature was remarkably critical in relation to the kind of reaction evoked. It is equally apparent (hat the time required to produce a given degree of reaction varied inversely with the temperature. it.6.3 Experiments on Human Subjects In order lo determine the extent lo which the re- ciprocal relationships of time and temperature in the production of cutaneous hums in pigs were applicable to human skin, a series of exposures similar to those described on pigs were made on human subjects. Some were made to the skin of the anterior thoracic region and others on the ventral aspect of the fore- SECRKT RECIPROCAL RELATIONSHIPS OF TIME \ ND TEMPER ATL’HE 333 arm. The applications were made with the apparatus shown in Figure 12. As in the case of the pig experiments, three de- grees of skin reaction were observed. Reactions char- acterized as first-degree were those that fell short of complete destruction of the epidermis. At one ex- treme a first-degree, reaction consisted of a faint and transient erythema. At the other, extreme erythema was severe and prolonged and miliary vesicles formed but failed to coalesce. Lesions in which there was complete destruction of the epidermis over the entire target area w ere designated second- or third-degree reactions, depending on the extent to which the dermis was involved. As in the ease of tfie pig, a threshold exposure represented the shortest time at any given temperature that caused complete de- struction of the epidermis. ~- That a given exposure of human skin had resulted in transepidermal necrosis was usually but not al- ways recognized by complete vesication of the target area. Although vesication resulting from heat indi- cates t hat the full thickness of the epidermis has been destroyed, absence of vesication does not necessarily indicate that the epidermis has escaped complete destruction. Transepidermal necrosis without vesica- tion was observed after certain suprathreshold ex- posures. The explanation of this phenomenon w ill Ire discussed in Section 17.8.8. The results of the human experiments have been summarized in Table 11. 17.6.1 Relative Vulnerability of Porcine and Human Skin to Thermal Injury To facilitate comparison of the data included in Tables 10 and 11, certain ol the more critical observa- tions have been depicted graphically in Figure 14. The solid line w as established by points representing the lime and temperature of exposures that caused minimal second-degree reactions of porcine skin. The points by which this line was established are repre- sented by crosses. Each cross represents the shortest time at (he temperature indicated that resulted in transepidermal necrosis of the entire target area. The more that (lie time of any given exposure placed it to the right or that the temperature of any given ex- posure placed it above the solid line, the greater the depth to w hich the skin was dest royed. All exposures t hat were situated a significant distance above and to the right of the solid line were suprathreshold and all those situated a significant distance below and to the Table 11. Time-surface temperature thresholds for thermal injury of human skin. Threshold Sub- and supra- — threshold 1 hrcshold exposures exposures 1 reac- 2° and 3° Term lions reactions at Duration Hyperemia sur- of without Complete face exposure loss of epidermal Sub- Xo. C Hr Min Sec epidermis necrosis ject Date 1 14 5 + BF 270 2* 5 + BK 2 23 3 0 .. + BF 2/0 4* 0 .. 4- BF 2/23 5* 45 2 + + KL 2/16 «• 3 .. KL 2/3 7 T-» 3 + HA 2 4 8* 47 .. 18 .. .... — f UKt 2/13 9* .. 20 .. + + KL 2 25 10* .. 20 .. 4" AM 2 26 n* .. 20 + Ft; 2 20 12 .. 25 + UKt 1 78 13* 40 _p AM 2/26 14 .. 40 .. + PC 2/26 15 rr- 45 + UKt 1/8 10 48 15 + PG 7/19 17 15 + AH 7/19 18 .. IS .. 4" AM 6 20 19* 49 8 .. + AM 2/10 20 8 + AM 0/26 21 9 30 4 AM 6/26 22* .. 10 .. . ±.. AM 0/20 23 .. 11 .. + AM 0/26 24 15 4- AM 6/26 25 51 2 .. + AM 6/26 20 4 .. + AM 6 20 27 fi + AM 6/26 28 53 .. .. 30 -F AM 6/26 29 1 30 + AM 6/26 30 55 + PG 7/19 31 .. .. 30 + AH 7/19 32* 00 .. .. 3 + FH 2/1 33* + FH 2/1 * Oil list'd instead of water as source of heat. t Subject RK was atypical in that his threshold for thermal injury was significantly lower than that of other experimental subjects. left of the solid line were subthreshold. The range of variation is shown in Table 10. The extent to which the results of human exposure corresponded with those of the more comprehensive animal experiments is indicated by the open and solid circles in Figure 14. The open circles represent the maximum exposure at the temperature indicated that failed to destroy human epidermis and the closed circles represent the minimum time at the temperature indicated that resulted in complete destruction of human epidermis. The broken line in Figure 14 represents the ap- proximate threshold at which the first morphological SECRET STUDIES OF THRUM A L lAJl'RY— CUTANEOUS AM) SYSTEMIC of epidermal injury 12 as determined I»y histological examination is produced. T, is the temperature in (' at the time, t. at the basal epidermal layer; A* is the gas constant and is equal to 2 calories per C per mole; and both .1 and AE are constants evaluated from the experimental data. Kquation (7) can also be expressed as an integral equation, namely i 12 . .4 fe-W*T‘+™\U (8) 0 where if 7’,, the dependence of the basal epidermal temperature on time, is known the integral can be evaluated. In all eases where the temperature of the basal layer of epidermis can be considered as independent of time of heat exposure,-equalion (8) can l>e inte- grated to equation (9). 12 = Ae ~ *K/KtT *■ 2T3)t. _ (9) An examination of the transepidermal threshold data depicted in Figure 14 and the epidermal time- temperature data given in Table 7 (Section 17.3.2) and illust rated in Figure 15 shows that equation (9) is applicable in all heat exposures where the skin surface temperature is less than 50 C; furthermore the skiti surface temperature can be substituted for the steady-state basal epidermal temperature since the differences (<0.3 C) between these two values are negligible in this temperature range. Thus, by using equation (9) in this temperature range, it is possible to evaluate numerically A and \E by standard graphical procedures from the data for the threshold of complete transepidermal necro- sis; and the following equations are obtained, IE » 150,000 cal/mole (10) and A = 3.1 X 10” sec"1. (11) This value of A depends upon the arbitrary choice of the value of unity for 42. Thus, when the threshold of complete epidermal necrosis is reached, it s 1. (12) By again making use of equation (9) a similar analysis can be made of the time-temperature rela- tionship depicted by the broken line of Figure I 1. Since these data arc not so complete as those used above, it Is best to use the same numerical yahies given by equations (10) and (11) for \E and A, and solve for the numerical value of 42. These data are found to be best represented by U = 0.53 (13) 1’ira RE 14. Graph showing thresholds for porcine skin at wliich microscopic evidence of spidermal injury is first apparent tbroken line) and at which transcpidermal necrosis is complete (solid line). Crosses indicate criti- cal individual experiments and show shortest time at temperature indicated at which transcpidermal necrosis of entire target area occurred. ()|>en and solid cir- cles show effects of heal on human skin.- Open circles represent longest exposure at temperature indicated that failed to destroy epidermis. Solid circles repre- sent shortest exposure at temperature indicated that re- sulted in transcpidermal necrosis. evidence of thermal damage to porcine epidermis was recognized. Exposures sit uated below t he broken line caused no appreciable change in the microscopic ap- pearance of the epidermis. Exposures lying between the broken and solid lines resulted in varying de- grees of epidermal damage short of transepidermal neci osis. Since the reactions of human skin to con- trol episodes of hyperthermia were not examined microscopically, no inferences can be drawn as to the reciprocal relations of time and temperature at which microscopic evidence of injury to human epi- dermis was first recognizable. lT.6.3 Mathematical Predictability of Epidermal Destruction by Exposure to Ileal ’ From a kinetic standpoint, the reciprocal relation- ships of time and temperature in the production of transepidermal necrosis follow the general pattern of rate processes. If the reaction leading to thermal death of epithelium conforms to t hat of most physical and chemical rate processes,*" it should be quanti- tatively predictable by the following equation: 4 -AE/lf(T, 1-273) (7) (It ~ K where dil dt is the rate at which an arbitrary function J By F. C. Henriques, Jr. SECRET RECIPROCAL RELATIONSHIPS OF TIME AND TEAIDER ATL'HE 335 when the upper limit of exposure which can be toler- ated without the occurrence of transepidermal ne- crosis is reached. Although the values given by equations (.10) to (13) for ,1, AE, and ft were obtained through the use of equation (9), which requires that the epidermal temperature can be considered constant during the entire heat exposure, those numerical values should permit the computation of the two thresholds of transepidermal injury under all conditions by means of equation (8), so long as T, is known. Under the experimental conditions that, the data depicted in Figure 14 were obtained, namely, con- stant skin surface temperature during the entire heat exposure, it is possible to ascertain the time de- pendence of basal epidermal temperature by means of equation (tie). Referring to equation (6e), it is found that the evaluation of T, depends upon two parameters, T , and y. An examination of the equa- tion show s that T, is very insensitive to variations in T„, the original epidermal temperature; 35 (’ is taken as the original skin surface temperature (see Table 6 of Section 17.3.2); In view of the uncertainties which enter into this direct experimental evaluation of y by means of equation (6b), it is best to evaluate it empirically by obtaining the best fit to the complete transepidermal necrosis data. — It is then found that 7 = 0.15 (14) if I, the lime during the heat exposure, is expressed in seconds. This numerical value checks well with that obtained by direct substitution of the experi- mental values for the thermal conductivity, heat capacity, density, and thickness of epidermis (see Section 17.3.2) into equation (6b). A consideration of equations (6a), (6c), and (8) together with the requisite numerical values given by equations (10), (11), and (11) shows that the experi- mental data given in Table 10 and depicted in Fig- ure 14 are completely described by the following equation; < ft = 3.1 X 10-f.e ~ 75000 T‘+ m 0.5 and T, < 50 C the time dependence of T, can be ignored and T, put equal to T,; equa- tion (15) can then be integrated and takes the form of equation (9), which greatly facilitates the compu- tation of ft. For all T, > 50 C and ft < 1, the time dependence of T, cannot In* neglected, and the evalu- ation of ft by means of equation (15) requires one of the standard methods of numerical integration.17 This numerical determination of ft from the two experimental parameters, t and T„ permits the pre- diction of the degree of epidermal injury, since mi ft < 0.53 results in a time-temperature relationship that can IxT tolerated without the occurrence of < ransepidermal necrosis, and ft > 1.0 results in a time-temperature relationship which produces com- plete epidermal necrosis. The success of equations (15) and (15a)In predict- ing these time-temperature relationships is shown in Table 12. It can be seen that the agreement of the experi- mental data of Section 17.6.3 with this equation is, in general, excellent, and, thus, that t he tacit assump- tion throughout this section of the applicability of equation (7) is justified. In the four cases where there is appreciable variance between experiment, and prediction, either the experimental data are in- sufficient or the duration of heat exposure was too short to preclude considerable experimental error. Thus, equations (15) and (15a) probably give a more accurate prediction of epidermal injury thresholds than the dotted and solid lines of Figure 14. The numerical computations resulting from equa- tion (9) are also included for comparative purposes. For the reasons stated above there is no appreciable difference, under these specific experimental condi- tions, between this equation and equation (15) for all surface temperatures below 50 (Kquation (9) cor- responds to an experimental condition in which the basal epidermal layer is immediately brought to and maintained at a constant temperature. If this were feasible at 70 (', complete epidermal necrosis would result in 3 ten-thousandths of a second. The 2,000- fold difference between this value and 0.5 second predicted by equations (15) and (15a) indicate the extreme importance of the heat capacity of t he skin during the early period of heat exposure. y = 0.15 (14) SECRET 336 STL DIES OF THERMAL INJURY — CUTANEOUS VND SYSTEMIC Tabi k 12. A comparison of the experimental time-temperature Figure 14 with those obtained from equations (9) and (15). relationship for (ransepidermal injury as depicted by Minimum time in seconds for complete Maximum time in seconds for subthreshold t ransepidermal necrosis I ransepidermal necrosis U se I SI = 0.53 Experimental Surface Experimental Equation Equation solid line temp dotted line Equation Equation (»)* (15) Figure 14 C Figure 14 (15) (9)* 23,000 23,000 25,000 44 I8,000t 12,000 12,000 11,000 11,000 11,000 45 7,200f 5,900 5,800 5,100 5,200 5,000 46 3,000 2,800 2,700 2,400 2,500 2,400 47 1,300 1,380 1,300 1,100 1,200 1,100 48 560 6.50 t»0 580 630 570 49 260 340 310 270 325 300 50 130 165 110 130 165 160 51 75 90 68 65 91 90 52 44 52 35 is 31 35 54 IS 19 8 4.4 13 16 56 8.3 8.1 23 0.25 3.0 5 60 2.6 2.3 0.13 0.009 1.0 2t 65 1.0 0.7 0.005 0.0003 0.5 It 70 0.4 0.0002 ♦ Above 50 C equation (9) has no experimental siunifieanca?. t Experimental value uncertain. These tabulated values, resulting from the solution of equation (15), are of course only valid under spe- cific experimental conditions, namely, only when the skin surface temperature is immediately brought to and maintained at a constant value during the entire heat exposure. However, equation (15) should ac- curately predict the epidermal injury to l>e expected from all conceivable kinds of heat exposures, so long as the temperature dependence of the skin surface temperature as a function of time is known, since the time-temperature relationship at the basal epidermal layer can be predicted quite accurately by making use of the “infinite body” heat theory u implicit in equation (15a). (See Section 17.3.1.) 1T.6.6 Vulnerability of Ischemic Skin to Thermal Injury One of the reasons that a running stream of hot water was employed as the source of heat in these ex- periments was that by this technique there would be no mechanical interference with the circulation of blood through the dermal capillaries. The exposures were made at atmospheric pressure. It was felt, that circulation of relatively cool blood through the der- mal capillaries would probably tend to protect the skin against burning and that any method employed for the production of uniform burns should be one which did not cause mechanical interference with capillary circulation. — The following experiments were undertaken for the purpose of determining the extent to which local im- pairment in blood flow may increase the vulnerability of the epidermis to thermal injury. A control series of burns were made on each of three pigs by exposing various skin sites to hot water at atmospheric pressure. The predetermined time and temperature of each exposure were such that severe first-degree or mild second-degree reactions could he anticipated. See Table 13. Tablk 13. Effects of thermal exposures with and pressure ischemia. without Ani- mal num- ber Kxpt temp C Expo- sure dura- tion (min) Excess pres- sure on skin (mm I Ik ) Xo. of exp made Number of lesions Without With transepi- traasepi- dcrmal dermal necrosis necrosis 887 40 7 0 5 5 0 40 9 0 5 0 5 49 7 80 5 5 0 899 49 7 0 4 4 0 49 8 0 4 2 2 40 9 0 4 0 4 40 7 80 4 4 0 49 8 80 4 3 1 !K)1 51 2 0 3 3 0 51 3 0 3 2 1 51 4 0 3 0 3 51 2 80 3 3 0 51 3 so 3 I 2 It was found that all 7-minute exposures at 49 C and all 2-minute exposures at 51 C made at atmos- pheric pressure were subthreshold in the sense that SECRET REC1PROC A1, RELATIONSHIPS OF TIME WO TEMPERATURE 337 they failed to cause complete transepidermal ne- crosis. That they were close to threshold was indi- cated by the fact that all 9-minute exposures at 4!) (' and all 1-minute exposures at 51 C did cause (rans- epidcrmal necrosis. After it was established that the position of the threshold for transepidermal necrosis in these ani- mals was between 7 and 9 minutes at 19 (' and lx- t ween 2 and 4 minutes at 51 C for exposures made at atmospheric pressure, a second series of exposures were now made during which the water pressure was increased by an amount corresponding to 80 mm of mercury. With this amount of pressure on the sur- face of the skin during the time that it was exposed to heat, there was no instance in which the reaction to a 7-minute exposure at 19 C or to a 2-minute ex- posure at 51 (' was increased in severity. It is apparent from the data summarized in Table 13 that the application of pressure sufficient to collapse superficial dermal capillaries during a period of exposure does not cause appreciable aug- mentation in the vulnerability of epidermis to ther- mal injury. In view of the extreme thinness of the epidermis, these results-were to be expected, since, for reasons given in Section 17.3.2, the epidermal temperature is primarily determined by the skin surface tempera- ture. Thus the dermal temperature gradients, which may be appreciably altered in ischemic as compared with normal skin during thermal exposure, would have little effect on the time-temperature relation- ship that exists at the epidermal-dermal interface. 17.6.7 Latent Thermal Injury and Cumulative Effects of Repeated Subthreshold Exposures If the data summarized graphically in Figure 1 I are recalled it is apparent that recognizable damage to the epidermis occurred only during (he terminal phase of the subthreshold exposures represented in these experiments. Xot until the duration of any given episode of hyperthermia was such as to bring it. to the level indicated by the interrupted line in Figure 14 was there recognizable evidence of epi- dermal injury. This phenomenon is even more readily apparent in the photographs shown in Figure 13. In these, it may lie seen that the 7-minute exposure at 49 C on the left side of the animal shows only a trace of residual erythema, whereas both of the sites of 9-rninute exposures at that temperature show t ransepidermal necrosis. Does this indicate that no epidermal injury had boon sustained during (lie first 7 minutes, or does it. mean that injury was present hut morphologically latent ? In order to gain more information concerning this point, the experiments summarized in Table 11 were undertaken. Thermal exposures were made with a running stream of hot water at 19 C and at atmos- pheric pressure. Three young pigs were used. The first series of exposures (1 to IS) were for con- trol purposes and served to establish the reproduci- bility of reactions to single exposures at this temper- ature. It may be seen that there was not a single in- stance in which an exposure for less than 7 minutes caused recognizable necrosis of the epidermis and that in every instance in which exposures as long as 9 minutes were given, there was complete necrosis of the epidermis. Skin sites receiving 7-minute expo- sures recovered without loss of epidermis, whereas skin sites receiving 9-minute exposures underwent complete ulceration. The control exposures were followed by a series (19 to 39) in which repeated exposures, individually incapable of causing recognizable epidermal injury, were applied to the same area. It was found, for in- stance, that, although a single 3-minute exposure at 19 C caused no recognizable change in the epithelial cells, three such exposures separated by recovery periods as long as 21 minutes had the same total de- structive capacity as a single continuous 9-ininutc exposure. It was clear that a certain amount of epidermal in- jury was sustained during the first 3 minutes and that at least 21 minutes were required before there was an appreciable recovery from this injury. That complete recovery occurred after between 2 and f hours was indicated by experiments 30 and 31. Experiments 31 to 39 showed what might have been expected, namely, that recovery from the latent injury of a 2-minute exposure was more rapid and that from a 5-minute exposure less rapid than was the case after a 3 minute exposure. Further discussion of the implications of these ex- perimental results will be found in Section 17.8 of this chapter. 17.6.8 Summary The reciprocal relationships of time and temper- ature in the causation of transepidermal necrosis are similar for similar skin areas of man and pig. Thermal injury or burning of the skin was ob- served to occur at surface temperatures as low as SECRET 338 STUDIES OF THERMAL INJURY — CUTANEOUS VND SYSTEMIC Table 14. The cumulative effects of repeated suhthreshold thermal exposure on the skin of the pig. All exposures were made to water at 4!) C. Effect of exposure on skin No evidence of Duration epidermal injury Epidermal necrosis of each No. of Interval Mild Severe Complete exposure exposures at 1 ict ween vase. vase. and Expt (min) same site exposures react ion reaction Focal irreversible No. 3 1 + 1 .. — 1 + 2 1 + 3 4 1 + 4 5 1 + 5— 6 1 + 6 1 +• 7 1 + * . 8 . 7 1 + .-r ■ 9 1 + 10 8 1 + 11 - I 12 I . . - + 13 9 1 + 14 1 . . — + 15 1 + 16 1 +■ 17 1 + IS 3 3 3 min + 19 3 3 min f 20 3 3 min . + 21 3 3 6 min + 22 3 3 12 min r. + 23 3 3 24 min + 24 3 3 48 min + 25 3 48 min + 26 3 3 72 min . . + 27 3 72 min + 28 3 3 96 min - + 29 3 3 120 min + 30 3 3 240 min + 31 3 3 24 hr + 32 3 3 48 hr + 33 2 5 2 min . . + 34 5 30 min + 35 5 60 min + 36 3 2 12 min + 37 5 2 60 min 38 - 2 240 min + 39 14 C and it, can be inferred from the shape of the lime-temperature curve that the threshold at which hyperihernial cellular injury is first sustained is not far above the level that is normal for the blood. The rate at which injury occurs increases rapidly if the temperature is raised. The progress of injury at any given temperature is determined by the dura- tion of the hyperthermic episode. Thus, the amount of time required to convert a reversible into an irre- versible cellular injury is different for each degree of temperature and in the case of the epidermis can be computed if the surface temperature as a function of time is known. The existence of latent or morphologically unrecog- nizable cellular injury after certain apparently harm- less thermal exposures and the fact that the time re- quired for recovery from such latent injuries becomes longer the nearer they approach the threshold of microscopic visibility were demonstrated experi- mentally. t7.7 PATHOLOGY OF CUTANEOUS BURNS AND THEIR PATHOGENESIS In the foregoing section, measurements of the re- ciprocal relationships of time and surface tempera- ture with respect to the capacity of thermal exposures SECRET PATHOLOGY OF CUTANEOUS BIHXS AM) THEIR PATHOGENESIS 339 to destroy the epidermis were reported. The following studies concern the pathological characteristics of cutaneous injuries caused by thermal exposures at different temperatures and for different durations, and a comparison of the pathogenesis of cutaneous burns in man and pig. 17.7.1 Experimental Procedure Much of the material used in this investigation was derived from the experiments described in Sec- tion 17.6 of this chapter. It was added to from sev- eral sources (see Table 15)7 Since most of the lesions produced in the experiments reported in Sect ion 17.6 were not excised until they had been under clinical observation for days or weeks, many duplicate ex- posures were made and excised in order to observe in (lie various types of lesions the sequence of micro- scopic changes that took place between injury and repair. To~aequire this material, approximately 60 additional hot water exposures of pigs were made and examined microscopically after recovery periods ranging between a few seconds and several weeks. Additional material comprised a series of bums of porcine skin made by exposure to hot air at temper- atures varying between SO and 1)00 ('. There were ( wo series of human burns, one comprising 33 experi- mentally produced lesions which were studied clini- cally but were not excised for microscopic examina- tion, and the other comprising a collection of skin specimens obtained after death from victims of con- flagrations. Sections of tissue for microscopic examination were cut from specimens that had been fixed in Zenker- formol or JO per cent formaldehyde. Phloxine- methylcne blue stains were made routinely and were augmented by sections stained with hemotoxylin and eosin or by Poliak’s modification of Masson's tri- chrome method. Many sections were stained by (he Feulgen technique. IT.7.2 (General Consideration of Quantitative and Qualitative Effects of Ileal on Skin A cutaneous injury caused by hyperthermia may be characb rized quantitatively according to the depth to which (he tissue has been destroyed, or qual- itatively according to the nature of the changes that have occurred. The characterization in Section 17.0 of hyperthermic episodes as subthreshold, threshold, and suprathreshold referred to their quantitative capacities for injury production, the determining factor being the capacity of the exposure to cause complete destruction of the epidermis. Thus, any exposure that failed to cause complete destruction of the epidermis was designated as sub- threshold and any reaction short of t ransepidermal necrosis was one of the first-degree. A second-degree reaction was one caused by the shortest exposure at any given temperature, or by the lowest temperature at any given time, that resulted in full-thickness'de- struction of the epidermis. Although it was not pos- sible to destroy the entire thickness of the epidermis without some damage to the underlying connective tissue, dermal necrosis was a relatively insignificant feature of a truly threshold exposure. A third-degree reaction was one caused by an exposure that was suprathreshold in respect to time or temperature and was accordingly one in which a significant degree of dermal necrosis usually accompanied the destruc- tion of t he epidermis. Slope of Transcutaneous Temperature Gradi- ent in Relation to Depth and Quality of Burn If account is taken of the potential variations in the intensity and duration of the different thermal exposures that are capable of producing burns of similar depth, if becomes apparent why thermal lesions of approximately the same depth may be qualitat ively dissimilar. This fact is more readily appreciated by reference to Figures I a and lt>. The critical temperature, so far as the ultimate fate of the epidermis is concerned, is that attained at the interface between epidermis and dermis rather than that of the surface. In Figure 15 are shown the estimated changes" in temperature that would occur at the basal cell level during the course of thermal exposures at three different surface temperatures if each were terminated at a time calcu- lated to be just adequate to destroy the epidermis. In Figure 16 are shown the temperatures that would Table 1 5. Sources and kinds of material used for study of the pathogenesis of cutaneous burns. Sub- ject Source of heat flange of temp C Hangeof exjxtsnre llange of recovery period Pin Water 44-100 0.5 sec -7.5 hr 1 min-1 weeks PiR Air SO-000 0.5 min-45 min lmin-3davs Man* Water 44- 00 3 sec 0 hr 1 min 4 weeks or oil Man Air 7 ? Less than 1 hr • f-rsions not excised for microscopic study. "Calculations based on data presented in Section 17.3. 