### '6- 22-06 A09 :56 IN 

# ImmuQues

# t L aboratories, Inc. S'FQ`'~N~ 1600 East Gude Drive FEB 619~ Rockville, Maryland 20850 01,11r"I of 61(' 301-- 762- 7193 i:or ►1esticiutss arm ! ox::;
January 26, 198 4

Dr . John Moore Assistant Administrato r Office of Pesticide and Toxic Substances TS788 EPA 401 M Street, S .W . Wasington, D .C . 20460 Dear jack , 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 cop y of the draft to Gene McConnell . for internal review . Jack, good luck to you at EPA and I look forward to seeing you at some point in the future . Sincerely,

~' Ji McCo y Director of Research JLM :jwc Enclosure
cc : Dr . Charles Bowles



# 0 0D 002p. 2

. . ,

LIMITED TOXICITY OF INHALED ACETONITRIL E ON THE IMMUNE SYSTEM OF MICE I

Bowl I s4 C .A .2, McCartney, D .M .2, Coate, W .B .3, and McCoy, J .L . '

1 . Supported in part by Contract No . NOl-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 221GG .
4 . F ormerly with the National Toxicology Program, National. Institute of Environmental Health Sciences, Research Triangle Park, North Carolina .

t! ' {~ . 

# 0 0 0Q

~ _L_____• i I' - '~'~ ~_ ~p . 3

SUMMAR Y Hematological, cellular and humoral immune responses as well as clinical chemistry arameters of mice exposed to acetonitrile by inhalation were examined follo .wing observations that 800 ppm (10 treatment days) of this agent induced thy :nic atrophy in mice and rats . B6CSF1 mice received ten 6-hour exposures to 0, 100, 2 0 0, or 400 ppm of acetonitrile during . "ourteen day period and were evaluated by a battery of immunological assays . Hematologic parar,~ters including hematocrit, henoglobin, RBC and WBC were significantly depressed (P<0 .05) at increasing concentrations of the chemical . Lymphocyte proliferative responses to T-cell millogens were depressed along with a somewhat depressed host susceptibility to tra^zi, . .z itable tumor cell cha-lenge, although neither of these rcsponses 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 cortcentrations of IgA and IgM were unchanged . Other assays of B-cell ftsn, :tion, including Cunningham plaque assay responses to sheep RBC and B-cell proliferative responses to mitogens, were not significantly altered by acetonitrile . Delayed hypersens :. t-•i`-' :wonses 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 seriim clinical chemistry evaluation appeared to remain intact in mice

r :ece,fving 65 6-hour exposures at up to 400 ppm inhaled acetonitrile .

~~. F'~u :tIt3hQt, no significant lesions were observed by histopathological evaluation of these tissues or other body organs . Some treatmen t vo ~-~s~~ad M-1stchanges were observed in female animals receivin g 

# 0 0 0004p . 4

400 ppm acotonitrile consisting of slight cytoplasmic vacuolization of hepatocytes . 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 molecu l ar w eight volatile a gent that is used as a catalyst in chemical re a ctions and as a solvent in extractive distillation and crystalization of pharmaceutical an d .agricultural products including vitamins, bacter :cides, hormones, insecticides, plant growth regulators and fungici t- es (1) . The production volume of acetoriitrile is estimated at 80 million pounds
per year in the United States and it is estimated that 23,000 occupational workers may be expo t ed 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 acetunitrile . Marian et al . (3) observed that daily intramuscular inje c tion of acetonitrile induced exophtthalmos in rabbits, the severity of which was proportional to the degree of thyroid h,vperplasia . Daily subcutaneous injections of acetonitrile into infantile Z acs have been reparted to cause . , -rest in growth an d
, .~ e nr:l

.ar°g;emeint of thyroids, histological changes of the hypophysis, fatty gse!ii*,r ;ati.o-n of the liver and kidney and atrophy of the thymus (4) . Rat~s exposed to acetonitrile vapor by inhalation for ninety day s
~-~r :O hoiwO'O na~~ tdrAc effects at 1 6 6 or 330 ppm but did demonstrate 

# oooao:=5 

### rkp . 5

pulmonary inflammatory changes and minor abnormalities in the kidne y and liver at a concentration of 665 ppm . Rats inhaling 2800 ppm of stetonitrile v apors two hours per day for five days displayed difficulty in breathing, impaired renal function, paralysis of extremities , severe loss of body weight and hemorrhages in the lung and brain ( 5) . Finally, Drew et al ., (7) observed increases in ser u m glutara ine ox3lacetic transaminase (SGOT), glutamic pyruvic transaminase (SCPT) and ornithine carbamyl transferase activity in rats receiving a single for hour inhalation exposure to 3000 ppm of scetonitrile, indicating acute hepatic injury .

