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4 IRRITANTS ANT) ~JESICANTS BACKGROU?~O The compounds discussed in this chapter ( see Table 4-1 ) are mustard gas (H), chloroacetophenone (CN) ~ dibenz [b ~ f ] [ 1~4] oxazepine (CR) ~ o-chlorobenzylidene malononitrile (CS)' brombenzyl cyanide (CA), diphenylaminochlorarsine (DM), chloropicrin (PS), nonanoyl morpholide (EA 1778), a cycloheptatriene (EA 4923), ant 123 com- pounds involved in 1 imitet test ing . H is a ves icant, pulmonary irri- tant, and systemic poison, depending on dose ant route of entry into the body. The other compounds are irritants. Of the irritants, DM is a sternutator (a substance that causes sneezing), whereas the others are lacrimators. All are called gases in warfare use, although they may be administered as vapors, liquid droplets, smokes, or mixtures thereof. Mustard gas and irritant chemical agents, the latter often called harassing agents, were introduced to the battle- field in World War I to confuse, harass, and disable enemy troops. Since the 1920s, irritants have been used as riot-control agents by civil authorities. Of these, CN came into widespread use ant is now commonly supplied in a formulation under the name Mace. Although CS was synthesized in 192S, it became known as a riot-control agent only in the 1960s, when it appeared safer than CN. For military use, harassing agents are intended to reduce or destroy the ef feet iveness of enemy troops . For this purpose, rapid onset of effects is usually, but not always, desired. Rapid recovery facilitates the handling of prisoners, whereas men injured by mustard gas require intensive care ant weeks for recovery. There were no plans for studying long-term effects of World War I-harase- ing agents. Riot-control agents are also designed to have a rapid onset of effects, produce a high degree of immediate disability, and require a short recovery time as soon as the rioters are dispersed from the area. With the increasing use of such agents as CN and CA in recent years, their possible long-term effects have aroused concern. At the end of World War I, medical thought was turning to the possibility thee soldiers who had been gassed with mustard, chlorine, pho~gene, and other agents would develop tuberculosis. In the early postwar years, publications described efforts to identify cases of tuberculosis among gas casualties. The expected epidemic failed to appear, and attention subsided. More extensive studies, such as that of Beebe, were initiated. 1 Gradually, mustard gas became the -101-

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L,— - ~ ~ - ~ . r4 rat At - 0 rat ~ cr. ~ lo ~ ~ ~ ~ or cr. ~ _ rat _ ~ _ _ _ \0 fir lo O ~ ~ ~ ~ Lit ~ a o o o o o ~ c ~ ~ 3 ~ ~ ~ ~ U. ~ ~ ~ ~ ~ - c so c ~ ~ ~ =~ U ~ ~ .~-5 ~ ~ (~1 ~ C ~ O V ' ~ ~ a, e o. U. ~ I: ~ ~ ~ ~ ~ 815 ~ _' ~ e ~. ~~ :~. . -4 0 ~ ~ 0 ·1 o c ^- c ~ ~ ~ ~ 0 0 '- ~ ~ I ~ ~ _ ~ U :^ ~ O ~ ~ O O C :^ (J I P o c 0 N ~C —t ~ O U ~ O ~ ~ ~ ~ ~ C a ~ ~ ~ ~ 0 .2 ~ U aO ~ a ol. ~ ~ I ~ ~ z I S I ~ ~ ' ~ ~ ' O O ~ ~ ~ ~ ~ 1 1` \0 ~ Z \0 Cn 1 ~J O 1 10 1 1 40 1 1 ~ 1 1 ~ O ct. u~ CO ~ ~ ~ ~4 1 0` <: O r~ ~ ~ ~ ~ ~ ~ ~ U Zl ~ ~ ~ ~ O ~ ~ \0 {~~ 0 0 ~ ol ~ ~ I I `~ `~: ~,: e ~ I V 0` ~ :~: b |— 0 Y ~ ~ r~ ~ ~ 0 ~Z ~ ~ ~ ~ ~ O ~ ~ 1 _ ~ ;5 o~ e ~ 1= x~ ^ ~ ~ ~ ~ ~ ~ S -102-

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center of interest; the discovery of its mutagenic properties stimu- lated further he search . Suf f ic lent ~ ime has now passed for long-term effects to become clear. Mustard gas has become the subject of a large body of reports, which have disclosed a multifaceted problem. On the other agents discussed here, there is no such volume of informal ion. Their e f fects have generally been regarded as tran- sient, ant only in the Himsworeh reports on the use of CS in the Lontonderry riots of 1969 is there even a proposal to study the long- te.- effects of the riot-control agents . Indeed, on several com- pounts, practically no useful information is available, except with regird to chemistry, acute toxicology, and pathology. Because information on possible long-term ef fects of the other irritant chemicals used in the Edgewood tests is sparse, this chapter focuses on the effects of mustard gas and two lacrimators, CS and CN. Information on the potential long-term adverse effects of these chemicals is derived from several sources: first, observation of long-ter~ disabilities in soldiers who were exposed to a single ~ in most cases) toxic concentration of irritant during World War I and in persons exposed in peaces ime ace idents or riot-control procedures; second, studies of morbidity in workers chronically exposed to chemi- cal irritants during their manufacture; and third, studies in which experimental laboratory animals were exposed to selected chemicals by topical application, injection, or aerosol inhalation. A review of the literature on experiments to assess possible chronic effects, especially mutagenic activity and carcinogenicity, of the irritant and vesicant agents reveals that these ef fects have not been studied systematically by current standards and techniques. The current view is that a carefully selected battery of tests involving prokaryotic and eukaryotic organisms can be used to assess the mutagenic ity or care inogenic ity of a chemical . Mutagenic ity tests should include asesys for gene mutations and chromosomal aber- rations. No such systematic investigation has been conducted of any of the agents reviewed in this chapter, except possibly mustard gas. In the case of any agent for which a risk assessment is desires with respect to past human exposure or that continues to be used for riot control or relates purposes, it seems desirable to use a battery of tents, such as those recommended in the National Research Council report on chemical environmental mutagens.4 Under the sponsorship of the National Cancer Institute and the National T Toxicology Program, two chronic tests of CN and CS adminis- tered by inhalation are unter way. 5,6 Preliminary data obtained in the subehronic studies preparatory to the definitive inhalation car- cinogenicity tests are presented in the sections of this chapter dealing with CS and CN. One probable adverse effect, to judge from -103-