340 STUDIES OK THERMAL INJURY—CUTANEOUS AND SYSTEMIC would extend to, hut not far beyond, the basal coll layer. That qualitative differences in the resulting reactions might exist despite their quantitative simi- larity can be inferred from the fact that in the ex- posure shown in Figure ISA the epidermis was de- stroyed by a 3-hour episode of hyperthermia the in- tensity of which at no time rose above 14.8 t’ at the basal cell level. Approximately the same amount of irreversible change would l>e sustained as the result of the exposure depicted in lot'. In I lie latter in- stance, the epidermis was destroyed in approxi- mately 0.1 second by an episode of hyperthermia in which the temperature at the basal cell level rose sharply and briefly to 70 C. The exposure depicted in I5B falls about midway between these extremes. Although the total amount of irreversible injury is about the same in each, it is not surprising that tlie three lesions produced in (his manner differed quali- tatively. Since certain qualitative characteristics of thermal reactions arc dependent on the degree to which the temperature of the tissue has been raised, it follows that the longer any given episode of tissue hyper- thermia is maintained, the greater the likelihood that the qualitative attributes of the reaction will reflect the intensity of the exposure. Such was found to be true: The more severe t he ex- posure, the greater were the qualitative differences between the reactions produced at high and low sur- face temperatures. An additional reason for the occurrence of quali- tative differences in quantitatively similar reactions to thermal exposures of different intensity is shown in Figure Ki. There are depicted the calculated trans- cutaneous thermal gradients to a depth of 2 mm that would exist at the moment of completion of the same three episodes of hyperthermia illustrated in Fig- ure lo. In each instance, irreversible thermal injury would extend to, but not appreciably beyond, the basal cell layer. In the exposure depicted in A (Fig- ure 10), the temperature of the dermis to a depth of about 2 rnm was elevated above normal for at least 2 hours. In the exposure depicted in (', the transcu- taneous thermal gradient, was so steep that the re- sulting temperature changes in the dermis were ex- ceedingly brief and superficial. It is apparent why the epithelial cells would be destroyed in C with rela- tively little disturbance of the dermis, whereas, in .4, the same or even a lesser degree of epidermal injury might, be accompanied by a severe and persistent vascu lar distu rbance. Figure 15. Curves depicting changes in temperature at interface between dermis and epidermis during sur- face exposures of 15 (A), 55 (TV>, and 100 (C) C. Kacli of these was threshold exposure in that 3 hours, 0.4 minute, and 0.1 second, respectively, arc estimated to lx* shortest time at indicated temperature that would cause transepidermal necrosis, (Estimates derived from measurements reported in Section 17.3.2.) Fim re 16. Solid line traversing each chart from left to right depicts tcm|>eraturcgradient through first 2nun of skin at conclusion of exposures estimated to Ire just sufficient to cause transcpidermal necrosis. Inter- rupted line traversing each chart from left to right de- pic's original pre-exposure temperature gradient through skin to depth of 2 nun. The 0 vertical line in each represents surface of skin. Interrupted vertical line at depth of approximately 0.1 min indicates depth of dermal-epidermal interface. In A, surface tempera- ture of 45 C had liecn maintained for 3 hours. In It, surface temperature of 55 (' had licen maintained for 0.4 minute. In C, surface temperature had been maintained at 100 C for 0,1 second. (Estimates de- rived from measurements reported in Section 17.3.2.) prevail at different depths below the surface of each at the moment that the duration of the exposure was just sufficient to cause irreversible injury of the en- tire thickness of the epidermis. In each instance, the effects would be quantita- tively similar, in (hat irreversible cellular injury PATHOLOGY OF CU TANEOUS BURNS AND THEIR PATHOGENESIS 341 lT.7.4 First-Degree Reactions HvCKRKMiA, Edema, and Cyanosis Sufficient dilatation of the superficial capillaries to cause erythema characteristically accompanied and frequently preceded damage to the epithelium. One exception to this generalization represented in the material upon which (his investigation was made was provided by the effects of heat on the skin of animals suffering from circulatory failure. In the presence of circulatory failure, there was frequently such a pro- found depression of vasomotor irritability that injuri- ous episodes of either high- or low-intensity hyper- thermia failed to elicit vascular reactions even though extensive epidermal injury was sustained. Other cir- cumstances in w hich thermal damage of the epider- mis was sustained with little or no accompanying vascular reaction included exposures of sufficient in- tensity to burn the stratum eorneum, but so brief as to cause little or no rise in dermal temperature. Attention has already l>een called to the fact that the duration of an episode of low-intensity hyper- thermia must be greatly prolonged if it is to produce an injury quantitatively comparable to one resulting from a high-intensity exposure. Since the dermal blood vessels are far more responsive to temperature changes than are the epithelial cells, it can be under- stood why severe and persistent vascular reactions were often elicited by protracted episodes of low- intensity hyperthermia that failed to harm the epi- dermis (see Figure 13). There was considerably greater individual vari- ation among human subjects than there was among pigs in respect to the vascular reactions to cutaneous hyperthermia. The variability of dermal vascular re- actions in human subjects was so great and tbc num- ber of reactions studied in this investigation was so few that little could be inferred as to the extent to which the animal data apply to human skin. The im- pression was gained that the thermal stimulus neces- sary to cause visible erythema in most human sub- jects was substantially lower than that required to elicit erythema in pigs. In man the change in skin color was usually more intense and of longer duration than that in the pig after an identical exposure. That an active circulation of blood was maintained through the dilated capillaries of an evanescently erythematous skin was indicated in part by the pink nr red color of the surface and in part by the fact that the surface temperature during such a reaction was characteristically between 0.5 and 1.5 degrees higher than that of the adjacent skin. An evanescent erythematous reaction to heat could not as a rule lie recognized in sections prepared for microscopic examination. Vessels, the seat of physi- ological dilatation, usually contracted during or im- mediately after excision, and it was difficult or im- possible to distinguish a sample of physiologically hypcremic skin from one that was normal or ischemic. If cutaneous hyperthermia was prolonged to be- tween 40 ami 00 per cent of (he minimum time re- quired for the production of transcpidermal necrosis in cither man or pig, ir characteristically resulted in a more severe and pathological vascular disturbance which led to edema and cyanosis and which persisted for flays rather than minutes or hours. That the How of blood through the dilated capillaries was slowed was Indicated by the blue or purple color of the sur- face in cont rast to the pink or red color caused by the moit' evanescent active hyperemia. The surface tem- perature of such a lesion during the first few hours was frequently found to be from 0.5 to 2 degrees be- low that ol-thc adjacent normal skin. That the re- action was pathological rather than physiological was also indicated by the fact that in both man and pig it was almost invariably accompanied by cutaneous edema. Within the first hour after (he onset of a vascular injury of this grade, the water content of the dermis was observed to increase by as much as 100 per cent. Microscopic examination of reactions of this type at varying periods after exposure in ■ pig confirmed the clinical observation that heat may cause a severe disturbance of the dermal blood vessels in both pig and man without causing recognizable damage to the epidermis. The capillary loops of the dermal papillae became dilated and elongated and filled with closely packed masses oferythrocytes. Separation of collagen fibers by edema fluid was obvious and perivascular mantles of extravasated erythrocytes were often seen. The escape and extravascular deterioration of erythrocytes in such a lesion was often sufficient to result in brown discoloration of the target area for as long as a week. Ext ravascular fibrin was not encoun- tered nor di«l collagen fibrils appear to be swollen. Between 12 and 24 hours after such an injury was sustained, occasional polymorphonuclear leucocytes were found in the edema fluid. Neither thrombosis nor visible alteration in the vascular endothelium was seen, despite the fact that superficial vessels were filled by static, sausagelike agglomerates of red blood cells. 342 STUDIES OF THERMAL INJURY — CUTANEOUS AND SYSTEMIC Reversible Impairment of Epidermal Antiiorau.e During most, and possibly all, injurious episodes of cutaneous hyperthermia in which the temperature of the dermis was maintained for a sufficient time at 49 C or higher, there was a brief interval subjacent to the threshold for transepidermal necrosis in which the adhesion of epidermis to dermis was impaired. The attainment of this degree of injury was recog- nized by the ease with which the epidermis could l>e dislodged by friction. If the exposure was discontin- ued before further injury was sustained and if the loosened epidermis was not dislodged either by trauma or vesication, the change was often reveisible in the case of the pig and after 12 or 18 hours the original firm anchorage of the epidermis was usually regained. Unless the exposure had been excessive, such injuries subsided without further evidence of cell death. If skin altered in this manner was protected against mechanical artifact, there was no microscopic evi- dence either in the basal epithelial cells or in the un- derlying dermis by which impairment of the epi- dermal anchorage could be recognized. If, however, sufficient friction was applied to the temporarily in- secure porcine epidermis to cause its detachment, microscopic examination revealed a fringe of up- rooted or fractured lonofibrils protruding from the lower ends of the detached basal epithelial cells. The protruding fibrils appeared to have been pulled out of their anchorage in the superficial dermal felt work of collagen fibers. It was not determined whether the essential change responsible for such epidermal in- stability was a deterioration of the extracellular ex- tensions of the tonofibrils which predisposed them to rupture or a softening of the dermal collagen in which they were embedded. The latter was considered the more plausible explanation of the phenomenon. In man it is doubtful that the tonofibrils of the epi- dermal cells have much if anything to do with the attachment of epidermis to dermis. In human skin, the epidermis appeared to be cemented to, rather than rooted in, the dermal collagen. It has already lieen indicated that when porcine skin sustained this type of cutaneous bum, recovery sometimes took place within 24 hours without death of cells, providing (he damaged area was protected against mechanical disturbance during that period when its anchorage to the dermis was insecure. Too few appropriate specimens of human bums were available for microscopic examination to permit conclusions regarding the threshold at or the fre- quency with which this particular type of first-degree thermal injury occurs in man. The opinion was gained from clinical observations of human burns that ther- mal exposures insufficient to cause primary epider- mal necrosis may result in a temporary impairment in the adhesion between epidermis and dermis. If such a temporarily insecure layer of epidermis is de- tacher! by friction or vesication, the detached cells would undoubtedly die. Thus, it is entirely possible that the phenomenon of vesication results, in some instances, in secondary destruction of human epi- thelial cells that would otherwise survive. If this be true, and if the thermal exposure has been insuffi- cient to cause primary transepidermal necrosis, the immediate institution of pressure to prevent epi- dermal displacement by vesication should predispose to early and uncomplicated healing of what might otherwise become an open lesion. Irreversible Thermal Injury of Epidermal Cells Material for microscopic study was available from almost every conceivable kind, grade, and stage of thermal injury of the skin of the pig. All hough a wide range of experimental thermal injuries of human skin was studied clinically, most of the burns that were available for microscopic examination were ob- tained from autopsies. Thus, (here was no direct in- formation regarding the intensity or duration of the thermal exposures that were responsible for most of the burns of human skin that were studied micro- scopically. The impression was gained, however, that, apart from the phenomenon of vesication, the cytological changes induced by heat in the epidermis of man were similar, if not identical, to those ob- served in (he pig (see Figures 17 to 22). Attention lias already been directed to the fact that the time- temperature threshold for the destruction of epi- dermisJs almost identical in human and porcine skin (see Figure 14). The first manifestation of irreversible thermal in- jury of the epidermis was a change in the distribu- tion of water and solids within the nuclei of the cells of the intermediate zone. As the nuclei swelled, their chromatin granules coalesced to form compact eres- centric masses immediately beneath and attached to one side of the nuclear membranes (Figure 20). When the swollen nucleus ruptured, the peripherally dis- tributed chromatin contracted to form a dense and irregularly shaped central mass which remained sep- arated from the surrounding cytoplasm by clear fluid. SECRET PATHOLOGY OF CUTANEOUS BURNS AND THEIR PATHOGENESIS 313 Figure 17 Figure 18 Photographs of severe first-degree burns of porcine (Figure 17) and human (Figure 18) skin showing degenerative changes in In Figure 17, there is generalized pyknosis of nuclei and it is not likely that any epidermal cells included in picture would have recovered. In Figure 18, changes are focal rather than general and most of altered nuclei are swollen and show periphraal displacement of chromatin. This tyjx* of nuclear change precedes that shown in Figure 17. Both specimens were excised within an hour after injury was sustained. In both instances, epidermal attachment to dermis was insecure and lesion shown in Figure 18 would probably have gone on to vesication in normal course of events. Magnification 100 X. _ Photographs of early second-degree burns of porcine (Figure 19) and human (Figure 20) skin showing early spontaneous detachment, of epidermis from dermis. Vacuolar cytoplasmic disintegrat ion of basal cell layer has been added to nuclear changes similar to those shown in Figures 17 and 18. In Figure 19, lonofibrils that were uprooted from their anchorage in dermis can lx* seen projecting from detached basal cells. Magnification 400X. Figure 19 Figure 20 This fluid, whether extruded into the nuclear lacuna or contained within the distended nuclear membrane, was faintly basophilic and sometimes contained a few fine Feulgen-positive fragments of chromatin. Pyknosis of nuclei was by no means pathogno- monic of thermal injury. Spontaneous nuclear pykno- sis was sometimes seen in the upper zone of normal unheated epidermis and was caused by injuries other than heat. — In the ease of subthrcshold exposures sufficient, to injure tin* upper layers of epidermal cells but insuffi- cient to cause t ransepidermal necrosis, the types of nuclear change which have 1 teen described were fre- quently focal and difficult to distinguish from quali- tatively similar changes in control material. Even though it could be plausibly assumed that all of flic cells at a given level were exposed to the same degree of hyperthermia, il was not uncommon to find groups of cells with normal appearing nucleuses interspersed among those that showed advanced degenerative change (Figure 18). The reason for this apparent dif- ference in the susceptibility of cells in the same layer to heat was not apparent. If the thermal exposure was of sufficient intensity 344 STL DIES OF TIIEUMAL 1 NJIRY — CUTANEOUS AND SYSTEMIC Photograph *if pseudo-vesicle of |H>reiue skin ( Figure 21) and early true vesicle of human skin (Figure 22). In each, trails* epidermal necrosis apjx'ars to lie complete. In porcine skin, detached epidermis would have remained in situ as flaccid membrane. In human skin, detached epidermis would have Iieen elevated by collection of edema fluid between it and dermis. Magnification IOOX. Fkjcbk 21 Fionite 22 or duration to cause irreversible cellular injury, nuclear changes of the kinds described in the fore- going paragraphs were usually apparent immediately after the conclusion of the episode of hyperthermia. This was not invariably the case, however, and after certain exposures at relatively low temperature (un- der 47 C) a post exposure interval of between (» and 12 hours was sometimes required for the develop- ment of recognizable nuclear alterations. Moreover, if the exposure was of sufficient severity to cause pseudo vesication in the pig or I rue vesication in man, many of the nuclei which were apparently undam- aged at the conclusion of the exposure disintegrated during the next 21 hours. If the episode of hyperthermia was such as to cause visible alterations in nuclear structure, there was inhibition of mitotic division throughout the en- tire area of exposure for many hours. No evidence was derived from the microscopic study of subthresh- old exposures to indicate that hyperthermia predis- posed to acceleration of mitotic activity. The impres- sion was gained that nuclear swelling with coales- cence of chromatin granules constituted evidence of an irreversible cellular change and invariably led to premature death and desquamation of the altered cells. In the pig, the irreversibly damaged epidermal cells were gradually desquamated over a period of a week or 10 days in the form of thin brown scales. Alterations in the appearance of nuclei in the upper and intermediate layers of epithelium wen* thought, to provide the earliest morphological evidence of primary thermal injury of (he epidermis and were frequently encountered will tout perceptible damage to the cells of the basal layer, ('baracteristieally, the earliest change in the basal cell layer caused by hy- perthermia. was cytoplasmic rather than nuclear. The injured basal cells swelled and their cytoplasm became vacuolated and increasingly acidophilic (Figures ID and 20). The vacuolization appeared to be due in part to imbibition of fluid and in part to redistribution of water and solids within the cells. The fluid contained within the cytoplasmic vacu- oles was clear, nonsudanophilic, and sometimes con- tainer] a delicate mesh of granular amphophilic debris. With severe* injury there was widespread rupture and disintegration of the lower ends of t lie basal cells. 17.7.1 Second-Degree Reactions TUANSEPIULKMAL NECROS1S The time-temperature characteristics of exposures just sufficient to cause transcpidermal necrosis in both man and pig arc indicated in Figure 14. In man, whether or not a thermal exposure has destroyed the epidermis can usually be determined by the occur- rence or nonoccurrence of vesication within the first 24 hours. To recognize with certainty during the first day or two after a near-threshold exposure whether or not porcine epidermis has been destroyed, the skin must be excised and examined microscopically. When the area of injury was 7 mm in diameter and when the duration and intensity of the exposure was at or not far above the threshold required for transepi- dermal necrosis, the time usually required for com- plete healing was between 5 and 10 days in the pig and between 1 and 2 weeks in man. PATHOLOGY OF Cl TA N FOPS BURNS AND THEIR PATHOGENESIS 345 In the pig, microscopic evidence that an exposure had been sufficient to cause transepidermal necrosis Wits provided by the changes that had occurred at the basal cell level. With the disintegration of the cytoplasm of the proximal or lower ends of the in- jured basal cells, there was at first focal and later extensive spontaneous detachment of the epidermis from the dermis. In the pig, the amount of fluid that collected beneath the loosened epidermis was never sufficient to produce true vesication. With still more severe hyperthermia, the cyto- plasmic disintegration of (he basal cells was followed by nuclear changes similar to those seen in the more superficially located cells. If the epidermal detach- ment was incomplete, stretching and attenuation of the remaining bridging cells and their nuclei was often seen. Such at tenuated masses of chromatin were often stretched to two to three times the original length of the entire cell. — In the event that the surface temperature of the epidermis was brought rapidly to a level of 55 C or higher and maintained at that level for a period longer than that necessary to cause cell death, trans- epidermal coagulation was likely to occur so quickly as to inhibit recognizable redistribution of intra- and extracellular fluids. In such an event, neither the cytoplasm nor the nucleuses of the epithelial cells appeared swollen (Figure 25), On microscopic ex- amination, both appeared shrunken, the former being intensely and uniformly acidophilic and the latter small and homogeneously basophilic. Vksicatiox Attention has already been called to the fact that a common effect of heat on the skin of both man and pig is impairment of the attachment of the epidermis (Figures 21 and 22) to the dermis, and the opinion expressed that this may be due either to a change in the physical state of the superficial dermal collagen or to disruption of the basal layer of epithelial cells. A common collateral effect of cutaneous hyperther- mia, and one that is essential to tme vesication, is an outpouring of fluid from the dermal capillaries. When a thermal exposure of human skin was suffi- cient to impair the attachment of the epidermis, the amount of edema fluid that collected between it and the dermis was usually sufficient to elevate and stretch the entire layer of dead, dying, and living cells and to form a vesicle. Although vesication of human skin was usually an almost immediate re- sponse to a thermal exposure of sufficient severity to cause primary epidermal injury, there were several circumstances in which it was either delayed or in- hibited. Delayed vesication was most frequently seen after long-time exposures at low temperatures or after flash exposures at high temperatures. In both cir- cumstances it seemed likely that the delay was due to the fact that the vascular damage was relatively mild, and that hours rather than minutes were re- quired for enough fluid to collect beneath the dam- aged epidermis to form a vesicle. Another circumstance in which vesication was de- layed or prevented was when the injury was so over- whelming that the dermis and its superficial capil- laries were almost immediately coagulated. With such-thermal injury, the level at which edema de- veloped was too deep to result in vesication. Thus, in man, the nonoccurrence of vesication after a thermal exposure sufficient to cause severe injury of the epidermis may mean that the dermal hyperthermia was either inadequate to result in edema or that it was so overwhelming that the super- ficial capillaries were almost immediately occluded. In no instance was true vesication of porcine skin observed. This was true despite the fact that many of the injuries met at least two prerequisites to vesicle formation: namely, sufficient vascular injury to re- sult in dermal edema and sufficient, impairment of the adhesion between epidermis and dermis to per- mit easy mechanical detachment of the former (Fig- ure 21). Failure of the pig to vesicate appeared to be due to the fact that the amount of edema fluid that penetrated the surface of the dermis was never suffi- cient to elevate the epidermis. In the absence of evi- dence to the contrary, a tenable explanation for non vesication in the pig is that an episode of hyper- thermia that is sufficient to impair the attachment of the epidermis to the dermis characteristically al- ters either the superficial felt work of dermal collagen fibers or the walls of the capillaries contained by it in such a way that they become virtually imperme- able to edema fluid. The nature or, for that matter, the existence of (his theoretical alteration in the permeability of the collagen or the capillary walls was not disclosed by microscopic examination. When the severity of an exposure was considerably in excess ot that required to destroy the epidermis, swelling of the superficial dermal collagen and occlusion of its capillaries could be recognized. There was, however, a wide range of exposures between the threshold tor epidermal ne- 346 STUDIES OF THERMAL INJURY' CUTANEOUS AND SYSTEMIC Figure 23 Figure 24 Photographs«f mild (Figure 23) and moderately severe (Figure 24) third-degree thermal reactions in porcine epidermis 24 hours after injury. Both injuries were produced by episodes of hyperthermia that were of low intensity (under 55 C) and long duration. In both instances, irreversibly injured dermal tissue will undergo autolysis and organization. Magnification 4tX)X. — crosis and that for recognizable swelling of collagen or occlusion of capillaries in which the microscopic examination of the pig’s skin disclosed no explana- tion for the failure of porcine skin to vesicate. 