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# FYr-©7~ .~-a~~y-o~9a 12 Rockville, Maryland20850 0111ceofVie!+a.rtan..4'- 301-762-7193 1:or11esuciac ;s;Anacux:::zj,..... 

## --....... -- January 26, 1984

Dr . John Moore Assistant Administrator Office of Pesticide and Toxic Substances TS788 EPA 401 M Street, S.W . Wasington, D,C. 20460 Dear jack, p. 1

":~~ I 

# .020

First, congratulations on your new position. I know NIEHS feels your loss but your contributions there, especially with NTP, will undoubtedly be appreciated for years. Enclosed is a draft manuscript describing the immunotoxicology studies on acetonitrile. As you recall, when I left the program I briefly discussed the possibility of some manuscript generation with you on the compound. I have forwarded a copy of the draft to Gene McConnAll for internal review . Jack, good luck to you at EPA and I look forward to seeing you at some point in the future . Sincerely, JiW' McCoy Director of Research

.-



# M R1I13o1% 000002LIMITED TOXICITY OF INHALED ACETONITRILE

W ON THE IMMUNE SYSTEM OF MICE1

BowlJpsQ C.A.2, McCartney, D.M .2, Coate, W

.B.3, and McCoy, J .L.

1 . Supported in part by Contract No. NO1-CP-95655-02 with the National Toxicology Program . 2. ImmuQuest Laboratories, Inc ., 1600 East Gude Drive, Rockville, Maryland 20850. 3. Hazleton Laboratories, America, Vienna, Virginia 22180. 4. Formerly with the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina.

### SUMMARY Hematological, cellular and humoral immune responses as well as clinical chemistry ;arameters of mice exposed to acetonitrile by inhalation were examined following observations that 800 ppm (10 

### treatment days) of this agent induted thyttic atrophy in mice and rats . B6C3F1 mice received ten 6-hour exposures to 0, 100, 200, or 400 ppm of acetonitrile during ;, 'ourteen day period and were evaluated by a battery of immunological assays. Hematologic parar~,-~ters including hematocrit, hemoglobin, RBC and WBC were significantly depressed (P<0.05) at increasing concentrations of the chemical . Lymphocyte proliferative responses to T-cell mi :ogens were depressed along with a somewhat depressee host susceptibility co rra^zpLaitable tumor cell, cha::lenge, although neither of these responses were dose dependent . Studies of B-lymphocyte function indicated that levels of serum IgG were significantly depressed (P<0.05) with increasing concentrations of acetonitrile while concentrations of IgA and IgMwere unchanged . Other assays of B-cell fun, :tion, including Cunningham plaqiie assay responses to sheep RBC and B-cell proliferative responses to mitogens, were not significantly altered by acetonitrile., Delayed hypersens: tj "" i`y :,Rsponses to keyhole limpet hemocyanin responses were not affected b; acetonitrile treatment . In a concurrent 90-day prechronic study, liver, kidney and thyroid functions as evidenced by serum clinical chemistry evaluation appeared to remain intact in mice e4vn,eri:ving 65 6-hour exposures at up to 400 ppm inhaled acetonitrile. 'F-u~~~*-e%r, tto significant lesions were observed by histopathological 1!#,Wtio.n of Oese tissues or other body organs. Some treatment t;e`ILg*+ed- Astol,og-ical changes were observed in female animals receiving 

# 000004400 ppm acetonitrile consisting~of slight cytoplasmic vacuolization of

ttepatocytes. The data demonstrate that with these doses, inhalation of acetonitrile in mice appears to induce limited toxic effects on lymphoid cell function and significant alterations in WBC and IgG levels in a dose response relationship generally not seen in other organs . INTRODUCTION Acetonitrile is a low molecular weight volatile agent that is used as a catalyst in chemical reactions and as a solvent in extractive distillation and crystalization o.' pharmaceutical and agricultural products including vitamins, bacter :cides, hormones, insecticides, plant growth regulators and fungicit:es (1) . The production volume of acetonitrile is estimated at 80 million pounds , per year in the United States and it is estimated that 23,000 occupational workers may be exposed annually by dermal and inhalation routes (2) . A series of studies have demonstrated a variety of toxicological dysfunctions following injection or inhalation of high ,doses of acetonitrile. Marian et al . (3) observed that daily intramuscular injection of acetonitrile induced exopht.thalmos in rabbits, the severity of which was proportional to the degree of thyroid hyperplasia. Daily subcutaneous injections of acetonitrile into Mtf3tttile zacs have been reported to cause -, -rest in growth and T *d:la~C°g,!V