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observation of the subehronic Bests, is irritation of the upper res- piratory tract and tunas. A potential consequence of inhalation of these chemicals by humans is pulmonary damage that might readily develop into pneumonia. Prediction of any other chronic effecte, including carcinogenesi~, awaits the generation of appropriate data, including in vitro and in viva bioassays. REFERENCE S 1 . Beebe, G. Lung cancer in World War I veterans: Poss ible rela- tion to mustard-gas injury and 1918 influenza epidemic. J. Natl. Cancer Ins t . 25: 1231-1252, 1960. 2. Himsworth, H. Chairman. Report of the Enquiry into the Medical and Toxicological aspects of CS (Orthochlorobenzyl itine Malono- nitrile) Part I, Enquiry into the Medical Situation Following the Use of CS in Londonderry on 13th and 14th August, 1969. London : Her Ha je~ty's Stationery Office. Command 4173. 1969. 13 p. 3. Himsworth, H., Chairman. Report of the Enquiry into the Medical and Toxicological Aspects of CS (Orthochiorobenzylidene Malono- nitrile). Part II. Enquiry into Toxicological Aspects of CS and its Use for Civil Purposes. London: Her Ma jesty' ~ Stationery Off ice. Command 4775. 1971. 82 p. 4. National Research Council, Committee -on Chemical Environmental Mutagens. Identifying and Estimating the Genetic Impact of Chem- ical Mutagens. Washington, D.C.: National Academy Press. 1983. 5. Tracor Jitco, Inc. Subchronic study report on CS2. Rockville, Md.: Tracor Jitco, Inc., 1982. 286 p. 6. Tracor Jitco, Inc . Study Report on alpha-Chloroacetophenone . Rock~ille, Md.: Tracor Jieco, Inc., 1982. 177 p. MUSTARD GAS CHARACTERI STICS Mustard gas (H)--also known as yellow cross, yperite, sul fur mustard, Schwefellost, bis(2-chloroethyl) sulfide, and dichlor- diethylaul f item-is a chemical-warfare agent with both ~resicant and systemic effects. H is colorless and almost odorless and is an oily 1 iquid at 14-215°C with a molecular weight of 159.08. Except in extremely colt weather, the low vapor pressure (0.072 mm Hg at 20°C) and low~olatility of H are sufficient to make contaminated surfaces a source of danger to anyone nearby. H is slightly soluble -104-

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in water and soluble in oils, fats, and organic solvents. It pene- trates clothing readily. Moist skin of armpits, groin, and inner surfaces of thighs is especially vu' nerable. i5 ~ 7 ~ 77 Its garlicky odor, faint at first, is soon imperceptible. E2po- sure to H does not cause immediate discomfort; rather, the onset of effects is delayed and -insidious. Troops have been known to remain in contaminated areas until their eyes, skin, and respiratory organs were affected. Exposure of skin produces erythema, then blisters that are painful and slow to heal. Such eye injuries as conjunc- tivitis, keratitis, and corneal ulcers cause temporary or permanent blindness. The respiratory effects of H include rhinitis, larya- gitis, bronchitis, and, in severe cases, destruction of mucous men branes. The bone marrow and digestive system are affected by sys- temlc administration of H. The multiple effects of this insidious agent make it among the most potent used on the battlefield. TOXICOLOGY Mutagenicity Mutations are heritable changes in genes or chromosomes. Although mutations occur spontaneously as rare events in all organ ems, their rates of occurrence can be markedly increased by exposure to mutagenic agents. Geneticists generally agree that the effects of human exposure to mutagens are deleterious and that such exposure should be minimized. 54 ~ 53 The basis for concern about hen exposure to mutagens is that increases in the rates of muta- tion in human germ cells and somatic cells may lead to art increased incidence of genetic diseases and cancer, respectively. After H. J. Mum er showed in 1927 that Tic rays induce sex-linked recessive lethal mutations in the fruit fly Drosophila me~ano- gas~cer,5\ efforts were made to determine whether chemicals can also be mutagenic. Unequivocal evidence of chemical mutagenesis was not obtained unt i' the 1940~. Among the first demonstrations of chemical mutagenesis was a report by Auerbach and Robson6 that mustard gas induces mutations in Drosophila. Over several years, Auerbach and her colleagues found that mustard gas causes genetic alterations ranging from gene mutations to chromosomal breaks asked rearrange- ments. ~ lDhe mechanism of mutagenesis by sulfur mustard (and other mus- tarda) involves the alkylatlon of DNA. As a bifunctional alkylating agent, sulfur mustard. causes cross-linkage of DNA strands, as well as monofunctional alkyla~cion producta.~9 Sulfur mustard and nitrogen mustard have been used in mutation studies in a variety of organisms, but data on the relative frequencies of induction of dif fe rent alky- —105—