17.7.5 Third-Degree Reactions The more a thermal exposure exceeded the thresh- old required for destruction of the epidermis, either in respect to temperature or time, the deeper the in- jury and the longer the recovery period necessary for repair and regenerat ion. In both pig and man, several weeks represented the minimum healing time if a significant degree of dermal injury had been sus- tained. Further Changes l\ Epidermis In the case of the pig, prolonged exposure at a rela- tively low surface temperature (under 55 C) caused relatively litlle additional change in the microscopic appearance of the epidermis. In the higher range of surface temperatures, significant prolongation of the rate of duration of exposure beyond the time neces- sary lo destroy the epidermis modified the quality of (lie superficial changes both in human and porcine skin. In man, vesication was permanently inhibited and in both man and pig I he loosened epidermis be- came reattached to the damaged dermis. Early solidi- fication and contraction, both cytoplasmic and nu- clear, occurred before there was opportunity for the development of the retrogressive changes observed in first- and second-degree reactions. The higher the temperature, the shorter the time required to cause transepidermal coagulation. With exposures to super- heated air, desiccation was superimposed on the effects of heat, and, soon after the temperature rose above 300 C, carbonization of the dry tissue began to take place. Red axd Pale Burns The surface color of third-degree burns ranged from pale gray through red, purple, and brown to SECRET PATHOLOGY OF CUTANEOUS BURNS AND THEIR PATHOGENESIS 347 Figure 25. Third-degree thermal reaction in porcine skin showing coagulation of epidermis and dermis 24 hours after injury. Bundles of denatured dermal colla- gen apjH'ar swollen and homogeneous and become in- creasingly basophilic. Thermal reactions of this type were encountered only where surface temperature had heen brought to and maintained at level of 55 C or higher. Magnification 400X. Figure 26. Photograph of third-degree thermal reac- tion in porcine skin 72 hours after injury. Exudative cells have migrated into interstices between bundles of coagulated collagen. Precise level at which this injury will be stabilised is not yet apparent. Healing will be slow because of resistance of denatured collagen to autolysis and organization. Magnification 400 X. black, depending on certain attributes of the ex- posures responsible for their production. A black’or carbonized surface resulted from ex- posures at temperatures in excess of 200 C (Fig- ure 28). The precise temperature at which carboniza- tion began was not determined. A red, purple, or brown surface, due to the presence of blood in (he superficial layer of the skin, resulted from exposures in which the dermal temperature was raised slowly enough to permit advanced engorge- ment of the superficial capillary plexus before the occurrence of coagulation. A gray or ischemic surface indicated that the upper portion of the dermis had undergone thermal coagu- lation before the superficial capillaries had become fully engorged. The reciprocal relationships of time and tempera- ture as they relate to the visibility of hemoglobin beneath (he surface of a third-degree thermal re- action is shown in Table 10. It was found that at at- mospheric pressure and at surface temperatures of 65 C l or lower bums appeared superficially hyperemie regardless of the duration of exposure. When a 70 C surface exposure was interrupted at the end of 2 sec- onds, the lesion remained red, but, if it were pro longed to 3 minutes, (he zone of reactive hyperemia became overlaid by so thick a layer of coagulated tissue that it was no longer visible. Above 70 C all exposures of a second or longer coagulated the super- ficial plexus of dermal capillaries so rapidly that most or all of the blood contained in them was displaced to a level too deep to bo seen from the surface. POOUNG OF BloOO IX 11 VJ’KKEMK' BURNS A qualitative impression of the pooling of blood in the dilated cutaneous vessels after an injurious epi- sode of hyperthermia was derived from (lie photo- micrographs shown in Figure 11. In order to learn something of the actual amount of blood that was present in such lesions, samples of both normal and hyperemie skin were excised for chemical examina- tion. Samples of skin and subcutaneous tissue having an area of 25 cm2 and extending to the deep fascia were taken from the lateral thoracic area of each of nine pigs and their iron content was determined. Two of the samples represented normal skin and the other throe were from areas of hyperemie burn- ing. It is apparent from the results of the experiments shown in Table Hi that a relatively large proportion SECRET 34 H STl DIES OF THERMAL INJIRX - CFTVXF.OFS \MD SYSTEMIC Florins 27. Transcutaneous coagulation resulting in deep ischemic burn. Five-minute exposure to air at 200 C. Surface temperature of skin not known but probably in excess of 55 C. Epidermis h:is become re- al Inched to dermis. Magnification H5X- of the total circulating blood of an animal may be pooled in the skin and subcutaneous tissue as a re- sult of thermal injury. Calculations based on the amount of recoverable iron per unit of surface area Fioitkk 28- Carbonization of surface and intense baso— pliilia of coagulated dermis due to 2.5-ininufe exposure of skin at I (Vi ('. Effects of ambient heat augmented by radiant energy. Magnification 85X.' posiife to heat did not increase the vulnerability of the epidermis to thermal injury. It was found, how- ever, that compression of the skin was capable of modifying the superficial color of the burn even though there was no quantitative increase in its severity. To determine the circumstances in which compression of the skin during an episode of hyper- thermia may modify subsequent surface color of the lesion, the experiments summarized in Table 17 were undertaken. In some, hot water was applied at .at- mospheric pressure; in others, it was applied with a compressive force of 120 mm of mercury. The results of these exposures indicated that the color of burns resulting from surface temperatures lower than 55 C was not affected by pressure, hut that an increase in pressure during exposures at sur- face temperatures of <50 C or higher determined whether the surface of the resulting burn would he ischemic or hyperemic. Thus, an exposure at at- mospheric pressure at 00 C produced a red burn even though it was extended for as long as 5 minutes. With increase in pressure, a 2-minute exposure at the same temperature resulted in a pale burn and yet the Table 16. Poe due (o thermal iling of blood in the injury. sal m it a neons vessels Condition of Mr iron in Est. cc of blood skin 25 em2 sample in 25 em* sample Normal 0.06 0.11 0.10 0.18 Moderate 0.14 0.25 hyjieremia 0.20 0.51 Severe 0.56 1.00 liypcremia 0.60 1.10 0 40 0.70 0 JO 0.70 o:j7 0.67 of humeri skin in relation to the body weight indi- cated that as much as 30 per cent of the erythrocytes of an animal suffering from generalized cutaneous hyperemia could he accounted for in the skin and subcutaneous fat. Effect of Compressive Hypkktiikrmia ox Cot-or of a Bfkx In a preceding section attention was called to the fact that compression of the skin surface during ex- PATHOLOGY OF CITANEOUS ni l!AS AMI THEN! PATHOGENESIS 319 Table 17. Experiments to determine the circumstances in which compression of 1 lie skin during an episode of hyperthermia may modify subsequent surface color of the lesion. Temp of surface (C) Duration of hyperthermia (seconds) Pressure on skin (nun Tig) Kxlernal appearance of burn 24 hours after exjMisure Ischemic ITyperemic 70 5 0 5 120 + 65 30 0 4" 30 120 + GO 120 -F 1.200 0 + GO GO 0 + GO 120 4- 120 120 + 300 0 + 55 1,800 0 + 1,800 120 + bundles tended to collapse the dermal blood vessels and the loose areolar tissue that surrounded them. Visible edema- receded in advance of this type of al- teration as though the fluid were imbibed or dis- placed by the denatured collagen, Xot until 24 or IS hours had elapsed was it possible by microscopic examination to recognize the line of demarcation be- tween reversible and irreversible dermal injury (fig- ures 23 and 21). From the intact blood vessels of the deeper and relatively uninjured tissues, leucocytes migrated up- ward through the perivascular interstices and into the zone of denatured collagen. A frontier was even- tually established between the tissue capable of re- generation and repair and that destined to be seques- tered in the form of a desiccated crust. The deeper the lesion, the longer the time required for the stabi- lization of such a frontier. The transition between the obviously necrotic tissue of the upper dermis and the least disturber! tissue of the deepest portion of the zone of hyperthermia was a gradual one. Exudation of leucocytes occurred within a few hours and within 24 hours usually served to delineate the zone within which the plane of irreversible injury would eventu- ally become st abilized. Within 2 or 3 days fibroblasts and new capillaries began to push up toward the sur- face in the interfascicular interstices of the denatured collagen. The least affected connective tissue at the base of the reaction zone recovered quickly and with- out apparent loss of fixed t issue cells. The fate of the more severely injured collagen varied according to the extent to which it had been denatured. Thermal denaturation of collagen at temperature levels under 55 C did not usually result in the kind of coagulalive change that made the collagen resistant to subse- quent autolysis and organization (Figure 2(>). Col- lagenous denaturation at temperatures over 55 C often resulted in an irreversible type of coagulation which resisted lysis and eventually led to sequestra- tion en masse. Thus, deep and severe burns resulting from surface exposures lower than 55 C were likely to remain soft and red and the necrotic tissue was susceptible to organization. Deep burns resulting from higher temperatures were characteristically firm and pale and the necrotic tissue was seques- tered rather than organized. After exposures to temperatures between these two extremes the dead and damaged connective tissue was infiltrated by leucocytes and pcnel rated by granulation tissue and its necrotic elements were gradually resorbed and replaced by new connective tissue. depth to which the tissue had been destroyed in the latter was less than (hat to which it had been de- stroyed in the former. At 70 C a 5-second exposure at atmospheric pres- sure resulted in a red burn, but, with an additional pressure of 120 mm of mercury, the resulting burn appeared ischemic. Microscopic examination of these lesions provided evidence that the color of a burn was not a reliable criterion by which to judge its depth. After hyper- thermic episodes of comparable duration and at the same surface pressure, a red surface color usually indicated that the lesion was less severe than one- having a gray surface. Without knowledge of time, temperature, or surface pressure during the period of exposure, it is not possible to estimate the relative severity of burns on a basis of surface color. Other Effects of Heat on Dermis After edema and pericapillary extravasation of erythrocytes the earliest recognizable extravascular alteration of the dermis was swelling of the collagen fibers. Tins occurred first in its most superficial layer where, in the case of porcine skin, the projecting tonofibrils of the basal epithelial cells were imbedded in the collagen of the subjacent connective tissue. As the intensity and duration of the hyperthermia increased, the corium tended to lose its fibrillar char- acter and was converted into a thin lamella of homo- geneous acidophilic material as though its individual fibers had been converted to a gel. With increasing exposure the swelling of collagen became apparent at greater and greater depths in the underlying con- nective tissue (Figure 25). Expansion of collagen SECRET 350 STUDIES OF THERMAL INJURY CUTANEOUS V M) SYSTEMIC During the time required to establish the level of irreversible injury, tentative tonguelike masses of new epithelial cells grew out from the margins of the lesion and from the viable roots of partially destroyed hair follicles as thoi igh they were seeking a suffi- ciently well-stabilized layer of connective tissue to provide support and nutrition. Repeated crops of such new epithelial rolls extended over or into the granulation tissue and failed to survive, for reasons not disclosed by microscopic examination. The number of experimentally produced deep burns of human skin was not great enough to draw any definitive conclusion regarding the relative rates of healing of such lesions in man and pig. The impres- sion was gained, however, that lesions of similar area and depth heal more rapidly in the pig. 17.T.6 Summary Comparison of effects of heat on human and por- cine skin; In a previous section of this chapter it was shown that the quantitative, relationships between temperature, duration of hyperthermia, ami depth of injury were similar in human and porcine skin. In this section it has been shown that there is a strik- ing qualitative similarity between the microscopic alterations that are caused in human and porcine skin by hyperthermia. The most important quali- tat ive difference is that true vesication was not ob- server! in the pig, whereas in man it is a character- istic cutaneous reaction to certain types of thermal injury. The reason for this difference has been dis- cussed. Attention was called to (he fact tliat vesica- tion is an undesirable phenomenon in that it may re- sult in the separation and death of viable epidermal cells and that there is reason to believe that healing of certain burns in man would be hastened if vesica- tion could be prevented. Sequence of changes caused by harmful episodes of hyperthermia: The earliest changes are latent, in the sense that they are not associated with visible altera- tion in the appearance of the damaged cells. Such changes are reversible. Beyond the stage of latent injury, (he pathological changes produced by exposure to heat are of two kinds: those that represent the reaction of living tissue to injury and those that represent the effects of excessive heat on cells and intercellular substances that have already sustained irreversible injury. The former may or may not Lie reversible and differ in nature according to the type of cell or tissue in which the reaction has occurred. The latter are of impor- tanco principally with respect to the extent to which such postvitill thermal cleiiaturation interferes with t lie organization and disposal of the necrotic tissue. Both types of reactions have been described in detail. 17.« CONSIDERATION OF THE N\TUHE OF PHYSIC\L \ND CHEMICAL CH \NGES INDUCED IN TISSUE BY IIVPERTHERMl \ f IT.».i Introduction Ideally, an attempt to elucidate the precise nature of the changes produced by heat on the skin should be based on a knowledge of the various physical and chemical phenomena that are normally essential to the survival and functional integrity of the living cells that comprise cutaneous tissue. If it were then possible to observe the alterations of each of these physical and chemical functions with temperature, a direct solution of the problem of how heat injures the skin might be readied. Unfortunately, detailed in- formation regarding the basic physical and chemical properties of the skin or the effects of temperature thereon does not exist. In fact, very little qualitative and almost no quantitative data arc available on even the general physical and chemical attributes of skin constituents as of to date. It is apparent, then, that any consideration of temperature-induced physi- cal and chemical changes which may lead to thermal death must be based on the known in vitro effects of temperature on substances that are akin in function and/or properties to those which probably occur in cutaneous t issues. Since nearly all the quantitative experimental data derived from this investigation deal with epidermis, the ensuing discussion will be limited primarily to this1 tissue. In Sections 17.0.5 and 17.9.3, these data are shown to be quantitatively predictable by the standard form of a rate equation,19 specifically, equa- tion (7). In this equation there appear two empirical and experimentally determinable constants, namely, -4 and YE; any theoretical consideration of the cause of thermally induced transepidermal necrosis should take into account, at least qualitatively, the numeri- cal values of these quantities. Aside from certain general conclusions regarding the entropy of the overall process,20 little specific information can be obtained from the numerical value of A, since this constant is intimately connected with the as yet un- f By F. C. Ilcnriques, Jr. SECRET PHYSICAL VXD CHEMICAL CHANCES INDUCED RV HYPERTHERMIA 351 known detailed physical and chemical properties and functions of the epidermal constituents. This i.-> not the case, however, with SE, and thus, before proceed- ing further, a brief general consideration of the na- ture of IE, the activation energy in calories per mole, is in order. 17.8.2 Thermal Injur) and Energy of Activation 20 In general, the kinetics of any given physical amt Dr chemical process depends upon the total energy content of the constituents involved. If this energy content is less than a certain critical value, known as the activation energy, the process cannot take place; if the energy content is equal to or greater than this critical value, the process may take place. Thus the rate of the process will be proportional to the fraction of these constituents which, collectively considered, possess an energy content at least equal to the activation energy. This fraction is deduced from the Maxwell-Boltzmann energy distribution law, which states that f==e-*S/K(T + 2n) (1(}) where / is this fraction, and the remaining symbols have heen previously defined. Equation (16) deter- mines only the temperature coefficient of a rate process, since, as shown by equation (7), the rate of a process is also proportional to one other factor that is essentially nondependent on temperature, namely A. Thus, the rate of any conceivable process that may result in cell death, whatever it may be, depends upon a critical energy content of the participants. The fraction of the participants, collectively consid- ered, having this energy is determined by the activa- tion energy and the temperature [equation (Hi)]. The availability of this fraction is requisite but not in itself sufficient to allow the process to proceed. An inspection of equation (16) shows that the temperature coefficient of any kinetic process is a strong function of the activation energy; for example, in the neighborhood of 50 C, the rate of a process with an activation of I, 10, or 100 kcal mole will be altered by alxmt 0.4 per cent, 7 per cent, or 70 per cent, respectively, per unit change in temperature in C. The kinetics of a considerable number of physical and chemical phenomena have been studied in detail and it is possible to classify all rate processes and, hence, in particular, those mechanisms which may lie of considerable importance in the general considera- tion of thermal injury, according to the order of magnitude of their activation energy. During the past 50 years, numerous theorieshave been proposed to explain thermally induced injuries in living organisms. Before applying the above cri- teria to the mechanisms involved in these theories, it is necessary briefly to characterize the attributes of a living cell.12 _ The living cell appears to consist of a semirigid relatively nonsoluble framework (e.g., nucleus, nu- clear wall, and cell wall) that is pr imarily protein in nature. This aggregate is bathed in an aqueous intra- cellular fluid which contains both particulate (e.g., micellar) and soluble constituents ranging from simple ions to proteins of extraordinary complexity. Aside from certain purely physical attributes (e.g., permeability, contraetibilify, elasticity, rigidity, and tensile strength), this protoplasmic entity respires, excretes, synthesizes all imaginable types of molecules, utilizes ami liberates energy, and reproduces in a manner that perpetuates its own kind. This exceedingly complex metabolic activity is apparently both catalyzed and precisely controlled by a multiplicity of enzymatic proteins and function- ally allied molecules which contribute to both the structural framework and the cytoplasmic, fluid. In view of this complex picture, no theory of ther- mal injury can be considered tenable unless it takes into account these completely integrated and pre- cisely balanced phenomena, which, taken as a whole, comprise cell life. Unfortunately our knowledge of these phenomena is as yet meager and is limited to isolated observations on living protoplasm (e.g., cell respiration, mitosis, diffusion of a few substances through cell walls) and to certain chemical and physi- cal properties and functions of a few of the molecules that can be extracted in a presumably unaltered state from dead cell brei. Nevertheless, even on the basis of this limited in- formation, it is interesting to speculate with regard to the general kinds of mechanisms that may be of importance in explaining the quantitative time- temperature relationship that results in irreversible epidermal injury as judged morphologically. These injury data (Sections 17.6.5 and 17.9.3) showed that episodes of transepidermal injury are quantitatively predictable by a rate equation with an activation energy of 150 kcal/mole over the entire experi- mental skin temperature range (44 to 70 C). The theories* that have been advanced to explain SECRET 352 STIOI CS OF Til Fit MAL INJI'KY CITAYEOUS VXD SYSTEMIC thermal injury may be classified into three general groups. 1. Thermal alterations in proteins. In view of the many varied functions of proteins in the maintenance of normal cell life, it is obvious that even minor ther- mally induced alterations of these molecules may result in profound irreversible injuries. Thus, for example, these thermal protein changes could pro- duce an increased permeability of the nuclear and or cell wall, structural alterations in the nucleus itself, disintegration of the protein mitochondria present in the cytoplasm, inactivation of enzymes. .Many quantitative studies 44 have been made of the effects of temperature on proteins, and altera- tions that proceed at a measurable rate between 0 to 100 C with activation energies in excess of 50 kcal mole are not unusual. The heat inactivation of in- verbase (A A' — lit) kcal at pH 1 and A A’ — 52 kcal at pll 5.7) and of peroxidase (AK ~ 189 kcal), and the heat denatoration of egg albumin — 132 kcal at ydl 5) and of hemoglobin (AE = 70 kcal at />H 0.8) are a few of the many examples. Thus, the morphological observations of protein dissolution and or coagulation on which the quanti- tative judgment of transepidermal necrosis is based may well be directly due to the thermal alterations of as yet unknown proteins present in epidermal cells. 