No. 447 

Technical Report Series 

NATIONAL TOXICOLOGY PROGRAM 

(CAS NO. 75-05-8) 

STUDIES OF ACETONITRILE 

TOXICOLOGY AND CARCINOGENESIS 

IN F344/N RATS AND B6C3F, MICE 

(INHALATION STUDIES) 

National Institutes of Health 

Public Health Service 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 
FOREWORD 

applied research and to biological assay development and validation. 

relevant programs, staff, and resources from these Public Health Service agencies relating to basic and 

Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the 

Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control. In July 1981, the 

National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National 

the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the 

Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; 

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of 

prevention of disease. 

chemicals. This knowledge is used for protecting the health of the American people and for the primary 

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous 

quality assurance audits before being presented for public review. 

Laboratory Practice Regulations, and all aspects of the chronic studies were subjected to retrospective 

and chronic studies were conducted in compliance with Food and Drug Administration (FDA) Good 

accordance with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic 

exceed all applicable federal, state, and local health and safety regulations. Animal care and use were in 

were conducted in compliance with NTP laboratory health and safety requirements and must meet or 

The studies described in this Technical Report were performed under the direction of the NIEHS and 

studies. Selection per se is not an indicator of a chemical’s carcinogenic potential. 

other species and quantitative risk analyses for humans require wider analyses beyond the purview of these 

this Technical Report are based only on the results of these NTP studies. Extrapolation of these results to 

human exposure, level of production, and chemical structure. The interpretive conclusions presented in 

Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of 

carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). 

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including 

NTP’s World Wide Web site: http://ntp-server.niehs.nih.gov. 

Management. The Abstracts and other study information for 2-year studies are also available on the 

Listings of all published NTP reports and ongoing studies are also available from NTP Central Data 

Management, NIEHS, P.O. Box 12233, MD El-02, Research Triangle Park, NC 27709 (919-541-3419). 

copies of this Technical Report are available without charge while supplies last from NTP Central Data 

U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). Single 

These NTP Technical Reports are available for sale from the National Technical Information Service, 

\ 
TOXICOLOGY AND CARCINOGENESIS 

ON THE 

NTP TECHNICAL REPORT 

STUDIES OF ACETONITRILE 

(CAS NO. 75-05-8) 

IN F344/N RATS AND B6C3Fl MICE 

(INHALATION STUDIES) 

Research Triangle Park, NC 27709 

P.O. Box 12233 

NATIONAL TOXICOLOGY PROGRAM 

April 1996 

NTP TR 447 

NIH Publication No. 96-3363 

National Institutes of Health 

Public Health Service 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 
2 

CONTRIBUTORS 

Acetonitrile, NTP TR 447 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

K.L. Witt, M.S., Oak Ridge Associated Universities 

D.B. Walters, Ph.D. 

G.S. Travlos, D.V.M. 

J.H. Roycroft, Ph.D. 

G.N. Rao, D.V.M., Ph.D. 

H.A. Herbert, D.V.M., Ph.D. 

J.K. Haseman, Ph.D. 

T.J. Goehl, Ph.D. 

M.R. Elwell, D.V.M., Ph.D. 

J.R. Bucher, Ph.D. 

D.A. Bridge, B.S. 

G.A. Boorman, D.V.M., Ph.D. 

C.J. Alden, Ph.D. 

Battelle Pacific Northwest Laboratories 

B.J. Chou, D.V.M., Ph.D., Principal Investigator 

Conducted studies, evaluated pathology findings 

R.B. Westerberg, Ph.D. 

S.E. Rowe, D.V.M., MS. 

H.A. Ragan, D.V.M., Ph.D. 

R.A. Miller, D.V.M., Ph.D. 

Experimental Pathology Laboratories, Inc. 

Dynamac Corporation 

E.T. Gaillard, M.S., D.V.M. 

J.F. Hardisty, D.V.M., Principal Investigator 

Provided pathology quality assurance 

Analytical Sciences, Inc. 

S. Brecher, Ph.D., Principal Investigator 

Prepared quality assurance audits 

Provided statistical analyses 

S. Rosenblum, MS. 

N.G. Mintz, B.S. 

R.W. Morris, MS., Principal Investigator 

NTP Pathology Working Group 

Evaluated slides, prepared pathology report on rats 

J. Cullen, V.M.D., Ph.D. 

M.P. Jokinen, D.V.M., Chair 

(30 June 1992) 

North Carolina State University 

C. David, D.V.M. 

North Carolina State University 

K. Takahashi, D.V.M., MSc., Ph.D. 

R.C. Sills, D.V.M., Ph.D. 

C.C. Shackelford, D.V.M., MS., Ph.D. 

J.R. Hailey, D.V.M. 

E.T. Gaillard, M.S., D.V.M. 

Pathology Associates, Inc. 

National Toxicology Program 

National Toxicology Program 

National Toxicology Program 

National Toxicology Program 

Experimental Pathology Laboratories, Inc. 

Evaluated slides, prepared pathology report on mice 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

K.T. Morgan, B.V.Sc., Ph.D. 

J.R. Hailey, D.V.M. 

E.T. Gaillard, MS., D.V.M. 

D. Dixon, D.V.M., Ph.D. 

M.P. Jokinen, D.V.M., Chair 

(14 July 1992) 

National Toxicology Program 

Chemical Industry Institute of Toxicology 

National Toxicology Program 

Experimental Pathology Laboratories, Inc. 

National Toxicology Program 

Pathology Associates, Inc. 

Biotechnical Services, Inc. 

Prepared Technical Report 

S.R. Gunnels, M.A. 

G. Gordon, M.A. 

N.F. Fisher, M.A., M.P.H. 

J.R. Beverly, B.A. 

D.D. Lambright, Ph.D., Principal Investigator 
3 

CONTENTS 

ABSTRACT ................................................................... 5 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTMTY ............... 9 

................................... 10 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS ........... 11 

INTRODUCTION .............................................................. 13 

MATERIALS AND METHODS .................................................... 21 

RESULTS .................................................................... 31 

DISCUSSION AND CONCLUSIONS ................................................ 55 

REFERENCES ................................................................ 59 

APPENDIX A Summary of Lesions in Male Rats in the 2-Year Inhalation Study 

of Acetonitrile ..................................................... 65 

APPENDIX B Summary of Lesions in Female Rats in the 2-Year Inhalation Study 

of Acetonitrile .................................................... 107 

APPENDIX C Summary of Lesions in Male Mice in the 2-Year Inhalation Study 

of Acetonitrile .................................................... 143 

APPENDIX D Summary of Lesions in Female Mice in the 2-Year Inhalation Study 

of Acetonitrile .................................................... 177 

APPENDIX E Genetic Toxicology ................................................. 219 

APPENDIX F Organ Weights and Organ-Weight-to-Body-Weight Ratios .................... 229 

APPENDIX G Hematology and Thyroid Hormone Assay Results .......................... 237 

APPENDIX H Chemical Characterization and Generation of Chamber Concentrations ......... 241 

APPENDIX I Ingredients. Nutrient Composition. and Contaminant Levels 

in NIH-07 Rat and Mouse Ration ..................................... 259 

APPENDIX J Sentinel Animal Program ............................................ 265 
4 Acetonitrile, NTP TR 447 
5 

ABSTRACT 

CH,CN 

ACETONITRILE 

CAS NO. 75-05-8 

Chemical Formula: C,H,N Molecular Weight: 41.05 

Synonyms: Cyanomethane, ethanenitrile, ethyl nitrile, methanecarbonitrile, methyl cyanide, nitrile of acetic acid 

13 weeks. 

blood of B6C3Fl mice exposed to acetonitrile for 

cultured Chinese hamster ovary cells, and peripheral 

studies were conducted in Salmonella typhimurium, 

tion for 13 weeks or 2 years. Genetic toxicology 

exposed to acetonitrile (at least 99% pure) by inhalaMale and female F344/N rats and B6C3Fl mice were 

cyanides, and because of widespread worker exposure. 

lack of information on the carcinogenicity of alkyl 

drinking water supplies and the environment, due to 

National Cancer Institute due to its presence in 

cal reactions. It was nominated for testing by the 

and agricultural products and as a catalyst in chemitive distillation and crystallization of pharmaceutical 

Acetonitrile is used primarily as a solvent in extracreceived 800 ppm died during the study. At exposure 

rats that received 1,600 ppm and one male that 

per week for 13 weeks. Six male and three female 

acetonitrile by inhalation for 6 hours per day, 5 days 

(equivalent to 0, 168,335,670, 1,340, or 2,681 mg/m3) 

exposed to 0, 100, 200, 400, 800, or 1,600 ppm 

Groups of 10 male and 10 female F344/N rats were 

13-WEEK STUDY IN RATS 

cyanide-induced anoxia. 

rats that died early. These lesions are consistent with 

hemorrhage in the lung and brain were seen in some 

although pulmonary congestion and edema and 

no clear exposure-related histopathologic effects, 

liver weights than those of the controls. There were 

cantly greater absolute and relative heart, kidney, and 

females. Females exposed to 1,600 ppm had signifithe controls in the 800 and 1,600 ppm males and 

thymus weights were significantly lower than those of 

trations in 1,600 ppm females. Absolute and relative 

females, and decreased triiodothyronine (T3) concen1,600 ppm males and females and in 800 ppm 

nonresponsive, normocytic, normochromic anemia in 

convulsions. Clinical pathology findings included 

week 1 were ataxia, abnormal posture, and clonic 

clinical findings in 1,600 ppm males that died during 

males and females receiving 1,600 ppm. Additional 

week of the study in males receiving 800 ppm and 

activity and ruffled fur were observed during the first 

significantly lower than those of the controls. Hypomean body weights of both males and females were 

1,600 ppm, body weight gain was lower and the final 

generally similar to those of the controls. At 

mean body weights and body weight gains were 

concentrations up to and including 800 ppm, the final 
6 Acetonitrile, NTP TR 447 

mice. 

of the adrenal cortex of 800 and 1,600 ppm female 

fatty degenerative change was observed in the X-zone 

and females exposed to 400 or 800 ppm. A lack of 

cytoplasmic vacuolation occurred in the liver of males 

hemosiderin deposition. An increased incidence of 

hyperplasia and ulceration, sometimes asociated with 

raised lesions consisted of areas of focal epithelial 

Histologically, these focal or multifocal pale to dark 

and of females that received 200 ppm and above. 

stomach, of males that received 400 ppm and above 

observed in the stomach, predominantly the foreclearly associated with acetonitrile exposure were 

were greater than that of the controls. Lesions 

weights of females that received 400 ppm and above 

greater than that of the controls and relative liver 

800 ppm females, the absolute liver weight was 

weights were greater in all exposed groups. In 

greater than that of the controls and relative liver 

200 ppm and above, absolute liver weights were 

and a hunched, rigid posture. In males that received 

week in 800 and 1,600 ppm mice were hypoactivity 

controls. Clinical findings observed during the first 

mean body weight was slightly lower than that of the 

mice except the 800 ppm males, for which the final 

similar to those of controls for all surviving groups of 

before the end of the study. Body weight gains were 

four females from the 800 ppm groups also died 

In addition, one 400 ppm female and one male and 

1,600 ppm died during the first 3 weeks of the study. 

per week for 13 weeks. All mice exposed to 

acetonitrile by inhalation for 6 hours per day, 5 days 

(equivalent to 0, 168,335,670, 1,340, or 2,681 mg/m3) 

exposed to 0, 100, 200, 400, 800, or 1,600 ppm 

Groups of 10 male and 10 female B6C3Fl mice were 

13-WEEK STUDY IN MICE 

hematology parameters. 

were evaluated at 15 months for histopathology and 

male and eight female rats from each exposure group 

6 hours per day, 5 days per week for 2 years. Eight 

168, 335, or 670 mg/m3) acetonitrile by inhalation for 

exposed to 0, 100, 200, or 400 ppm (equivalent to 0, 

Groups of up to 56 male and 56 female rats were 

1,600.ppm males and females in the 13-week study. 

were based on reduced survival of 800 ppm males and 

The doses selected for the 2-year study of acetonitrile 

%YEAR STUDY IN RATS 

minor and of no biological significance. 

controls. The hematologic effects observed were 

male and female rats were similar to those of the 

behavior, general health, and appearance of exposed 

Two-year survival, mean body weights, organ weights, 

and Hematology 

Survival, Body Weights, Clinical Findings, 

female rats. 