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ration products in DNA by the two agents are limited. Nevertheless, sulfur mustard seems to exhibit greater SN1 character50 as an alkylating agent than does nitrogen mustard.l9 Because agents involved in SNI reactions attack oxygen sites in DNA more readily than do agents whose reactions are almost exclusively of the SN2 type, sulfur mustard may differ substantially from nitrogen mustard in the spectrum of alkylation products formed in UNA. Such dif fe- rences in mechanisms of alkylat ion and in alkylat ion products can feat to considerable differences in mutagenicity.30~7t Therefore, although nitrogen mustard and sul fur mustard are both alkylating agents, one must be caut ious in assuming they are comparably muea- genic . A comprehens ive review of the mutagenic ity of sul fur mustard and nitrogen mustard has been publishes by Fox and Scoet.l9 The muta- genicity data base on nitrogen mustard is more extensive than that on sulfur mustard. Nevertheless, results have been reported regarding the genetic activity of sulfur mustard in tests for forward mutations and reversions in bacteria; 12 differential killing of DNA-repair- deficient strains of microorganisms and their repair-proficient counterparts; 34 ~ 38 revere ions in fungi; 38, 68 chlorophyl 1 mutations in vascular plants 19 gene mutations and chromosomal aberrations in Drosophila;5, t9 reversions in cultured L517BY mouse lymphoma cells ;7Z~st-mediated assays involving bacteria or mammal fan cells in mice; 12 and dominant lethal mutations in mice .62 Clastogenic ef fects of sul fur mustard have been studied in plant root tips and microsporocytesl9 and in cultured mammalian cells.65 Data from a mouse dominant-lethal test suggest that sulfur mustard reaches germinal tissue ant induces dominant lethal mutations.62 However, the data are inadequate for prediction of genetic risk to human germ cello. Uncertainties stem from the lack of data on defined genetic events induced by sulfur mustard in mam- mal fan spermatogonia or oocytes and from the variat ion in mutagenic potency that has been observed for the mustards in various assay sys- teme. Nevertheless, the possibility that sulfur mustard is a human ge.,u cell mutagen cannot be disregarded, part icularly because it is mutagenic in diverse assays, including tests for germ cell mutations in Drosophila and dominant lethals in mice; moreover, other direct- acting alkylating agents are known to induce mutations63 ant chro- mosomal alterations 1 in mammalian germ cells. Pathogen IS iS of Skin bee ions Vogt et al.72 recently studied the pathogenesis of lesions caused by the appl ices ion of H to the skin of guinea pigs and rabbi to; their methods inc luded l ight and e lee tron microscopy, h i s to- -106-

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chemistry, ant the use of contrast ing Evans blue dye. Cutaneous applications of H at 25-250 ~g/cm2 caused severe injury to the skin of both species and resulted in nonve~icating, necrotic, encrusted inf lavatory lesions . Cutaneous response to H had immediate and delayed phases. Within the first hour of exposure, injury to superficial capillaries and venules, vascular leakage, and infiltration by granulocytes with a high percentage of basophile were obeemed. The delayed phase was evident after ~ h; was manifested by nuclear pyknosis, an increase in lysosomal enzymes, and autophagic vacuoles in basal epideneal cells; and was accompanied by diffuse vascular leakage, infiltration by polymorphonuclear leukocytes, and ulceration. After the peak of inflammatory reaction at 24-72 h, the superficial, encrusted ulcers healed in about 10 d. Topical and systemic administrat ion of gluco- cort icosteroids decreased the extent of edema during the immediate phase, but d id not af feet the rate of heal ing. Care inc,~eni~ i tv Because of the corre let ion between mutagenic ity ant care inogeni- city, one would expect sulfur mustard to be carcinogenic on the basis of mutagenicity data alone. This expectation in borne out by carcin- ogenicity tests in experimental animals and by data from human expo- sures. The International Agency for Research on Cancer classifies sulfur mustard a. one of relatively few chemical agents on which the data are adequate to show an association with the induction of cancer in humans. 7 H-induced carcinogenic effects have been demonstrated in mammals. Heston26 reported that in two experiments the intravenous injection of aqueous H into strain A mice resulted in pulmonary tumors in 93X and 68Z of the surviving mice. In 1953, Heston27 documented the appearance of a variety of tumors in strain A, C3H, and C3Hf mice after the subcutaneous injection of H in olive oil. At the injec- tion site (middorsal area), there were sarcomas. At other sites, there were mammary and pulmonary tumors and hepatomss, and one mouse developed lymphocytic leukemia. Heston28 found significant increases in tuna eumore of ter inhalat ion exposure of mice . Heston26~28 reported many separate experiments, some of which were performed with nitrogen mustard. Heston concluded that both mustard compounds were mutagenic and care inogenic . Chrc nic exposure of rats to H ( at 1 or 100 ug/m3, 6.5 in/d, S d/wk) at Edgewoot produced an increase in epithelial cell tumors, but no evidence of systemic injury.45 —107—

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The investigations discusses above show a clear progression from the d~isco~rery of a mutagenic action of H in Drosophila' through the studies of alkylation reactions of H with TUNA, to the experimental production of tumors in mammals, including humans. INDUSTRIAL AND MILITARY EXPOSURE S A few clinical observations may be instructive before discussion of the major studies of H carcinogenicity in humans. Jackson and Adamo31 studied 33 cases of extensive basal cell carcinoma, two of which involved mustard-gas burns sustained during World War ~ ~ One of those deve loped 35 yr at ter the burn, but 2 yr after irradiation with cobalt-60. In the other, basal cell carcinoma developed at the site of three separate burns, 3 yr after exposure. Some of the mustard burns did not lead to basal eel 1 cancer. I'lig _ al.35 treated nineteen patients suffering from peo- riasis vulgaris once or twice with 50 g of 0. 005X H in petrolaeum. They cone luded that whole-body inunct ion with H presents a low car- cinogenic risk. That is likely to be erroneous, in view of the low dose and brie f treatment used . When examining reports of exposures to chemical agents, one should note the different conditions involved. After July 1917, during World War I, H was used often by both Germany and the Allies. Some areas of French battler ield. became so badly contaminated that they were abandoned by both ~ides. 13 Thorpe69 estimated atmos- pheric concentrations of H during gas shelling as averaging 3 ppm, with a maximum of 5 ppm (about 19 and 32 mg/m ~ respectively). Prentiss estimated thee exposure at 23 ppm ~ 150 mg/m3) for 10 min. giving a Ct ~ product of concentration and duration of exposure) of about 1, 500 ma. minims, would be lethal for an unprotected man. 60 In military and riot-control situations, exposure to agents is acute, but relatively brief. the clinical cases citedl7.40 involved "Iong-eerm" exposure, meaning a few months to a few years. Nakamura,53 in a 1956 paper, reported working conditions in a Japanese mustard-gas factory operated secretly in Hiroshima from 1930 to 1945. Workers alternately worked 1 h in gas product ion and 2 h in a gas-free env ironment over a 10-h workday. They wore gasmasks and complete protective clothing, including rubber boots, and were often rotated. Nevertheless, many workers showed a darkening of skin; some developed ulcers ~ diarrhea, ant jaundice and later coughed blood and developed tuberculosis. The concentration of mustard gas78 may have reached 50-70 mg/m3, as determined by bioassay. The bioassay involved exposure of unprotected birds in the work areas that resul- -108—