2. Other passable alterations in metabolic processes. Since temperature affects, to a greater or lesser de- gree, the kinetics and thermodynamics of all chemi- cal and physical phenomena, he at may cause altera- tions in metabolism irrespective of its effect on pro- teins. For example, the entire metabolic equilibrium may be upset because of concent ration changes in some of (he individual constituents as a result of temperature variations both in rate of diffusion and formation and degradation of the chemical reactants comprising the process; in fact, because of this ab- normal functioning, certain metabolites normally present may completely disappear and or others abnormal and toxic in character may arise. There can lie no doubt that these phenomena do take place and that they may cause cell death. Many of these metabolic reactions,91' both en- zyme- and nonenzyme-catalyzed, have lieen studied as in vitro processes, and activation energies usually between 10 and 20 kcal mole are found. In certain instances the activation energies are less than 10 kcal mole but none have been found to exceed 50 kcal. Thus, to date, there is no experimental evidence (hat these typos of reactions can lead to a tempera- ture coefficient for thermal injury which corresponds to that found experimentally for transepidermal necrosis. 3. X on prate in-inti need alterations in the physical characteristics »f cells. In this group are placed all physical phenomena that are characteristic of proto- plasm but are not primarily effected by the thermal alterations of proteins contained therein. For exam- ple, diffusion of me(abolites.through a cell wall that has not undergone chemical alteration is a member of this class, whereas changes in diffusion rates that are the result of an increased cell wall permeability due to the degradation of structural protein are spe- cifically excluded, since this phenomenon is classified under group (1). ~ All of the biophysical rate processes that have been studied, such as diffusion through liquids and mem- branes, viscosity, rigidity, tensile strength, lique- faction, possess activation energies that are usually less than 5 kcal mole, and never in excess of 15 kcal mole. Although these types of mechanisms arc undoubt- edly potentially capable of causing cell death, they are not the instigators of the morphological changes that are observed in irreversible epidermal injury. Since many fat like substances are known to melt around 45 C, the liquefaction of lipoids has received considerable consideration as a potential instigator of thermal injury.* From a kinetic viewpoint, the rate of melting is a physical process with essentially a zero activation energy. This theory would predict a sharp temperature threshold for injury, with the injury rate becoming nearly a linear function of the increment in temperature above threshold value. Hence, although liquefaction might account for the quantitative epidermal thermal relationships at skin surface temperatures between 45 C and 48 C, there would be extreme variance with the experimental data at the higher skin temperatures. The extent to which thermal liquefaction of lipoid substances may contribute to cell death in tissues other than the epidermis was not investigated. In view of the preceding discussion, it can be con- cluded that the only biokinetic phenomena known to date that can account for epidermal cell death are the thermally induced changes in protein structure which have an activation energy in the neighborhood of 150 kcal mole. This in no way excludes the in- jury propensity of the innumerable mechanisms im- plied above, but merely states that all quantitative SECRET PHYSICAL AND CHEMICAL CHANCES IMJECED BY IIYFEUTIIBHM1A 353 studies made in this investigation indicate that the morphological changes (see Section 17.7.3 and Sec- tion 17.7.4) observed in the epidermal tissue can lx* ascribed to these protein alterations. As to the number anti kinds of proteins involved, the specific nature of thermally induced reactions,* and the individual rate of each protein alteration, nothing can be stated. Further, it is probable that at any given hypothermic level any one of these numerous protein alterations is potentially capable of producing cell death. 1T.K.3 Thermal Injury and Entropy and Free Energy of Activation With no intention u'hal.smver of implying that the thermal effects on living protoplasm can be ascribed to the alteration of any single protein, it is of value to make for the moment this extreme oversimplifi- cation in order to interpret the significance of the numerical value of A in the empirical rate equa- tion (7) which predicts completely the thresholds of transepidermal necrosis. In vitro studies on both enzymatic and uonenzy- matic proteins have shown that the rate of thermally induced changes is first-order and the quantity of degraded protein is given by 2,1 hi(—) = -T/’ *' 273) (*«"“) ( SE < 20 kcal can lead to any overall phenomena with an activation energy less than 10 kcal or greater than 20 kcal). Thus, the interpretation in the text is valid. SECRET 354 STl'DIES OF THERM VI, 1NJI RV - CI TWEOI S AND SYSTEMIC cytoplasmic proteins which have activation energies for thermal degradation in the neighborhood of 150,000 cal, mole. iT.a.t talent Thermal Injury In Section 17.6.7, the existence of latent or mor- phologically unrecognizable epidermal cellular in- jury after certain apparently harmless thermal ex- posures was proved by (he repeated applications of subthreshold exposures. Furthermore, the time re- quired for recovery from these latent exposures be- came longer the nearer they approached the thresh- old of microscopic visibility. The concept of an unknown but definite fraction of certain of the cellular proteins that must lx1 thermally altered in order to result in morphologically recog- nizable injury is in accord with these experimental dat a. During a heat exposure that results in latent in- jury, a noncritical fraction of these proteins is al- tered. At the termination of the heat exposure, the epidermis rapidly approaches normal temperature (Section 17.3.2) and at least partial cell function is resumed. Thus, during the recovery period, the ther- mally altered proteins arc replenished to a degree which depends, in part, upon the length of the re- covery period, and, in part, upon the duration of the heat, exposure which produced unrecognizable injury. 17.8.5 Summary The numerical constants of an experimental equa- tion, w hich quantitatively predict s ) he morphological episodes incident to 1 ransepidermal necrosis, have been subjected to theoretical analyses. It is deraon- st rated that, of all of the known biokinetic phenomena, only thermal alterations in cellular proteins that have an energy and entropy of activation of 150 keal mole and 395 entropy units, respectively, will account for the experimental observations. This theory is also in agreement with the latent thermal injury data given in Section 17.6.7. 17.9 E\post UK TO HOT AIR \N1) RADIANT IIKAT 17.9.1 Introduction In preceding sections of this chapter it. has I>een shown that a very brief exposure of an animal to ex- cessive circumambient heat may cause rapid circula- tory collapse and death. It was found that transfer of heat to and through the skin was a more important causo of such casualties than was the effect of heat on ♦ lie respiratory tract. In a quantitative and patho- logical study of the effects of hot water on the skin it was shown that certain predictable and reproducible reciprocal relationships exist between the intensity and duration of an episode of hyperthermia and its capacity to destroy the epidermis. These findings suggest that similarly reproducible and predictable relationships may exist between the intensity and duration of an episode of hyperthermia and casualty production by exposures to hot air and radiant heat such as may occur incident to a con- flagration or to a (lame thrower attack. To determine whether or not such is the case a series of experiments was undertaken in which pigs received generalized cutaneous exposures for varying periods of time to circumambient (and circumradi- ant) temperatures that varied between 70 and 550 C. The cutaneous and systemic effects of these expo- sures on animals were correlated with exposure time and source temperature. 17.9.2 Experimental Procedure Previously clipped and anesthetized pigs were fastened on a platform in the manner shown in Fig- ure 29 and a preheated oven was lowered over them. In most of the experiments the snout of the animal protruded through an aperture in the bottom of the platform. There were two advantages in this arrange- ment, one being protection of the respiratory tract and the other being that it was possible thereby to determine the time of death of animals that suc- cumbed during the |x*riod of exposure. The source of heat was a bottomless oven con- structed of iron and firebrick and having a capacity of approximately 1,100 1. The box weighed 2,700 kg and its internal measurements were 89x91x130 cm. Chromel alumel (10 gauge) thermocouples welded onto the inside plate of the box provided in- formation as to the source temperature during the period of pre-exposure heating as well .os during the period that the animal was living exposed. To heat the box, it was lowered into a vertical gun annealing furnaceh (Watertown Arsenal). When it had become thoroughly heat-soaked and was at a slightly higher temperature than that at which it was desired to ex- pose the animal, the oven was quickly withdrawn from the furnace by an overhead crane and lowered over the platform on which the animal was sus- b These facilities at the Watertown Arsenal were made available through the courtesy of the War Department. EXPOSURE TO HOT AIK AND RADIX XT MEAT 355 temperature of the right auricular blood was taken for comparison with that, of the rectum. In a numlter of experiments a 2S gauge iron-con- stantan thermocouple contained in a venipuncture needle was inserted into the dermis to record the temperature of the subepithelial connective tissue during and after exposure. Temperature of air in different parts of exposure chamber: Values given in the text for ambient tem- perature refer to the mean temperature of the air in which the-animal was enveloped. The thermocouples by which the ambient temperature was measured were routinely placed in approximately the same po- sitions in relation to the animal in all experiments. One was fastened to the skin just below the base of the tail and one on each side of the mid-portion of the body. It was regularly observed that the mean ambi- ent temperature was approximately 20 per cent lower than that measured by the thermocouples incor- porated in the wall of the exposure chamber. Al- though the rate of cooling of tlie exposure chamber (and the air contained by it) varied according to the magnitude «>1 the initial difference between its tem- perature and that of the room, the drop was never in excess of 5 per cent in experiments last ing 15 minutes or less. Because of the Convection currents that resulted from the difference between the temperature ot the surface of the animal and that of the air surrounding it, the temperatures recorded in various parts of the exposure chamber showed remarkably little vari- at ion. Thus in the mid-horizontal axis of the chamber difference in temperature was less than 5 per cent from a point 15 cm internal to the wall to a point. 15 cm external to the animal. In the mid-vertical axis there was less than 15 per cent difference in the temperature of the air between a point 15 cm below the roof and a point 15 cm above the floor of the ex- posure chamber. — Measurement of Heat Transfer Under these conditions, there were three mecha- nisms by which heat could be transferred from the hot walls of the box to the surface of the animal, namely, air conduction, air convection, and infrared radi- ation. The energy transferred by conduction and con- vection is hereafter designated as ambient, and that, transferred by radiation as radiant. Although the relative importance of these two types of heat trans- fer can be directly computed by means of equations (1) and (2) of Section 17.3, it was decided to verify FiciTRE 29. Method of exposing animals to hot air and radiant heat at Watertown Arsenal. Heavy iron and firebrick box was preheated in gun annealing furnace and lowered over platform. pended. The interval required for the descent of the box from the top of the tripod to the floor ot the plat- form was bet ween 3 and 4 seconds. The platform supporting the tripod upon which the animal was suspended was elevated 75 cm above the floor and covered by a layer of dry sand. In addi- tion to the aperture to accommodate the snout of the animal, there were other openings in the platform through which wires could be passed to the temper- ature recording equipment. Three 28 gauge iron-constantan thermocouples connected in parallel wen- fastened to the surface of the animal in such a way that the junctions were separated from the skin by a distance of between 2 and 5 cm. These prov ided for a continuous recording of ambient temperature. Rectal temperatures were taken routinely. In some experiments a rectal thermocouple provided for a continuous record. In others the temperature was taken by thermometer before and at intervals after exposure. On several occasions the postexposure SECRET 356 STL DIES OF THERMAL INJURY CUTANEOUS AND SYSTEMIC these calculations under the conditions that pre- vailed in these experiments. Unfortunately, direct determinations of the ambient and radiant caloric uptakes of animals were not feasible and it was neces- sary to measure these values by means of calorim- eters suspended in the center of the exposure cham- ber preliminary to animal experimentation. The calorimeters consisted of copper cylinders which measured 2.5 cm in diameter and 5 cm in length. ( )ne of each pair of cylinders was gold-plated and the other blackened with colloidal graphite faquadag). Thus, the former measured only ambient energy, whereas the latter determined both ambient and radiant energy. The caloric uptake rate of (he calorimeters was readily calculated from their known heat, capacity and surface area and the experimentally determined rate of temperature rise as measured by an iron- constant an thermocouple soldered within the calorim- eter. Because of the discrepancy between the size of these calorimeters and that, of the pigs (approxi- mately 30x75 cm), it was necessary to multiply the ambient calorimetric measurements by a numerical factor equal to 0.5. Since the skin is known to lx* a nearly perfect black body for the radiation emitted under these experimental conditions and since the dimensions of the exposure chamber were large with respect to those of the animal, the radiant caloric measurements are directly applicable. Actually, these data, so corrected, apply to a metallic cylinder of dimensions similar to those of a pig. Since it has been shown that under the condi- tions of experimentation these data would be equally applicable to both smooth and rough and to metallic and nonmetallic surfaces, it is believed that they represent a true estimation of (he caloric uptake rate of pig skin. The data given in 'Fable IS are an estimation of the radiant and ambient caloric uptake rate per square centimeter per minute of pig skin when the surface temperature is 35 C. It is obvious that during the heat exposure the surface temperature increases with time, resulting thereby in a corresponding de- crease in the rate of caloric uptake. For skin surface temperatures not greater than (IOC, caloric uptake rates are directly proportional to the difference be- tween the temperature of the surrounding air and that of the surface of the animal. Thus, for surface temperatures below 00 C the requisite caloric uptake rates can be computed from these data. Further ex- amination of Table IS shows that the infrared radi- at ion from the inside walls of the box was the princi- pal source of heat energy absorbed by the animals. Under conditions that produced an air temperature of 70 C, this contribution was 50 per cent, whereas at 500 C it was 85 per cent. These percentages remained nearly invariant throughout the entire time of a given heat exposure. As previously indicated, these values for the nonradiant and radiant contribution to calorie uptake rate can be directly computed from equations (1) and (2) and, if this is done, it will be found that they agree with the experimental values to within about 15 per cent. Tabi.e 18. . Estimated e aloric uptake for pitr when skin surface temjierature is 3.1 C. Air Caloric uptake in cal cm- min Per cent tem(H*rature Nonradiant of total C (ambient) Radiant * Total radiant 70 02 0.2 0.4 50 100 0.5 0.0 1.1 Hi) 150 1.0 1.4 2.4 _ 58 200 1.7 2.6 4.3 61 2,50 2.2 4.2 6.4 65 300 3.0 6.2 9.2 68 350 3.8 9.8 13.6 72 400 4.5 17.0 21.5 79 450 5.5 24.0 29.5 81 300 6.5 35.0 41.5 85 * BiTausf of tin* difference bet ween the air and source temperature when animals an* placed in the exposure chamber, these radiant data refer to a source tern lx* nature 20 j>er cent in i excess of the tabulated ambient tempera- ture. 17.9.3 Effects on Animals The results of 71 individual exposures of pigs are shown in Figure 30. Il was at first intended to present in this chart only the data derived from 49 experi- ments in which pigs of uniform weight (7 to 18 kg) received generalized (approximately 90 per cent) cutaneous exposures to heat. The additional 22 ex- periments included those in which large animals (in excess of 15 kg) were used, those in which hot air was breathed during the time that the skin was being exposed, and those in which the animals were anes- thetized after rather than before exposure. When it was found that there were no significant differences in the experimental results that could be related to the body weight of the animals (7 and 32 kg) or to anesthesia it was decided to present all experimental data in one chart. The temperature and duration of each exposure is indicated by the position of the individual experi- ments on the grid. The vertical points of reference on the left are in logarithmic progression and represent the internal temperature of the exposure chamber, SECRET EXPOSURE TO HOT AIR AM) RADIANT HEAT 357 ing. The second line (II) represents the approximate threshold at which generalized second-degree burning occurred. The third line (III) represents the approxi- mate threshold at which (he burned skin and sub- cutaneous tissue underwent ischemic coagulation. The skin of most pigs that received exposures lying above this threshold was pale and the loss of elas- ticity of the coagulated superficial tissues resulted in the formation of deep fissures when the extremities were Hexed. The uppermost line (IV) represents the approximate threshold at which rapidly fatal sys- temic hyperthermia occurred. Most pigs receiving exposures in excess of this threshold died within a few minutes (usually under 15 and occasionally as long as 30) after the oven had been lifted from the plat form. Comparison of effects of hot air and hot water ex- posures: Injury by heat is determined by the degree and duration of the rise in tissue temperature. It will be shown that for the same kind of skin the produc- tion of a given degree of thermal injury depends only on the time-temperature relationships within the tissue irrespective of the source of (he heat. Since threshold II in the hot air experiments (see Figure 30) depicts the occurrence of transepidermal necrosis, it can be inferred that for the same source temperature actual tissue temperatures attained were consider- ably lower than those in hot water experiments (sec Figure 11). In Figure 31 are depicted the source temporature- time relationships that were required to produce 1 ransepidcrmal necrosis in both I he air and water ex- posures, where in the latter case the surface of the skin was maintained at essentially the same temper- ature as that of the source. A comparison of (he two curves shows that a 15-minute exposure to water at 48 C was sufficient to produce approximately the same degree of injury as that w hich resulted from a 15-minute circumambient and radiant exposure at 75 C. A hot wafer exposure for 1 minute at 53 C pro- duced about the same degree of injury as resulted from a 1-minute exposure at 100 C to ambient and radiant heat. It is apparent, therefore, that the actual surface temperatures responsible for the kind of irre- versible injury observed at threshold II in Figure 30 were considerably lower than the recorded ambient temperatures at which they were produced. In the hot. water exposures the change in tissue temperature with time was determined by the rate of heat flow- through the skin, whereas in the oven exposures it was limited by the rate of heat I ransfer to the surface. INDIVIDUAL ANIMALS # GEN BU»N;NC AND FATAL rtTR upper limits of exposures which pigs survived without either cutaneous injury or severe physiologi- cal disturbance are indicated by the line (I) that traverses the grid from left to right. Exposures lying below this line failed to cause cutaneous burning. Exposures lying between the first and second lines characteristically resulted in mild or localized burn- SECRET 358 STUDIES OF THERMAL INJURY - CUTANEOUS AND SYSTEMIC of numerically integrating 17 equation (15) following the .substitution of the epidermal time-temperature relationships which result from an exposure to ambi- ent and radiant heat as computed by equation ((») and recorded in Table 7B. These calculations were made for air temperatures of SO, 100, 125, 150, and 175 C, respectively. It is to be observed that the con- cordance of these computations with the experi- mental data is excellent . Considerable confidence can thus be placed both in the statement of the previous paragraph and in the “infinite body picture” (Sec- tion 17.3.1) which permitted the estimation of the temperatures attained in the epidermis as a function of time. Probaiu.e Effects ok Comcvkaiu.k Exposmts ox M ax So far as the skin effects of ambient and radiant, heat are concerned, the reactions in man and pig should be similar if the time-temperature relation- ships within the epidermis were t he same in each in- stance (see Figure 14, Section 17,6.4). However, a predictable difference in these relationships during identical heat exposures of this type arises from the fact that sweating of human skin can undoubtedly increase the time threshold at which cutaneous burn- ing occurs. That sweating can afford considerable protection in the case of relatively low-intensity hot air exposures can be assumed, from the fact that man may lose moisture by this mechanism at the rate of approxi- mately a liter j>er hour. This could result in heat loss at the rate of bet ween 0.5 to 1.0 cal/min/cm2 of skin surface. Heat loss by porcine skin through vaporiza- tion of moisture is relatively slight (approximately 0.1 cal cm2 min). See Section 17.5. Thus, in view of the caloric uptake data presented in Table 18, it is possible that the time threshold for cutaneous burn- ing in man is appreciably longer than that for the pig for all circumambient and radiant temperatures lower than about 120 C. That such a degree of pro- tection would l>e afforded at higher air temperatures is unlikely since it would be necessary to assume that sweating was already established at a significant level at the moment of exposure and that all of the sweat excreted was vaporized. No experiments were con- ducted to establish the quantitative extent to which sweating may be capable of protecting human skin against thermal injury to either low or high ambient and/or radiant temperatures. It should be emphasized tliat these data refer only Fiocre 31. Solid curve depicts time-source temperature relationships requisite to complete trausepidermal necrosis when skin site is exposed to flowing water of constant temperature. Dotted curve shows time-air temporal ure relationships (curve 11 of figure 30) that produce similar degree of injury when skin surface is surrounded by an envelope of radiant and ambient heat (oven experiments). Often circles show values which were computed by means of equations (I), (2), (Section 17.3) and (15) (Section 17.7). The actual time-temperature relationships within the epidermis under these experimental conditions have Ikh'H computed by equation (6), which results from the application of the general theory of heat to this problem (Section 17.3.1), and are reported in Table 7H (Section 17.3.2). These data show the rate of increase in the epidermal temperature incident to an exposure to an envelope of radiant and ambient heat. It is apparent in the case of a generalized ex- posure that long before the temperature of the sur- face of the skin would approach (hat of the air, the animal would have succumbed to a generalized by- pert hermia. In Section 17.6.5, the degree of epidermal destruc- tion was shown to be mathematically predictable by means of equation (15), so long as T,, the time de- pendence of the basal epidermal temperature, is known, 'this equation was developed empirically from data pertaining to the degree of epidermal in- jury when the skin surface was immediately brought to and maintained at a constant temperature (hot water experiments). It was stated that equation (15) should predict the time required to produce all ther- mally induced transepidermal injuries which result, from any conceivable type of heat application, so long as the time dependence of the temperature at the dermal-epidermal junction during the heat ex- posure is known. The