There were no exposure-related liver lesions in 

controls, suggesting hepatotoxicity of acetonitrile. 

400 ppm males were marginally greater than in 

basophilic, eosinophilic, and mixed cell foci in 

controls (2%-8%). In addition, the incidences of 

(combined) slightly exceeded the range of historical 

dence of hepatocellular adenoma or carcinoma 

the range of historical controls. However, the inciadenoma and hepatocellular carcinoma were within 

(one carcinoma). The incidences of hepatocellular 

in male rats exposed to 400 ppm than in the controls 

adenoma or carcinoma (combined; 5/48) were greater 

hepatocellular carcinoma (3/48), and hepatocellular 

The incidences of hepatocellular adenoma (3/48), 

Pathology Findings 

ated at 15 months for histopathology. 

10 female mice from each exposure group were evaluday, 5 days per week for 2 years. Ten male and 

335 mg/m') acetonitrile by inhalation for 6 hours per ' 

to 0,50, 100, or 200 ppm (equivalent to 0,84, 168, or 

Groups of 60 male and 60 female mice were exposed 

groups of male and female mice in the 13-week study. 

histopathologic lesions in 400, 800, and 1,600 ppm ' 

study were based on reduced survival and gross and 

The exposure concentrations selected for the 2-year 

&YEAR STUDY IN MICE 

were clearly related to acetonitrile exposure. 

controls, and no clinical observations in any group 

male and female mice were similar to those of the 

body weights and organ weights of exposed groups of 

significantly greater than that of the,controls. Mean 

survival of male mice in the 200 ppm group was 

was similar to that of the controls, except that the 

Two-year survival of exposed male and female mice 

Survival, Body Weights, and. Clinical Findings 
Acetonitrile, NTP TR 447 7 

and females. 

lesion was dose related in all exposed groups of males 

females. At 2 years, the increased incidence of this 

significantly increased at 15 months in 200 ppm 

plasia of the epithelium of the forestomach was 

exposure in mice. The incidence of squamous hyperplasms that were considered related to acetonitrile 

There were no increases in the incidences of neoPathology Findings 

GENETIC TOXICOLOGY 

nucleated normochromatic erythrocytes was observed 

the absence, of S9. A significant increase in microtreated with acetonitrile in the presence, but not in 

observed in caltured Chinese hamster ovary cells 

small increase in chromosomal aberrations was 

chromatid exchange test without, but not with, S9. A 

produced a weakly positive response in the sister 

cultured Chinese hamster ovary cells, acetonitrile 

TA1537, with or without S9 metabolic activation. In 

typhimurium strain TA97, TA98, TA100, TA1535, or 

Acetonitrile was not mutagenic in Salmonella 

affected by exposure to acetonitrile. 

micronucleated erythrocytes in female mice was not 

with acetonitrile for 13 weeks; the frequency of 

in peripheral blood samples from male mice treated 

CONCLUSIONS 

200 ppm. 

or female B6C3Fl mice exposed to 50, 100, or 

evidence ofcarcinogenic activity of acetonitrile in male 

rats exposed to 100,200, or 400 ppm. There was no 

carcinogenic activity of acetonitrile in female F344/N 

adenoma and carcinoma. There was no evidence of 

marginally increased incidences of hepatocellular 

activity* of acetonitrile in male F344/N rats based on 

studies, there was equivocal evidence of carcinogenic 

Under the conditions of these 2-year inhalation 

stomach in male and female mice. 

male rats and of squamous hyperplasia of the foreincreased incidences of hepatic basophilic foci in 

Exposure to acetonitrile by inhalation resulted in 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 9. A summary of the Technical Reports Review 

Subcommittee comments and the public discussion on this Technical Report appears on page 11. 
8 Acetonitrile, NTP TR 447 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Acetonitrile 

F344/N Rats 

Male Female Male Female 

F344lN Rats B6C3Fl Mice B6C3F, Mice 

Doses 

335, or 670 mg/m3) 

(equivalent to 0, 168, 

inhalation 

400 ppm by 

0, 100, 200, or 

335, or 670 mg/rn3) 

(equivalent to 0, 168, 

inhalation 

400 ppm by 

0, 100, 200, or 

168, or 335 mg/m3) 

(equivalent to 0, 84, 

inhalation 

200 ppm by 

0, 50, 100, or 

168, or 335 mg/m3) 

(equivalent to 0, 84, 

inhalation 

200 ppm by 

0, 50, 100, or 

Body weights 

similar to controls 

Exposed groups 

similar to controls 

Exposed groups 

similar to controls 

Exposed groups 

similar to controls 

Exposed groups 

rates 

2-Year survival 

17/48 

11/48, 13/47, 9/48, 

43/50 

32\50, 32/50, 32/50, 

29/48 

23/48, 21/48, 26/48, 

32/50 

28/50, 33/50, 29/50, 

effects 

Nonneoplastic 

25/48, 31/48) 

focus (15/48, 22/47, 

Liver: basophilic None 

1 2/5 0) 

(3/49, 3/50, 6/48, 

squamous hyperplasia 

Forestomach: 

19/48) 

(2/49, 7/50, 9/50, 

squamous hyperplasia 

Forestomach: 

Uncertain findings 

Neoplastic effects None None 

None 

None None 

1/47, 1/48, 5/48) 

(combined) (1/48, 

carcinoma 

adenoma or 

hepatocellular 

0/48, 3/48); 

carcinoma (1/48, 0/47, 

hepatocellular 

1/48, 3/48); 

adenoma (0148, 1/47, 

Liver: hepatocellular 

Equivocal evidence 

None None 

activity 

of carcinogenic 

Level of evidence No evidence No evidence No evidence 

Genetic toxicology 

Salmonella typhimurium gene mutations: 

TA1535, and TA1537 

Negative with and without S9 in strains TA97, TA98, TA100, 

Chromosomal aberrations 

Sister chromatid exchanges 

Mouse peripheral blood in vivo: 

Cultured Chinese hamster ovary cells in vitro: 

Cultured Chinese hamster ovary cells in vitro: 

Micronucleated erythrocytes 

Negative in female mice; positive in male mice 

Negative without S9; equivocal with S9 

Weakly positive without S9; negative with S9 
Acetonitrile, NTP TR 447 9 

EXPLANATION OF LEVELS OF EVIDENCE4OF CARCINOGENIC ACTIVITY 

analysis that extends beyond the purview of these studies. 

Thus, the actual determination of risk to humans from chemicals found to be carcinogenic in laboratory animals requires a wider 

available evidence, including animal studies such as those conducted by the NTP, epidemiologic studies, and estimates of exposure. 

such as the International Agency for Research on Cancer, assign a strength of evidence for conclusions based on an examination of all 

the conditions of the study and indicate that exposure to the chemical has the potential for hazard to humans. Other organizations, 

conducted under a limited set of conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under 

neoplasia than control animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are 

evidence for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of 

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the 

potency or mechanism. 

five categories is selected to describe the findings. These categories refer to the strength of the experimental evidence and not to 

evidence of carcinogenic activity. For each separate experiment (male rats, female rats, male mice, female mice), one of the following 

and then revised in March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual weight of 

evaluated because of major flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 

findings (equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be 

observed in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain 

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence 

l Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related 

malignancy. 

increase of benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to 

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked 

that required for clear evidence. 

increased incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than 

l Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related 

limitations, cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity. 

increases in malignant or benign neoplasms. 

increase of neoplasms that may be chemical related. 

l Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative 

l No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related 

* Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal 

borderline between two adjacent levels. These considerations should include: 

current understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the 

actual boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and 

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the 

l adequacy of the experimental design and conduct; l occurrence of common versus uncommon neoplasia; 

l progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; l some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, 

organ or tissue; 

course is to assume that benign neoplasms of those types have the potential to become malignant; 

it is impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent 

e combining benign and malignant tumor incidence known or thought to represent stages of progression in the same 

l supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments 

l metastases; 

l multiplicity in site-specific neoplasia; 

0 latency in tumor induction; 

l in some cases, genetic toxicology. 

l structure-activity correlations; and 

l survival-adjusted analyses and false positive or false negative concerns; 

l concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm; 

l statistical significance of the observed tumor increase; 

l presence or absence of dose relationships; 

(same lesion in another sex or species); 
10 Acetonitrile, NTP TR 447 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS 

company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing NTP studies: 

June 21, 1994, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, 

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on acetonitrile on 

l to ascertain that all relevant literature data have been adequately cited and interpreted, 

l to determine if the design and conditions of the NTP studies were appropriate, 

l to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, 

l to judge the significance of the experimental results by scientific criteria, and 

l to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses. 

Arnold L. Brown, M.D., Chair 

Madison, WI 

University of Wisconsin Medical School 

Paul T. Bailey, Ph.D. 

Princeton, NJ 

Mobil Oil Corporation 

Environmental and Health Sciences Laboratoly 

Meryl H. Karol, Ph.D., Principal Reviewer 

Pittsburgh, PA 

University of Pittsburgh 

Department of Environmental Occupational Health 

Kansas City, KS 

University of Kansas Medical Center 

Department of Pharmacology and Toxicology 

Curtis D. Klaassen, Ph.D., Principal Reviewer 

Claudia S. Miller, M.D. 

San Antonio. TX 

University of Texas Health Sciences Center 

Janardan K. Reddy, M.D. 

Chicago, IL 

Northwestern University Medical School 

Department of Pathology 

Philadelphia, PA 

Fox Chase Cancer Center 

Irma Russo, M.D. 

Louise Ryan, Ph.D, 

Boston, MA 

Dana-Farber Cancer Institute 

Harvard School of Public Health and 

Division of Biostatistics 

Robert E. Taylor, M.D., Ph.D., Principal Reviewer 

Washington, DC 

Howard University College of Medicine 

Department of Pharmacology 

Matthew J. van Zwieten, D.V.M., Ph.D. 

West Point, PA 

Merck Research Laboratories 

Department of Safety Assessment 

Mary Jo Vodicnik, Ph.D. 

Belgium 

Lilly MSG Development Center 

Jerrold M. Ward, D.V.M., Ph.D. 