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ted in death in 12-15 he Exposed rabbits refused food, coughed, and tied within a 3-d period. Some adverse health effects experienced by workers may have been due to the inadequacy of protective equipment. Yamada et al.79 found 97 deaths during the period 1946-1957 among worker. exposed to H in a Japanese factory before and after the war. Of the 97 deaths, 20 were from malignant tumors, 13 of them in the respiratory system. In 1963, Yamada78 further reported 172 deaths, extending the survey to 1933-1962. Yamada gave no figure on the total number of men, but Wade et al .73 seated that there were 495. Forty-eight deaths (2BX) were caused by cancer, including 28 (161 of the 172) involving the respiratory tract. Among 5,030 deaths in the nonexposed general population, 406 (81) were from cancer, including 25 (0.5X of the S,030) involving the respiratory tract (Table 4-21. Wad a et al. extended Yamata' ~ observations on the same men, 73 f inding 33 deaths from respiratory tract cancers for 1952-1967, com- pared with an expected 0.9, a relative risk of nearly 37. For 960 employees not exposed to H. Wada et al. found only three deaths from respiratory tract cancer, compared with 1. ~ expected. These data point to a connection between long, low-dose exposure to H and later cancer, especially in the respiratory tract. Weiss ant Weiss75 found a statistically signif Leant increase in malignant tumors, especially bronchial and bladder carcinomas and leukemia, among 245 German workmen employed in the manufacture of H ant nitrogen mustard (HN) in the period 1935-1945. The 245 men, studied over 20 ye, all had verified case histories. By 1974, 114 of these men hat died, 40 of malignant tumors ant 38 of chronic respira- tory ailments. From 1951 to 1972, there were 32 deaths from various cancers among the exposed workmen, more than expected in a comparable nonexposed population; only bronchial carcinoma showed a statis- tically significant difference:- 11 observed vet 5 expected, a re let ive risk of more than 2 . Hellmann25 studied German munitions workers and reported 20 deaths from cancer among 157 former workmen. It is not clear whether these were included in the 245 of Weiss and Weiss. Morgenstern et al.48 reported on over 200 workmen in an American chemical plant making H during World War II, focusing on 10 case histories that illustrated the immediate and delayed ef fects of daily exposure to small quantities of H vapor. Exposure for 3 wk.to 6 ma 'ed these men to the tispeneary for treatment of respiratory distress . Typically, a man developed some or all of the following symptoms: ret eyes, photophobia, lacrimation, impaired vision, blepharospasm, loss of taste ant smell, nosebleed, sore throat, chest pain, wheezing, and dyspnea. After several such occurrences, a worker was removed from further contact with H. -1 09-

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TABLE 4-2 Characteristics of Male Mustard-Gas Workers Who Died from Cancer (1955-1967)a Case ___ Interval f ram Exposure Employment Age at Durat ion, to Death, Death, Site of yr ma ~ ma yr Neoplasm Histolo~ Type 3 7 2 22 4 62 Pharynx Undif ferent iated 4 5 3 22 0 40 Pharynx Squamou ~ ce 1 ~ 5 8 0 24 5 57 Paranasal ~ inus Squamous ce 11 6 4 11 23 2 44 Paranasal sinus Squamous cell 7 7 5 28 1 65 Paranasal ~ inus Squamous ce 11 10 ~ 0 16 10 62 Larynx Squamous cell 11 10 0 19 2 52 Larynx Squamous cell 12 12 10 22 11 58 Larynx Squamous cell 13 7 11 20 10 58 Larynx Squamous ce 11 15 8 0 25 5 48 Larynx Squamous cell 16 7 0 23 4 59 Trachea Squamous ce 11 18 1 4 10 1 1 30 Bronchu ~ Squamou ~ c e 11 21 6 1 23 1 62 Bronchus Squamous ce 11 22 ~ 1 17 4 62 Lung Squamous cell 23 2 0 33 0 54 Bronchus Undifferentiated 24 17 0 27 0 54 Bronchus Undifferentiated 26 16 4 27 10 61 Bronchus Undifferentiated 27 4 10 17 9 61 Bronchus Squamous cell 29 2 2 19 3 58 Bronchus Undifferentiated 30 ~ 5 22 5 63 Bronchus Undif ferent fated 32 5 ~ 20 ~ 50 Bronchus Undifferentiated 38 7 11 27 7 74 Lung Squamous cell 40 2 0 27 6 55 Bronchus Squamous ce 11 43 0 3 26 1 47 Lung Undif ferent iated 44 7 3 29 0 63 Bronchus Squamous ce 11 __ —^ a Data from Wada _ alms -1 10-