`, Metabolism and pharmacok• .netic investigations of acetoni* .rile are complex and have been somewhat unre l iable . It was initially presumed that in vivo, acetonitrile degrades to formic acid and thiocyanate (8), but the finding of serum hydrogen cyanide (HCN) by a number of investigators suggests other pathways may be inv o lved . Major toxic df fec :t' may result from the production of HCN by its action in a~r~fiibiting cytochrome oxidase and impairing cellular respiration . Thr ;~i~`o;;t
-lms.~-tioin of cyanide after acetonitrile exposure is supported by ;`alut.bps3~ reports of human poisoni iig victims which showed high levels o f d-y,a# id o

_ ii~ i ar ious organs, blood and urine

- (9) and by studies in rats, v,

, oi i ke

- is 41h d, dog s showing urinary thiocyanates in all three specie ;; !~a;fter . inhalation of acetonitrile and free cya• , ide in the blood oi

~ . 1 nke ;vs and< : 'dbg s , (5) . After intraperitoneal injection of o r i.nti All atiap exposure to acetonitrile, free and conjugated hydrogen cyanide,~, as we 11 ~si -a+e e t onit t ile, were found in urine and variou s
or ga As Highest concentrations of ac :etonitrile accumulated in tlhe kidney, testes, 4-pledr., liver, intestine and lung o nd free an d i , • bound H CN ;were found in the apleen
. 

# U4-0,00 6

. . k ' . . .-. . . , . .•~ : .,t . ;p . 6

Recent interest in specific toxic effects of chemical agents o n 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 *ipidly proliferating populations of lymphoid cells . Further, chemical damage to the immune system may result in hosts with depressed abilities to combat infPctious 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 Cuucar 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 wer e ° 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 . Che,miqal Acetonitrile was obtained from Vistron Corporation (Lot No . V041381) . Gas chromatography analysis verified the purity of the compound was 99 .67 . The chemical was stored at 4°C until use
. 

# 00.0007 ~~~" 4p . 7

Chemical Exposure 

## and Generatio n 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 concentration of 750 ppm resulted in death of 4/10 animals and caused significant body weight decreases in survi~,ing 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 mi c e , 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-str .el inhalation chambers by sweeping the head space of glass flasks containing the liquid compound . The chambers were ventilated with filtered air at a flow of 1200L/r:in . All flasks were set in water baths and held at approximately 26°C with immersion heaters . Control animals were exposed to filtered air only . Acetonitrile vapor concentrations were monitored every 30 minutes during 6-hour exposures using a Miran 80 1

' ;Tnfttr,ed Vapor Analyzer calibration checked daily . An analytical wa~e 3 :'07a$th of 11 .1 microns was us?d for all analyses . Animals werY ooetl-ved twice daily for evidence of clinical signs of rhemical toa~ic,~ty 

# 000008
f F

x.Y ._._i

.. .. _ _ . . 1
. .~ .p . 8

Tumor Susceptibility Assa y 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-t-,-nor chall-nge . Tumors were confirmed after sacrifice on day 67 . Clinical Chemistrv Evaluation s Serum from each mouse was evaluated for liver, kidney and thyroid function including SGOT, SGPT, SDH, BUN and creatinine as measured 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 acetonitrile 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 the immunology experiments were performed concurrentlv with a larger 90-day p .rechronic inhalaticn study of acetonitrile, clinical chemistry evaluations of liver, kidney, and thyroid function as well as necropsy a°nd histopathological evaluation findings from the 90 day study ar e also included in this report
. 0000 1 1 

# ~ .s,~~ Vp . 9

A . Clinical Signs of Chemicel Toxicity All chemically treated mic e remained normal in physical 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 . $ . Necropsy and Histopathology No gross lesions were observed in animals in any treatment group during the 14 or 9 0 day exposure periods . Histologically, thymic atrophy was observed in the 400 and 200 ppm treated mice during these time periods . Further, some slight vacuolization of hepatocytes and hydropic degeneration of li,rer tissue was observed in animals exposed to 400 ppm acetonitrile over 90 days
. 

### C . Clinical Pathology 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 T5 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 WBi2 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 suppie3sions 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 . 

# 000012p .10

D . Organ/Brain Weights Table 2 presents organ weight data (mea n values of 6 animals per treatment group) . 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 pp m dose levels . Spleen/brain weig h t ratios tended to increase . C%A
a36 9S g p r esented in Table 3 . Mea ti 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 d epressed 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 were not observe d between treated and untreated animals . Mean IgM concentrations were depressed by 11 .5% and 6 .0% at the 100 and 200 ppm levels and although these reductions were
s ignificant, linear regression analysis showed no dose related effe c ts of acetonicrile . 