Frederick, MD 

National Cancer Institute 
Acetonitrile, NTP TR 447 11 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMI'M'EE COMMENTS 

Health Sciences, Research Triangle Park, NC. 

was held at the National Institute of Environmental 

Reports Review Subcommittee. The review meeting 

Program's Board of Scientific Counselors' Technical 

received public review by the National Toxicology 

toxicology and carcinogenesis studies of acetonitrile 

On June 21, 1994, the draft Technical Report on the 

carcinogenic activity in male or female B6C3Fl mice. 

activity in female F344/N rats, and no evidence of 

in male F344/N rats, no evidence of carcinogenic 

studies were equivocal evidence of carcinogenic activity 

female mice. The proposed conclusions for the 

male rats and nonneoplastic lesions in male and 

on possible chemical-related neoplastic lesions in 

on survival and body weight effects, and commenting 

study, describing the experimental design, reporting 

cussing the uses of the chemical and rationale for 

and carcinogenesis studies of acetonitrile by disDr. J.R. Bucher, NIEHS, introduced the toxicology 

the levels of acetonitrile in cigarette smoke. 

literature that could be cited with data quantifying 

contains acetonitrile and wondered if there was 

Dr. Taylor noted the statement that tobacco smoke 

and mixed cell foci, and Dr. Bucher agreed. 

based upon the findings of basophilic, eosinophilic, 

in male rats there might be some hepatotoxic effects 

be added to the conclusions that in the 2-year studies 

proposed conclusions. He suggested that a sentence 

Dr. Taylor, a principal reviewer, agreed with the 

perhaps 800 ppm. 

2-year studies hould have been higher in rats, 

highest exposure concentration of acetonitrile in the 

with the proposed conclusions. He thought the 

Dr. Klaassen, the second principal reviewer, agreed 

best conclusion. 

studies, equivocal evidence was considered .to be the 

neoplasms in the control groups in some inhalation 

preneoplastic lesions or atypical foci, and up to four 

lack of a strong dose-related response, no increase in 

could be made for some evidence, but based on the 

ship. Dr. Bucher acknowledged that an argument 

relevant and likely would support a causal relationhistorical data from inhalation studies would be more 

studies. She said that concurrent controls and the 

showing a 10% incidence of liver neoplasms in feed 

that appeared to be based on historical control data 

acetonitrile exposure and liver neoplasms in male rats 

mented on the "uncertain" association between 

that is the cause of death. Dr. Karol further comthere is good evidence for a pharmacologic action 

lethal dose determined in the 13-week studies unless 

concentration slightly greater than a quarter of the 

lethality, the aim is to set the highest exposure 

that when setting exposure concentrations based on 

should be added. Dr. Bucher disagreed and explained 

sure concentration for the 2-year studies, a statement 

rationale for choosing 400 ppm as the highest expoobserved in rats exposed to 800 ppm were part of the 

Dr. Karol said if gross and histopathological changes 

studies based on survival in the 13-week studies. 

appropriate exposure concentration in the 2-year rat 

Dr. Klaassen that 800 ppm would have been an 

the proposed conclusions. She concurred with 

Dr. Karol, the third principal reviewer, agreed with 

11 votes. 

motion, which was accepted unanimously with 

carcinogenic activity. Dr. Klaassen seconded the 

female rats and male and female mice, no evidence of 

equivocal evidence of carcinogenic activity, and for 

and with the conclusions as written for male rats, 

acetonitrile be accepted with the revisions discussed 

Dr. Taylor moved that the Technical Report on 
12 Acetonitrile, NTP TR 447 
13 

INTRODUCTION 

CH,CN 

ACETONITRILE 

CAS NO. 75-05-8 

Chemical Formula: qH,N Molecular Weight: 41.05 

synonym: Cyanomethane, ethanenitrile, ethyl nitrile, methanecarbonitrile, methyl cyanide, nitrile of acetic acid 

(Reynolds and Prasad, 1982). 

sition or when reacted with acids or oxidizing agents 

produces hydrogen cyanide when heated to decompoToxicology, 1982; Merck Index, 1989). Acetonitrile 

(petroleum fractions) (Putty's Industrid Hygiene and 

It is immiscible with many saturated hydrocarbons 

methyl acetate, and many unsaturated hydrocarbons. 

acetamide solutions, ethylene chloride, methanol, 

carbon tetrachloride, ethanol, ether, ethyl acetate, 

readily miscible with water, acetone, chloroform, 

74.0 mm at 20" C, and a vapor density of 1.42. It is 

760 mm, a density of 0.78745, a vapor pressure of 

a sweet, ether-like odor, a boiling point of S1:6O C at 

Acetonitrile is a volatile, clear, colorless liquid with 

CHEMICAL AND PHYSICAL PROPERTIES 

temperatures in the presence of a catalyst (e.g., Ag, 

the reaction of ethanol and ammonia at moderate 

ammonia mixture, acetamide, or ammonium acetate; 

methods involve dehydration of an acetic acid and 

between propylene and ammonia. Other production 

involving a high-temperature catalytic reaction 

commercial synthesis of acrylonitrile by a process 

Acetonitrile is produced as a minor by-product in the 

AND HUMAN EXPOSURE 

PRODUCTION, USE, 

epoxides, and paraffins or olefins (WHO, 1993). 

chloride with methane, ketones, ethanol, alkylene 

Cu, MOO, or ZnS); or the reaction of cyanogen 

1990). 

for DNA synthesis and peptide sequencing (Borman, 

performance liquid chromatography and as a solvent 

mon laboratory solvent, and is widely used in highused for removing artificial fingernails. It is a commolding plastics. It is also a component of products 

as a solvent for spinning fibers and for casting and 

superior solvency with polymers, acetonitrile is used 

products uch as crude wood resin. Due to its 

industry ; and for isolating components from crude 

mixtures and C,-hydrocarbons in the petrochemical 

(Merck Index, 1976); for separating olefin-diolefin 

insecticides, plant growth regulators, and fungicides 

products, such as vitamins, steroids, bactericides, 

crystallization of pharmaceutical and agricultural 

1993). It is used for extractive distillation and 

fatty acids and animal and vegetable oils (WHO, 

dielectric constant and is used primarily to extract 

Acetonitrile is a highly polar solvent with a high 

potentially exposed to acetonitrile for more than 

Revelise, 1952); United States workers have been 

amounts in the United States since 1950 (Gergel and 

Acetonitrile has been available in commercial 
14 Acetonitrile, NTP TR 447 

tion may also be exposed (NIOSH, 1979). 

noncaptive uses of acetonitrile, the general populaHautala, 1983). In addition, because of the many 

excess of 100 ppm in air is possible (Amoore and 

air, and exposure of workers to concentrations in 

nitrile will allow concentrations of about 170 ppm in 

exposure potential, At 25" C, the volatility of acetorelatively high air odor threshold influence worker 

is used in open systems where its high volatility and 

However, for many industrial processes, acetonitrile 

during production could only occur accidentally. 

closed system; therefore, occupational exposure 

Synthesis of acetonitrile is usually carried out in a 

in the United States was 70 mg/m3 (40 ppm). 

exposure limit for acetonitrile during an 8-hour shift 

female (NIOSH, 1994). The 1991 occupational 

greater than 31,000 workers, 25% of whom were 

potential human exposure in 1991 estimated at 

10.2 million kg (USITC, 1993), with the extent of 

nitrile in the United States during 1991 was 

40 years. The reported annual production of aceto- 

(Woolley, 1972). 

thermal decomposition of flexible polyurethane foams 

literature. Acetonitrile vapor is also released by the 

tions in cigarette smoke were not found in the 

et al., 1962). Estimates of the acetonitrile concentra- 

(117.6 pg/L) than in nonsmokers (2.9 pa) (McKee 

level of acetonitrile was 40 times higher in smokers 

40 smokers and 20 nonsmokers. The average urinary 

smoke has been confirmed by urinalysis in 

smoke contains acetonitrile, and its absorption from 

exposed to moderate levels of acetonitrile. Tobacco 

Smokers have been identified as a group chronically 

ENVIRONMENTAL IMPACT 

wastewater sludge (Ludzack et al., 1961). 

effective means of removing the compound from 

is limited or absent and does not appear to be an 

rate of aerobic degradation. Anaerobic degradation 

able to degrade these compounds, thus increasing the 

these bacteria to acetonitrile selects strains that are 

sludge, natural waters, and soil; regular exposure of 

several strains of bacteria common in wastewater 

(Lyman et al., 1982). It is readily biodegraded by 

Acetonitrile volatilizes from water and soil surfaces 

Japan reported acetonitrile concentrations between 

Although .the Environmental Survey of Chemicals in 

showing contamination of food by acetonitrile. 

knowledge, no report was found in the literature 

detected in 72 water samples (OHS, 1990). To our 

sampled in all 47 prefectures, acetonitrile was not 

0.02 and 0.54 mgkg in 11 out of 60 aquatic sediments 

2 weeks (WHO, 1993). 

biodegradation studies to be -approximately 1 to 

25" C has been estimated based on volatility and 

The half-life of acetonitrile in natural waters at 20" to 

cant factors for removal of acetonitrile from water. 

and microbial degradation appear to .be the signifiin the aquatic environment or in air. Volatilization 

and photodegradation is not expected to occur either 

Hydrolysis of acetonitrile in water is extremely slow, 

(Nishiuchi, 1981). 

sensitive species (48-hour LC,, of 730 ma) 

common carp (Cyprinus carpio) being the most 

invertebrates are more than 700 ma, with the 

Kuhn, 1980). The LC,, for freshwater fish and 

with thresholds at 520 mg/L or more (Bringmann and 

(bacteria, cyanobacteria, green algae and protozoans) 

Acetonitrile has low toxicity to microorganisms 

1985). 

occur, with subsequent removal by rainfall (USEPA, 

that dissolution into clouds and rain droplets may 

The complete water solubility of acetonitrile suggests 

air, which reduces the half-life to less than 20 days. 

is 10 times faster than normal in moderately polluted 

mately 42 days. However, the hydroxyl reaction rate 

atmospheric half-life in air is estimated to be approxireaction with singlet oxygen (USEPA, 1985). The 

radicals; reaction with ozone is slow, as is the 

from the troposphere is reaction with hydroxyl 

The major mechanism for removal of acetonitrile 

(Becker and Ionescu, 1982). 

anthropogenic source of atmospheric acetonitrile 

straw, and vegetation appears to be -the only nonfrom 4.0 to 34.9 ppb. The combustion of .wood, 

showed a 9-fold increase in acetonitrile concentration 

after burning of bush and grass by farm workers 

tored for acetonitrile in a rural area both'before and 

the city of Wuppertal, Germany. Air samples moniconcentrations of 7.4 rf: 2.4 ppb were reported for 

values reported in urban areas than in rural areas; air 

11,960 pLg/m3 (2 to 7 ppb by volume), with higher 

contain acetonitrile at concentrations of 3,360 to 

Air monitored close to the.ground has been shown to 
Introduction 15 

ABSORPTION, DISTRIBUTION, 

Experimental Animals 

METABOLISM, AND EXCRETION toxicity may be due to the parent acetonitrile moleresults of this study suggest that acetonitrile neurothe methyl group, which cannot penetrate the blood- Although no quantitative analytical data are available, brain barrier (Ahmed et 1992). There are no 

. cule rather than to any of its metabolites containing 

in systemic toxicity. Most of the systemic toxic effects 

gastrointestinal tract and through the skin, resulting nitrile result in its accumulation in animal tissues. 

acetonitrile is readily absorbed from the lungs and indications that repeated administrations of acetoSmith, 1981). 