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Buscher,ll MorgensterD et al.~48 and others emphasized the lingering bronchitis, bronchial asthma, hoarseness, aphonia, and hypersensitivity to "oke ~ dust, and fumes that develop especially in men working in ludustri~ situations tat expose them to mustard at constant low concentrations. Even after discontinuing such work, they may be subject to continuing respiratory and systemic disablli- ties with a general deterioration of health that leaves them partial or complete invalids . These men recovered partially af ter leaving the mustard plant, but were sub ject to bronchitis, were susceptible to respiratory infections, and were like By to develop bronchlectasis. Many men were partially or completely disabled by 1945, when these observations ended. The implication of the observed disabilities is that complete recovery would probably not occur. It should be empha- sized that the sequelae outlined here resulted from the chronic, long-term exposure to H in the working environment. Most important, these long-ter~ sequel ae ~ except the malignancies ~ generally const i- tuted extensions or continuations of acute probe ems experienced during exposure to H; they did not sudden1 y appear years af ter exposure. MEDICAL EFFECTS Immediate Ef fects An unprotected person exposed to H vapor will suffer simultane- ously from skin burns, eye injury, and irritation of the respiratory tract. 20 The acute effects of H depend on the concentration of the gas, the duration of exposure, the ambient temperature, the extent of protection, and the susceptibi~ ity of the person. 23 Clothing will be contaminated and become a secondary source of poisoning even af ter a gas cloud has blown away. Onset of action may occur within several hours of exposure or after a ~ atent period of up to 24 h. The immediate effects of H within 0.5-3 h of espo sure include sneezing, acute conjunctivitis, lacrimation, rhinorrhea or nasal bleeding, soreness and burning of the the throat, hoarseness and dry, hacking cough, and erythema of exposed skin. Within 4-16 h of exposure, the effects of H include eye pain with acute conjunctivitis, corneal edema, lacri~tion, photophobia, blepharospasm, and edematous erythema of the eyelids; nausea, vomiting, diarrhea, and epigastric pain; and erythema and vesication of the skin with coalescence of vesicles to form bul- lee. 2~47~77 Between 24 and 48 h, effects on the eyes and skin pro- gress and are manifested by eyelid pain and edema with blepharospasm, lacrimati=, photophobia, and blludness; erythema, vesication, and edema of the skin; persistent cough and hoarseness or aphonia due to membranous laryagotracheobronchi~cis; increased temperature, pulse, and respiratory rate, as well as granulocytosis resulting from sec- —111—

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T4BLE 4-44 P ~ r c n t e r ~ 1 Tox i c i t y 0 f CHT in Rat, Mouse, and Rabb it Spec ies Roueeb LD,o, mg/kg . Mou Oral 78 (412-555) M o u I n t r ~ p e r i e o n e a l ~ 3 ~ ~ 2 - 1 5 ~ Rae Intreperitoneal 21 ~16-27) Re: Intrevenous 13 ( 11-15) Rabb i t Intrevenous ~ ~ 1-9 ~ Data from Biskup et ·~. 1 b Administered in polyethylene glycol 200. TABLE 4-4 5 Toxicity of CHT Atministeret Cutaneoualy to Doge~ Amount Appt ied, b Time, Acutc Acute mI/kg h _ Signs Mortality 1.0 (O) I 1/8 (neurologic) 0/8 0.5 (O) 16 or 24 Noe recorded 8/8 0.5 (O) 2 No to%ic signeC -- 0.5 (O) 4-6 Toxic signeC ~~ 0.5 (O) 1 ever, 4 d 1/3 (neurologic) 0/3 for 4 doses 0.25 (O) 1 every 4 d 0/3 -- for 4 doses 0.125 (0) 1 ever~r 4 d 0/3 -- for 4 doses 0.5 (N) 24 0/4 0/4 ~ Dats froa Dilley ee a1. 3 b 0 ~ occluded, ~ ~ nonoccluded. c Number of aniaele not stated. —245-

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REFERE:tJCES l 1. Blskup, R.K. ~ Loire, S.C. ~ Jr. ~ and Snodgrass, H.L. ~ Jr. Parenteral toxicity of riot control agents in mice ~ rats ~ and rabbits. U.S. Army Medical Research Laboratory, Edgewood Arsenal, Md. EATM 100-22. 1971. 11 p. 8. Brown , ~ . K. H ., Ferrigan, L . W., and S te~renson , D . E . The acute toxicity and skin irritant properties of tropilidene ~ cyclo- hepta-1, 3, 5-triene ~ . Ann. Occup . Hyg. 10: 123-126, 1967 . 3. Dilley, J. V., Newell , G.W., and Sasmore , D . P. The pharmacologic and toxicologic properties of CHT in dogs and monkeys. Flaal report, Contract No. 03-4772. Stanford Research Institute, Metro Park, Ca. January 17, 1977. 79 p. Dll fey, J.V., Newell, G.W., and Sasmore, D.P. The pharmacologic and toxicologic properties of CHT in dogs and monkeys. Final report, Contract No. 03-4771. Stanford Research Instltute, 'enio Park, Ca. May 1978. 35 p. Heddle , J.A., Hite, M., KIrkhart, B., Mavournin, K., MacGregor ~ J.T., Newell, G.W., and Salamone, M.~. The induction of micro- nuclei as a measure of genotosicity: A Report of the U.S. Envi- ronmental Protection Agency Gene-Tos Program. Mutat. Res. 123: 61-~8, 1983. 6. Jorgenson, T.A., and Rushbrook, C.~. Study of the mutagenic ef fects of CHT by the dominant lethal tent in rats. Final report, Contract No. DAAK-~-77~-0029. Stanford Research Institute, Menio Park, Ca. April 1978. 22 p. McNamara , B. . P ., Weimer , J . T ., Biskup , R . IC., Thomas , W . IJ ., Hopcus, M.W., Stude, H., Jr., Merkey, R.P., and Clark de Wal, A., Jr. Status report of the toxicity of EA 4923. U. S. Army Bio~ medical Laboratory, Aberdeen Proving Ground, Md. EAIR 4697. 1973. 55 p. Na~clonal Academy of Sciences-National Research Council. Identifying and Estimating the Genetic Impact of Chemical Muta- gens. Washington, D.C.: National Academy Press. 1983. 316 p. 9 . S inmost , T . C ., S inger , A ., Koviak , T . A., Shuely , W. ~ ., Sultan , W. E., and -King, J.W. Studies of potential irritant agents . I . A comprehensive investigation of EA 4923. U.S. Army Chemical Laboratory, Aberdeen Proving Ground, Md. EC-TR-76018. 1976. 139 p. -246—

