'6- 22-06 A09 :56 IN

ImmuQuest 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 ► 1 estic i utss a rm ! 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 0D002
p. 2

. . ,

LIMITED TOXICITY OF INHALED ACETONITRIL E

ON THE IMMUNE SYSTEM OF MICE I

Bowl

I

s

4

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 00, 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 0004
p. 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;eme int 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 :OhoiwO'O na~~ tdrAc effects at 1 6 6 or 330 ppm but did demonstrate

oooao:= 5

rk
p. 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

dffec: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,

,

oiike- 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< :'d bg 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 tile, were found in urine and variou s

or gaAs Highest concentrations of ac:etonitrile accumulated in

tlhe kidney,

i , •

testes, 4-pledr., liver, intestine and lung o nd free and

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

~~~" 4
p. 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

f F 000008

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,~~ V
p. 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 .

000012
p.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 9Sg

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 of

PFC 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 n

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

depression in responsiveness to the three mitogens suggesting

some possible functional loss of T'and B lymphocyte activity,

000014
p. 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 . 000015
p. 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

000017
p.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

WAf
p. 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 .

REFERE 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 cc

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

tn o \ 0~ ~ p r N

. „~,. INI

~ I~• ~C C~ w 0 R

do --- ~ O 0

°'

0

t-I

do dP

~,',• ' w v N• H

v V ~ \ C)

(D ~ ' ( _ • ro

v Q

W :L9

.•

dp H

. ~

H

o O co F-~ F-- tJt z

. • • O ti7 h+

w(+ ► + (+ I+ W -J Zn 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

v

• O

w V ~11 N ~

• N -

. • H H

.l 7q

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

Ln W ► + ► + ► + + o ~ ► ~ n t~

( + ~ + 0 0 o c, ( + ( + o ~• [7

O O • • • O o o M~

O O -J CJ

• F-' O N O (D

% N J O W

W r.. .-. `J co ~ CJ

O O Ln w Ci

M F- '

~, ~

.

.t u~ tn w E-' z

.. .

. fD O

..,. .,. y

ro r

~ 0

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 O O O 'Tj

O O(+ O ~+ t O W

• • N !-+ H • M-''

O O Ln N h-j 1-j V1

.~

N W

. pD H

.+ k

W f7

D W ds %C ~

O O

iJl

v V •l

•

V

10 .•~ tr~

F

t+ O d+ CO H ds

.

C

. V1 ~

•

.> 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 fR

O 0

13 '~U t~Y 1~~•

m ~- m n

3 L4 r►'

e, n ~•

~• ~ ~

cl

r O~ N N F~•~ I-~

} tf C h+ ~1 b0

~ ~ r• ~ cri vn •a o z

• • • • in Fi

f~ U! H

tD N ~ ~ ~ I (D

N rn

IN

' fD a w v

T1 ~' n t

0

n

~ 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 riW y ~• o ~• v m

r• w ~7 N

LO

~• v ~ ty

(D C7

F. .

~r ~r d

v co lt •. ,

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~

oN to

f

r~ ; . ... .~ a

'•.' H F-.

(AI N

Fi

Al

Carr . w

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 .

000o 23