# 0000 13 ~~`P. 1 1

~- • ~ .
F . Cunningham Plaque Assay The antibody plaque forming response to SRBC is presented in Table 4 . Geometric and arithmetic means ofPFC and PFC per 106 spleen cells were analyzed with Dunnett's ttpst . There was no significant difference between any of th e 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 increas-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 Blastogenesis 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 i nlymphocyte 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 depression in responsiveness to the three mitogens suggesting
some possible functional loss of T'and B lymphocyte activity, 

# 000014p . 12

; • , . linear regression analysis showed no dose-related trends in these responses . H . Delaved Hypersensitivity 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 groups of mice . Further, doseresponse effects were not demonstrated using the TerpatraJonckheere test for trend . Mean cpm for saline inoculated ears (from six mice per group) ranged from 1193 to 2107 while KLH inoculated ear mean cpm for the acetonitrile treated and control groups ranged from 2964 to 4178 . 1 . Tumor Susceptibility Susceptibility of acetonitrile-treated and untreated animals to PY$6 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 one-tailed exact test for proportional comparison revealed no significant difference between treated and control groups . Mean latency time to tumor detection and mean tumor volume were nit significantly different in comparisons made between control and chemically treated animals as determined by a two-sample independent t-test . There was a trend, however, for the mean latency period to tumor detection to be somewhat deceased in the acetonitrile-treated mice . 

# 000015p . 13

DISCUSSIO N 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 ac :etonitrile . Our initial prechronic inhalation studies of exposures to acetonitrile 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 eariie~ 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 concenLrations . 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 of in vivo and in vitro assays of humoral and cellular immunity to thoroughly evaluate possible acetonitrile toxicity on various immune compartments as previously described (13 , 

# 000016



# . ; ; ~ .p . 14

14) . These assays have been successfully used in investz3atin g suppressive immuno-modulation by such agents as polybrominated byphenyls (15), benzene (16), diethylstilbestrol (14, 1 7 ) and heavy metals (18) . With the concentrations of acetonitrile used here, we were able to quickly demonstrate relatively specific toxicity on the immune system, with no other major target organ toxicity, even after the 90- day exposure period, with the exception of some slight to moderate hydropic degeneratioj of the liver . Short-term toxicity to lymphoid tells was manifested by statistically significant decreased total WB C ,and neutrophils and IgG levels . WBC counts and neutrophils were not significantly depressed in exposed 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, lymphocyt- 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 deprersion in neutrophil (considered the first line of defense against microbial infections (19)) numbers an V 

# 000017p .15

proportions following the 10-day . series of acetonitrile exposure s 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 eithor be directl'y--q'_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 l~ _- k 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, although apparent life-threatening offects were not found, due to the limited immune toxicity of acetonitrile, this study suggests that by using short-term . immunological measures of toxicity that a more generalized toxicity of chemicals may be predicted . This would not be surprising since lymphoid tissue revresents a population of rapidly dividing cells that may be one of the f? .rst target populations to be damagid by many chemical or en- .iro, :mental agents . 

# 000018

WAfp . 1 6

. ` . Whether such a battery of immunological tests as employed in this study will allow predictior of more generalized chemical toxicity in other cases is a question calling for further application of t :is approach . RE FERE NCES 1 . Hawley, G .G ., The Condensed Chemical Dictionary . 8th Ed . Van Nostrand Reinhold Company-, New York, New York, 1 .971 . 2 . NIOSH, Criteria for Recommended Standard Occupational Exposure to Acrylonitrile . DHEW Rpbl . 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 Rabbits 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 ., 1 : 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 : 8 09-819, 1978 . 8 . Williams, R .T ., Detoxication Mechanisms, 2nd Ed . John Wiley and Sons, Inc ., New York, 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 . Taxicol . 2 : 1, 1979 . 12 . Lucier, G .W . and Hook, G .E .R ., Target Organ Toxicity : Immune System, Environ . Health Perspect . 43 : 1-135, 1982 . 13 . Vos, J .G ., Immune Suppression as Related to Toxicology CRC Crit . Rev . Toxicol . 5 : 67-101, 1977 . 

# 000019 

# .~;~ ,p .17

14 . Dean, J .H ., Luster, M .I ., Boorman, G .A . and Lauer, L .D . , Procedures Available to Examine the Immunotoxicity of Chemicals and Drugs, Pharm . R e views, 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 Marrow Cell 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 . Reticuloend o thel . Soc . 28 : 547-559, 1980 . 18 . Koll e r, 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 .



### .~



# 000020

~k~~~ ~~ ~ . N ~ft < ( ~~ ✓'p . 18

~ o o ~ ~ 7 (D m m c c w N A ~o 5 fi CL o a w

:3 n Zr ro tD W W rt -r ro rt 0 0 ~ . w o 0 00 °

° ►- ~' r cron rt a o °n . °- P• 0 c ► ~ -, m 0 r* ~11 fD E' ~ w a - czo m w o .