of this species to acetonitrile toxicity (Willhite and 

the mouse appears to account for the high sensitivity 

nitrile; the rapid rate at which cyanide is produced in 

the relative speed of cyanide formation from acetovariations in toxic response are probably related to 

cyanate excretion is more efficient. Interspecies 

slowly and, consequently, detoxification via thionitriles, because it is converted to cyanide more 

dependent toxicity of acetonitrile is less than other 

was higher. These data indicate that the cyanidethan that for other nitriles, even when the initial dose 

excreted in the urine as thiocyanate was also lower 

Hayes, 1985a). The percentage of acetonitrile 

other nitriles or potassium cyanide (Freeman and 

levels 1 hour after dosing with similar amounts of 

nitrile dosing and were comparable to blood cyanide 

blood cyanide were achieved 7.5 hours after aceto- 

(Ahmed and Farooqui, 1982). Peak concentrations of 

proceeds at a slower rate than that of other nitriles 

1959a,b). The conversion of acetonitrile to cyanide 

nitrile via inhalation or injection (Pozzani et al., 

cyanate in the urine following exposure to acetohave documented cyanide in the blood and thioExperimental studies with rats, monkeys, and dogs 

eliminated in the urine (Freeman and Hayes, 1987). 

conjugated with thiosulfate to form thiocyanate and 

through metabolism to cyanide, which is subsequently 

of acetonitrile and other nitriles are mediated 

organs and, to a lesser extent, in the brain. The 

nitrile was demonstrated in the male reproductive 

delayed accumulation and retention of 2-['4C]-acetoretained in the liver and gastrointestinal tract, and 

dosing. At 24 and 48 hours, radioactivity was still 

tract, gallbladder, and urinary bladder 5 minutes after 

its metabolites in the liver, kidney, gastrointestinal 

demonstrated heavy localization of acetonitrile and 

body autoradiography with 2-['4C]-acetonitrile in mice 

tion exposure (Haguenoer et al., 1975a,b). Whole 

organs following intraperitoneal injection or inhalaacetonitrile, were detected in various tissues and 

conjugated hydrogen cyanide, as well as unchanged 

In pharmacokinetic studies in male rats, free and 

exposure levels. 

important pathway of elimination, especially at high 

unchanged acetonitrile via exhalation is also an 

(Haguenoer et al., 1975a,b). Pulmonary clearance of 

thiocyanate excretion occurred for up to 11 days 

up to 4 days following intraperitoneal administration; 

small amounts were recovered in the urine of rats for 

is greatest during the first 24 hours after dosing, but 

free and bound hydrogen cyanide. Urinary excretion 

urinary excretion of the unchanged compound and 

Elimination of acetonitrile occurs primarily through 

Hayes, 1985b). 

dependent and inactivated by heat (Freeman and 

acetonitrile to cyanide was found to be NADPH- 

(Koop and Casazza, 1985), microsomal metabolism of 

acetone, an inducer of cytochrome P450 isozyme 2E1 

liver microsomes isolated from rats pretreated with 

hydrogen cyanide and formaldehyde. In a study with 

hydrin, which then spontaneously decomposes to 

a cytochrome P450 monooxygenase system to cyanoand Hayes, 1987). Acetonitrile is biotransformed via 

Willhite, 1983; Tanii and Hashimoto, 1984; Freeman 

of in vitro preparations (Ohkawa et al., 1972; 

thiocyanate have also been demonstrated in a variety 

Biotransformation of acetonitrile to cyanide and 

poisoning during an industrial exposure that resulted 

a clinical study of 15 cases of accidental acetonitrile 

that acetonitrile is well absorbed by both routes. In 

humans; however, human poisoning cases indicate 

experimental studies of oral or dermal absorption in 

the mouth (Dalhamn et al., 1968b). There are no 

significant portion of this could have been retained in 

rette smoke was retained (Dalhamn et al., 1968a); a 

that 91% 2 4% of the acetonitrile inhaled in cigaacetonitrile in humans, studies on smokers showed 

there is little information on absorption of inhaled 

the skin (Fassett, 1963; Losek et al., 1991). Although 

occur via ingestion, inhalation, or absorption through 

rapidly distributed through the body. Exposure can 

Acetonitrile is readily absorbed by all routes and 

Humans 
16 Acetonitrile, NTP TR 447 

patient prior to death (Michaelis et al., 1991). 

were calculated during the hospitalization of the 

32 hours for acetonitrile and 15 hours for cyanide 

acetonitrile ingestion, elimination half-lives of 

of thiocyanate in the serum. In a case of suicidal oral 

ence of cyanide in the blood, urine, and tissues, and 

in one fatality, Amdur (1959) documented the presTOXICITY 

after oral administration in other animal species. 

nitrile) are similar to or lower than those obtained 

applications in rabbits (1.25 mL/kg undiluted acetoCarpenter, 1948). The LD,, values for dermal 

tion exposure in rats is 8,000 ppm (Smyth and 

The lowest published LC,, for a single, 4-hour inhalasensitive to exposure to acetonitrile by inhalation. 

exposure in rats. Mice appear to be the species most 

tion exposure or 12,000 ppm for an 8-hour inhalation 

exposure in mice to 16,000 ppm for a 4-hour inhalation exposure or 2,300 ppm for a 2-hour inhalation 

from approximately 2,700 ppm for a 1-hour inhalain acute inhalation studies with acetonitrile range 

et al., 1959a,b; Kimura et al., 1971). The LC,, values 

administration (Smyth and Carpenter, 1948; Pozzani 

weight) depending on the species and route of 

acetonitrile vary widely (175 to 5,620 mgkg body 

gavage, or dermal application. The LD,, values for 

mals following administration by inhalation, injection, 

Acetonitrile toxicity has been demonstrated in aniExperimental Animals 

tachypnea, gasping, tremors, convulsions, and corneal 

of 500 to 5,000 ppm by inhalation displayed dyspnea, 

et al., 1975b). CD-1 mice exposed to concentrations 

tion, and paralysis of the extremities (Haguenoer 

5 days had difficulty breathing, impaired renal funcacetonitrile by inhalation for 2 hours per day for 

(Verbrugge, 1899). Rats exposed to 2,800 ppm 

and the death of two out of seven animals 

in rabbits, followed by immobilization, convulsions, 

occurred 1 to 3 hours after a subcutaneous injection 

and seizures. Rapid and irregular respiration 

respiratory symptoms, often followed by prostration 

different routes of administration always exhibit 

Smith, 1981). Animals exposed to acetonitrile via 

cyanide or other nitrilcs (Willhite, 1981; Willhite and 

tinguishable from those observed after exposure to 

are similar in different animal species and indisSigns and symptoms of acute acetonitrile intoxication 

99-day inhalation period (Pozzani et al., 1959a). 

reflexes and hyperexcitability toward the end of the I 

was exposed to 330 ppm and showed overextension 

day 23 and the other on day 51. The last monkey 

ordinated from the second week; one monkey died on 

Two monkeys exposed to 660 ppm appeared uncothe second day; death occurred a few hours later. 

followed by prostration and labored breathing during 

day of inhalation, but showed poor coordination 

2,510 ppm acetonitrile appeared normal after the first 

four Rhesus monkeys, one monkey exposed to 

2 hours (Willhite, 1981). In an inhalation study in 

exposed to 5,000 ppm for 60 minutes died within 

60-minute exposure was 2,693 ppm. All mice 

opacity within 30 to 300 minutes; the LC,, for a 

sperm motility (Morrissey et al., 1988). 

had no effect on body weight, testicular weight, or 

or 400 ppm acetonitrile by inhalation for 13 weeks 

sure of F344 male rats and B6C3Fl mice to 100,200, 

3,104 and 10,485 mdm’ (Roloff et al., 1985). Exporespiratory and/or ocular iritation occurred at 

observed at the highest exposure level, and 

for 1 month, lower body weight and death were 

acetonitrile vapor 6 hours per day, 5 days per week 

In rats exposed to 1,038, 3,104, or 10,485 mdm’ 

(Pozzani et al., 1959a). 

sinuses were found at necropsy in all three monkeys 

Hemorrhages of the superior and inferior sagittal 

7 hours per day, 5 days per week for 91 days. 

were exposed to 350 ppm acetonitrile by inhalation 

hepatic injury (Drew et al., 1978). Three monkeys 

ornithine carbamyl transferase), indicating acute 

transaminase, glutamic pyruvic transaminase, and 

increased serum enzymes (glutamic oxaloacetic 

3,000 pp‘tn acetonitrile for 4 hours and had markedly 

inhalation study, CD-1 rats were exposed to

brain that were characteristic of anoxia. In another 

study were sporadic, focal hemorrhagic lesions in the 

lung (Pozzani et al., 1959a). Also observed in this 

edema with or without bronchial inflammation in the 

liver and alveolar capillary congestion and/or focal 

at 665 ppm were cloudy swelling of the kidney and 

for 7 hours per day, 5 days per week, lesions observed 

acetonitrile concentrations of 166, 330, or 665 ppm 

90-day inhalation study in which rats were exposed to 

and congestion (Haguenoer et al., 1975b). In a 

acetonitrile inhalation exposure showed hemorrhage 

Histopathologic examination of rat lungs after 
Introduction 17 

(Pozzani et af., 1959a). 

blood cyanide or urinary thiocyanate occurred 

later, despite the fact that no significant changes in 

exposure, and a feeling of bronchial tightness 5 hours 

flushing of the face in one subject after 2 hours of 

two subjects to 160 ppm for 4 hours caused a slight 

not increased. Nine days later, exposure of the 

not detected, and urinary thiocyanate excretion was 

4 hours with no adverse effects. Blood cyanide was 

later, two of the subjects were exposed to 80 ppm for 

no cyanide was detected in the blood. One week 

cyanate level was observed in one subject, although 

olfactory fatigue. A slightly elevated urinary thio3 hours of exposure, and then experienced some 

detected the odor of acetonitrile for the first 2 to 

ness of the chest a few hours later. All three subjects 

period, although one subject reported a slight tightproduced no adverse effects during the exposure 

teers, exposure to 40 ppm of acetonitrile for 4 hours 

air. In an inhalation study in three human volunbut are probably in excess of 840 mg/m’ (500 ppm) in 

The levels causing toxicity in humans are unknown, 

Humans 

1974). 

and he died 6 days after the exposure (DeQuidt et aZ., 

and acetonitrile were found in his blood and urine 

convulsions. Large amounts of cyanide, thiocyanate, 

The following day he became comatose and had 

epigastric pain and nausea and vomited repeatedly. 