;me,tit of thyroids, histological changes of the hypophysis, fatty 

### e~c~,artioz of the liver and kidney and atrophy of the thymus (4) . Jc,OoO,ie:d to acetonitrile vapor by inhalation for ninety days ,no' . ' Fac~~c effects : at 166 or 330 p:pm but did demonstratep. 5

ptlbondry inflammatory changes and minor abnormalities in the kidney htaid liver at a concentration of '665 ppm . Rats inhaling 2800 ppm of Adetonitrile vapors two hours per day for five days displayed difficulty in breathing, impaired renal function, paralysis of eittremities, severe loss of body weight and hemorrhages in the lung and brain (S) . Finally, Drew et al ., (7) observed increases in serum glutaroine oxalacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT) and ornithine carbamyl transferase activity in tats receiving a single four hour inhalation exposure to 3000 ppm of ,$cetonitrile, indicating acute hepatic injury.

. '.Metabolism and pharmacok~inetic investigations of acetonirritz~ are tomplex and have been somewhat unreliable. It was initially presumed that in vivo, acetonitrile degrades to formic acid and thiocyanate but the finding of serum hydrogen cyanide (HCN) by a number of Investigators suggests other pathways may be involved. Major toxic r±~~f:f:ec:t~ may result from the production of HCN by its action in

..,- Th ~hibxting cytochrome oxidase and impairing cellular respiration. c r,va-t.iotz of cyanide after acetonitrile exposure

ze :.ports of human poisonilig victims which is supported by showed high levels of kh ,ra7rious organs, blood and urine ~(9) and by studies in rats, :~!~tid, .~d.~gs showing urinary thiocyanates in all three speciez! ~1:#,?'t-ion of acetonitrile and free cya." ide in the blood of d=ags. :~'S)

. After intraperi :oneal injection of or o-~~-tf~~e t~Q d,4eto-ritrile, free and conjugated hydrogen

~OFt-,.a.tiittile, were fvu.nd in urine and various -ghost tancentrartlob-A of accumulated



### riive.r;t: :5t?fit* Wnd lung iind free and 3.n 1Recent interest in specific toxic effects of chemical agents on

the immune system (11-14) has arisen since it may be possible by using short-term assays to provide predictive toxicity information of chemicals using r-Apidly proliferating populations of lymphoid cells . Further, chemical damage to the immune system may result in hosts with depressed abilities to combat infectious disease or carcinogenic processes. The early work of Dessau (4) who demonstrated atrophy of thymus tissue coupled with the finding that acetonitrile accumulates in the spleen (6, 10) suggested to us that this chemical may express toxic action on normal lymphoid cell functions . The importance of possible acetonirrile~toxicity to lymphoid tissue is obvious considering its diverse use and the possible exposure of workers during its synthesis and use or possible exposure to others outside these operations. MATERIALS AND METHODS Mice Female B6C3C1 mice (6 weeks of age) wer_ received from the Frederick Cancer Research Facility, Frederick, Maryland, quarantined for 14 days prior to initiation of exposures. and held for an additional 6-8 weeks. Between chemical exposures, the animals were housed in stainless-steel cages suspended in 6-M3 inhalation chambers with food pellets (Ziegler NIH-07 diet) and tap water available ad libi tum

. Chemical Acetonitrile was obtained from Vistron Corporation (Lot No. 70413g1) . Gas chromatography analysis verified the purity of the compound was 99.6%. The chemical was stored at 4oC until use. 

# 000007Chemical Exposure and Generation

In preliminary studies we demonstrated that over a two week period, 10 repeated 6-hour inhalation exposures to acetonitrile were lethal to B6C3F1 mice at 3000 and 1500 ppm concentrations . A concentras.ion of 750 ppm resulted in death of 4/10 animals and caused significant body weight decreases in survi~ring mice. These concentrations, as well as 375 and 188 ppm exposures, induced thymic atrophy and scme hydropic degeneration of the liver .. In view of these toxicological data we further tested inhaled acetonitrile in immunotoxicity studies at 100, 200 and 400 ppm where no or only subtle toxicity might be expected in other organs. Test mice received 10 exposures of acetonitrile (100, 200 or 400 ppm) six hours/day during a 14-day period in the immunology study. Other mice, evaluated by clinical chemistry and histopathology, were exposed to these same concentrations of compound six hours/day, 5 days per week, during a concurrent 90-day prechronic study . Acetonitrile vapor was generated into 6-M3 glass and stainless-st(.el inhalation chambers by sweeping the head space of glass flasks containing the liquid compound . The chambers were ventilated with filtered air at a flowof 1200L/cin . All flasks were set in water baths and held at approximately 26oC with immersion heaters . Control animals werc e.xposed to filtered air only. Acetonitrile vapor concentrations were ti7clifored every 30 minutes during 6-hour exposures using a Miran 801 ~T,wfft=rvd Vapor Analyzer calibration checked daily. An analytical