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10. Stanford Research Institute. Excerpts from Monthly Progress Reports, #15 (dated 1 Sept. 1978), #14 (dated 1 Aug. 1978), SRI Project t6465, Contract DAAR-11-77-C-0029. Menlo Park, Ca. And. ] 11 p. 11. U. S. Army Biomedical Laboratory. Evaluation of EA 4923 for mutagenicity and chromosome damaging potential. IN EA 4923 - A volat ile Sensory Irritant , Part 2 - Source Documents . Edgewood Arsenal, Md. November 8, 1977. p. 217-223. - 247-

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EFFECTS OF ~ ~S~S IRRITANT CHEMICALS AT . . . EDGEWOOD IN l-WO-MAN TESTS RL SOP No. 70-3, dated June I, 1967, describes methods used at Edgewood for searching for and selecting toxic chemicals. Some of the details in connection with exposure of hen volunteere to experimental irritant chemicals are described. Human volunteers were exposed to compounds after review of animal screening data and appro- val by committees based on a conclusion that the experimental chemi- c~s were safe for human use. Generally, two volunteers were esposed to each substance. Subjects were exposed in a wind tunnel at an alr- speed of 5 mph and were asked to resist leaving the test atmosphere (up to ~ mind until exposure was unbearable. During 1962-1972, 123 irritant chemicals were tested. Further details on the chemicals are available from the MRC repository of Edgewood data. The substances were classified as irritants on the basis of the preliminary animal studies. Except where noted below, exposures were for ~ min or less in an aerosol chamber; each subject was exposed to a chemi Cal on' y once. AGENTS THAT CAUSED SLIGHT: OR NO EFFECI S Of the 123 irritant chemicals, 64 caused might or no effects on the exposed subjects. Two subjects were exposed to each of the 64 chemicals, except CS 40806, to which only one subject was exposed. The following 64 irritant chemicals caused slight or no effects: 301021 CS1086 CS4659 CS15442 CS20409 CS23653 CS27474 CS29780 CS30800 CS36650 CS36667 CS36722 CS3.~149 CS37200 CS38355 CS38731 CS39241 CS39242 CS39666 CS39715 CS40320 CS40325 CS40332 CS40679 CS40683 CS40686 CS40781 CS40785 CS40804 CS40805 CS40806 CS40841 CS40850 CS41462 CS41468 CS41592 CS41623 CS41725 CS42055 CS42057 CS42213 CS42216 CS42740 CS42824 CS43000 CS43001 CS43013 CS43014 CS43166 CS43168 CS43169 CS43945 CS43974 CS43988 CS43989 CS44854 CS45514 CS45659 CS46345 CS47137 CS47148 CS47563 CS48418 CS61804 -248- -

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CS38731 might have been contaminated with doodle. Despite a lack of complete toxicity data, it 8eem8 unlikely that the short, single exposures to the 64 irritant chemicals that caused slight or no acute effects on the exposed subjects will cause long-term health effects. LACRIMATORY AGENTS The predomloant ef facts of 42 of the 123 irritant chemicals were ocular, including eye irritation, eye closing, lacrimation, and con- junctivitis . Of the 42 lacrimatory agents, 34 caused very mild effected generally eye irritation, sometimes associated with dermal and upper respiratory passage irritation. The 34 mild lacrimatory agent o were tested on two subjects each, except ll 9135 (four subjects), CS46398 (one subject), EA 2542 (17 subjected, EA 3365 (17 subjects), EN 4922 (sis subjecta), and CSS15799 (ylidinea~ine, 21 subjects). The mild lacrim~eory agents include: 118055 CS30749 119135 CS30785 302049 CS30799 CS2127 CS31979 CS5146 CS37270 CS5616 CS38756 CS18692 CS40331 CS30747 CS40849 CS30748 CS41377 EA 2305 CS41722 EA 2329 CS43331 EA 2413 CS43981 EA 2433 CS46398 EA 2542 CS48861 EA 3176 EA 2284 EA 3365 EA 2302 EA 4922 CS815799 CS38756 and CS40849 might have been contaminated with dioxin. Addltional details are available on EA 2542 and CS815799. In 1963, two subjects underwent one espo sure each to EA 2542 at Ct's of 39 and 57 mg min/m3. No effects were described. In 1969, EA 2542 was tested further on 15 subjects who had one exposure each. Ct's ranged from 29.S to 65.S mg.D~in/~5, but no exposure durations are available. The subjects experienced irritation of eyes, nose, throat, and periorbital sites. Results of postesposure laboratory analyses were normal. In 1972, 21 subjects underwent exposure to CS815799. Seven subj- ec~cs had aerosol exposures at a Ct of 50 mg min/m3; one was exposed twice. CSSI5799 exposure caused mild ocular and respiratory irrita- tion. 'aboratory analyses 7 ~ after exposure retreated increased SCOT (43.8 and 42.7 lU) in two subjects, both of whom had normal pre-espo- sure SOOT. One of these two subjects also had 6-10 white cells in urinary sediment--a finding not seen in preexposure urlaalysis. -249_