Four hours after leaving work he complained of 

nitrile and boiling water on the floor to clean it. 

occurred when a laboratory worker poured acetoAnother fatal case of acute acetonitrile poisoning 

convulsions, coma, and death (Amdur, 1959). 

headache, extreme weakness, semiconsciousness, 

tension, ausea, emesis, respiratory depression, 

chest pain, tightness in the chest, tachycardia, hypoplant included irritation of the nose, throat, and skin, 

the accidental poisoning of 15 workers at a chemical 

Symptoms of acute acetonitrile intoxication following 

of the patient after vomiting, convulsions; coma, 

resulted in death (Michaelis et aZ., 1991) or recovery 

1988). Cases of suicidal ingestion of acetonitrile have 

the death of another child (Caravati and Litovitz, 

ingestion of artificial fingernail remover resulted in 

emesis, hypotension, and tachycardia. Accidental 

have been reported in children, resulting in nausea, 

acetonitrile contained in artificial fingernail remover 

Accidental dermal and inhalation exposures to 

et aZ., 1991; Turchen et af., 1991). 

reported following acetonitrile ingestion (Geller 

sodium nitrate and sodium thiosulfate has been 

In other cases, successful treatment of patients with 

and stomach contents (1.3 g/L) (Jones et af., 1992). 

was detected in the blood (0.8 g/L), urine (1.0 g/L), 

two fatal cases of accidental ingestion, acetonitrile 

acidosis, and cardiac arrest (Jaeger et af., 1977). In 

acute respiratory insufficiency, severe metabolic 

mPRODUCTIVE AND 

(Willhite, 1983). 

genic effects than when administered intraperitoneally 

dose given by gavage elicited greater toxic and teratobody weight compared to controls. Thus, the same 

caused a significant increase only in the average fetal 

single intraperitoneal injections of 200 to 400 mgikg 

number of malformed fetuses or resorptions, while 

400 mgikg produced a significant increase in the 

demonstrated that single gavage doses of 300 to 

gestation. Additional studies in pregnant hamsters 

8,000 ppm acetonitrile by inhalation on day 8 of 

fetuses in Syrian golden hamsters exposed to 5,000 or 

dose-dependent increase in the number of abnormal 

recent studies have demonstrated a significant and 

embryo system (Levene, 1961). However, more 

Acetonitrile had no teratogenic potential in a chick 

Experimental Animals 

DEVELOPMENTAL TOXICITY 

(WHO, 1993). 

fetuses at doses below those causing maternal toxicity 

sequently, acetonitrile is not considered to be toxic to 

was observed only at the 30 m@g dose level. Congain was also reduced at 15 m@g. Embryotoxicity 

25 animals receiving 30 mg/kg died. Body weight 

showed decreased body weight gain, and five out of 

acetonitrile orally on gestation days 6 through 18 

Pregnant rabbits administered 2, 15, or 30 m@g 

litters at 300 and 500 mgikg (Smith et al., 1987). 

500 mgikg, as well as fewer dams delivering viable 

strated maternal body weight reduction and death at 

by gavage on gestation days 7 through 21 demonA similar study in Long-Evans rats given acetonitrile 

and post-implantation losses (Berteau et aZ., 1982). 

effects which included increases in early resorptions 

body weight reduction and death and embryotoxic 

through 15, a dose of 275 mgikg caused maternal 

received acetonitrile by gavage on gestation days 6 

In a study with pregnant Charles River rats that 
18 Acetonitrile, NTP TR 447 

(Klaassen et al., 1986). 

be responsible for the detoxification of cyanide 

due to induction of rhodanese, the enzyme thought to 

in the maternal blood cyanide level may have been 

tially constant during the same period. The decrease 

while the acetonitrile concentration remained essention day 8 to approximately 0.8 mg/mL on day 18, 

rats declined from approximately 2 mg/mL on gestaconcentration of cyanide in the blood of 1,200 ppm 

each time point, but it could not be quantified). The 

was also detected in the blood of one 400 ppm rat at 

cyanide in the blood of the 1,200 ppm group (cyanide 

blood of all exposed groups and the presence of 

exposure-related concentrations of acetonitrile in the 

400 ppm. Analysis results indicated significant, 

at the 1,200 ppm concentration, and one occurred at 

fetal malformations, a few maternal deaths occurred 

body weight, reproductive indices, or the incidence of 

significant exposure-related effects upon maternal 

through 19 (NTP, 1994a). Despite the absence of 

1,200 ppm for 6 hours per day on gestation days 6 

acetonitrile at concentrations of 100, 400, or 

inhalation study in Sprague-Dawley rats exposed to 

This conclusion is supported by a more recent 

reported in the literature. 

opmental toxicity of acetonitrile in humans has been 

No information related to the reproductive or develHumans 

to acetonitrile (Mirsalis et al., 1983). 

observed in rat hepatocytes exposed in vivo or in vitro 

induction of unscheduled DNA synthesis was 

judged to be equivocal (Galloway et al., 1987). No 

soma1 aberrations with S9; these responses were 

be critically evaluated. 

the "induced" levels, and the data, therefore, cannot 

control micronucleus frequencies for comparison to 

1988). However, this particular study did not include 

to male and female NMRI mice (Schlegelmilch et al., 

acetonitrile administered by intraperitoneal injection 

reported in a bone marrow micronucleus test with 

1989). In addition, weakly positive results were 

this effect (Zimmerman et al., 1985; Whittaker et al., 

mately 5%) of acetonitrile were required to produce 

although relatively high concentrations (approxition, in a diploid strain of Saccharomyces cerevisiae, 

aneuploidy, but not point mutations or recombinaAcetonitrile was also found to be a potent inducer of 

either as larvae or as adults (Osgood et al., 1991a,b). 

melanogaster fed an aqueous solution of the chemical 

mosome gain) in oocytes of female Drosophila 

somal aneuploidy (both chromosome loss and chroaneuploidy events. Acetonitrile induced sex chromoreported in assays that measure the induction of 

described previously, positive results have been 

were obtained with acetonitrile inthe assays 

In contrast to the essentially negative results that 

CARCINOGENICITY 

mental animals or humans have been reported in the reported in assays designed to detect chromosomal 

No carcinogenicity studies of acetonitrile in experi- cultured mammalian cells, but positive results were 

mutation assays conducted either in bacteria or 

In conclusion, acetonitrile was not active in gene 

literature. aberrations. 

GENETIC TOXICITY 

sister chromatid exchanges without S9 and chromoovary cells, acetonitrile induced slight increases in 

In cytogenetic tests with cultured Chinese hamster 

with and without S9 metabolic activation enzymes. 

Systems Corp., 1984). These tests were conducted 

or in cultured Chinese hamster ovary cells (Bioassay 

in L5178Y mouse lymphoma cells (Rudd et al., 1983), 

Mortelmans et al., 1986; Schlegelmilch et al., 1988), 

typhimurium (Florin et al., 1980; Maron et al., 1981; 

Acetonitrile did not induce mutations in Salmonella 

(Osgood et al., 1991a,b). 

female Drosophila exposed via inhalation or feeding 

1990; Zimmermann et al., 1989) and in oocytes of 

Saccharomyces cerevisiae (Whittaker et al., 1989, 

et al., 1988), but there was induction of aneuploidy in 

no mutagenicity in Salmonella was observed (Zeiger 

same pattern of activity demonstrated by acetonitrile; 

from the acetonitrile analogue propionitrile show the 

TAlOO (Kushi et al., 1983). Mutagenicity test data 

for S9 activation) in Salmonella typhimurium strain 

found to be a direct-acting mutagen (no requirement 

A metabolite of acetonitrile, hydrogen cyanide, was 
Introduction 19 

STUDY RATIONALE 

it occurs as a byproduct in the synthesis of acrylo- exposure to acetonitrile. 

turing processes, its disposal by manufacturers when of exposure to mimic the principal means of human 

for study because of its widespread use in manufac- . noncaptive uses. Inhalation was chosen as the route 

The National Cancer Institute nominated acetonitrile as exposure of the general population due to its many 

nitrile, its high potential for worker exposure, as well 
20 Acetonitrile, NTP TR 447 
21 

MATERIALS AND METHODS 

in Appendix H. 

The methods and results of these studies are detailed 

Institute of Environmental Health Sciences (NIEHS). 

the acetonitrile studies are on file at the National 

MO). Reports on analyses performed in support of 

laboratory, Midwest Research Institute (Kansas City, 

analyses were conducted by the analytical chemistry 

of the 2-year studies. Identity, purity, and stability 

Corporation, Cleveland, OH) were used for a portion 

Baker, Phillipsburg, N.J.) and VO41381 (Vistron 

majority of the 2-year studies. Lots BO82889 (J.T. 

used throughout the 13-week studies and for the 

E.I. DuPont de Nemours (Wilmington, DE) and was 

B082889, and VO41381). Lot 2485 was obtained from 

Acetonitrile was obtained in three lots (2485, 

CHARACTERIZATION OF ACETONITRILE 

PROCUREMENT AND 

purity for all lots was determined to be at least 99%. 

than 0.1% relative to the major peak. The overall 

one major peak and no impurities with areas greater 

gas chromatography using a second system indicated 

with an area of 0.14% relative to the major peak, and 

system indicated one major peak and one impurity 

For lot VO41381, gas chromatography using one 

relative to the major peak for lots 2485 and B082889. 

and no impurities with areas greater than 0.1% 

raphy using two systems indicated one major peak 

indicated less than 35 ppm free acid. Gas chromatogindicated less than 0.2% for all lots. Titration 

values for acetonitrile. Karl Fischer water analysis 

hydrogen, and nitrogen agreed with the theoretical 

chromatography. Elemental analyses for carbon, 

Fischer water analysis, free acid titration, and gas 

all lots was determined by elemental analyses, Karl 

nuclear magnetic resonance spectroscopy. Purity of 

as acetonitrile by infrared, ultraviolet/visible, and 

The chemical, a clear, colorless liquid, was identified 

2 weeks when stored in tightly sealed containers 

nitrile was stable as a bulk chemical for at least 

chromatography. These studies indicated that acetoby the analytical chemistry laboratory using gas 

Stability studies of the bulk chemical were performed 

approximately 22" C in the original containers. 

ensure stability, the bulk chemical was stored at 

protected from light at temperatures up to 60" C. To 

detected. 

titration. No degradation of the bulk chemical was 

bulk chemical using gas chromatography and free acid 

The study laboratory monitored the stability of the 

Appendix H. 

vapor generation and monitoring are given in 

Figures H6 through H11. Detailed descriptions of 

bers during the 2-year studies are presented in 

monthly mean exposure concentrations in the chamthroughout he 13-week and 2-year studies. The 

Chamber atmosphere uniformity was maintained 

standards using an off-line gas chromatograph. 

ples were analyzed against gravimetrically prepared 

brated critical-orifice sampling system. These samformamide-filled, fritted-glass bubblers and a calisamples from each exposure chamber using dimethylCalibration was accomplished by acquiring rab 

studies) or 12-port (2-year studies) sampling valve. 

using an automated, multiplexed, 8-port (13-week 

monitor was coupled with the inhalation chambers 

single on-line HP-5840 gas chromatograph. The 

Chamber concentrations were monitored with a 

OF CHAMBER CONCENTRATIONS 

GENERATION AND MONITORING 

studies. 

12 minutes was used in the 13-week and 2-year 

and 14 to 17 minutes in the 2-year studies. A T, of 

ranged from 12 to 14 minutes in the 13-week studies 

tration after vapor generation was terminated (Tlo) 

concentration to decay to 10% of the target concen2-year studies. The time for the chamber 

the 13-week studies and 10 to 15 minutes in the 

tion (T,) was determined to be 15 to 17 minutes in 

concentrations after the start of vapor generathe chambers. The time to achieve 90% of target 

were determined with and without animals present in 

Buildup and decay rates for chamber concentrations 
22 Acetonitrile, NTP TR 447 

line as a result of test chemical generation. 

occurred in the chambers or in the vapor distribution 

analyses indicated that no test chemical degradation 

in the vapor distribution line. The results of these 

2-year studies in the 50 and 400 ppm chambers and 

the 100 and 1,600 ppm chambers, and during the 

tion were conducted during the 13-week studies in 

and 2-year studies. Studies of acetonitrile degradaexposure chambers was confirmed during the 13-week 

Uniformity of vapor concentration in the inhalation 

be used in the 2-year studies. 

and to determine the appropriate concentrations to 

tive toxic effects of repeated exposure to acetonitrile, 

These studies were conducted toevaluate the cumula13-WEEK STUDIES 

Sentinel Animal Program (Appendix J). 

on sentinel animals using the protocols of the NTP 

end of the studies, serologic analyses were performed 

3 weeks after their arrival for viral screening. At the 

additional five male and five female rats and mice 

disease. Serology samples were collected from an 

for parasite evaluation and gross observation of 

and five female rats and mice were randomly selected 

first day of exposure. Prior to study start, five male 

14 days and were approximately 6 weeks old on the 

4 weeks old. Animals were quarantined for 12 to 

Upon receipt, the rats and mice were approximately 

obtained from Simonsen Laboratories (Gilroy, CA). 