five circles depicted in Figure 31 are the result SECRET EXPOSURE TO HOT AIR VXD RADIANT II GAT 359 to unclothed animals. It is possible to estimate the degree of protection afforded by clothing by a knowl- edge of their impedance to the heat reaching the skin surface, but since this thermal impedance is so de- pendent upon the physical characteristics of the fabrics involved, upon tightness of fit, and upon the type of heat exposure, further consideration of this problem is not warranted in this chapter. The method of obtaining these thermal protect ivities of clothing under specific experimental conditions is given in detail elsewhere.u Death ok Bigs ' It may be seen from Figure 30 that rapidly fatal physiological disturbances resulted from a wide range of thermal exposures and that at any given temper- ature within the range investigated survival or death was determined by (he duration of the exposure period. Observations were made on the various patho- logical and physiological changes resulting from sub- let hal and lethal cutaneous exposures to heat. Pathological Changes There was no apparent relationship between the occurrence of early deat h and the severity of the cu- taneous injury. Some animals that died during or soon after exposure at relatively low temperatures showed remarkably little evidence of cutaneous in- jury. Others that received extensive third-degree burns at higher temperatures survived many hours alter exposure and showed no systemic evidence of impending death at the time they were sacrificed. T( was obvious that t he cutaneous lesion per se was not responsible for early collapse and death. Apart from cutaneous burning there were no sig- nificant differences in the pathological changes ob- served in animals that died following short exposures at high temperatures and in those that died following longer exposures at lower temperatures. The most constant post-mortem finding in all animals that died within 30 minutes after exposure to heat was the presence of widely disseminated small and large focuses of hemorrhage throughout the internal vis- cera. These were seen most frequently and promi- nently beneath the endocardium of the right and left ventricles. Another site of predilection for such hemorrhages was (he gastric and duodenal mucosa. The right auricle was characteristically dilated and filled with dark red unclutted blood. The impression was gained that the ventricles were more frequently found in the state of contraction after high- than after low-intensity exposures. The lungs of pigs (hat died during or soon after cutaneous exposures to excessive heat rarely showed more than a mild degree of pulmonary edema, in con- trast to those of dogs and goats, in which systemic hyperthermia characteristically led to moderate or severe pulmonary edema. Animals sacrificed 12 to 21 hours after seveie cu- taneous burns had been sustained frequently showed severe parenchymatous degeneration of adrenal cortex, liver, and renal tubular epithelium. Hemo- globin easts were sometimes observed in the collect- ing tubules of the kidneys and the urine of burned animals regularly contained large amounts of blood pigment. Changes in Blood Examination of the blood of burned animals regu- larly showed intravascular hemolysis. That intra- vascular hemolysis was not a determining factor in survival was indicated by its absence in animals that died after low-intensity exposures. A more complete discussion of the relationship between intensity of thermal exposure and hemolysis will bo found in Section 17,10. Examination of wet and dn smears of blood of severely burned animals disclosed microspherocytosis and disintegration of erythrocytes (sec Figure 32). These changes were similar to those observed in the blood of burned human subjects, by Shen, Ham, and Fleming.42 They were not observed in the blood of animals that died after low-intensity thermal ex- posures. In severely burned animals there was an increase both in the clotting time and in the fragility of erythrocytes. Plasma Turbidity. The observation of turbidity of the plasma toget her with the finding in some fatally burned animals of small agglomerates of protein and enmeshed cells in wet smears of blood led to a re- invest igation of a phenomenon described by Rabat and Levine.27 These observers reported that t he in- travenous injection into a cat of 4 ml of heated cil- rated plasma caused immediate death. After een- (rifugalization, they found (hat the supernatant fluid of such plasma produced no ill effects, whereas death resulted from the intravenous injection of the resuspended sediment. A repetition of the experiments of Ivabat and ■ Scvcrnl goats and dogs received exposures estimated to he lethal or sublet hat for pigs and the impression was gained that their susceptibility to fatal systemic hyperthermia did not differ significantly from that of the pig. SECRET 360 STUDIES OF THERMAL INJURY—CUTANEOUS AMI SYSTEMIC aortic pressure (Ilg manometer), systemic minute output (total output less coronary flow), ventricular volume (Henderson cardiomelcr with a Kie.se volume recorder), and oxygen consumption (spirometer). The preparation had been list'd earlier fora study of the metabolic effects of alloxan; the heart was failing spontaneously at the time the blood from the burned animal was introduced into the perfusing system (about 3 hours after the preparation had been isolated). Fifty milliliters of the blood of the heated animal were injected into the venous return during a period of 1 minute. The minute output was about 130 ml min at the time of i\f injection and the in- jected blood reached the heart diluted two or three times with the original blood of the preparation. Six minutes later 100 ml of the blood of the burned animal were injected again in a period of 1 minute. This was diluted no more than once wit h the blood of the preparation. Six minutes later the blood in (he venous reservoir was removed and replaced with 200 ml of blood of the burned animal. Following this last addition the heart-lung preparation was living perfused almost entirely by the blood of the heated animal. In none of the three t rials was there any significant change in the pressure, the minute output, the heart rate, or the oxygen consumption. Although coronary How was not recorded, any great increase in it such as might have been expected if the blood had con- tained as much as 0.5 mg of histamine would have been recognized by an increase in the discrepancy between the stroke volume as recorded by the cardi- ometer and the stroke volume as calculated from minute output and heart rate. Such a change was not observed. No deleterious effect resulted from perfusing the heart-lung preparation with t he blood of the burned dog. Actually there was slight evidence of a beneficial effect, such as would be expected from the addition of any fresh blood after 3 hours of perfusion. Relation ok Systemic Hyperthermia to Survival There appeared to be a definite correlation be- tween survival and the height to which the internal body temperature was raised. Most of the animals that died soon after exposure were found to have a marked elevation of rectal temperature. In the case of exposures of long duration and low intensity the rectal temperature was only slightly lower than that of the blood within the right auricle. In animals that died within a few minutes after exposures of short Flue re 32, Blood smears of pig No. 856 ( 0.1 kg) 1*>- fore ( A) and 3 minutes afti*r(B)5-minute exposure to hot air and radiant heat at ambient tenifierature of 180 C. Animal received third-degree hums of about 85 per cent of hody surface and died 3 minutes later-witli rectal temperature of 43.5 C. Examination disclosed intra- vascular hemolysis, plasma potassium concentration of 10.1 milliequiv 1 and disintegration of erythrocytes ns shown in (BP During exposure, temperature at inter- face In'tween dermis and suMermal fat, as recorded by needle thermocouple, rose to maximum of 03 C. Levine resulted in the observation that blood presr sure fell rapidly and (hat sometimes animals died following the intravenous inject ion of a small amount of heated citraleA plasma. However, when heparin was used as an anticoagulant instead of cit rate, ani- mals tolerated relatively large intravenous injections of heated plasma without ill effects and without sig- nificant change in blood pressure. Slight lowering of blood pressure was observed in a few animals alter injection of heated heparinized plasma or the sedi- ment of heated plasma. No deaths occurred, how- ever, even when amounts as great as 15 ml were used. It was concluded (hat (he particulate masses in preheated blood described by Rabat and Levine may lie deleterious to a slight degree and in combination with sodium citrate (250 mg 10 ml of blood) may cause death if injected rapidly. It is not believed, however, that these masses contributed significantly to the hyperthermic deaths observed in these ex- periments. Perfusion Experiments. A heart-lung preparation (Starling method) was perfused with the blood of a dog that had died of circulatory failure 7 minutes after lining immersed in hot water at 70 C. Continu- ous records of the heart-lung preparation included SECRET EXPOSURE TO HOT AIK AND RADIANT HEAT 3«I duration and high intensity there was characteristi- cally a difference of several degrees between rectal and blood temperature (see Section 17.11). The correlation between severity of systemic hy- perthermia and the occurrence of early death is shown in Figure 33. With one exception all pigs that Pathological Physiology 1 Prior to the exposure of several pigs to hot air, in- sulated electrocardiographic loads wore connected with the extremities and a carotid cannula was in- (roduced. The effect of the exposure on the rale of respiration, the pulse rate, the arterial blood pres- sure, and the conduction system of the heart of those animals was observed. Within a few seconds after exposure, there was a sharp increase both in blood pressure and in rate of respiration. The respiratory rate continued to in- crease and remained rapid for some time after the exposure was terminated. Soon after the initial rise there was a tall in blood pressure to or slight ly below the pro-exposure level. In some animals, the pressure was well maintained at that level until within a few minutes before death, whereas, in others, there was a gradual and progressive decline beginning immedi- ately at the conclusion of the initial rise, inability to control the movements of the animal during the period of exposure made it impossible to secure satis- factory records of venous pressure. Elect rocardiographic abnormalities were observed in some animals soon after the beginning of exposure, whereas, in others, such changes did not develop un- til well after the onset of circulatory failure. Abnor- malities observed in a few instances soon after the beginning of the exposure (within 2 or 3 minutes) in- cluded increase in rate, reduction in t be voltage of the (>RS complex, and inversion of the T waves. Ventricular extra-systoles were observed and as the exposure was prolonged there were greater disturb- ances in rhythm. Such animals developed vent ricular tachycardia followed by fibrillation and death. Although abnormalities in the electrocardiogram were sometimes observed before there was evidence of respiratory failure, the. terminal and agonal fall in blood pressure usually occurred at about the same time that tachypnea gave way to intermittent peri- ods of apnea. — Although the results of these experiments indicated that there were two types of hyperthermic circula- tory failure, one central and the other peripheral, it was obvious that further and more rigidly cont rolled physiological experimentation was required. Such studies were not feasible in the circumstances in which the hot air experiments were conducted (see Section 17.11). Changes in filood Potassium. Samples of blood were withdrawn by cardiac puncture before, during, and after lethal exposures of four pigs to hot air. It was Ficcke 33. Distribution of animals according to maxi- mum 30-inimitc rise in rectal temperature following exposure to hot air and radiant heat. Initial tempera- tures were low liccause of pentobarbital sodium an- esthesia. Open portions of columns represent animals that survived; shaded portions, animals that died dur- ing or within 30 minutes after exposure. It is apparent that there is close correlation between systemic hyper- thermia and death. died during the early postexposure period were those that developed rectal or heart's blood temperatures of 42.5 C or higher. Xo pig whose rectal temperature rose to 44 C or higher survived for more than a few minutes. Eleven of the 15 that developed rectal temperatures between 13 and 14 C and 4 of the 13 with rectal temperatures between 12 and 43 C died during the episode of hyperthermia. SECRET 362 STUDIES OF THERMAL INJURY —CUTANEOUS AND SYSTEMIC found that the potassium concentration of pig’s blood is approximately 50 milliequiv 1. The partition of potassium between erythrocytes and plasma is approximately 50 to 1.5. The post exposure plasma levels in these four animals were 7.3, 10.0, 17.4, and 19.0 milliequiv 1, respectively. The observation that cutaneous hyperthermia was capable of causing the plasma potassium to, rise to 17 milliequiv ! and higher suggested acute potassium poisoning as a po- tential cause of death. Further investigation of the importance of potassium release to the occurrence of circulatory failure and death following exposure to heat will be discussed in Sections 17.10 and 17.11. I7.«).t Summary The time-temperature relationships responsible for varying degrees of cutaneous injury and for acute circulatory collapse and death incident to exposures to circumambient and circumradiant heat similar to those that may result from a conflagration or from a flame t hrower attack have been determined for t he pig. At relatively low air temperatures (under 120 C) man, because ofhis ability to sweat, is undoubtedly less susceptible to injury than the pig. It is doubtful, however, that sweating provides a significant degree of protection at higher temperatures in which the rate of heat transfer to the skin is considerably more rapid than the rate at which it can be dissipated by vaporizat ion of sweat . It should be borne in mind that the relationships of source temperature to injury production derived from these experiments apply to unprotected skin and are not valid for exposures in which the skin is protected by hair or clothing. It has been shown that the time-tissue tempera- ture relationships responsible for t ranscpidermal necrosis (second-degree burning) by exposure to hot water as given in equation (15) (Section 17.6) are equally applicable to exposures to circumambient and eireumradiant heat. The severity of the immediate physiological dis- turbances resulting from exposure to excessive heal is frequently disproportionate to the severity of cu- taneous burning. Rapid circulatory collapse and death may result from exposures of such low intensity that little or no burning of the skin is sustained. Ex- posures ot short duration at. higher temperatures may cause severe and generalized cutaneous burning with remarkably little systemic physiological reaction during the early postexposure period. The severity of the immediate physiological dis- (urbanees resulting from exposure to excessive heat bears a quantitative relationship to the extent to which the body temperature is increased. Pigs in which the recta! temperature failed to rise above 12 0 rarely and those in which it rose as high as 44 C invariably died of acute circulatory failure. In ani- mals that died within a few minutes after exposure to excessively high environmental temperatures, the temperature of heart's blood was consistently higher than that recorded by a rectal thermometer. The shorter the interval between onset of exposure and death, the greater was the difference between the temperature in the rectum and that in the heart. Although the precise physiological mechanisms re- sponsible for hyperthermic circulatory failure were not fully elucidated by these experiments, it was ap- parent that the early death of some burned animals was caused or contributed to by hyperpotassemia. Perfusion experiments failed to disclose the pres- ence of injurious humoral agents (other than po- tassium) in the blood of recently burned animals. Pathological examination of the bodies of animals that died during or soon after an episode of acute systemic hyperthermia disclosed evidence of capil- lary endothelial damage in tin’ form of disseminated visceral petechiae. Int ravascular hemolysis and al- terations in the form and fragility of erythrocytes were observed in animals that had sustained severe cutaneous burning. 17.10 HYPEKPOTASSEMU CAUSED RY EXPOSURE TO HEAT it.to. l Introduction In Section 17.1), it was observed in some experi- ments that cutaneous exposure of pigs to excessive heat resulted in rapidly fatal circulatory failure that was associated with marked electrocardiographic ab- normalities and a sharp rise in plasma potassium to levels ordinarily considered incompatible with life. The implication of t hese observations was such as to warrant further study of the effect of cutaneous hyperthermia on the concentration of potassium iu the blood. 17.10.2 Experimental Procedure Samples of blood for chemical analysis were ob- tained from the heart by means of an inlying jugular cannula. Potassium determinations were carried out on t he trichloroacetic acid filtrate of plasma and lysed blood according to the method of Lowry and 1 fast- SECRET HVPERI’OTASSEMIA CAUSED BY EXPOSURE TO HEAT 303 ings ® as modified by Cohn and Tibbetts. Hema- tocrit was determined in \\ hit robe tul>es after cen- trifuging for 30 minutes at 2500 rpm. The method of Bing,33 et al, as modified by Ham,34 was used for de- termining plasma hemoglobin. Whole blood hemo- globin was determined on 0.1 ml of 1 5 dilution of blood in 6 ml of dilute ammonia by the Ivlett-Sum- merson colorimeter. appears that the observed decrease in the concen- tration of intracellular potassium from 132 to 128 mil- licquiv I was probably due to swelling of red cells rather than to loss by leakage. Since the actual po- tassium content of the erythrocytes did not appear to have dropped and since there was no hemolysis, it was inferred that (he porassium in the plasma had been increased by diffusion from extravasoular sources. The need for taking blood promptly after death, if reliance is to be placed on analytical results, is illus- trated by the rise in plasma potassium that occurred during the first hour post mortem. At death, the plasma concentration of potassium was 9.3 milli- equiv 1, wheivas 1 hour later it was 1(5.8. Although there is no evidence in the data presented in Table 19 as to the source of this increment, other observations indicated that both leakage from red blood cells and diffusion from extra vascular tissues may cause a post-mortem rise in plasma potassium. So far as the significance of this experiment in providing control data is concerned, it is apparent that a twofold rise in plasma potassium may occur as a result of severe systemic anoxia. In order to” correlate chemical data with known degrees of cutaneous hyperthermia, it was decided to submerge animals in hot water rather t han expose them to hot air. By the former method, the tempera- ture of the surface of the skin could be controlled wit h greater precision than was possible by the lat ter. The experimental procedure that was followed in submerging animals in hot water is described in de- tail in Section 17.11. The animals were anesthetized with pentobarbital sodium and between 00 and 75 per cent of the total body surface was raised to the desired level. The effects on the blood of exposing four pigs to water at 47 C and eight pigs to water at 75 C are shown in Table 20. Exposure at 17 C: Although all these animals de- veloped an acute and rapidly fatal systemic hyper- thermia, none showed a rise in plasma potassium sig- nificantly greater than that which may result from anoxia independently of hyperthermia. In none of these was the magnitude of the increase comparable to that which was observed in some of the severely burned animals reported in Section 17.9. In the first two animals, it appeared that the po- tassium increase in the plasma was derived from ex- travascular sources. In the third animal the increase was due to leakage in only one sample. In the fourth animal it may have been due in part to leakage from 17.10.3 Animal Experiments 1 Before undertaking further investigation of the relationship of hyperthermia to the development of hyperpotassemia, an experiment was undertaken to determine the effect, of systemic anoxia on the po- tassium concentration of the plasma independently of hyperthermia (Table 19). Tabi.e 1ft. Changes in the blood of a pig during and after death by strangulation Hemoglobin in Vol- Hemo- plasma Blood ume globin % Potassium Potassium with- packed in cells heinoly- in red cells in plasma drawn cells g 100 ml sis millicquiv/1 milliequiv/l Control 45 83 0 132 5.2 0 min Trurhm damped 4 min 4ft 31 0 128 ft.l 8 min 48 34 0 130 9.3 8 min Animal died 68 min ? ? 0 ? 16.8 A cont rol sample of blood was taken from an S.2-kg pig. The trachea w as then exposed and clamped and after 4 and 8 minutes additional samples of blood were obtained. The animal died at the end of 8 min- utes and was allowed to remain on the operating table at room temperature for an hour thereafter, at w hich time, the fourth and last sample of blood was withdrawn. The analytical results are shown in Table 19. It may be seen that the plasma potassium level was almost doubled during the 8 minutes that elapsed between the onset of asphyxia and death. Most of the increase occurred during the first 4 minutes of this period. There are two obvious sources from which the increment may have been derived, one being the erythrocytes and the other the extravascular tissue. A comparison of hematocrit and hemoglobin content of cells at the end of the 1-minute period indicates that swelling of erythrocytes had occurred. The hematocrit rose from 45 to 49, whereas the hemo- globin dropped from 33 to 31 g per 100 ml of cells. It SECRET 364 STUDIES OF THERMAE INJURY — CUTANEOUS AND SYSTEMIC intact erythrocytes, and in part to diffusion from kind produced in those animals did not result in a extravascular tissue. Cutaneous hyperthermia of the significant amount of intravascular hemolysis. T MILE 20. Effects on the blood of exposing pigs to hot water. Potassium in plasma — milliequiv/l Hemoglobin Increment lltood He mo- in Potassium Potential from Thermal Body Time samples Volume globin plasma in red increment source* I*i(5 Time ex pusure temp of time parked in cells % hemol- nils from other than No. . min c c death taken erlla g/100 ml ysis milliequiv/1 Total Change hemolysis hemolysis 877 Control 34.3 Control 32 29 0 145 3.8 0 Started 1 10 10 min 33 30 0 1.58 6.2 +2.1 0 2 4 14 ‘47 14 mm 33 30 0. 154 6.9 +3.1 0 3 1 24 44 3 21 min 31 32 0. 1.58 8.2 +4.4 0 44 20 Stopped + 1(157 Control 37.0 Control 35 35 0.1 115 4.1 0 Starter! 1 — - 20 47 20 min 36 35 0.0 125 7.0 + 2.6 0 2.6 30 Stopped 15.5 + 36 min 36 35 0.2 120 10,2 +5.8 0.2 5.6 1056 Coni rol 37.8 Control 33 37 0.1 118 4 7 0 Started ' 10 10 min 33 36 0.1 114 59 + 1.2 0 1.2 15 47 15 min 35 33 0.1 113 7,2 , +2.5 0 2.5 34 34 min 36 35 0.2 US 7.1 + 2.4 0.1 2.3 43 Stopped 45.5 + 923 Control ? Control 48 33 0.0 f 3.8 0 Started [ — 13 13 min 47 14 0.1 124 5.5 + 1.7 0.1 1.6 23 23 min 46 40 0.2 120 5,5 + 1.7 0.2 1-5 34 ■1/ 34 min 55 32 . 0,1 113 6.2 +2.4 0.2 2.2 42 42 min 55 32 0.1 112 6.5 + 2.7 0.2 2.5 47 47 min 36 33 0.1 7.5 +3.7 0.2 3.4 SO Slopped ? + 809 Control 37.4 Control 38 34 0.4 139 3,6 0 Started u. 1 Stopped 75 — 5 5 min 48 31 3,0 109 10.2 + 6.6 3.7 2.9 10 16 min 37 33 80 117 6.9 +3.3 0.5 40 46 min 39 32 7.5 IIS 4.2 +0.6 6 2 76 39.2 76 min 37 35 6.7 122 7.4 +3.8 5.2 918 Control 30.0 Control 34 41 0.0 131 3.7 0 Started >75 7* _ 3 Stopped 4 4 min 51 30 2.5 98 11.0 + 7.3 2.6 4.7 a 11 min 45 42 4.4 no 9.5 +5.8 4.2 1.6 17 17 ruin 44 35 5 9 102 9.5 + 5.8 5.1 0.7 37 406 37 min 10 48 5.6 103 9.4 +5.7 4 0 1.7 55 + 919 Control Control 43 33 0,8 118 4 2 0 Started 37.1 — . . . 4 >75 1 min 36 29 7.8 81 25.5 +21.3 8.6 12.7 3 Stopped 8 8 min 47 26 255 67 21.4 + 17.2 20.2 10 10 min 40 32 22.2 *» 18.3 + 14,1 T M 14 min 35 37 23.1 77 17.0 + 12 8 12.6 17 44.3 17 min 33 31 30.1 72 17.5 + 133 15.2 18 + 913 Control 380 Control 26 37 0.0 no 3 5 .... 0 Started - — 2 1.5 2 min 35 32 12.3 103 14.2 +10.7 7.7 3.0 6 6 min 32 33 24.5 06 17,7 + 14.2 14.7 7 Stopped] 8 10.8 + 8 min 30 32 25.3 in 17.4 + 13.9 16.0 ... 907 Control 37.3* Control 12 34 0.6 125 3,5 0 .Started 8 „o 8 min 53 31 2.7 100 17.4 + 13.9 3.1 10.8 10 Stopped] 42.5* 4- * Right heart temperature. SECRET HYPERPOTASSEMI V CAUSED UV EXPOSURE TO HEAT 305 Table 20 (Continued). Pi* Ko, Time min Thermal exposure c Body temp C Time of death Blood samples time taken Volume parked fells Hrmo- idobin in eells *100 n a Hemoglobin in Potassium plasma in red % hemol* eells j sw mitliequiv. I Potassium in plasma milliequiv/l Increment Potential from increment sources from other than Total Change hemolysin hemolysis 910 Cont rol 36 8 Control r 7 3.0 0 Stalled 2 2 min 7 7 19.1 + 16.1 5 5 min ? 7 181 + 15.1 7 40 . . . 7 min ? J ... * 24.0 1 21.0 13 + . 14 Stopped 13.7 14 min ... - 17.3 + 14.3 908 Cont rol * Cont rol 32 f 7 106 3.8 0 Started — — 4 4 min ? 7 7 * 16.7 + 12.9 » i75 9 min 33 ? 7 98 18.5 + 14.7 . . 11 11 min 32 f 7 90 17.1 + 13.3 H Stopped 7 + 912 Control 36.0 Control 33 37 0.0 125 4.1 0 Started 1 I rain 4 a 31 1-9 102 16.7 + 12.6 1.6 11.0 4 ... 