### .:'0 ;i~rgth of 11 .1 microns was used for all analyses. Animals wer- -v~bd twice daily for evidence of clinical signs of chemical 

# 000008Tumor .Susceptibility Assay Twenty control mice and 10 mice per test group were inoculated by the subcutaneous route with a TD10-20 dose of transplantable PYB6 tumor cells two days following the last exposure to acetonitrile. Animals were individually housed and palpated for tumor formation twice weekly. Tumor incidence and latency period to tumor detection were determined up to sixty days post-ti .nor chall~nge. Tumors were confirmed after sacrifice on day 67. Clinical Chemistry Evaluations Serum from each mouse was evaluated for liver, kidney and thyroid function including SGOT, SGPT, SDH, BUN and creatinine as measurPd by Centrifi Chem(R) autoanalyzer and determination of T3 and T5 hormone levels using radioimmunoassays with specific anti T3 and T5 sera. RESULTS Our initial prechronic studies of acPtonitrile demonstrated that mice exposed to a concentration of 750 ppm or higher (inhaled six hours daily for ten days) resulted in significant weight losses, atrophy of thymus, some hydropic degeneration of liver ti~,sue or death. Further, lower concentrations of compound (375 and 188 ppm) also induced thymus atrophy. In studying possible toxicological actions of inhaled acetonitrile on the immune system we chose concentrations of 400, 200 and 100 ppm administered ten times (six hours/day) during a fourteen day period. Since :.he immunology experiments were performed concurrently with a larger 90-day P .teehronic inhalaticn study of acetonitrile, clinical chemistry evalu.ations of liver, kidney, and thyroid function as well as necropsy wmd hi .stopathological evaluation findings from the 90 day study are 6lso included in this report . . 0000A. Clinical Signs of ChemicPt Toxicity All chemically treated mice

remained normal in phvsical appearance during the 14 day, as well as the 90 day, exposure periods of the compound . No significant weight losses were seen in any treatment group up to 90 days of chemical exposure. B. Necropsy and Histopathology No gross lesions were observed in animals in any treatment group during the 14 or 90 day exposure periods. Histologically, thymic atrophy was observed in the 400 and 200 ppm treated mice during these time periods . Further, some sliqht vacuolization of hepatocytes and hydropic degeneration of lirer tissue was observed in animals exposed to 400 ppm acetonitrile over 90 days. 

### C. Clinical PatholoR Y 1 . Liver, Kidney and Thyroid Function Evaluation All animals exposed to acetonitrile, including the 400 ppm levels up to 90 days, had SGOT, SGPT, SDH, BUN, creatinine and T3 and TS hormone levels within normal ranges and demonstrated no significa differences from control animal values in this study . 2. Hematology The results of hematology profiles of animals treated 10 times during a 14-day period are presented in Table 1 . Suppressions of hematocrit, hemoglobin, RBC and WBi, counts, as well as proportional numbers of neutrophils and monocytes were significant as analyzed by Dunnett's two-tailed t-test with the 400 and 200 ppm concentrations. The suppiessions appeared compound-related as indicated by further analysis by linear regression. Hematocrits and WBC counts at the 100 ppm concentration were not significantly different from the untreated 