Male and female F344/N rats and B6C3Fl mice were 

Table 1. 

design and animal maintenance are summarized in 

and at the end of the. studies. Details of the study 

The animals were weighed initially, weekly thereafter, 

ally. Clinical examinations were recorded weekly. 

ad libitum. Both rats and mice were housed individuduring exposure periods, and water was available 

holidays). Feed was available ad libitum; except 

5 days per week for 13 weeks (excluding two. 

exposed for 6 hours plus Tw (12 minutes) per day, 

335, 670, 1,340, or 2,681 mg/m3). The animals were 

200, 400, 800, or 1,600 ppm (equivalent to 0, 168, 

exposed to acetonitrile at concentrations of 0, 100, 

Groups of 10 male and 10 female rats and mice were 

the 13-week studies. The rats were anesthetized with 

and 10- female rats per exposure group at the end of 

Clinica1,pathology studies were performed on 10 male 

evaluated are listed in Table 1. 

The hematology and thyroid hormone parameters 

tion was measured using a double-antibody technique. 

were used for the T, and T, assays. TSH concentramercial reagent kits (NML Organon Teknika Corp.). 

Downers Grove, IL). Tri-Tab and Tetra-Tab comspectrometer (Packard Instrument Company, 

methods using a Packard Auto-Gamma scintillation 

trations were determined by radioimmunoassay 

(T4), and thyroid-stimulating hormone (TSH) concenquantitation. Serum triiodothyronine (T,), thyroxine 

methylene blue and a Miller disc for reticulocyte 

incubating equal volumes of whole blood and new 

mined by light microscopy, using smears prepared by 

Wright-Giemsa. Reticulocyte counts were determicroscopic examination of blood films stained with, 

evaluation of blood cells were determined by light 

Leukocyte differential counts and morphologic 

tology analyzer (Ortho Instruments, Westwood, MA). 

tions were performed on an Ortho ELT-8/ds hemaanalyses were performed. Hematology determinaplastic containers for storage at -70" C until the 

centrifuged. The serum was separated and placed in 

coagulant, allowed to clot at room temperature, and 

hormone analyses was placed in tubes with .)ut antisium EDTA as the anticoagulant. Blood for thyroid 

determinations was placed in tubes containing potasfrom the retroorbital sinus. Blood for hematology 

a 70% CO,:30% air mixture and blood was drawn 

lists the tissues and organs that were examined. 

rats and mice, and on 800 ppm female mice. Table 1 

control and 1.,600 ppm animals, on 800 ppm male 

histopathologic examination was performed on all 

stained with hematoxylin. and eosin. A complete 

paraffin, sectioned to. a thickness of 5' to 6 pm, and 

buffered formalin, processed, trimmed,. embedded in 

nation were fixed and preserved in 10% neutral 

study were weighed. Tissues for microscopic examiand thymus of, animals surviving until the end of the 

heart, right and left kidneys, liver, lungs, right testis, 

A necropsy was performed on all animals. The brain, 

60 female mice were exposed to acetonittile by 

335, or 670 mglm3) and. groups of 60. male and 

tions of 0, 100,200, or 400 ppm (equivalent to 0,168, 

exposed to acetonitrile by inhalation at concentraGroups of up to 56 male and 56 female rats were 

Study Design 

%YEAR STUDIES 
Materials and Methods 23 

15 months. 

mice from each exposure group were evaluated at 

and eight female rats and 10 male and 10 female 

per day, 5 days per week for 103 weeks. Eight male 

(equivalent to 0, 84, 168, or 335 mum3) for 6 hours 

inhalation at concentrations of 0,50,100, or 200 ppm 

Program (Appendix J). 

using the protocols of the NTP Sentinel Animal 

the studies, the health of the animals was monitored 

3 weeks after their arrival for viral screening. During 

additional five male and five female rats and mice 

disease. Serology samples were collected from an 

for parasite evaluation and gross observation of 

and five female rats and mice were randomly selected 

first day of exposure. Prior to study start, five male 

studies and were approximately 6 weeks old on the 

quarantined for 15 days before the beginning of the 

animals were 4 weeks old. Both rats and mice were 

for use in the 2-year studies. Upon receipt the 

obtained from Simonsen Laboratories (Gilroy, CA) 

Male and female F344/N rats and B6C3Fl mice were 

Source and Specification of Animals 

Appendix I. 

feed composition and contaminants is provided in 

maintenance are given in Table 1. Information on 

racks were rotated weekly. Further details of animal 

sure, and water was available ad libiturn. Cages and 

available ad libitum, except during periods of expoRats and mice were housed individually. Feed was 

Animal Maintenance 

and clinical findings were recorded every 2 weeks. 

During the final 13 weeks of the study, body weights 

first 13 weeks, and at 4-week intervals thereafter. 

The animals were weighed initially, weekly for the 

clinical observations were recorded every 4 weeks. 

and signs of toxicity or moribundity. Individual 

The animals were observed twice daily for mortality 

Clinical Examinations and Pathology 

Ortho ELT-8/ds hematology analyzer. Leukocyte 

Hematology determinations were performed on an 

containing potassium EDTA as the anticoagulant. 

hematology determinations was placed in tubes 

thetized with a 70% CO,:30% air mixture. Blood for 

was taken from the retroorbital sinus of rats anesnated for interim evaluation at 15 months. Blood 

and 10 female mice per exposure group were desigEight male and up to eight female rats and 10 male 

weighed. 

Table 1. The liver, right kidney, and lungs were 

parameters evaluated at 15 months are listed in 

films stained with Wright-Giemsa. The hematology 

determined by light microscopic examination of blood 

differential and nucleated erythrocyte counts were 

microscopically are listed in Table 1. 

organ were examined. Tissues that were examined 

adrenal gland, kidney, and ovary), samples from each 

microscopic examination. For all paired organs (i.e., 

6 pm, and stained with hematoxylin and eosin for 

embedded in paraffin, sectioned to a thickness of 5 to 

10% neutral buffered formalin, processed, trimmed, 

all major tissues were fixed and preserved in 

tissues were examined for grossly visible lesions, and 

were performed on all rats and mice. All organs and 

A complete necropsy and microscopic examination 

kidneys of mice. 

rats; the liver of female rats; and the brain and 

gland of rats; the adrenal medulla and testis of male 

for specific lesions included the pancreas and thyroid 

male and female mice. Additional tissues reviewed 

female mice, and the forestomach of female rats and 

rats and mice, the liver of male rats and male and 

quality assessment pathologist reviewed the lung of 

technique was evaluated. For the 2-year studies, a 

slide and tissue counts were verified, and the historecords and tables were compared for accuracy, the 

quality assessment laboratory. The individual animal 

pathology tables were evaluated by an independent 

The slides, individual animal data records, and 

for inventory, slideblock match, and wet tissue audit. 

residual wet tissues were sent to the NTP Archives 

System. The microscopic slides, paraffin blocks, and 

entered into the Toxicology Data Management 

laboratory pathologist, and the pathology data were 

Microscopic evaluations were completed by the study 

presented by the chair to the PWG for review. The 

pathologist, or lesions of general interest were 

between the laboratory and quality assessment 

tration, examples of disagreements in diagnoses 

ing examples of lesions related to chemical adminisexisted. Representative histopathology slides containthe laboratory and quality assessment pathologists 

tissues for which a disagreement in diagnosis between 

chair, who reviewed the selected tissues and any other 

ted to the NTP Pathology Working Group (PWG) 

The quality assessment report and slides were submit- 
24 Acetonitrile, NTP TR 447 

McConnell et al. (1986). 

separately or combined according to the guidelines of 

diagnosed lesions for each tissue type were evaluated 

subsequent analyses of the pathology data, the 

and Boorman (1982) and Boorman et al. (1985). For 

procedures have been described, in part, by Maronpot 

pathologists and the PWG. Details of these review 

final diagnoses represent a consensus of contractor 

pathologist, the diagnosis was changed. Thus, the 

differed from the opinion of the laboratory 

rendered diagnoses. When the PWG consensus 

without any knowledge of dose groups or previously 

logic pathology. This group examined the tissues 

and other pathologists experienced in rodent toxicoPWG consisted of the ‘quality assessment pathologist 

survival analyses are two sided. 

dose-related trends. All reported P values for the 

equality and Tarone’s (1975) life table test to.identify 

Cox’s (1972) method for testing two groups for 

for possible dose-related effects on survival used 

natural causes were not censored. Statistical analyses 

sored from the survival analyses; animals dying from 

found dead of other than natural causes were cenand is presented in the form of graphs. Animals 

product-limit procedure of Kaplan and Meier (1958) 

The probability of survival was estimated by the 

Survival Analyses 

Statistical Methods 

group and each site-specific neoplasm, i.e., the 

also give the survival-adjusted neoplasm rate for each 

necropsy was performed. Tables A3, B3, C3, and D3 

inators consist of the number of animals on which a 

occurrence (e.g., leukemia or lymphoma), the denomor when neoplasms had multiple potential sites of 

and mammary gland) before microscopic evaluation, 

certain tissues (e.g., skin, intestine, harderian gland, 

examination was required to detect neoplasms in 

ined microscopically. However, when macroscopic 

the numbers of animals affected at each site examand D3) and all nonneoplastic lesions are given as 

the incidences of most neoplasms (Tables A3, B3, C3, 

scopically. For calculation of statistical significance, 

number of animals with that site examined microsuch lesions at a specific anatomic site and the 

and D5 are given as the number of animals bearing 

as presented in Tables Al, A5, B1, B4, C1, C5, Dl, 

The incidences of neoplasms or nonneoplastic lesions 

Calculation of Incidence 

competing risks (Kaplan and Meier, 1958). 

study in the absence of mortality from all other 

that would have been observed at the end of the 

Kaplan-Meier estimate of the neoplasm incidence 

Crowley, 1984). 

the time-specific neoplasm incidences (McKnight and 

neoplasm prevalences also provides a comparison of 

incidental, this comparison of the time-specific 

Dime and Haseman (1986). When neoplasms are 

Lagakos (1983), further described and illustrated by 

mortality is the prevalence analysis of Dime and 

dose. This method of adjusting for intercurrent 

likelihood score test for the regression coefficient of 

control groups were compared on the basis of the 

enhanced. The neoplasm incidences of exposed and 

nated if the fit of the model was not significantly 

porated initially, and the quadratic term was elimiBoth linear and quadratic terms in time were incora logistic function of chemical exposure and time. 

this approach, neoplasm prevalence was modeled as 

cause and thus did not affect the risk of death. In 

covered as the result of death from an unrelated 

assumed that the diagnosed neoplasms were dismethod used was logistic regression analysis, which 

not rapidly lethal. Thus, the primary statistical 

considered to be incidental to the cause of death or 

The majority of neoplasms in these studies were 

Analysis of Neoplasm Incidences 

neoplasm-bearing anin‘als. 