4 min 33 37 19.2 7 7 ? 5 5 min 34 31 21 2 100 164 + 12.3 16.5 10 10 min 40 31 19 9 85 16,4 + 12.3 14.2 14 Stopped 43.1 + Exposure at 75 C: The chemical changes in this group were of a different order of magnitude from those observed in animals exposed at 47 C. All ani- mals exposed for 5 minutes, or longer, at 75 C de- veloped plasma potassium levels in excess of 10 milli- equiv/1. In most instances, such levels were reached during the first few minutes of exposure and were either maintained or increased as the period of ex- posure was prolonged. If the pig survived for more than a few minutes after the termination of the ex- posure, there was a slow decline in plasma potassium concentration. Thus, in animal 919 the plasma po- tassium rose from 4.2 to 25.5 rnilliequiv during the first 4 minutes of exposure, and during the next 4 minutes declined to 17.4. The rapidity with which an excessively high plasma potassium level may be lowered by extra- vascular diffusion is indicated by the discrepancies that were observed between estimated increments by hemolysis and total amounts present. Thus, it may be seen in the case of pig 913 that with an incre- ment by hemolysis of 7 milliequiv/1 between the 2- and 0-minute samples, the actual plasma level rose by only 3.5 rnilliequiv. Similarly, in pig 912 the in- crement by hemolysis between the 1- and 5-minute samples was 14.9 rnilliequiv 1, whereas the total plasma potassium actually changed from 10.7 to 10.4 during this period. In most of the animals exposed at 75 t', there was some increase in the volume of packed cells. The comparison of cell volume and hemoglobin content indicated that most, if not all, of the early increase in cell volume was due to swelling of erythrocytes rather than to loss of plasma or mobilization of cells from storage depots. It is of interest to note that plasma hemoglobin values as high as 24 per cent hemolysis were observed as early as 5 minutes after the onset of cutaneous hyperthermia. It was estimated that during this period the temperature in the vicinity of the most superficial blood vessels probably rose to approxi- mately 70 C. Chemical changes in the blood of dogs caused by cutaneous hyperthermia; It was inferred from the foregoing experiments on pigs that most of the po- tassium responsible for these potentially fatal plasma levels either leaked out of intact red blood cells or escaped from hemolyzed cells. If this inference is cor- rect, fatal hyperpotassemia due to cutaneous hyper- thermia would occur only in animals having a high concentration of potassium in the erythrocytes, such as man or pig. Its occurrence could not be expected in an animal having a low cellular concentration of potassium, as is the case in dog’s blood. To test this assumption, samples of blood were taken from each of five dogs before and during im- mersion in hot water. The results of these experi- ments are shown in Table 21. The animals were exposed at temperatures ranging between 55 and 75 C until death occurred. The high- SECRET 366 STUDIES OF THEUMAL INJl RY — CUTANEOUS AM) SYSTEMIC Table 21. Changes in blood of dogs caused by immersion in hot water. Blood Time Thermal Body Time samples Volume Hemoglobin I lemoglobin Potassium Potassium Hog in exposure temp of time packed in cells in plasma in red cells in plasma Xo, min c C death taken cells g 100 ml % hemolysis milliequiv 1 milliequiv ! 931 Control 35.4 Control 35 37 0 9.4 2.8 0 Started 5 5 min 41 36 0 8.1 5.2 13 55 13 min 57 32 0 10.7 4.7 21 41.4 21 min 57 33 0 11.2 6.9 23 Slopj)ed) + 930 Control 36.9 Control 49 34 0.1 4.3 4.0 0 Started 5 . - . 5 min 66 27 17.9 6.4 33 8 60 8 min 65 28 J 20.2 5.5 4 7 11 39.1 11 min 62 28 / 23.8 6.1 5.3 17 Stopped + 929 Coni rol 37.2 Control 49 34 0.3 6.3 3.9 0 Started 3 3 min 57 29 26.1 7.0 4.8 9 7 5 9 min 42 37 31.8 5.7 6.1 13 44.1 13 min 39 34 35.8 7.9 8:2 14 Stopj)cd + .. — * * * * 922 Control — 37.9 Control 42 35 0,2 8.8 3.1 0 Started 3 3 min _ 47 30 22.9 8.9 5.8 7 75 , 7 jnin 47 30 29.5 12.6 6.4 10 10 min 43 29 33.5 7.9 5.8 15 Sloped 39.3 + 15 min 45 30 31.4 8.0 6.8 931 Coni ml 34.6* Control 41 35 0.1 5.6 3.1 a Started 1 25 Stopped/ 43.5* + 25 min 40 34 31,9 6.5 6.9 ♦ HiKht heart temperature. 17.10.1 In I iiro Effects of Heat on Pig’s Blood It was thought (hat more precise information re- garding the reciprocal relationships of temperature, time, and the release of potassium from erythrocytes could be obtained by heating samples of pig’s blood in vitro. Heart’s blood was collected from normal pigs by cardiac puncture in a heparinized syringe, where it was mixed and then discharged into heparinized glass-stoppered vials. One vial was kept at room temperature as a control; the others were strapped to a mechanical mixer and immersed in a constant temperature bath. Exposure temperature's ranged be- tween 44 and 63 C; during exposure the blood was mechanically decanted from one end of the vial to the other at a rate of six times per minute. It re- quired approximately 2 minutes for the temperature of the blood to reach that of the water bath. As soon as a sample was removed from the water bath, it was immediately cooled in ice water and analyzed. It is apparent that there was a progressive increase in the rate at which potassium passed out of the est potassium concentration observed in the erythro- cytes in control samples of blood from these animals was 9.4 millieqniv ly hi contrast to the pig, whose erythrocyte concentrations ranged I»etween 106 and 145 millieqniv 1. The greatest potassium increase that occurred in the plasma of the dogs that died as a result of cutaneous exposure to heat was from 3.9 to S.2 milliequiv/1. T1 ie increments to the plasma potassium that were observed in these animals could not be accounted for by loss of potassium from the erythrocytes. The po- tassium content of the red blood cells of the dogs characterist ically rose during exposure in contrast to the loss of potassium that occurred from the erythro- cytes of the pig. As in the case of the pig, there was severe intravascular hemolysis in animals exposed at 75 C until death occurred. It can be inferred, therefore, that the development ot a potentially fatal level of hyperpotassemia fol- lowing cutaneous exposure to heat results from the rapid release of potassium from thermally injured red blood cells and that a high erythrocyte content of potassium is essential to its occurrence. SECRET HYPERPOTASSEM1A CAUSED BY EXPOSURE TO HEAT Tabi.k 22. In vitro effects of heat on pig's blood. Potassium in plasma — milliequiv/1 Time Volume Hemoglobin Hemoglobin Potassium Increment Increment S|)oci- Temp in packed in cells in plasma in red cells from from mm C minutes cells g 100 ml "% hemolysis miliicqiiiv/1 Total Change hemolysis leakage 1 047 Control Control 30 34 0.1 105 3.2 40 15 30 34 0.1 113* 3.5 (0.3 0.3 30 30 32 0.3 111* 3.5 + 0.3 0.1 0.2 60 30 34 0.1 102 3.8 +0.6 0.6 2 049 Cont rol Cont rol 32 33 0 99 3.2 44 15 31 33 0.1 107* 3.9 +0.7 0.7 30 31 34 0.1 102* 4.0 + 0.8 0.8 60 31 33 0.3 97 4.8 + 1.6 0.1 1.5 3 940 Control Control' 31 34 0 lot 4.6 48 15 32 31 0.1 101 7.5- +2.9 2.9 30 32 32 0.1 90 9.2 + 4.6 4.6 00 32 29 0.4 90 11.0 +6.4 0.1 6.3 1-930 Control Control 33 32 0.0 109 4.3 51 15 35 81 0.8 96 10.2 +5.9 0.4 5.6 30- 34 34 0.5 98 11.8 +7.5 0.2 7.3 (40 36 31 0.7 92 10.7 +6.4 0.4 6.0 5 050 Control Control 34 35 0.1 120 4.2 52 15 35 34 0,8 103 10.0 +5.8 0.5 5.3 — . . .» 30 35 32 2.7 101 10.4 +6.2 1.5 4.7 60 - 36 32 2.7 100 10.9 +6.7 1.6 ~5.1 6-1*47 Control Cont ml 31 33 0.1 109 4.2 55 15 40 28 13 85 7.5 +3.3 0.7 2.6 30 37 30 5.7 87 12.1 + 7.9 3.0 4.9 60 37 30 9.6 71 18.5 -t 14.3 4.4 9.9 7-1052 Control Control 38 33 0.0 119 3.6 60 5 48 28 1.4 S3 12.6 +9.0 1.2 7.8 8-1052 Control Control 36 35 0.1 121 4,1 61 5 45 28 3.5 76 20.8 + 16.7 2.4 14.3 9 10.52 Cont rol Control 34 36 0.0 122 3.9 62 5 33 34 11.7 69 30.8 +26.9 4.5 22.4 10-1052 Control Control 30 36 0.1 121 4.1 63 5 26 34 31.6 58 40.2 +36.1 9.6 26.5 ♦ These values must be due to analytical errors. erythrocytes and into the plasma of the blood as its temperature was raised ('fable 22). The amounts of the plasma increment at the end of 1 hour’s exposure at ft), Id, 18, 51, 52, and 55 C wen* respectively 0.G, l.G, 6.1, 6.4, 6.7, and 14.3 milliequiv 1. At the lower temperatures (51 C and under), the increments were due almost entirely to leakage from intact cells. At the end of 30 minutes of exposure at 52 and 55 0, the proportion of the plasma increment contributed by hemolysis was 24 and 38 per cent, respectively. Unequivocal ev idence of swelling of erythrocytes was first observed at 55 C, although there may have been some swelling in all specimens exposed for more than 30 minutes at IS O and higher. The rate of change in the blood was much more rapid during exposures at 60 C and higher. In these experiments the blood remained in the bath for only 5 minutes and the actual time during which it was at the temperature of (he water was approximately 3 minutes. The rise in plasma potassium after such brief periods at GO, 61, 02, and 63 C was, respectively, 9.0, 10.7, 20.9, and 30.1 imiiiequiv/1. The biood was totally hemolyzed at 05 0'. Not until blood was heated at GO or higher in a test tube, were the observed increases in plasma potas- sium comparable with those that occurred in living pigs after cutaneous exposures at 75 C. This is not to imply that the effects of hyperthermia on blood in a test tube are necessarily similar to those effects in a living animal. Attention has already been called to the fact that asphyxia without rise in temperature may cause hyperpotassemia in a living animal. Al- though the mean temperature of the blood of a living pig is never raised to GO C, most or all of its blood may in the course of its circulation through the over- heated dermis be brought to a much higher temper- ature than would be recorded by a rectal thermom- eter or intracardiac thermocouple. It will be recalled from the calculations made in Section 17.3 that the superficial portion of the dermis of a living pig SECRET STUDIES OF THERMAE INJURY — CUTANEOUS AND SYSTEMIC reaches a temperature of GO C within a second after the surface of the skin has been brought to 75 ('. It would appear quite possible then that the temper- ature of most or all of the blood of an animal that had received an extensive cutaneous exposure to water at 75 (’ for as long as 5 minutes would be raised briefly during its passage through the sub- cutaneous tissue to the neighborhood of 60 C. Not until the temperature of the bath was raised to 62 C did a 5-minute exposure of blood in a test, tube result in hemolysis comparable with that ob- served in living pigs exposed at 75 C. Attention has already been directed to the fact that unequivocal swelling of erythrocytes was first observed in a test tube after a 15-minute exposure at 55 C. So far as could he judged by the hemoglobin-, hematocrit ratios, swelling of erythrocytes continued through 61 C, beyond which if was not observed. 17.10.5 Summary These experiments have established that severe and extensive cutaneous burning may result in a rapid rise in plasma potassium to levels ordinarily considered incompatible with life. Such levels are obtained when a large proportion of body surface is maintained at 75 C for more than a few minutes. That lower surface temperatures may also be re- sponsible for fatal hyperthermia is suggested by the fact that potassium is released rapidly from blood cells in vitro at temperatures of 60 C or over. In pari because of the slowness with which potassium is re- leased at lower temperatures and in part because of the rapidity with which excess potassium leaves the blood stream, it is not likely that thermal exposures of insufficient intensity to cause severe cutaneous burning could cause sufficient damage to t he erythro- cytes to produce dangerously high plasma levels. In vitro experiments on pig’s blood indicate that rapid leakage of potassium from erythrocytes oc- curred when its temperature was raised over GO C and that rapid hemolysis occurred when its temper- ature was raised above 62 C. Leakage was accom- panied by swelling at temperatures ranging between 55 and til C. Above that temperature, so far as could be judged by the hemoglobin content of cells, rapid release of potiissium occurs without cell swelling. It was demonstrated that leakage from and lysis of red blood cells were the principal sources of the potassium increments of plasma. At the lower tem- peratures (47 C in vivo and 4S C in vitro) hemolysis was negligible. The increase in plasma potassium in vivo at these tcnqKTaturcs was due either to dif- fusion from extra vascular sources or to leakage from erythrocytes. It was obvious in the lower-temper- ature in vitro experiments that leakage from erythro- cytes was the only source, of the plasma increment. Although leakage alone could be sufficient to account for potentially fatal plasma levels (in excess of 16 milliequiv 1), no such increases were observed without accompanying hemolysis. When blood was heated in vitro leakage contributed more than hemol- ysis to the attainment of such levels. In thermal exposures in vivo of sufficient duration and intensity to produce comparable levels, hemolysis was the more important factor. . I7.il PHYSIOLOGICAL DISTURRANCKS FROM KXCKSSIVE I1K\TJ 17.11.1 Introduction 9 In Sect ion 17.9 of 1 his chapter, at ten tion was callci 1 to the fact that acute hyperthermic circulatory fail- ure in some animals was accompanied by, and un- doubtedly contributed to by, large increases in the potassium concentration of the plasma. An investi- gation of (he circumstances in, and the sources from which, thermally induced rises in plasma potassium occur has been described in Section 17.10. Although it appeared that central circulatory fail- ure caused by hyperpot assemia was one of t he mech- anisms responsible for death incident to cutaneous exposure to heat, it was apparent that this was not the sole cause of death during hyperthermia. The following investigations2 were undertaken for the purpose of determining the precise nature of the various kinds of circulatory disturbances which may result from cutaneous exposure to excessive heat. The acute physiological disturbances caused by systemic hyperthermia have at tracted the attention of a number of investigators. 1 leymans 26 injected methylene blue into dogs anesthetized with chloral- ose. This produced a gradually mounting rectal tem- perature which reached the lethal level of 43.7 to 44.8 C in 1 to 1} _> hours. The heart rate rose gradu- ally from 90 120 to 300-330 per minute. At first the respirations were deep and rapid (less than 200 per minute); after the temperature had risen to 41.5- 43.5 C they became very shallow and even more rapid (over 300 per minute). Systolic pressure rose and diastolic pressure fell. Respiration almost always failed first, and artificial respiration enabled the J By Alticrl Rons. SECRET PHYSIOLOGIC AL DISTURBANCES FROM EXCESSIVE MEAT heart to continue for a longer time. Reflexes per- sisted up to the time of respiratory standstill. IJveno45 produced hyperthermia in eats, anesthetized with urethane, by exposing them to water of 11-42 C or to a high environmental air temperature. During the 30 minutes of exposure the rectal temperature rose from 35 to 39 C. There was little increase in heart- rate, but a pronounced rise in minute-volume out- put, Shortly after exposure the respiratory rate in- creased to an average of 2tH) per minute. This breath- ing was very shallow (tidal air 2 3 ee per minute) and sometimes resulted in a 29 per cent, drop in arterial oxygen saturation, (’beer11 placed dogs anesthetized with morphine and barbital in a cabinet heated hy electric light bulbs. In 2 to 3 hours a lethal (rectal?) temperature of 43 15 C was reached. The heart rate increased progressively unt il a temperature of 42 1IC wjls reached, when the heart slowed rather suddenly. Before this stage electrocardio- graphic abnormalities were limited to slight abbrevi- ation of the 1*1! interval, slight changes in the QRS complex, and Inversion of the T wave. The terminal bradycardia was due to the development either of nodal rhythm or of various other types of ventricular rhythm. Systolic and diastolic pressures remained fairly constant up to 41 C, then both dropped;"the former more than the latter. The respiratory rate also increased. Respiratory standstill usually oc- curred before cardiac arrest, vagotomy delaying respiratory failure. A progressive decrease of the blood carbon dioxide was found associated with slight alkalosis and rise of oxygen content, which were ascribed to the increased pulmonary ventila- tion. From the same laboratory, Wiggers and Orias 47 reported observations on the effects of short radio waves on dogs. The cardiac acceleration, increase in rate and depth of 1 lie respiration, and primary fail- ure of the respiration were identical with the findings of Cheer.13 However, instead of a decrease in blood pressure, a rise of systolic and diastolic pressure was observed which progressed until death. Clinical observations on the effect of hyperthermia were made by Ferris et al,15 Patients with heat stroke whose rectal temperatures varied from 39.9 to 11.0 C exhibited a hot dry skin, a normal or elevated sys- tolic pressure, which dropped to low levels only in the terminal stage, and venous pressures of from 2 to 12 cm of saline. Their respiratory rate was 28 to 50 per minute. Of 29 patients (all comatose) whose temperatures exceeded 41.5 C, 17 died; all others recovered. Attempts to analyze the disturbances observed in the intact organism by elevating the temperature in one organ have been made since 1872. Fiek 16 heated the bio nl as it passed through the carotid arteries of the dog and noticed marked hyperpnea without change in heart rate or blood pressure. Cyon 14 iso- lated the circulation of a dog’s head. Perfusion of the head with heated blood produced bradycardia and a drop in blood pressure. Kahn 2* warmed the carotid arteries of unanesthetized dogs without producing a rise in rectal temperature. He observed the develop- ment of tachycardia and a moderate rise in blood pressure. Moorhouse M heated the carotids and simul- taneously cooled the jugular veins in dogs. Phis re- sulted in tachycardia, rarely preceded by brady- cardia, ascribed respectively to increased sympa- thetic and vagal activity. Coincidentally, tachypnea and peripheral vasodilatation were observed. liey- mans and Ladon severed all connections except the vagal nerves between head and trunk of dogs anes- thetized with chloralose. Artificial respiration was applied and the circulation in the head maintained by connecting it to a donor dog. The sublingual tem- perature of the preparation rose to to C in 1J o hours. There was no change in the heart rate which had risen to I GO after severance of the cervical cord. The head exhibited a progressive and pronounced in- crease in respiratory rate which persisted until a sublingual temperature of 15 C was reached, when the rate rapidly decreased and the reflexes of the head, which had been active up to that time, dis- appeared. . The effect of hyperthermia on the heart was in- vestigated by Kuowlton and Starling,*1 using the innervated heart-lung preparation perfused with heated blood. From 2G C t o approximately 45 C the heart rate was a linear function of the blood temper- ature, the rate at 45 C being 180 per minute. Above this temperature marked slowing occurred and the heart soon stopped. Arrhythmias occurred above 40 C. To summarize these data, it can be said that in the dog the highest rectal temperature compatible with life lies between 43 and 15 C, when this temperature is reached in I to 3 hours. Respiratory failure often seems to precede circulatory failure. Tachypnea, tachycardia, and peripheral vasodilatation seem to be, in part at least, of cerebral origin. The physiological changes of rapidly developing hyperthermia leading to death within half an hour have not been heretofore studied. As high environ- SECRET 370 STUDIES OF THERMAL INJURY — CUTANEOUS AND SYSTEMIC Tabi.k 23. Rectal temperature, arterial pressure, and electrocardiogram of 12 pigs immersed in hot water. A - Normal sinus rhythm (normal rate, tachycardia, or bradycardia). Normal duration of QRS complex. A' — First or second degree A V block. Normal duration of QHS complex. A" — Complete A V block. Normal duration of QliS complex. H — Slight BB Moderate Widening of QRS complex without P wave. HUB — Pronounced j BBB -- Can often be interpreted as ventricular fibrillation. Time rnin sec Rectal temp C Arterial pressure (mm Hg) F.CG Rectal Arterial Time temp pressure min sec C (mm Hg) KCCi Pig 876 (7.7 kg) 48 C. Died after 26.5 min. Control 34.3 118 A 1G .. 44.0 GG A 24 :t0 45.2 42 A 26 30 45.7 7G BB Pig 875 (6.4 kg) 48 50 C. Died after 35 min. Control 35.0 130 A 27 30 42.2 64 A 29 .. 42.8 64 A 34 15 43.7 26 Pig 878 (12,0 kg) 47 C. Died after 50 min. Control ... 410 A 29 .. ~ ... 70 A 37 20 ... ■ 50 A 49 .30 44.9 30 A Pig 879 (11.8 kg) 44-47 C. Died after 106.5 min. Control 36.8 106 A 33 .. 43.1 5-1 A Out of hot bath* from 33.5 to 48.5 min. 49 53 420 116 79 30 44.1 86 A 105 .. 44.5 14 A Pig 895 (18.0 kg) 49 C. Curare. Died after 32 min. Control 37.8 148 A 15 .. 41.9 ~*172 A 25 30 43.7 76 A 31 30 44.0 10 A'f Pig 943 (8.3 kg) 47 C. Curare. Died after 36 min. Control 37.7 126 A 17 .. 42.6 126 A 29 .. 44.5 136 A Pig S!17 (16.4 kg) 47 C. Curare. Died after 56 min. Control 37.9 — 146 24 .. 43.5 146 A 47 .. 44.0 90 A 55 ., ... 36 A' Pig 946 (9.5 kg) 47 C for 23 min. Curare. Died after 42 min. Control 40.1 82 A 17 .. 43.0 — 120 A 26 .. 44.4 40 A 34 30 44.6 26 A Pig 944 (10.4 kg) 47 C for 25 min. I>icd after 99 min,— Control 38.1 108 A 14 .. 43.5 120 A 26 ,r~ 45.4 100 A 37 ... 44.1 90 A Pig 867 (7.3 kg) 64 65 C. Died after 15 min. Control ... 146 A 5 30 ... 72 A 10 30 ... 72 — A 15 .. 46.0 12 _ BB Pig 872 (7.3 kg) 64 65 C. Died after 11 min. Control 150 A 7 ... 50 A 10 30 ,.. 50 BB 10 45 ... 40 BBB Pig 871 (9.1 kg) 70 73 C. Died after 12 min. Control ... 100 ... 5 30 ... - - 74 A 6 10 ... 74 BB 9 30 ... 54 BBB 12 .. 44.5 24 BBB * Skin temperature lowc t Occasional ventricular red hy exposure to cool water between two cpisod extra-systole. s of cutaneous hyperthermia. mental temperatures am needed for such experi- ments, the results are necessarily complicated by the damaging effect of heat, on the skin directly. More- over, these high temperatures will produce damage to the red blood cells that are circulating in the small vessels of skin and underlying (issues.42 iT.li.2 Experimental Procedure Young pigs weighing from (>.4 to 18 kg and adult dogs weighing from 7.1 to 8.5 kg were used as ex- perimental animals. They were anesthetized with pentobarbital sodium (32 mg kg intraperitoneally), shaved, and tied to a wooden animal board. This was lowered into a galvanized iron tank (92x10x41 cm). 4'he head of the hoard rested on a metal bar" in the tank, so that it was slightly higher than the loot. A similar tank, placed on a high table, partly projected over Ihe former. This tank was filler! with water steam-heated to the desired temperature. In the bottom of the projecting part was a circular opening 13 cm in diameter that could be closed with a heavy rubber and metal stopper, resulting in full immersion in 8 to 10 seconds. During im- mersion, the temperature ot the water, which was continuously stirred, was kept within narrow limits by intermittent introduction of steam. Drainage of SECRET PHYSIOLOGICAL DISTURBANCES FROM EXCESSIVE HEAT 371 Table 24. Rectal temperature, arterial pressure, electrocardiogram, hematocrit, and hemoglobin and potassium content of plasma md of red bio id cells of 15 pigs immersed in hot water. A — Normal sinus rhythm (normal rate, tachycardia or bradycardia). Normal duration of QitS complex. A First or second degree A- V block . Normal duration of QRS complex. A "— Complete A-V block. Normal duration of QRS complex. B — Slight ) BB — Moderate Widening of QRS complex without P wave. HUB — Pronounced 1 BBB —- Can >ften lx interpreted as v entrieular fibrillation. Rectal Rectal Time temp Arterial K plasma Time temp Arterial K plasma min sec C pressure i:cc. milliequiv/1 min see C pressure KCG millicquiv/1 Pi a S77 (7.0 kg) 47 C. Died after 26 min. Pig 905 (12 7 kg) 75 C. Curare. Died after 23 min. Control 34.3 96 A 3.8 Control . . . 94 A 1.8 10 20 41.6 136 A 6.2 16 30 41.6 78 BB_ 11 5 42.5- 112 A" 6.9 22 40 42! 32 BBB 17.3 24 10 44.3 56 A" 8.2 Pig 921 (16.8 kg) 75 C. Curare. Died after 27 min. Pig 923 (13.6 kg) 47 C. Died after 50 min. C’ont rol 122 A 3 2 Control 116 A 3.8 3 30 66 A 5.1 13 15 146 A 5.5 8 58 BB 11.6 22 30 146 A 5.5 IS 36 B 11.9 34 15 102 A 6.2 26 45 28 B 10.2 42 56 A A 6.5 Pig 906 (13.0 kg) 70-7. C. ('urare. Died aftr r 70 min. 40 33 66 — 7.5 Control 38.6 102 A 4.0 Pit? 057 (8.0 kg) 17 C. Died after 36 5 min. 10 50 41.4 112 BBB Control 37.0 .. t A 4.4 16 35 42.3 62 BB 17.4 19 50 A 7.0 25 20 43.0 92. BBB 15.2 30 15 : T., ■ A 10.2 44 35 44.6 72 BB 13.3 30 30 45.5 O 46 40 44.8 16 A Pin 1056 ( 7.0 kg) 47 C. Died ifter 44.5 min. 48 29 45.0 46 BB Control 37.8 A 4.7 65 46.8 46 BBB 9 30 A 5.9 Pig 913 (8.2 kg) 75 C or 6.5 rnin Died after 7.5 min. 15 7 . A 7-2 Control 38.6 1 CM) A 3,5 34 A 7.1 2 25 37.9 100 B 14.2 14 30 45.5 O 6 15 40.5 50 BBB 17.7 Pic 910 (9.5 kgi 72 75 C. Died after 12,5 min. 7 45 40.8 15 0 17.4 Control 36.8 148 A 3.0 Pig 919 (9.1 kg) 75 C for 5 min. Died after IS min. 2 15 40.7 100 A 19.1 Control 37.1 138 A 4.2 4 40 10,7 86 BB 18.1 I 15 41.1 78 BB 25.5 7 20 41.5 71 BBB 24.0 7 15 42.3 28 A 21.4 13 52 43.7 10 O 17.3 10 10 43.2 26 A 18.3 Pie 912 (10.0 kg) 7 2-75 C. Died after 14 min. 14 44.2 30 B 17.0 Control 36.0 88 A 4.1 16 45 14.3 14 B 17.5 1 20 35.4 154 A 16.7 Pig 918 (8.7 kg) 75 C for 3 rain. Died after 55 min. 3 35 37.0 '.18 BB Control 36.6 70 A 3.7 5 7 37.1 74 BB 16.4 4 25 38.7 56 A 11.0 9 45 40.8 74 BBB 16.4 II 39.7 62 A 9.5 13 10 43.1 30 BBB 17 5 40.3 70 A 9.5 Pig 908 (9.1 kg) 75 C. Died : ifter 13.5 min. 37 40.6 70 A 9.4 Control 96 A 3.8 Pig 899 (13.6 kg) 75 Cf >r 1 min. Sacrificed after 77 min. 3 40 96 BB 16.7 Control 37.4 142 A 3.6 S 55 . . . 