### control group. 

# 000012D. Organ/Brain Weights Table 2 presents organ weight data (mean

, values of 6 animals per treatment qroup) . The data are expressed as organ/brain weight ratios (the brain being one of the most constant organs in terms of weight loss-gain in chemical treated animals) . Although no statistically (as evaluated by the twotailed Dunnett's t-test) significant depression in spleen, liver, kidney, or thymus/brain weight ratios were observed, there was a trend for the thymus weights to be depressed at the 200 and 400 pQm dose levels. Spleen/brain weight ratios tended to increase, ~~~~cca~~a2~~2~.cc ~ra~~c4~-3Q .n ~-`~~LAbY7D2SDTi9 Df S~G~ ~g4aS. as~d ~gA 1~SC Cc~w- tCCQ ¢z D3,~ 2 .,S-- presented in Table 3. Mead IgG levels in the sera of animals exposed to 100, 200, and 400 ppm of acetonitrile were decreased by 25.8%, 33.1%, and 48.1% respectively, compared to untreated animals. The IgG levels were demonstrated (by Dunnett's twotailed t-test) to be significantly depressed as compared to , controls and a dose-response effect of the compound was indicated by linear regression analysis . Mean IgA concentrations in the sera of acetonitrile-treated animals were not significantly different from untreated animals. Dose effects in linear regression analysis weiE not observed between treated and untreated animals. Mean IgMconcentrations were depressed by 11 .5% and 6.0% at the 100 and 200 ppm levels and although these reductions were significant, linear regression analysis showed no dose related effects of acetonicrile. 

# 000013



# 16.,. F. Cunningham Plaque Assay The antibody plaque forming response to

SRBC is presented in Table 4. Geometric and arithmetic means of PFC and PFC per 106 spleen cells were analyzed with Dunnett's ttpst . There was no significant difference between any of the chemical exposed and control mice. It is of interest, however, that the number of plaques formed per spleen was reduced by 12.9%, 22.2% and 18.5% for treatment groups at 100, 200, and 400 ppm, respectively. These decreases in PFC per spleen is contrasted to the increa&ed mean spleen weights for animals in these respective groups (Table 2) and is somewhat suggestive of a depression in this immune function . G. Lymphocyte B_lastogenesis Response Results of lymphocyte blastogenesis studies are presented in Table 5. Dunnett's two-tailed t-test was performed on the mean (total) cpm for each mitogenic response assessed. There was no significant difference in lymphocyte responses to PHA, Con-A, or LPS between acetonitrileexposed and control animals . Analysis of the percentage change in net cpm revealed a depression in all groups examined at each exposure level . At 100 ppm of acetonitrile, lymphoproliferative responses to PHA, Con-A, and LPS were depressed by 23.3%, 26.4% and 11 .4%, respectively, as compared to untreated animals. Lymphoproliferative responses to the three mitogens by spleen cells of mice exposed to 400 ppm were also depressed although to a lesser extent than observed in the 100 ppm group. Although there was a trend for some dap~,.Ossion in responsiveness to the three mitogens suggesting tame possible functional loss of T' and B lymphocyte activity,



# .,:. 

# 11 000014linear regression analysis showed no dose-related trends in these

responses. H. Delaved Hy" rsensitivitv Responses Results of the delayed hypersensitivity response assay are presented in Table 6. Mean response indicies calculated from counts per minute of KLH challenged ear versus saline challenged ear of six mice per group ranged from 1 .52 to 2 .19.' Dunnett's two-tailed t-test analysis of these indices failed to reveal a significant difference between treated and control gro?!ps of mice. Further, doseresponse effects were not demonstrated using the TerpatraJoncfcheere test for trend .

for saline inoculated ears (from six mice per from 1193 to 2107 while KLH inoculated ear mean cpm Mean cpm group) ranged



### I . for the acetonitrile treated and control to 4178. Tumor Suscepti bilitv Susceptibility of

significantly different in chemically treated animals independent t-test . There one-tailed exact test for proportional comparison revealed significant difference between treated and control groups. latency time to tumor detection and mean tumor volume were groups ranged from 2964 acetonitrile-treated and untreated animals to PYB6 tumor challenge is presented in Table 7. Tumor incidence in untreated mice challenged with an LD10-20 of tumor was 11% . Tumor incidence in animals exposed to 100 or 400 ppm of acetonitrile was 44% and 33%, respectively . Fishers

comparisons made as determined by no Mean n.)t between control and a two-sample was a trend, however, for the mean latency period to tumor detection to be somewhat deceased in the acetonitrile-treated mice. 