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Fourteen CS815799 subjects were exposed cutaneously with 1: solu- tion, which caused mild irritation. One subject was exposed talcs. Glared the absence of followup data, it is not possible to pre- dict whether short exposures to the 34 mild lacrimatory agents will have long-term health effects among the esposed subjects. The increase in SCOT in CS815799-exposed subjects might have represented hepatic reactions to the chemical, but recovery was probably complet e. Eight of the 42 t acrimatory agents caused more severe ef facts than the other 34, namely more prolonged incapacitation in asso- ciation with lacrimation asked eye closing: llB539, 123175, 126312, CS encapsulated in gelatin, CS36579, FOGOlLCS, EA 2366, and CS-DM mixtures . The CS-~H mixtures were tested on 88 subjects. The CS:DM ratio in the mixture was ~ :10. Thirty-eight CS-DM exposures occurred in 195B, and half the 1958 subjects had two exposures each. Fifty of the CS-DM exposures occurred in 1966. Ct's were 0.5-40 me min/~3. Exposure durations, when recorded-, were 16 s to 2 min. The principal effects were ocular irritation, lacrimation, and conjunctivitis, sometimes associated with upper respiratory tract irritation and cough. One Subject required analgesic therapy after exposure because of severe discomfort. Results of laboratory analyses 7 ~ after exposure, were available for all the 1966 CS-DM sub Sects, as weU as some of the earlier subjects. One subject had an increase in SCOT (to 60 IU) in postesposure laboratory analysis, but his pre-esposure SCOT had also been slightly high ~ 33. 6 IU) . In 1963, two subjects underwent short, single exposures to 118539, which caused lacrimation and severe conjunctivitis, sali- vation, nasal irritation, chest constriction, and cough. In 1965 eight sub] ecus underwent single exposures to 123175, which caused lacrimatlon and eye closing for several minutes after cessation of exposure. Ir1 1966, 12 subjects underwent single exposures to CS36579, which caused eye pain, profuse lacrimatlon, eye closing and incapacitation that lasted several minutes after cessation of espo- sllre, nasal irritation, and. dyspnea. In 1963, two subjects underwent short, single exposures to EA 2366, which caused lacrimation and con- junctivitis, respiratory tract irritation and dyspnea, and nausea. Given the available information on subjects exposed to 118539, 123175, 126312, CS36579, and EA 2366 and their short, low~dose exposures, one carrot predict long-term health effects of these agent'. The discomfort associated with the exposures was marked, but exposures were short and recovery appeared complete. -250-

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RESPIRATORY IRRITANTS Seventeen of the 123 irritant chemicals caused predominantly respiratory effects, which were generally mild and transient. Ten caused nasal and throat irritation, including cough. These upper respiratory irritants were tested on two subjects each, except CS5635, CS42818, and EA 2129, which were tested on four subjects each. Other upper respiratory irritants were CS24302, CS41458, CS42822, CS42984, CS43010, CS43109, ant EA 3437. Seven respiratory irritants caused primarily a sensation of chest constriction and dyspnea. EA 2097 (CS14632) was tested on 19 sub- jects, 15 of whom had two exposures each, and EA 2214 was tested on four subjects. Other lower respiratory irritants were tested on two subjects each: IlB609, 119400, CS36273, CS42985, and CS43329. Giver the lack of followup information on the primary respira- tory irritant chemicals, it is not possible to predict whether they will have long-term health effects. Because these were short, low- dos~ exposures in which the acute effects were generally mild, with complete recovery, the Committee believes that Iong-term health ef fects are unlike ~ y. CONCLUST ONS The Committee analyzed published studies describing the in vitro and in vitro properties of the agents used and reviewed short-term data collected by the U.S. Army on volunteers. The ability to pro- vide definitive answers to the questions raised by ache charge to the Committee was limited by the absence of long-term followup studies of the sold iers and by the sparsene ss of chronic studies of these compounds in animals or in humans after industrial exposure. In general, the Committee found insufficient evidence deco eYal- ua~ce these chemicals, except mustard gas. Mustard gas is an esperi- mental mutagen asps human carcinogen at high doses. Data on the other irritants are insufficient to evaluate their mutagenicity, carcino- geniclty, or other long-te~ effects. Tests of all scheme chemicals involved few exposures and low doses. MUSTARD GAS (H) . Mustard gas is highly reactive and has vesicant and systemic toxic effects. It is an alkylating agent that is mutagenic in various laboratory test systems, including mammalian germ cells, but data are inadequate to predict the extent of its genetic risk in human. Mustard gas is also carcinogenic in experimental animals -251-

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and hours . Other possible long-tem ef fee to of mustard gas are related to its systemic toxicity; 8peclfically, it can cause blind- 11e88, permanent scarring of the skin. (which may lead deco skin tumors), and chronic bronchitis. Reported instances of long-term injury such as carcinogenesis in workers in a Japanese mustard production plant, were associated with exposure at high, long-term dosages. Infor- mation is insufficient to project risks associated with smaller exposures to mustard gas; however, serious long-term adverse effects in the small number of soldiers who received one or a few low-dose exposures at Edgewood seem unlikely (except for some cases of perma- nent scarring). Some of those exposed at Edgewood suffered skin injuries that took several weeks to resolve. However, in view of the small number of persons tested (about 150 healthy men) and the very low dosages involved, it is unlikely that a statistically significant increase in the risk of cancer or other chronic disease can be detected in those exposed to mustard gas at Edgewood. When exposed, the Edgewood subjects were wearing gasmasks and impregnated clothing--an ensemble being tested for efficacy against toxic contamination. o-CHLOROBENZ TRIDENT MALONONITRIr~ (CS) Results of experimental studies in microorganisms and short-term experiments in laboratory animals suggest that long-ter~ medical abnormalities in soldiers exposed to CS are unlikely. Acute tissue changes produced in animals and humans seem re~reraible and not likely to become chroni c in the absence of recurrent exposures. Follownp information on the long-term state of health of exposed soldiers is not yet available, but no reports have indicated that Edgewood sub- jects have experienced any long-term sequelae. CHLOROACETOPHENONE (CN) ON, a moderately toxic irritant, h88 immediate effects on the eyes, skin, and respiratory tract. ON is a strong skin-sensitizlug agent, but is rarely lethal. The Committee found no evidence of lasting ocular or respiratory effects in 99 volunteers esposed experimentally at Edgewood between 1958 and 1972 when subjects evaluated 2 wk after cutaneous administration or inhalation of sol. Alleraic contact dermatitis or hypersensitivity in these were aero- vo lun— , . - teers on re-exposure to CN is possible. There ha8 been no systematic study of the possible mutagenic and neoplasm-promoting ef facts of CN with current scientific methods. -2 52 -