, ► ~ ~ w~ ~ m 1-- ~ a, v w ~ p r ~ tn o \ 0 N

. „~, . INI w 0 ~ I~• ~C C~ R do --- ~ O 0 °' 0 t-I do d P v V ~ \ C) ~,',• ' w v N• H (D ~ ' ( _ • r o W :L9 v Q .• . ~ dp H
H o O co F-~ F-- tJt z

. • • O ti7 h+ w(+ ► + (+ I+ W -J Z n o n (+ o c~ o o(+ + (+ tj ; o a o o q • O is N tJ~ • • • 0

. o d

► t71 0 co O h' ~ 'Z N .- . .- . .• . ~f \D T H h-+ 00 1•~ ~3 • N - .l 7q v • O . • H H w V ~11 N ~ d

. . .r .~ ~ tri v' ro ~ ct y (D 0 z 0 0 0 % H ti tT1 F-' • . .~ ~a ~• . v 0 0 'v o a .) oo • rt' y o ~ ► ~ n t~ Ln W ► + ► + ► + + ( + ~ + 0 0 o c, ( + ( + o ~• [7 O O • • • O o o M ~ • F-' O N O (D O O -J CJ % N J O W ~ CJ W r.. .- . `J co M F- ' O O Ln w Ci

. . fD O .t u~ tn w E-' z ~, ~ .. . ..,. ., . y ~ 0 ro r 0 Co Ln 0 arn w N ib
. 3

. . • • o~ a~ ~- td O O 00 tJ~ ~ N t!t J 1+ 1+ ( J1 1+ N N C~ 1+ 1+ O O(+ O ~+ t O W O O • • O O O O 'Tj • • N !-+ H • M-'' O O Ln N h- j 1- j V1 N W .+ kD W ds % C ~
.~ . pD H W f7 O O 10 • .•~ tr~ iJl V v V •l F ds t+ O d+ CO H

. . V1 ~ • C
.> F J Ln Cl co N (+ (+ (+ (+ Cl rn .c~

► + (+ O O O O(+ (+ O O O • • • • O O O • CT O C~ F-~ • • O O 0 Ln Ln -3 N co F~ F-+ .••. - twJ F-0 .• . i-
. 

# co F• ~ 

# 0 

# ° -1 

# ~ aaau2~ 

# . . . .
ik4 L Y - V • .~ V •./ ~, . . ~ .a,P
. 19 i . !~ ~r• `' ~ ~
to , N ~' ~ G'> ~O O p f R O 0 13 '~U t~Y 1~~• 3 L4 r►' 

### m ~- m n e, n ~ • ~• ~ ~ cl

r O~ N N F~•~ I- ~ } tf C h+ ~1 b0 Fi • • • • in ~ ~ r• ~ cri vn •a o z f~ U! H 

### tD N ~ ~ ~ I (D

N rn IN ' fD a w v 0 T1 ~' n tn

~ N• O ID It M

w w w t

. , ~p s % co w Itb O ~ ~'~ ; ~ ~ w ~c ►-~ ~n ~ O ~ ~ v w v o (D
► '~ w q i rr o H+ w IN,\ l! H 0 3 N N r iW y m ~• o ~• v r• w ~7 N LO ~• v ~ ty (D C7 F. . 

### ~r ~r d

v co l t • ., co r nS .G

• a • • • • x rr 1 h N O~ N 01 r• F• j• rt :3 fN r) y .~. co w fd ds • `~ C . . pi • a► N ~ s~

o N t o

f 

### r~; . ... .~ a

'• .' H F- . (AI N Fi A l

. w Carr

cs 

### e,r, a ~ • ~ . ~i .~ ~ ~ :~~ N

r . 

# . . ; o~ ~ .

. . r . ~ . .§I~
. . _ . ~e . . , . ._ ,, . . . . . . p . - , .p . 20

Table 3
EFFECT OF ACETONITRILE INHALATION ON THE CONCENTRATION OF CIRCULATING IgG, IgA, AND IgM IN B6C3F1 MICE 1
Acetonitrile Mean Immunoglobulin Concentration + SEM2 (% Change) Treatment mg 100m1 ( % Chancre ) (PPM) IqG IgA IgM 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 % t ) (11 .5 $ 200 112 .5 + 7 .5 47 .2 + 1 .0 22 .8 + 0 .4

(33 .1 % 4) (6 .7 % +) (6 .0 % f ) 400 87 .2 + 7 .0 42 .2 + 0 .8 2 4 .8 + 0 . 3



### (48 .1$ + ) (4 . 1% y ) (2 .0%-+ )

1 . Numbers represent Mean + SEM Concentration of Immunoglobulin Levels from pools of 6 mice . 2 . Immunoglobulin Concentraticn expressed as mg/dl .



### 000

# o23