1979), procedures based on the overall proportion of 

Armitage trend test (Armitage, 1971; Gart et al., 

plasms, and the Fisher exact test and the CochranTarone, 1975), appropriate for rapidly lethal neomethods include the life table test (Cox, 1972; 

tests are summarized in the appendixes. These 

statistical analysis were used, and the results of these 

In addition to logistic regression, other methods of 

to Haseman (1984). 

further discussion of these statistical methods, refer 

to evaluate selected nonneoplastic lesions. For 

described in the preceding paragraphs were also used 

reported P values are one sided. The procedures 

were used in the analysis of neoplasm incidence, and 

overall dose-related trend. Continuity-corrected tests 

each exposed group with controls and a test for an 

Tests of significance included painvise comparisons of 
Materials and Methods 25 

proportion of affected animals. 

test was used, a procedure based on the overall 

detected at the interim evaluation, the Fisher exact 

function of chemical exposure and time. For lesions 

plastic lesion prevalence was modeled as a logistic 

was a logistic regression analysis in which nonneonot rapidly lethal, the primary statistical analysis used 

considered to be incidental to the cause of death or 

Because all nonneoplastic lesions in this study were 

Analysis of Nonneoplastic Lesion Incidences 

Whitney U test (Hollander and Wolfe, 1973). 

values were analyzed for significance using the Mannwere eliminated from the analysis. Average severity 

examined by NTP personnel, and implausible values 

the outlier test of Dixon and Massey (1951) were 

test). Prior to analysis, extreme values identified by 

monotonic dose-related trend (Dunnett’s or Dunn’s 

comparisons than a test that does not assume a 

Shirley’s test) was more appropriate for painvise 

mine whether a trend-sensitive test (Williams’ or 

significance of the dose-related trends and to deterJonckheere’s test (1954) was used to assess the 

methods of Shirley (1977) and Dunn (1964). 

using the nonparametric multiple comparison 

have typically skewed distributions, were analyzed 

Hematology and thyroid hormone assay data, which 

Dunnett (1955) and Williams (1971, 1972). 

parametric multiple comparison procedures of 

mately normal distributions, were analyzed using the 

Organ and body weight data, which have approxicontrol groups in the analysis of continuous variables. 

cance of painvise comparisons between exposed and 

TWO approaches were employed to assess the signifiAnalysis of Continuous Variables 

effects. 

neoplasms appearing to show compound-related 

et al., 1984,1985) are included in the NTP reports for 

from the NTP historical control database (Haseman 

certain instances. Consequently, neoplasm incidences 

the overall assessment of neoplasm incidence in 

evaluation, historical control data can be helpful in 

first and most appropriate control group used for 

Although the concurrent control group is always the 

Historical Control Data 

compliance with Food and Drug Administration 

The 13-week and 2-year studies were conducted in 

Quality Assurance Methods 

Report. 

addressed during the preparation of this Technical 

comments had been resolved or were otherwise 

were reviewed and assessed by NTP staff, so all 

reports and are on file at NIEHS. The audit findings 

Audit procedures and findings are presented in the 

draft of this NTP Technical Report were conducted. 

data, pathology specimens, final pathology tables, and 

covering completeness and accuracy of the pathology 

dent quality assurance contractor. Separate audits 

studies were audited retrospectively by an indepenstudies were submitted to the NTP Archives, these 

Part 58). In addition, as records from the 2-year 

Good Laboratory Practice Regulations (21 CFX, 

GENETIC TOXICOLOGY 

these studies and the results are given in Appendix E. 

erythrocytes in peripheral blood. The protocols for 

and to increase the frequency of micronucleated 

aberrations in cultured Chinese hamster ovary cells, 

and sister chromatid exchanges and chromosomal 

tions in various strains of Salmonella typhimurium 

testing the ability of the chemical to induce mutaThe genetic toxicity of acetonitrile was assessed by 

1981; Crawford, 1985). 

mutation theory (Miller and Miller, 1977; Straus, 

theory of chemical carcinogenesis and the somatic 

carcinogenicity in animals, based on the electrophilic 

of chemically induced DNA damage and to predict 

tests were originally developed to study mechanisms 

in vivo genetic toxicity tests. These genetic toxicity 

responses of the chemical in short-term in vitro and 

experimental animals from the structure and 

would permit the evaluation of carcinogenicity in 

a larger effort by the NTP to develop a database that 

The genetic toxicity studies of acetonitrile are part of 

types of DNA and chromosome effects that can be 

these other tests can provide information on the 

(Tennant et al., 1987; Zeiger et al., 1990), although 

tests do not correlate well with rodent carcinogenicity 

and Tennant, 1991). Other in vitro genetic toxicity 

rats and mice and/or at multiple tissue sites (Ashby 

correlated with the induction of carcinogenicity in 

philicity and Salmonella mutagenicity is highly 

genicity in rodents. The combination of electroreactivity), mutagenicity in Salmonella, and carcinopotential electrophilicity (structural alert to DNA 

There is a strong conelation between a chemical’s 
26 Acetonitrile, NTP TR 447 

induced by the chemical being investigated. Data genetic toxicity tests. That is, no battery of tests that 

from NTP studies show that a positive response in included the Salmonella test improved the predictivity 

Salmonella is currently the most predictive in vitro of the Salmonella test alone. The predictivity for 

test for rodent carcinogenicity (89% of the rodent carcinogenicity of a positive response in 

Salmonella mutagens were rodent carcinogens), and rodent bone marrow micronucleus tests is still being 

that there is no complementarity among the in vitro evaluated. 
Materials and Methods 27 

Experimental Design and Materials and Methods in the Inhalation Studies of Acetonitrile 

TABLE 1 

13-Week Studies 2-Year Studies 

(Richland, WA) 

Battelle Pacific Northwest. Laboratories 

Study Laboratory 

Mice: B6C3Fl 

Rats: M44/N 

Strain and Species 

(Gilroy, CA) 

Simonsen Laboratories 

Animal Source 

Mice: 14 days 

Rats: 12 days 

Time Held Before Studies 

6 weeks 

Average Age When Studies Began 

Mice: 10 April 1986 

Rats: 8 April 1986 

Date of First Dose 

2 holidays) 

6 hours per day, 5 days per week for 13 weeks (excluding 

Duration of Dosing 

Mice: 9-10 July 1986 

Rats: 8-9 July 1986 

Date of Last Dose 

Mice: 9-10 July 1986 

Rats: 8-9 July 1986 

Necropsy Dates 

(Richland, WA) 

Battelle Pacific Northwest Laboratories 

Mice: B6C3Fl 

Rats: F344/N 

(Gilroy, CA) 

Simonsen Laboratories 

15 days 

6 weeks 

Mice: 7 April 1988 

Rats: 31 March 1988 

1 day of nonexposure) 

2 holidays, 7 unscheduled early terminations of exposure, and 

6 hours per day, 5 days per week for 103 weeks (excluding 

Rats: 

Terminal: 

15-Month interim evaluation: 

23 March 1990 

28-29 June 1989 

Mice: 

15-Month interim evaluation: 

5-6 July 1989 

Terminal: 

30 March 1990 

Rats: 

Terminal: 

15-Month interim evaluation: 

2-4 April 1990 

29-30 June 1989 

Mice: 

Terminal: 

15-Month interim evaluation: 

9-12 April 1990 

6-7 July 1989 
28 Acetonitrile, NTP TR 447 

Experimental Design and Materials and Methods in the Inhalation Studies of Acetonitrile (continued) 

TABLE 1 

13-Week Studies 2-Year Studies 

19 weeks 

Average Age at Necropsy 

10 males and 10 females 

Size of Study Groups 

15-Month interim evaluation: 

111 weeks 

I2 weeks 

Terminal: 

Rats: 

Terminal: 

15-Month interim evaluation: 

up to 48 males and 48 females 

8 males and 8 females 

Mice: 

Terminal: 

15-Month interim evaluation: 

50 males and 50 females 

10 males and 10 females 

blocking variable. 

Systems Corp., Cedar Knolls, NJ), with body weight as the 

using the XYBION PATH/TOX SYSTEM (XYBION Medical 

Animals were randomly assigned to exposure and control groups Same as 13-week studies 

Method of Animal Distribution 

1 

Animals per Cage 

Toe clip and cage position during exposure 

Method of Identification 

available ad libifurn, except during. exposure periods. 

NIH-07 diet/pelleted (Zeigler Brothers, Inc., Gardners, PA), 

Diet 

1 

Mice: toe clip and cage position during exposure 

Rats: tail tattoo and cage position duringexposure 

Same as 13~week studies 

ad libitum 

watering system (Edstrom Industries, Waterford, WI); available 

Treatment Company, Rockford; IL) and supplied via automatic 

Tap water (Richland municipal supply) softened (Illinois Water Same as 13-week studies 

Water Distribution 

Cages 

MD); cage units changed weekly and rotated in chamber weekly 

Stainless steel wire bottom cages (Lab Products, Inc., Aberdeen, Same as 13-week studies 

during quarantine period; none used,during study period 

Sani-Chips (PI Murphy Forest. Products,.Rochelle Park, NY) Untreated,Shepard Specialty Papers (Kalamazoo, MI) 

BeddinglCage Board' 

charcoal.(RSE, Inc., New Baltimore, MI) 

Single HEPA (Flanders Filters, Inc., San Rafael, CA) and Same as 13-week studies 

Chamber Air Supply Filters 
Materials and Methods 29 

Experimental Design and Materials and Methods in the Inhalation Studies of Acetonitrile (continued) 

TABLE 1 

13-Week Studies 2-Year Studies 

changed weekly 

Stainless steel (Lab Products, Aberdeen, MD), 

Chambers 

Fluorescent light: 12 hours/day 

Relative humidity: 30%-75% 

Temperature: 19.1"-27.4" C (rats); 21.0"-27.4" C (mice) 

Chamber Environment 

670, 1,340, or 2,681 ms/m3) 

0, 100, 200, 400, 800, or 1,600 ppm (equivalent to 0, 168, 335, 

Doses 

13 weeks in rats and 12 weeks in mice. 

recorded weekly. Body weights were recorded weekly for 

Animals were observed twice daily; clinical observations were 

Type and Frequency of Observation 

Same as 13-week studies 

Fluorescent light: 12 hourslday 

Relative humidity: 55.3%-57.8% (rats), 54.0%-54.4% (mice) 

Temperature: 20.9"-26.8" C (rats), 20.5"-26.7" C (mice) 

335 ms/m3) 

Mice: 0, 50, 100, or 200 ppm (equivalent to 0, 84, 168, or 

670 mp/m3) 

Rats: 0, 100, 200, or 400 ppm (equivalent to 0, 168, 335, or 

every 2 weeks. 

13 weeks, clinical observations and body weights were recorded 

for the first 13 weeks, and monthly thereafter. During the final 

recorded monthly. Body weights were recorded initially, weekly 

Animals were observed twice daily; clinical observations were 

70% carbon dioxide asphyxiation Anesthetization with 70% carbon dioxide followed by 

Method of Sacrifice 

and 

were brain, heart, right and left kidneys, liver, lungs, right testis, the 15-month interim evaluations were liver, lungs, and right 

Necropsies were performed on all animals. Organs weighed Necropsies were performed on all animals. Organs weighed at 

Necropsy 

exsanguination via the brachial artery 

and thyroid-stimulating hormone (TSH) concentrations. 