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DIBENZ[b f] [1 4]0"ZEPINE (CR) . . CR, a alla lacrisetory irritant, manifests less acute toxicity than EN and CS. At low doses, it causes transient effects. There are a few studies on long-tene health effects, including potential mutagenicity and teratogenicity. The available data are insuffi- cient to predict long-tem health effects. The small number of espo- sures and the small number of subjects exposed to CR at low doses at Edgewood make the occurrence of demonstrable ef facts in these sub- jec to unlikely. CHLOROPICRIN ~ PS ~ PS is acutely toxic and has a variety of sensory ef facts tn animal. It has not been evaluated thoroughly for mutagenicity or carcinogenicity. Like those exposed to mustard gas, the subjects exposed to PS were wearlog gasmasks, and small numbers of soldiers were e Spored to small doses. PS is urn ikely to have produced detect- able long-term health ef fects in volunteers exposed at Edgewood. BROMBENZYL CYANIDE ( CA), DIPHENY~NOCHLORARSINE ( DM), and 1-METHOXY-1, 3, 5-CYCLOHEPTATRIENE ( CHT ) CA, DM, and CHT are unlikely to have produced measurable long- term health effects in volunteers exposed at Edgewood. But there are no specific toxicologic data on the mutagenicity and carcino- genicity of these compounds. CHT is less toxic than CN or DM when admini stered acutely. NONAbJOYL MORPHOLIDE The Committee does not expect long-term health effects in volun- teers tested with nonanoyl morpholide at the dosages used at Edge- wood. As with C&, DM, and CHT, specific~tosicologic data regarding its potential in this regard are not avaliable. 123 IRRITANT CHEMICALS A total of 123 irritant chemicals were generally tested on only two subjects each. There are no data on their mutagenicity, carcino- genicit,r, or other loDg-term health effects. However, because the exposures were small, detectable adverse effects seem unlikely. -253-

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APPENDIX A Part 1 ~ Reproduced from Volume I, Anticholinesterases and Ant icholinergics HISTORY OF THE EDGEWOOD TESTING PROGRAM INTRODUCTION Human experimentation appears to have been an integral part of the history of the U.S. Army chemical warfare (COO) research efforts until its suspension in 1975. On June 2B, 1918, the President directed the establishment of the Chemical Warfare Service (CWS). Four years ~ ater, in October 1922, the CWS created a Medical Research Division to conduct research directed at providing a defense against chemical agents. No matter how exhaustively an agent was tested in snimaiS, it was felt that its efficacy in humane alto had to be Studied. In early 1941, the threat of war increased the urgency of the development of protection against CW agents and, consequently, engendered a need for a larger source of volunteers. Formal autho- rity to recruit and use volunteer subjects in CW experiments was initiated in 1942. The Secretary of War was asked to rule on the permissibility of using enlisted men for testing agents of the mus- tard-gas type. In Jul y 1943, the CWS was assigned responsibility for all medical research related to CW. This extension of the CWS mission included toxicological research and the study of hazards to the health of personnel in the COOS. The issue of the use of human volunteers was considered by the Armed Forces Medical Policy Council during the early 1950's. The Council concluded that es sent ial data could not be obtained unless Huron volunteers were used, and the use of hears ire medical research was authorized. By 1954, the Chemical Corps (formerly COOS) had estab- lished a framework within which to conduct human experimentation, but it lacked an adequate pool of vol''r~teers. In t955, it was decided that the most practical source of volunteers would be enlisted men stationed at Army installations in the vicinity of Edgewood Arsenal. It was emphasized that voluntary consent of each human subject was absolutely essential. It was also stated that, in all experiments involving volunteer subjects, the subjects would be thoroughly informed of all procedures and of what might be expected as a result of each test. Furthermore, each volunteer would be free to determine whether he desired to participate in a given experiment. In October 1959, approval was granted for the conduct of research on volunteers to investigate defense against incapacitating CW agents. -254-

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The search for incapacitating agents intensified when the Kennedy administration took office. The involvement with incapacitating agents represented a departure from an earlier period, begun in 1946, when interest in highly toxic (acute) anticholinesterase chemicals resulted from their development in Germany during World War II. The basic purpose of a military incapacitating agent is to produce tem- porary ineffectiveness without permanent injury or death. Incapaci- tating agents (anticholinergic chemicala) and highly toxic (acute) anticholinesterase chemicals produce functional and at Nctura' effects on the nervous system which cause rapid or delayed effects on an individual's performance and behavior. PROCEDURES USED AT THE EDGEWOOD CHEMICAL TESTING PROGRAM _ ~ A fairly extensive discussion of the procedures used is provided in the Inspector General's report, Use of Volunteers in Chemical Agent Research, prepared by Colonel James R. Taylor and Major William H. Johnson and dated March 1976 (listed in Appendixes C,D). RECRUITMENT OF VOLUNTEERS Recruiting teams (initially administrative officers, but later often including military physicians from the Edgewood laboratory) visited Army installations where a briefing, usually with a film and handouts, was presented to a large number of enlisted men. Gener- ally 10 to 20 percent of the audience expressed interest and these men were asked to complete a personal history, which included medical and psychologic items and the Minnesota Multiphasic Personality Inventory (MMPI). It was not unusual for 400-600 men to request assignment in the course of a tour of seven to ten installations. Of these, no more than 100 were selected and eventually assigned for a 1-to 2-month period of temporary duty at Edgewood Arsenal. The "incentives" for volunteering consisted of a small monetary allowance (approximately $lO50 a day for temporary duty), the assign- ment of only light duties while at Edgewood, and a ~ oat every weekend free. Some volunteers were genuinely interested in the scientific and experimental aspects; however, if curiosity or the desire to "test one ' a self " seemed too strong, the applicant was usually not accepted. As a group, the volunteers were above average in physical and mental qualifications, with a mean IQ near 110, good behavior records, deviations of the population mean on all scales. -255-