60 BBB 18,5 5 15 40.5 30 A 10.2 11 10 52 BBB 17.1 16 5 40.5 76 A 6.9 Pig 907 (10.4 kg) 75 C. Died after 10 min. 45 45 40.3 76 A A 4,2 Control 37.1 37.3* 116 A 3.5 76 39.2 76 7.4 6 39.0 42.7* 48 BBB _ 7 30 39.2 42.5* 32 BBB 17.4 ♦ High! heart temperature. SECRET 372 STUDIES OF THERMAL INJ LKY — CUTANEOUS AND SYSTEMIC (he water and termination of exposure could also be accomplished in S to 10 seconds. Temperatures rang- ing from I 1 to 75 C were used. Previous to exposure, all animals were heparinized (3 mg kg intravenously). Because ol spasmodic closure of the glottis on immersion, a tracheal can- nula was inserted. The carotid pressure was recorded with a mercury manometer. The rigid auricular pres- sure was measured by means of a rubber catheter introduced into the superior vena cava or right auricle by way of the external jugular vein and con- nected with a water manometer. The level of the right auricle as determined by opening (he chest at the end of the experiment was taken as the point of reference. In pigs the hydrostatic pressure did not influence the auricular pressure. In dogs immersion resulted in- a considerable rise in recorded auricular pressure, so t hat only changes occurring during ex- posure could be compared Pneumograms were ob- tained by means of a copper cannula thrust between the ribs Into the pleural space and connected by means of a rubber tube to a writing tambour. In other experiments, a tracheal cannula provided with a sealed-in side tube connected to the tambour was used. Electrocardiograms were taken with an ampli- fier type of electrocardiograph. It was only possible to take the first standard lead, as the hind legs of the animal were underwater. In some experiments, cu- rarized animals were used and artificial respiration was applied throughout the exposure. Intocostrin (Squibb) 1 mg kg diluted with saline was slowly in- jected intravenously. The side reactions were limited to a short (20 to 30 seconds) period of mild excitation. The drug had no effect on the arterial pressure. A second smaller dose usually had to be given 20 to 10 minutes later, A Palmer respiration pump for small animals, which allows the air to escape spon- taneously on expiration, was used. When venous pressures were recorded the animals were immersed in such a manner that most of the anterior thorax remained above the water level. This was sufficient to abolish artifacts produced by the increased re- sistance to the inflow of air. Temperatures were re- con led with a thermocouple introduced to a depth of 7 to 9 cm into the rectum, \Vhich had been cleaned by repeated enemas. The anus was closed around the couple. In three experiments, heart temperatures were also recorded by means of a thermocouple in- troduced through the external jugular vein into the right auricle. In some experiments only initial and final rectal and final heart temperatures were meas- ured with a sensitive thermometer. In a considerable number of animals blood was withdrawn from the jugular vein both before and during exposure for the determination of the hematocrit and of hemoglobin and potassium content of red cells and plasma (Sec- tion 17.10). In most instances, immersion was con- tinued until death. In some experiments exposure was temporarily interrupted, and, in a few cases, im- mersion was terminated at a time when the animal was still living. In addition to these observations, three pigs were infused with an isotonic (1.12 per cent) solution of KC1. Frequent electrocardiograms (lead I or 111 wen* taken. In one ol these pigs, the arterial and right auricular pressure and respirations were also recorded. The latter animal received the solution in the subclavian vein,.the other two in the jugular vein. Blood samples for the determination of potas- sium were taken from the carotid artery. 17.11.3 Results of Experiments In Table 2d are shown the results of 12 experiments in which pigs were exposed for varying periods of time at temperatures ranging between II and 73 C. Changes in rectal temperature, arterial pressure, and electrocardiogram arc indicated. In Table 21 are shown the resnltsof id experiments in which pigs were exposed at temperatures ranging between 47 and 75 C. The changes that occurred in (he potassium concentration of the plasma are indi- cated in relation to changes in rectal temperature, arterial pressure, and electrocardiogram. In Table 25 are shown the results of 5 experiments in which dogs were exposed for varying periods of lime at temperatures ranging between 55 and 75 C. The changes that occurred in the potassium concen- tration of the plasma are indicated in relation to changes in rectal temperature, arterial pressure, and electrocardiogram. In Table 2b are shown the results of 3 experiments in which pigs received intravenous infusions of isotonic potassium chloride. The changes in the plasma concentration of the plasma and the erythro- cytes are indicated in relation to changes in hemato- crit, arterial pressure, and electrocardiogram. Arterial blood pressure. The immediate effect of immersion in water of 00-75 G n[M»n the mean ar- terial pressure of pigs was a rise which sometimes amounted to as much as I 10 mm Hg. This rise also occurred in curarized animals or when hot water was SECRET PHYSIOLOGICAL DISTURBANCES FROM EXCESSIVE HEAT Fin cub 34. Plot of thermocouple recordings showing rate of change in rectal and right auricular blood temperatures during immersion in low (47 (') and high (73 C) temperature water Paths. 47 C Pig 882 (13.2 kg) 75 C Pig 1)07 (10.5 kg) It may l>e seen that, although right auricular blood temperature rises rapidly after immersion, there is considerable lag in temperature rise in rectum. The higher the temperature of the bath, the greater is the difference between the two. splashed on the skin. It was absent at immersion leni|)eratines of 45-47 C. At temperatures of 44-59 C the blood pressure was maintained at or above preimmersion level for 16 to 26 minutes. It began to fall at variable times during exposure, and reached half of the original value in 17.5 41 minutes. The rectal temperature at this time had risen from 31.3 40.1 C to 12-14 C. These ani- mals died after 25.5 to 50 minutes with rectal temper- atures of 43.9—15.8 C, the heavier pigs surviving somewhat longer than the lighter ones. Heart tem- peratures were within a few tenths of a degree of these values (Figure 34). In pigs exposed to water of 60-75 C, the arterial pressure was maintained for 1 6 minutes, and reached half of its original value in 5.5-11 minutes. The ani- mals died after S-15 minutes with rectal tempera- tures varying from 39.4-46.0 C. However, the dis- crepancy between heart and rectal temperature often was considerable (Figure 34). The possible reversibility of the fall in arterial pressure was investigated. Immersion of a pig at 17 (' for 33 minutes produced a fall in blood pressure from 104 to 40 mm Ilg (Figure 35). Exposure to cool water brought the pressure back to its original level and lowered the rectal temperature from 43.3 to 42.0 C. Re-exposure to 47 C again resulted in a fall in blood pressure, and death occurred at a rectal Tap us 25. Rectal 1 temperature, arterial pressure, electro- ran liogram , hematocrit, and hemoglobin and potassium content of plasma and of red blood cell.' s of i i dogs im- merged in hot water. A- - Xormal sinus rhythm (normal rate, tachycardia or hradv cardia) . Nonna 1 duration of i QRS complex. B- - Slight widen ing of QNS complex wit hont F wave. Rectal Arterial rime temp pressure K plasma niir i sec C mm 1 Ig 1 ;t (; millioquiv/1 Dog 031 (7.4 kg) 55 (’. Died after 23 min. C ontrol 55.4 112 A 2.8 5 10 37.0 02 A 5.2 13 15 40.0 58 A 4.7 20 45 41.4 IS A 0.0 Dog !1 i30 (7.5 kg) 00 C. Died after 10.5 min C ontrol 30.0 1(X) A 4.0 4 45 37.4 80 A 3.3 7 55 38.0 04 A 4.7 10 40 30.1 00 A 5.3 Dog 022 (8.5 kg) 75 C. Died after 15 min. C ontrol 37.0 118 A 3.1 2 55 37.0 00 A 5.8 6 30 38.4 08 A 6.4 It) 20 30.0 70 A 5.8 15 30.3 30 A 0.8 Dog 1) 20 (8,2 kg ) 75 C. Died after 13.5 min c ontrol 37.2 130 A 3.0 3 10 38.5 130 A 4.8 8 30 42.1 120 A 6.1 12 45 44.1 74 B 8.2 Dog 9 34 (7.0 kg) 75 C. Died after 25 min. Control 34.0* 148 A 3.1 15 16 41.7* 100 A 24 45 43.5* 72 A 0.0 ♦ Right heart temperature. SECRET 374 STUDIES OF THERMAL INJURY—CUTANEOUS VM> SYSTEMIC Figure 35. Effect of two episodes of cutaneous hyperthermia on pig 870 (11.8 kg) caused by immersion in water at 47 C. First period of immersion lasted for 33.3 minutes and is indicated by words on first and second segments of kymograph record. Fifteen minutes after end of first period of hot water immersion and bet ween second and third segments of record, animal was immersed again at 47 C and allowed to remain in hath until dead (56.5 minutes). Between two episodes of hot water immersion, skin temperature was lowered by exposure to cool water. Total duration of experiment was 105 minutes, t'pjier, middle, and lower tracings on the kymograph record represent resj>cctively pneumogram, carotid pressure, and right auricular pressure. The numlicrs under the electrocardiograms correspond to those under the kymograph tracings; C = control period. temperature of 44,5 C. In another instance exposure to water of 75 C for I minute reduced the pressure from 140 to 20 mm Hg in 5 minutes. During subse- quent exposure to room air the pressure recovered, and reached 130 mm Hg after 73 minutes. The ani- mal was still alive after more than 2 hours. Exposure of one animal to water of 75 C for 5 minutes resulted in a fall in blood pressure from 138 to 78 immedi- ately after immersion. The pressure continued to fall, and the animal died after 18 minutes. The arterial pressure in dogs behaved in a way comparable with that in pigs at the same tempera- ture. Animals immersed at 60-75 C survived for 13.6 25 minutes. Right auricular pressure; Intra-auricular pres- sures of pigs before immersion varied from +32 to — 66 mm H;0 (average —23 mm IbO). In only three out of fifteen animals was the pressure in the right auricle higher than atmospheric (+13, 20, and 32 mm 1FO). In most instances, a slight rise occurred following immersion, the control level being regained in 0.5 to 3 minutes. In five of the six animals im- mersed at 4-1—19C, this was followed by a gradual drop of 4-20 mm H20. There was no rise in venous pressure until 1 or 2 minutes before death. In the sixth pig, immersion did not influence the auricular pressure (Figure 35). In seven of the nine pigs exposed to water of GO- 75 C, a gradual rise of the right auricular pressure was observed, beginning in the middle of or even early in exposure and continuing until death. Tins rise amounted to 15-45 mm IT.O and occurred at a time when both arterial pressure and respiration were still adequate (Figure 36). In some instances, it SECRET PHYSIOLOGICAL DISTL’RRAXCES FROM EXCESSIVE HEAT 375 Kiouhk 36. Effect on pig H71 (9.1 kg) of immersion in water bath at 70-73 (' for 12 minutes, Upper, middle, and lower tracings on kymograph record represent respectively pneumogram, carotid pressure, and right auricular pressure. Sequence in which electrocardiograms were taken is indicated. was preceded by a fall of 20-30 mm H20 which rap- idly developed 1-3 minutes after the exposure had started. In two animals, this fall was the only change in auricular pressure that was observed until 1 min- ute before death, when it rapidly rose. One pig, exposed for only 1 minute to water of 75 C, showed an abrupt fall of 40 mm H20. During the following 70 minutes the auricular pressure grad- ually returned to the preimmersion level, coinci- dentally with recovery of the arterial pressure. The auricular pressure of four dogs was lower than that of the pigs. It ranged from —77 to —108 mm II2(). Because of hydrostatic effects the auricular pressures Indore and during immersion could not be compared. However, neither in the two dogs exposed to 73 C nor in those exposed to 55 and 00 C w as there observed any change in the recorded auricular pres- sure during the period of immersion. Because of the possible contributions of the type or rate of breathing to the observed pressure changes, some experiments were, performed on eurarized pigs. Artificial respiration was applied throughout the ex- periments. The course of the auricular pressure was found to be identical with that of the spontaneously breathing animals. At 17-49 C a slow and moderate fall was observed; exposure at 75 C resulted in a rise, beginning early during exposure. Respiration; In agreement with earlier writers it was found that a rise in body temperature was asso- ciated with a pronounced increase in respiratory rate. In the pig the immediate effect of immersion was usually a short period of very deep and fairly rapid respirations, followed by a variable episode of only moderately increased breathing Irate 20-40). In the animals exposed to the lower temperature range the onset of respiratory rates of 170-200 was often sudden, and occurred in t he first 10 minutes of exposure, at rectal temperatures of 39-41 C. Deep gasps interrupted this shallow tachypnea. The ar- terial blood maintained its bright red color. The tachypnea gradually increased, and rates of 300 were not infrequently reached. When the rectal temper- ature had mounted to 13-44 C, breathing abruptly slowed to 10 40 per minute and became much deeper. Additional slowing usually continued until death. In the dog, immersion was immediately fol- lowed by a tachypnea of 100 150 per minute, which gradually increased. Rates over 200 were not en- countered. It is difficult to estimate whether the respiratory SECRET STL DIES OF THERMAL INJURY — CUTANEOUS VNL) SYSTEMIC Table 26. Physiological and chemical changes in three pigs intravenously infused with an isotonic (1. 2%) solution of KC1. A — Normal sinus rhythm (normal rate, tachycardia or bradycardia). Normal duration of QKS complex. B — Slight ] HU — Moderate ) Widening of Qh'S complex without P wave — BBB — Pronounced 1 BBB — Can often be interpreted as ventricular fibrillation. Arterial Time pressure K plasma K cells min sec mm Hg ECG Hematocrit milliequiv/l milliequiv/l Pig 901 (14.8 kg) Bate of infusion 0.6 cc/kg min. Died after 50 min. font r«l A* (lead 1) 36 4.3 123 11 CHI A* 36 9.0 125 16 (Ml A* 37 9.5 124 IS 00 BB 38 11.2 121 26 00 BBB 37 15.5 132 Infusion stopjsed -I 26 10 O ... 35 00 O 36 00 A _ 11 00 A 38 8.7 139 41 30 Infusion started again. Bate 0.7 ee kg min. 50 00 BBB 35 17.7 136 Pig 911 (8.7 kg). Bate of infusion 0.9 cc/kg/min. Died after 22.5 min. Control A (lead II) 35 3.2 127 11 00 Af 34 8.7 122 14 00 B 35 lO.ti 122 16 00 BBB 35 12.7 125 20 00 BBB 31 27.0 22 00 0 28 38.0 127 Pig 925 (15.9 kg). Bate of infusion 0.6 cc/kg/min. Died ifter 39 min. Control 76 A (lead I!) 33 3.5 112 6 08 76 A 33 5.7 117 12 40 76 A 32 10.6 111 19 37 76 At 34 12.7 110 24 50 76 B 37 15.7 10!) 35 18 24 BBB 37 26.1 Ill ♦ P wave not clearly shown. t P wave getting blunt. X P wave very flat. or the circulatory system failed first in these animals. If hradypnea is considered as the first manifestation of failing respiration it might he said that the cardio- vascular system survived somewhat longer, as judged by the presence of an appreciable arterial blood pres- sure. However, at least in the beginning of hradyp- nea, the pulmonary ventilation certainly was as ade- quate as during the control period. If the onset of prolonged apnea is considered as the end point of adequate respiratory function, l>ot h systems failed simultaneously. In three animals, artificial respira- tion was applied at a time when the arterial pressure was st ill appreciable (80-1)0 mm Hg), without having the slightest effect upon its downward course. More- over, the final rectal and heart temperatures of the curarized pigs fell well within the range of those of spontaneously breathing animals. Exposure of pigs to (>0 75 C produced an increase in respiratory rate which did not exceed 80 00 per minute. The breathing remained deep until the terminal episode of hradypnea, ending in occasional deep gasps. In dogs the respiratory changes were essentially the same as those encountered at the lower temperatures. Electrocardiographic changes: In both pigs and dogs, the first, change, beginning immediately after immersion, consisted of a progressive increase in heart rate to levels of 300 350 per minute. Associ- ated with this increase, changes occurred in the (JRS complex, consisting of decrease in amplitude of the l{ wave and deepening of the S wave or vice versa with maintenance of the normal QffS interval; and inversion of the T wave. The changes in the initial vent ricular deflection might in part at least be due to SECRET PHYSIOLOGICAL DISTURBANCES FROM EXCESSIVE HEAT 377 Figcue 37. Helutionsliip hctwwn plasma potassium level and changes in electrocardiogram (lead 1) during immersion of pig 010 (0.5 kg) in water bath at 72-75 C. Plasma potassium values are given in milliequiv 1. DeaIh occurred 12.5 minutes after beginning of experiment. variations in typo of breat hing with resulting changes in the position of the heart. (I (arris,2*) They occurred only to a minor degree in curarized animals. In the pig, the abnormalities following this sinus tachycardia varied markedly with the temperature of exposure. Of all animals exposed to water at 44- 50 C (Tables 23 an I 24) only one showed appreciable widening of the QRS complex and loss of P wave. This occurred I minute before death. Another animal showed disappearance of the P waves. The changes in the remaining pigs were limited to sinus bradycardia and sinus arrhythmia, which be- came most pronounced 2 or 3 minutes before death (Figure 35). Occasionally, auricnloventricular block of varying degree was seen during this period. In contrast, eleven pigs continuously exposed to temperatures of 64-75 C (Tables 23 and 24) all showed the gradual development of exceedingly wide ventricular complexes with very large T waves, and the gradual disappearance of the P wave.k The gen- eral shape of these complexes resembled that of the original supraventricular ones. Their development was usually associated with definite slowing, al- though the heart rate remained regular, in some eases, the transitional phase consisted of salvos of fairly rapid and wide vent ricular complexes, which interrupted a still-existent sinus bradycardia. In the terminal stage, the initial ventricular deflection could not Ik- separated from the final one. The elec- trocardiogram consisted either of very slow, ex- tremely wide ventricular waves, separated from each other by isoelectric intervals of 0.2-1.0 second, or of more rapid variations at 100-240 per minute, in which one wave merged with the next. The latter state might be called ventricular fibrillation (see Figures 36 and 37). In nine of the eleven pigs, these changes made their first appearance early during immersion, at rectal temperatures of 37.0 to 41.6 C and at a time when the arterial pressure and respiration were still ade- quate. In four of these, the blood pressure at the k During tachycardia, actual ot enervation of this disap- pearance was impossible because of overlapping of P and preceding T waves. In these instances, it was assumed that the same changes had taken place as in the instances where the P wave could !«■ followed through it stage of decreasing amplitude to disappearance, as subsequent slowing of the teat similarly revealed the absence of auricular complexes. SECRET 378 STI DIES OF THERMAL IMIRY — CUTANEOUS \M> SYSTEMIC time of onset of the wide complexes was actually equal to or higher than that before immersion. In only two animals were the abnormalities first noticed when the pressure had fallen to low levels, and it is possible that they would have been demonstrated earlier if more electrocardiograms had been taken. Exposure for <1.5 and 5 minutes similarly resulted in marked widening of the QKS complex, whereas ex- posure for 3 minutes and 1 minute did not produce deviations other than those at lower temperatures. In the dog, the electrocardiographic changes at high temperatiires were in no way different from those encountered at 11-50 C (Table 25). They were limited to an increase in rate and to minor changes in the ventricular complex. No widening occurred and the auricular manifestations remained present until the end. Chemical Changes. For a complete discussion of the effect of temperature on the potassium concentra- tion of the plasma, see Section 17.10 of this chapter. The potassium concentration of the plasma of fifteen pigs in which physiological studies were made are shown in Table 24. 3’lie initial plasma levels ranged between 3.0 and 4.8 milliequiv 1. The potassium con- centration of the red blood cells ranged from 113 to 145 milliequiv/1. The course of these concentrations during immersion varied markedly with the tem- perature. Immersion of four pigs at 47 C produced a gradual and sustained rise in plasma potassium. Ten minutes exposure resulted in levels of about 6.0 milliequiv/l. During the rest of the exposure, the level increased by an additional I to 4 milliequiv. The highest level was 10.2 milliequiv 1 obtained 30 seconds before death. On the other hand, continuous exposure at 70 75 C characteristically resulted in an enormous rise in the plasma potassium level. This increase was found to take place with surprising rapidity. In five pigs, tlu- plasma after I to 4 minutes of exposure con- tained 1 1.2 to 25 5 millicquiv/1 of potassium. A sam- ple drawn in this period from one curarized pig was still essentially normal and the peak observed in this animal was only 11.0 milliequiv. Peaks from 1G.7 to 25.5 milliequiv were observed in six pigs during ex- posure. Curare did not prevent rises in this range in two pigs; however, no early observations were made on these animals. In some instances, the potassium level fell toward the end. However, it remained markedly elevated. In some experiments, the exposure was terminated before the animal had expired. Immersion for (>.5 and 5 minutes similarly resulted in a tremendous rise of plasma potassium. At the time of death, the level was still very high. Immersion for 3- and I-minute periods produced a less pronounced increase; at the time of death, the level was only 2 2.5 times the normal one. 17.11.4 Discussion These observations show that the physiological dis- turbances leading to death in pigs exposed to water at 4G 50 C are of a different nature from those en- countered in animals exposed to temperatures of GO 75 C, In pigs immersed at the lower temperatures, tlu1 occurrence of a gradual fall in right auricular pres- sure followed by a fall in mean arterial pressure indi- cates a progressive decrease in venous return to the heart. That this decrease, at least during a major part of the exposure, was due to an increase in ca- pacity of the peripheral vascular bed, rather than to loss of intravascular fluid, is evident, from the fact t hat the changes in circulat ory dynamics were found to be reversible to a considerable degree. As the ex- posure continued, the detrimental effects of the heated blood upon the heart muscle were added to the peripheral effects, and both factors undoubtedly contributed to the lethal ending. It is difficult to say whether cardiovascular failure or respiratory insufficiency was the immediate cause of death. Profound arterial hypotension and pro- nounced bradypnea were usually encountered at the same time. It can be said, however, that the mean arterial pressure fell considerably before any impair- ment in respiratory function was evident. Artificial respiration applied at a time when the arterial pres- sure was still appreciable had no effect upon its downward course. Moreover, curarized pigs did not survive longer than spontaneously breathing ani- mals; all but one animal died after 25 to 51 minutes of continuous immersion. The plasma potassium level increased by GG-250 per cent; the highest level found was 10.2 millieqiiiv/1. No profound changes in cardiac function, as judged by the electrocardio- gram, occurred. As will Ire shown, plasma potassium levels up to 10 milliequiv I do not produce significant changes in intraventricular conduction. At immersion temperatures of GO-75 C, the pigs survived for only 8 to 15 minutes. In the middle of the exposure, or even earlier, at a time when the respiration was still adequate and the mean arterial SECRET PHYSIOLOGICAL DISTURBANCES FROM EXCESSIVE HE YT 379 Figure 38. Effect of continuous intravenous infusion of 1.12 percent KClat the rate of 0.6 kg/rain. Upper, middle, and lower tracings on kymograph record represent respectively pneumograin, carotid pressure, and right auricular pressure. Time in minutes is shown at base of record. Time at which blood samples were taken is indicated by symbols K-, Kj, A», and K ,. The times at which the sequence of electrocardiograms (k to z) were taken are indicated by arrows. See pig 925, Table 26, for corresponding potassium levels. pressure was still considerable, pronounced changes in cardiovascular function made their appearance, They consisted of a rise in right auricular pressure, and electrocardiographic changes in (he form of dis- appearance of the P wave and progressive widening ot the QRS complex, often terminating in ventricular SECRET STL DIES OF THERMAL INJURY—El TWEOTS AND SYSTEMIC fibrillation. At the same time, the potassium concen- tration of the plasma reached values of Hi 19 milli- equiv 1. This was associated with