# 000015 11

### DISCUSSION

Previous studies employing large doses of acetonitrile have demonstrated thyroid (3), liver (4, 5, 7), kidney (4, 5, 6), and lung (6) abnormalities, as well as thymus atrophy (4) in experimental animals receiving compound intramuscularly (3), subcutaneously (4) or by inhalation (5, 6, 7) . Pozzani et al ., (5) further reported that rats exposed seven hours per day for ninety days to 665 ppm acetonitrile by inhalation exhibited pulmonary inflammatory changes and minor changes in the kidney and liver . No toxic lesions were obsPrved in their animals exposed to 330 or 166 ppm acetonitrile. Our initial prechronic inhalation studies of exposures to acetoni .trile over a fourteen day period are consistent with the findings of Pozzani et al . (5) . We observed some hydropic degeneration in the liver in animals receiving 750 or greater ppm inhaled acetonitrile. Furthermore, we also observed thymic atrophy in mice as earliec reported for rabbits (3) at levels as low as 188 ppm of compoun~., suggesting a high degree of sensitivity of this organ to the chemical . As in Pozzani's earlier studies (5) we observed no other toxicity at the 375 and 188 ppm concentrations . In investigating possible systemic immunotoxicological actions of inhaled acetonitrile, we selected levels (400 ppm as the highest level) that presumably would not induce general toxicity to other major target organs in the animal, but levels that might result in toxicity to the immune system . We chose a battery c.f in vivo and in vitro assays of humoral and cellsrLar immunity to thoroughly evaluate possible acetonitrile toxicity on various immune compartments as previously described (13,



# I) 00001614) . These assays have been successfully used in invest+3ating

suppressive immuno-modulation by such agents as polybrominated byphenyls (15), benzene (lb), diethylstilbestrol (14, 17) and heavy metals (18) . ' With the concentrations of acetonitrile used here, we were able to quickly demonstrate relatively specific toxicity on the immune aystem, with no other major target organ toxicity, even after the 90- day exposure period, with the exception of some slight to moderate hydropic degeneratio,j of the liver . Short-term toxicity to lymphoid cells was manifested by statistically significant decreased total WBC ,and neutrophils and-IgG levels . WBC counts and neutrophils were not significantly depressed in exposei groups at the end of the 90-day subchronic study, but were depressed after 10 exposures. In evaluations of thymus/brain weight ratios, Cunningham SRBC plaque, lymphocyto blastogenesis to T and B cell mitogens, and susceptibility to tumor challenges, although statisticaly significant changes were not observed, there were trends with each assay that suggested some immune suppression . It would appear that at the highest concentration . used (400 ppm) over a ten-day treatment period, toxic effects on some parameters measured (i .e ., WBC, neutrophils, and IgG Levels) vere evident, but that toxicity as measured by the other humoral and cellular immunity assays was marginal . Increased levels of acetonitrile might have magnified some of these effects. Such a treatment regimen, however, might have also caused a more pronounced general toxicity to the host, a situation we attempted to avoid . Even though there was a depre:sion in neutrophil (considered the first line of defense against microbial infections (19)) numbers anV 

# 000017

.~

.=proportions following the 10-day. series of acetonitrile exposures

there was no evident increased infecLious diseases in the animals . This disease-free status included the 90 day treatment period of the concurrent subchrc.nic study . The statistically significant short-term depression of neutrophil numbers was obviously not sufficiently severe to cause a recognized increased susceptibility to intercurrent infections. It is of interest that only IgG levels were depressed; a phenomenon not observed with IgM and IgA measures. One possible explanation is that plasma cells secreting IgG are more sensitive to toxic actions of aceLonitrile than are those cells that secrete IgM and IgA. Acetonitrile may either be directl-y-41-ytotoxic to the IgG secreting cells, disrupt their differentiation processes or may interfere with secretion of IgG. Delayed hypersensitivity reactions as measured by the ear swelling technique were,generally normal in acetonitrile treated mice. This finding coupled with tti- continual IPA of clinical signs of chemical toxicity and lack of any observable infections suggests that in vivo immune competence remained resonably intact in compoundtreated mice. Finally, found, due to suggests that although apparent life-threatening effects were not

the limited immune toxicity of acetonitrile, this study by using short-term immunological measures of toxicity that a more generalized toxicity of chemicals would not be surprising since lymphoid tissue

, may be predicted. This revresents a population of rapidly dividing cells that may be one of the f:.rst target populations to be damag"!d by many chemical or en:iro, :mental agents.Whether such a battery of immunological tests as employed in this

study will allowpredictior of more generalized chemical toxicity in other cases is a question calling for further application of thsis approach . 