a striking destruc- tion of red blood cells. These observations strongly suggest that (he hy- perpotassemia was responsible for the disturbances in cardiac mechanism and for the subsequent myo- cardial failure evidenced by the rise in auricular pressure. That t he damaging effects of a rising plasma potassium level first of all manifest, themselves in the heart is well known. In rabbits and dogs, the infusion of a solution of a potassium salt produces a sequence of electrocardiographic changes similar to those ob- served in pigs during ex posuretohigh temperatures.a-4* IT was found that an identical sequence of changes takes place in infused pigs (Table 20). In two animals, infusion rates were maintained that were likely to produce death in approximately the same time as in the burned pigs. It is evident (hat potassium levels of less than 10 milliequiv, 1 failed to produce either changes in the P wave or widening of the QPS com- plex, just as was the case in burned pigs. Higher levels resulted in a succession of changes which were similar in all respects to those observed at high tem- peratures (Table 24). In the one animal (Figure 38) in which arterial and right auricular pressure and respirations were recorded, the auricular pressure began to rise 19 minutes after the infusion had started. The potassium level was 12.7 millicqulvTTf the P waves had begun to flatten 3 minutes before and had disappeared. Three minutes later widening of the QRS complex began. The arterial pressure and respiration remained normal for another 10 minutes.1 That the cardiac changes due to the potassium ion are reversible to a remarkable degree is clear from experiment 901 (Table 26). The usual succession of electrocardiographic changes was observed until, some seconds after a potassium level of 15.5 milli- equiv/l had been reached, the string shadow re- mained resting. The infusion was stopped. No ('lee- trie or auscultatory evidence of cardiac activity could lx* demonstrated for the following 10 minutes, al- though the animal continued to breathe at a very slow rate. Then heart action returned and respira- tion became more rapid. The electrocardiogram had returned to normal. A plasma sample taken 5 min- utes thereafter contained S.7 milllequb' I of potas- sium. Infusion was started again, the well-known changes were again observed, ami the pig died with a potassium level of 17.7 milliecpiiv, 1. The rapidity with which potassium is removed from the plasma makts it imperative that the release of the ion into the circulation he intensive enough and he continued for a sufficiently long time to lead to death. This actually occurs in the burned pigs. The filtration of potassium often occurred at so rapid a rate that there was a lag between the rise in potas- sium and the electric changes. Thus, in pig 5)10 a level of 19.0 milliequiv 1 was reached in 2 minutes, whereas more than 4 minutes were required to pro- duce the typical widening. Animals exposed to high temperatures for only I or 3 minutes did not release sufficient potassium to produce a characteristic effect on the heart, whereas exposure for 0.5 minutes was adequate in this respect. Kxposure to 75 C for 5 min- utes resulted in a tremendous rise in potassium and in electrocardiographic changes, but even here both manifestations diminished in intensity during tin* following 14 minutes. Although it is clear that in pigs exposed to high (00 to 75 C) temperatures the most striking physi- ological disturbances are those which result from the release of excessive amounts of potassium, cont inued exposure results in a progressive and generalized rise Tn body temperature which undoubtedly causes dis- turbances other than those due to hyperpotassemia. Thus, the peripheral and central factors that were the cause of death at lower temperatures also come into play at t hese high temperatures. In order to evaluate the relative contributions of red blood cells and fixed body cells to the increase in plasma, potassium experiments were performed on dogs (Table 25). Whereas the potassium concentra- tion of (heir fixed cells is similar to that of the pig, their red cells contain only small amounts. Immer- sion at 75 C resulted in an intense hemolysis, but the potassium level did not rise above tha't encountered in pigs at 47 C and electrocardiographic changes characteristic of hyperpotassemia were not seen. The distribution of the potassium in human blood is similar to that in pig’s blood, the potassium con- centration of the red cells being approximately 110 milliequiv/1, that of the plasma approximately 4 5 millieqiiiv/l.*2-41 High plasma potassium levels should therefore be expected in human beings in whom a major part of the body surface has been ex- posed to high environmental temperatures. Several 1 The rate of infusion was slow enough so lhat the rise in venous pressure could not lie ascribed to the administration of the isotonic salt solution per se.h SECRET IMIVSIOEOEICA! DfSTl RIUNCES FROM EXCESSIVE HEAT 381 minutes of exposure would probably he required to result in the very high levels encountered in these experiments. It is also probable that, if the immedi- ate effects of the exposure were survived, a markedly elevated plasma potassium occurring immediately folk »\\ ing the injury would fall within the next hour. It should be remembered, of course, that a rise in plasma potassium is a normal post-mortem phe- nomenon. I7.li.r> Summary The re arc two principal mechanisms by which ex- posure of the surface of the lxidy to excessive heat may cause rapid circulatory failure and death. In one, the systemic hyperthermia due to con- duction of heat to the interior of the body by way of the blood stream leads to a rapid and progressive de- cline in blood pressure and failure of circulation due to peripheral vascular collapse. In the other, the circulatory failure is principally central and is due to the effect on the heart of an ex- cessively high concentration of potassium in the plasma. Centred-circulatory failure is likely to occur when the overheating of the skin and subcutaneous tissue is so intense, prolonged, and generalized that potassium js released from the erythrocytes with such rapidity and in such large amounts sis to main- tain plasma levels in excess of 11 milliequiv 1. In the case of thermal exposures of low intensity, peripheral circulatory failure may occur without suf- ficient rise in tissue (and blood) temperature to cause a functionally significant rise in plasma potassium. When a thermal exposure has been of sufficient sever- ity to cause fatal hyperpot assemia, the central circu- latory effects are likely to I>e complicated by periph- eral vascular collapse. It is essential to the development of acute hyper- thermic potassium poisoning that the erythrocytes have a high original concentration of this element. Thus, fatal hyperpot assemia, Hue to hyperthermia, occurs in (he pig but not in the dog. Since man and pig have similar potassium concentrations in erythro- cytes, it is inferred that they are probably similarly susceptible to the development of fatal hyperpo- tassemia following cutaneous exposures to excessive heat. Although thermally induced respiratory disturb- ances undoubtedly contribute to either type of circulatory failure, maintenance of pulmonary ven- tilation by artificial respiration does not prevent death or cause significant prolongation of the sur- vival period. SECRET Chapter 18 MISCELLA\E01TS TOXICOLOGICAL STUDIES Birdsey Remhaw I8.i 1NTRODLCTION Division' 9 has carried out, in its laboratories operated for toxicological and immunological studies on chemical warfare agents, a limited number of investigations with materials which were not con- sidered for use as war gases but whose toxicological properties were for other reasons of interest to the Army, Navy, or other National Defense Research Committee [NDRC] divisions. In this chapter are summarized the results of four such investigations: (I) the pathological changes caused by prolonged ex- posures to oil screening smokes, (2) the toxic effects of gasoline fumes, (3) (he toxicity of Salcomine dusts, and (1) the hypersensitivity and dermatitis caused by hexanitrodiphenylamine and enemy explosives containing it. 18.2 TOXICITY OF OIL SCREENING SMOKES With the development by NDRC Division 10 of generators for the production of oil screening smokes, the question arose whether personnel exposed for prolonged periods in such smoke clouds (consisting of fine droplets of unburned hydrocarbon oils) would 1)0 subjected to health hazards. Although no informa- tion was available concerning the toxicity of oil clouds for animals or man, there were on record marly 200 cases of “lipid pneumonia” attributed to aspiration of mineral oilA Inasmuch as lipid pneu- monia may occur whenever an exogenous oil reaches the pulmonary tissues and remains for a sufficient lime to cause irritation, the possibility existed that this potentially debilitating condition might result from the inhalation of the screening smokes. At the request of the Chemical Warfare Service, an experi- ment was performed in which mice were exposed for prolonged periods to clouds of atomized lubricating oil;1 the continuing interest of the Service led to the extension of the tests4 to include the exposure of monkeys to clouds both of lubricating oil and of fog oil standardized for use in the Langmuir-type gener- ator. The results of these tests with animals afforded no basis for supposing that prolonged exposures of military personnel to oil screening smokes in the field would In' dangerous. By now the actual use of oil screening smokos in military operations has lieen ex- tensive and no evidence has been forthcoming that, health hazards are involved. The experiments were pertormed with animals kept for 100 days in a large closed chamber into which for 30 minutes of every hour air containing oil fog was passed at a rate of 0.8 chamber volume per minute. In the experiments with lubricating oil (Penn Oil, SAE Xo. 10), the nominal concentration was 132 gg I and the droplets varied in diameter from about 0.3-1,5n; the mass median diameter was I I ju. In (he ease of fog oil (Texas Company, S(JF No. 1 Oil) I lie analytical concentration was do i. The death rate among the mice exposed to the clouds of atomized lubricating oil was not signifi- cantly different from that in the normal colony and the animals showed no serious pathological changes during or at the end of the exposure.* No free oil was ever seen in the alveoli or bronchi, and chemical analyses at the end of the 100 days revealed that relatively little had accumulated, there being in the lungs 1.05 mg per mouse (0. t per cent of the total lung weight). Occasional oil-containing macrophages could be seen after the experiment had been in prog- ress for a week. These increased in number during the first 35 days, after which time almost every alveolus contained at least one such cell, but they did not become significantly more numerous during the subsequent-two-thirds of the exposure period. The tracheo-bronchial lymph nodes of mice sacri- ficed after 3 weeks in the chamber showed accumu- lations of oil-containing macrophages, but there was no reaction to them. These essentially negative results with mice led to repetition of the experiments with the Rhesus mon- key— a species which, in terms of posture and size of respiratory passages, more closely resembles man.4 Chemical analyses of the lungs of exposed animals revealed a progressive accumulation of oil to a maxi- mum of about 10 per cent of the dry weight, or 2 per cent of (he wet weight, at the end of the 100 days; approximately one-half of this accumulation had dis- appeared a year after the start of the exposure. Microscopic examination revealed some free oil, and SECRET TO\H'lTV OF SU.COMIXK DUSTS 383 oil-laden macrophages were scattered throughout the lung, in the alveoli, snbpleurally, and in the bronchial and pleural lymphatics. However, little inflammatory reaction attributable to the oil occurred, and subse- quent to the exposure the fibroplastic reaction to the remaining oil was slight. The one conspicuous extra- pulmonary effect was loss ol hair during the pro- longed exposure, and its subsequent regrow th. In so far as the animal findings may be applied to man, the failure of large amounts of oil to accumulate and the absence ot severe acute and chronic reactions make it improbable that significant pulmonary ef- fects would bo produced by any exposures likely to be encountered. The results of exposure to fog oil (SCF No. 1) were similar, with one important exception. Six of seven monkeys died, apparently of starvation, dur- ing or shortly after the termination of the exposure. Examination of the stomachs revealed acute or hy- pertrophic gastritis and, in those dying after the greatest delays, the pict ure of an adenoma malignum superimposed on hyperplastic gastritis. These serious pathological changes are believed to have been in- duced by carcinogenic agents present in ingested oil; carcinogens have been found in petroleum oils 11 and presumptive evidence compatible with their presence in S(iF No. 1 oil was obtained. Thus, the possibility exists that cancer might result from prolonged ex- posure to oil smokes. However, it should be noted that the monkeys, their surroundings, and their food were continually covered with oil, and the animals therefore undoubtedly ingested much oil in addition to that w hich they breathed and swallowed. l« :i TOXICITY OF GASOLINE FUMES Early in 1911 reports were received that among the individuals killed in flame thrower attacks upon en- closed fortifications were some who had not sus- tained severe burns. The Chemical Warfare Service was interested in determining the cause of these deaths and, as a small part of a larger program, re- quested that the effects of short exposures to the vapors from unburned flame thrower fuel be de- termined. As it did not prove feasible to set up toxic concen- trations of vapor from thickened flame thrower fuel, the tests5* were limited to experiments with un- (hiekened gasoline compounded to meet Federal Specification VV.M-504. The gasoline was atomized into an airstream which was heated to vaporize the droplets Indore passing to an animal chamber in which the air temperature was about 35 C. The /J(('t);.,i’s for 5-minute exposures of mice, rats, and guinea pigs were very high, in (lie order of 3U0 mg I {('I = 1,500,000 mg min m:t, analytical). This con- centration was somewhat above the sat uration value at the temperature of the chamber, and a dense cloud formed. The mice and rats surviving the exposure period remained narcotized for 10 minutes but ap- peared normal within one-half hour; no gross patho- logical changes were produced. The guinea pigs ex- hibited rapid, shallow breathing with forced inspira- tions; autopsies revealed bronehospasm and emphy- sema. It is probable that the action of the gasoline fumes on mice and rats was purely narcotic, and that in guinea pigs this action was augmented by broneho- spasm. The above-mentioned concentration is above the upper explosive limit for gasoline and could not be built up in the presence of flame. There is no doubt, on the other hand, that concentrations of gasoline vapor rapidly lethal for man as well as animals can be attained in closed spaces in the absence of flame,10 and there is no information as to whether or not sensitivity to the vapors is markedly enhanced at greatly elevated ambient temperatures. 18.1 TOXICITY OF SALCOM INF DUSTS Karly in 1942 the success of NDRC Division 11 in developing Saleomine * oxygen generators for use on shipboard and elsewhere led to the need for an investigat ion of the possible industrial hazards which might be involved in manufacturing and working w ith this compound. * Salicylaldchyde ethylenediimine col wit, known as Sal- «nmne, has the following structure: -° '> <3 I V - I \ /• \ / IU \ :• i! I ICjC » a, This material has the property of absorbing oxygen (about 4 per cent by weight) when exposed to air, and of releasing the absorb'd oxygen when heated. Since this Cycle may l>e re- lated many times, it is possible to construct systems employ- ing Salcomine for the separation of atmospheric oxygen. For details the reader is referred to the Summary Technical Report of NDRC Division 11, Section 11.1. No doubt the Salcomine samples used in the toxicological studies were partially oxygenated. SECRET MISCELL V N EDITS TOMCOIOCICAL STL DIES UNCLASSIFIED Preliminary tests 2 revealed that Salcomine dust is toxic upon inhalation and clearly indicated the neces- sity of taking precautions to protect workers. Mice exposed for several hours to the dust at nominal con- centrations of 0.4 2.1 mg 1 (undoubtedly the actual, or analytical, concentrations were much lower) fre- quently died within Hi days. Autopsies revealed many pathological changes attributable to the Sal- comine: there was generalized degeneration and localized necrosis of the epithelium of the trachea and principal bronchi; the lungs were hyperemie and, particularly in the peripheral portions of the lobules, edematous; the thymus gland and lymph nodes con- tained fragmenting lymphocytes in moderate num- ber; and fat stains revealed fatty changes in the liver. In 1044 (he occurrence of a number of clinical eases of poisoning, presumably-due to Salcomine dusts, occurred *' and prompted further animal studies.''1’ The results left no doubt that Salcomine is both a respiratory and a systemic poison and that precau- tions must he taken against the inhalation of its dust. A single exposure to a high concentration killed guinea pigs immediately and mice after varying latencies. The lungs of (he guinea pigs were markedly distended with air and microscopic examination re- vealed that the bronchi and bronchioles were strongly constricted. Mice dying soon after such an exposure exhibited no visible changes which would account for death; those dying after 1 day or more exhibited a diffuse pneumonitis, suppurative tracheobronchitis, and occasionally jaundice and eoagulative necrosis of the liver. More important from (he standpoint of the health hazard and for revealing the generalized toxic effects of Salcomine are prolonged exposures to low concen- trations. Accordingly, animals were exposed for 1 hour daily in a chamber to air which contained the finer particles of Salcomine dust at concentrations in the order of 100 ag h Three to six such exposures, corresponding to a total dosage of about 20,000 mg min m:!, sufficed to kill approximately one-half of the exposed, mice and rats;c rabbits probably wen* not much more resistant, but guinea pigs proved to bo considerably less sensitive. The mice, developed a diffuse pneumonitis and t racheobronchitis, paren- chymatous degeneration of the renal tubular epi- thelium, jaundice, and liquefying eoagulative ne- crosis of the liver. Autopsies of rats sacrificed daily during the exposures revealed a gradually developing diffuse pneumonitis and tracheobronchitis; focal hepatitis with occasional necrosis also developed and was followed by the appearance of intracellular fat; parenchymatous degeneration of the. renal tubular epithelium occurred, followed by the appearance of severe fatty changes: and in the duodenum and jejunum the epithelial cells of the mucosal glands began to show vesiculation, swelling, and many mitoses. These changes subsided after the exposures were discontinued. Clinical pathological studies on rats revealed an increase in the urinary output after the first and subsequent exposures, the development of a mucoid diarrhea, a rise in hemoglobin and red cell count, and a 5 per cent loss in body weight. A leucocytosis also developed during the exposures and subsided rapidly upon their cessation. b Medical examinations of eleven men* exposed to small amounts of Salcomine dust revealed that the compound pro- duced irritation of the eyes, nose, larynx, and bronchi. The symptoms, which apjieared shortly after exposure and re- sembled those of an upper respiratory infection, cleared up after removal from exposure. Signs (xtssibly indicative of mild systemic effects — muscular aches, nausea, and vomit- ing — apjHXired after latency of 5-24 hours in some of the subjects. In general the respiratory symptoms disappeared within a day but the digestion was sometimes upset for 3 days. There may have been some slight cumulative effect , be- cause it was reported that chronic exposure led to anemia, lack of energy, and need for increased sleep. Xo permanent effects were noted and it was concluded that, with reasonable precautions including use of dust respirators, no marked industrial hazard was involved. One ease with much more severe systemic effects has l>een reported.5 An emergency obliged the subject to work without a mask for a short jteriod in an atmosphere laden with Salcomine dust. On the evening of the exposure there were no pronounced symptoms other than discomfort in breathing, but the following day abdominal pains of sufficient severity to require hospitalization and treat- ment with morphine developed. The subject had nausea, vomiting, and a fever. A tentative diagnosis of acute duode- nitis was made and his liver became progressively more en- larged and lender. Tests jierformed 48 hours after admission revealed definite liver damage. The liver condition with ac- companying jaundice gradually improved but the abdominal signs persisted. Penicillin was utilized. An exploratory lapa- rotomy 2 months after exposure revealed a retroperitoneal abscess in the left lower quadrant; this was removed, as was a second similar abscess which formed on the right side 3 months later. It was suspected that other abscesses were present deep in the liver tissue, but none required drainage. Definite hardening of the liver due *o scar tissue persisted. Attending physicians assumed that the inhaled and ingested dust was responsible for the acute digestive disturbances that followed exposure and led to disease of the liver, duo- denum, and retrojieritoneal tissues. Compare results of ex- [H-rimental animal exposures. _ c To illustrate the toxic potency of the dust it may be noted that for O-hour ex|Misures the Ij(('1)m of mustard gas vapor for mice is i,J(li\jmfnipi\ and for rats, 1.500 mg inin/m*.* ON i'h AiNolj' >r UNCLASSIFIED HYPERSENSITIVITY CYISED »Y HEX VNITRODIPHENYLAMI \E 18.3 11Y PKRSKNSITIVITY VND DERM V- TITIS CAUSED HY HKWNITRO- DlPIfENYLAMINE In 1943 the Navy Department reported the occur- rence of acute dermatitis in jrersonnel of both British and United States armed forces who had come in physical contact with enemy explosives containing hexanitrodiphenylamine, and requested NDHC to investigate the cause of the dermatitis and methods for its prevention and treatment. A survey revealed numerous statements in the literature that hexanitrodiphenylamine is a powerful dermal‘.tic agent, but the factual basis for this im- pression proved to lie weak. Furthermore, dinitro- chlorobenzene, a potent dermatitic and sensitizing agent for both man and the guinea pig,11-1* is em- ployed in the manufacture of hexanitrodiphenyla- mine, and there is the possibility that this or other intermediates or by-products may have been respon- sible for those cases of dermatitis which have been observed. At du Pont Company plants, which manu- factured limited quantities of hexanitrodiphenyla- mine in 1918 and 1910, care was taken to avoid ex- posure to the substance mid no noteworthy or severe cases of dermatitis occurred. Inasmuch .as no samples of enemy explosives known to have produced dermatitis were available, the investigation 3 was confined to studies with a highly purified laboratory-prepared sample of hexa- nitrodiphenylamine and with a preparation from a Japanese torpedo booster. Crystallographic analysis revealed the latter to contain about 75 per cent hexa- nitrodiphenylamine, about 25 percent trinitrotoluene (TNT), and no dinicrochlorobenzene; if any minor constituents were present, they totalled less than I per cent. After rigorous applications of both preparations had failed to produce irritation or sensitization in guinea pigs and swine, tests were carried out on inert. Neither the pure material nor the Japanese explosive proved to he a primary irritant when applied in hot weather to skin of the forearm as a saturated solution in acetone or as a powder covered by an occlusive dressing. In 2 of 29 men treated with the purifier! hexanitrodiphenylamine and in I of 31 treated with the Japanese explosive, however, ‘‘Hareup” derma- titis developed about a week after the second of two applications. The dermatitis cleared up under simple symptomatic treatment within 7 10 days. Patch tests later showed that these 3 men had become markedly hypersensitive, whereas the 57 other sub- jects had not . The residue from an incompletely detonated sam- ple of hexanitrodiphenylamine was innocuous to hypersensitive skin. Prolonged treatment with excess potassium sulfide likewise rendered the explosive harmless, but treatment of acetone solutions of it with sodium hydrosulfite did not alter its ability to cause inflammation of hypersensitive skin. Although the findings indicated that with gross contaminations of large numbers of men, instances of skin reaction of varying degree are to l>e expected, it was clear that hexanitrodiphenylamine is not a pri- mary skin irritant and that it occupies a low position among the skin-sensitizing substances. Practical pre- ventive measures were considered to be avoidance of unnecessary contact with the substance and, inas- much as the reactions are delayed, use of an organic solvent and soap and water as soon as possible after contamination to remove the substance from the skin and from objects with which the skin can come in contact.