### REFERENCES 1 . Hawley, G.G., The Condensed Chemical Dictionary . 8th Ed . Van Nostrand Reinhold Company-, NewYork, New York, 1971 . 2. NIOSH, Criteria for Recommended Standard Occupational Exposure to Acrylonitrile. DHEWRpbl . 78-116. U.S. Government Printing Office, Wash .ngton, D.C. 3. Marian, D., Rosen, S .H. and Cipra, A., Further Studies on the Exophthalmos in Rahbits Produced by Methyl Cyanide. Proc. Soc . E:.V. Biol . Med., 30: 649-651, 1933. 4. Dessau, F., Chronic Acetonitrile Poisoning in Rats . Acta Brevia Neerland . Physiol . Pharmacol . Microbiol ., 7: 57-60, 1937. 5. Pozzani, U.C., Carpenter, C.P., Palm, P.E., Weil, C.S. and Nai, J .H., An Investigation of che Mammalian Toxicity of Acetonitrile, J . Occup. Med., 1 : 634-642, 1959. 6. Haguenoer, J .M ., Dequidt, J . and Jacquemont, M .C., Experimental Toxicology of Acetonitrile: Acute Toxicity by Inhalation, Europ. J . Tox. 8: 102, 1975. 7. Drew, R.T., Patel, J .M. and Lin, F.N., Changes in Serum Enzymes in Rats After Inhalation of Organic Solvents Singly and In Combination . Toxicol . Applied Pharmacol . 45: 809-819, 1978. 8. Williams, R.T., Detoxication Mechanisms, 2nd Ed . John Wiley and Sons, Inc ., NewYork, 1959. 9. Amdur, M

.L., Accidental Group Exposure to Acetonitrile. J . Occup. Med . 1 : 627, 1959. 10. Haguenoer, J .M ., Dequidt, J . and Jacquemont, M .C., Experimental Toxicology of Acetonitrile: Acute Intoxication by the Intraperitoneal Route, Eur . J . of Tox. 8: 94, 1975. 11 . Moore, J .A., The Immunotoxicology Phenomenon, Drug and Chem . Toxicol . 2: 1, 1979. 12. Lucier, G.W . and Hook, G.E.R., Target Organ Toxicity : Immune System, Environ. Health Perspec:t . 43: 1-135, 1982. 13. Yos, J .G., Immune Suppression as Related to Toxicology CRC Crit Rev. Toxicol . 5: 67-101, 1977.



# 16 00001914. Dean, J .H., Luster, M .I ., Boorman, G.A. and Lauer, L.D., Procedures Available to Examine the Immunotoxicity of Chemicals and Drugs, Pharm . Reviews, 34: 137-148, 1982. 15. Luster, M

.I ., Faith, R.E. and Moore, J .A., Effects of Polybrominated Biphenyls (PBB) on Immune Response in Rodents . Environ. Health Prospect . 23: 227-232, 1978. 16. Irons, R.D., Heck, H., Moore, B.J . and Murihead, K.A., Effects of Short-Term Benzene Administration on Bone MarrowCell Cycle Kinetics in the Rat, Toxicol . Appl . Pharmacol . 51 : 399-409, 1979. 17. Boorman, G.A., Luster, M .I ., Dean, J .H. and Wilson, R.E., The Effect of Adult Exposue to Diethylstilbestrol in the Mouse on Macrophage Function . J . Reticuloendothel . Soc. 28: 547-559, 1980. 18. Koller, L.D., Immunotoxicology of Heavy Metals, Int . J . Immunopharmacol . 2: 269-279, 1980. 19. Gadebusch, H.H., Phagocytes and Cellular Immunity, CRC Press, Bora, Florida, 3, 1979.o o ~ cM a ~ ~ c~c m c~c a r- 0 '~o ~ rt a o ~ w

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### Table 3 EFFECT OF ACETONITRILE INHALATION ON THE CONCENTRATION OF CIRCULATING IgG, IqA, AND IcM IN B6C3F1 MICE1 Acetonitrile Mean Immuno lobulin Concentration + SEM2 (8 Change) Treatment m 100m ($ Chance) (PPM) IgG - Id `4-- - ,- - - -- gM ----- 

### 0 168.2 + 10.1 (-) 44 + 0 (-) 24.3 + 0.4 (-) 100 124.8 + 12.9 45.2 + 0.8 21 .5 + 0.3



## (25.8%-+) (2.7%-+) (11.5T+) 200 112.5 + 7.5 47.2 + 1 .0 22.8 + 0.4 (13.1$i) (6.78{ ) (6.0$i) 

### 400 87.2 + 7.0 42.2 + 0.8 24.8 + 0.3

(48.1$ F) (4.1%-+) (2.0%-+) 1. Numbers represent Mean + SEMConcentration of Immunoglobulin Levels from pools of 6.mice. 2. Immunoglobulin Concentraticn expressed as ma/dl .