Zhyroid hormone mqs: triiodothyronine (T3), thyroxine (TJ, erythrocyte counts. 

differentials, reticulocyte counts, and platelet counts. differentials, reticulocyte counts, platelet counts, and nucleated 

hemoglobin concentration, total leukocyte counts and hemoglobin concentration, total leukocyte counts and 

counts, mean cell volume, mean cell hemoglobin, mean cell counts, mean cell volume, mean cell hemoglobin, mean cell 

Hematology: hematocrit, hemoglobin concentration, erythrocyte HsMtologyr hematocrit, hemoglobin concentration, erythrocyte 

to study termination for clinical pathology. 15-month interim evaluation. 

Blood was taken from the retroorbital sinus of all rats surviving Blood was taken from the retroorbital sinus of rats at the 

Clinical Pathology 
30 Acetonitrile, NTP TR 447 

TABLE 1 

Experimental Design and Materials and Methods in the Inhalation Studies of Acetonitrile (continued) 

13-Week Studies 2-Year Studies 

ttistopathology 

Complete histopathologic examinations were performed on O, 

800 (excluding female rats), and 1,600 ppm rats and mice. In 

addition to gross lesions and tissue masses with regional lymph 

nodes, tissues examined included: adrenal gland, brain, bone 

and marrow, clitoral gland (rats), esophagus, gallbladder(mice), 

heart, kidney, large intestine (colon, cecum, rectum), larynx, 

liver, lung, lymph nodes (bronchial, mandibular, mediastinal, and 

mesenteric), mammary gland, nose (3 levels), ovary, pancreas, 

parathyroid gland, pituitary gland, preputial gland (rats), 

prostate gland, salivary gland, skin, small intestine (duodenum, 

jejunum, ileum), spleen, stomach (forestomach and glandular), 

testis, thymus, thyroid gland, trachea, urinary bladder, and 

uterus. Selected organs examined in rats included: bone and 

marrow, brain, lung, mesenteric lymph node, ovary, spleen, and 

thymus of 800 ppm females; and bone and marrow, testes, and 

thymus of 400 ppm males. Selected organs in mice included: 

adrenal gland of 200 and 400 ppm females; liver of 200 and 

400 ppm males and.females; lung of 400 ppm females; stomach 

of 200 and 400 ppm males and all females; and thymus of 

400 ppm females. 

Complete histopatho!ogic examinations were performed on all 

animals. In addition to gross lesions and tissue masses with 

regional lymph nodes, tissues examined included: adrenal gland, 

brain, bone and marrow, clitoral gland, esophagus, gallbladder 

(mice), heart, kidney, large intestine (colon, cecum, rectum), 

larynx, liver, lung, lymph nodes (bronchial, mandibular, 

mediastinal, and mesenteric), mammary gland, nose, ovary, 

parathyroid gland, pituitary gland, preputial gland, prostate 

gland, salivary gland, seminal vesicle, skin, small intestine 

(duodenum, jejunum, ileum), spleen, stomach (forestomach and 

glandular), testis, thymus, thyroid gland, trachea, urinary 

bladder, and uterus. 
31 

RESULTS 

RATS 

13-WEEK STUDY 

Six male and three female rats exposed by inhalation 

to acetonitrile concentrations of 1,600 ppm and one 

male exposed to 800 ppm died during the study; all 

but one of these deaths occurred during the first 

2 weeks of the study (Table 2). At exposure concentrations up to and including 800 ppm, final mean 

body weights and body weight gains were similar to 

those of the controls. At the 1,600 ppm concentration, body weight gains were lower than those of the 

controls; the final mean body weights were 81% of 

the control value in males and 91% of the control 

value in females (Table 2). Hypoactivity and ruffled 

fur were observed in 800 ppm males and 1,600 ppm 

males and females during the first week of the study. 

Additional clinical findings in 1,600 ppm males that 

died during week 1 were ataxia, abnormal posture, 

TABLE 2 

Survival and Body Weights of Rats in the 13-Week Inhalation Study of Acetonitrile 

Mean Body Weight b (g) Final Weight 

Dose Survival a Initial Final Change Relative to Controls 

(ppm) (%) 

Male 

0 

100 

200 

400 

800 

1,600 

10/10 115 ± 2 336 ± 4 220 ± 4 

10/10 118 ± 2 343 ± 5 225 ± 4 102 

10/10 121 ± 3 352 ± 5 231 ± 4 105 

10/10 118 ± 2 339 ± 5 221 ± 4 101 

9/10 c 117 ± 3 338 ± 7 222 ± 6 101 

4/10 d 119 ± 2 272 ± 11"* 153 ± 10"* 81 

Female 

0 10/10 97 ± 2 197 ± 4 99 ± 3 

100 10/10 97 ± 2 190 ± 4 92 ± 3 96 

200 10/10 98 ± 2 200 ± 4 102 ± 3 102 

400 10/10 96 ± 2 209 ± 5 113 ± 4* 106 

800 10/10 98 ± 2 203 ± 3 105 ± 3 103 

1,600 7/10 e 95 ± 1 180 ± 4* 85 ± 3* 91 

* Significantly different (P_<0.05) from the control group by Dunnett's test. 

** P_<0.01 

a Number of animals surviving at 13 weeks/number initially in group 

b Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the 

end of the study. 

c Week of death: 1 

d Week of death: 1, 1, 1, 1, 2, 4 

e Week of death: 1, 2, 2 
32 Acetonitrile, NTP TR 447 

biologically significant. 

No other organ weight differences were considered 

kidney, and liver weights than those of the controls. 

had significantly greater absolute and relative heart, 

controls (Table Fl). Females exposed to 1,600 ppm 

females were significantly lower than those of the 

thymus weights of 800 and 1,600 ppm males and 

clinical findings were observed. Absolute and relative 

and clonic convulsions. No other treatment-related 

triiodothyronine (TJ concentration occurred in the 

controls. In the 1,600 ppm female rats, decreases in 

tions in these groups were similar to those of the 

cell volume, and mean cell hemoglobin concentranormochromic because the reticulocyte counts, mean 

characterized as nonresponsive, normocytic, and 

800 ppm female rats (Table Gl). The anemia was 

occurred in the 1,600 ppm males and females and in 

count, hemoglobin concentration, and hematocrit, 

An anemia, evidenced by decreases in red blood cell 

unrelated to treatment. 

sporadic changes in other parameters were considered 

stimulating hormone (TSH) concentrations. Minor, 

absence of alterations in thyroxine (T4) and thyroidovary. 

(females), and depletion of corpora lutea in the 

thymic atrophy, lymphoid depletion of the spleen 

hemorrhage, cellular depletion of the bone marrow,. 

primarily in animals dying early that included. brain 

in alveoli; and a spectrum of lesions typically noted 

lesions consisting of congestion; edema; hemorrhage 

rats that died during the study. These included lung 

the 800 ppm male and 1,600 ppm male and female 

Gross and histopathologic changes were restricted to 

400 ppm. 

2-year inhalation study in rats were 100, 200, and 

acetonitrile exposure concentrations selected for the 

Dose Selection Rationale: Based on reduced survival, 
Results 33 

controls. 

rates of exposed rats were similar to those of the 

Kaplan-Meier survival curves in Figure 1. Survival 

and female rats are shown in Table 3 and in the 

Estimates of 2-year survival probabilities for male 

Survival 

%YEAR STUDY 

mean body weights (Figure 2, Tables 4 and 5), and 

or 2 years had no effect on body weight gain or final 

Exposure to acetonitrile by inhalation for 15 months 

Body Weights and Clinical Findings 

the controls throughout the study. 

exposed male and female rats were similar tothose of 

the behavior, general health, and appearance of 

cyte count was slightly greater than that of controls. 

minimally lower than controls; however, the erythromean cell hemoglobin in 400 ppm males were also 

of the controls (Table G2). Mean cell volume and 

400 ppm female rats were minimally lower than those 

mean cell volume, and mean cell hemoglobin in 

value, hemoglobin concentration, erythrocyte count, 

At the 15-month interim evaluation, the hematocrit 

Hematology 

Survival of Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE 3 

0 PPm 100 ppm 200 ppm 400 ppm 

15-Month interim evaluationa 

Animals initially in study 

Male 

Mean survival (days)c 

Percent probability of survival at end of studyb 

Animals surviving to study termination 

Natural deaths 

Moribund 

15-Month interim evaluationa 

Animals initially in study 

Female 

Survival analysisd 

Mean survival (days) 

Percent probability of survival at end of study 

Animals surviving to study termination 

Natural deaths 

Moribund 

Accidental deatha 

Survival analysis 

56 

613 

23 

11 

1 

36 

8 

P=0.141N 

56 

622 

50 

23 

0 

24 

1 

8 

P=0.166N 

55 

614 

28 

13 

8 

26 

8 

P=0.746N 

56 

641 

44 

21 

2 

25 

0 

8 

P=O.752 

56 

606 

19 

9 

4 

35 

8 

P = 0.667 

56 

638 

55 

26 

1 

21 

0 

8 

P=0.843N 

56 

641 

35 

17 

5 

26 

8 

P=0.137N 

56 

661 

61 

29 

5 

14 

0 

8 

P=0.290N 

a Censored from survival analyses 

b Kaplan-Meier determinations based on the number of animals alive on the first day of terminal sacrifice C Mean of all deaths (uncensored, censored, and terminal sacrifice) 

d The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table painvise 

is indicated by N. 

comparisons (Cox, 1972) with the controls are in the exposed columns. A negative trend or lower mortality in an exposure group 
34 Acetonitrile, NTP TR 447 

........... 

....... 

.......... 

................... 

0 15 30 45 60 75 90 105 

WEEKS ON STUDY 

by Inhalation for 2 Years 

Kaplan-Meier Survival Curves for Rats Administered Acetonitrile 

FIGURE 1 
Results 35 

500 

450 

IY 

2 400 

z 350 

0 

Q 

0 

I 

t300 

n 

3 

t 

p 250 

4 

W 

Z 

200 

150 

100 

500 

450 

* 400 3 

.. 

.. ..... 

................ ....... .................. 

a .............................. 

I 

rn o PPY 

A 200 PPY 

0 IOOPPY ..... 

! .. .. 

I I 

15 30 45 60 75 90 

WEEKS ON STUDY 

.. .... 

.. 

...... 

.. 

.. 

A 200 PPY 

0 IOOPPY 

rn o PPY 

. -- h 15 30 45 60 75 

WEEKS ON STUDY 

90 I5 

Growth Curves for Rats Administered Acetonitrile by Inhalation for 2 Years 

FIGURE 2 
36 Acetonitrile, NTP TR 447 

Mean Body Weights and Survival of Male Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE 4 

Study Survivors (9) controls) Survivors (9) controls) Survivors (8) controls) Survivors 

on Av. Wt No. of Av. Wt Wt (%of No. of Av. Wt Wt (%of No. of Av. Wt Wt (%of No. of 

Weeks 0 ppm LOO ppm 200 ppm 400 ppm 

(9) 

9 

8 

7 

6 

5 

4 

3 

2 

1 

103 

101 

99 

97 

95 

93 

91 

89 

85 

81 

77 

73 

.69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29 

25 

21 

17 

13 

12 

11 

10 

458 

459 

465 

467 

479 

479 

479 

486 

489 

498 

494 

497 

493 

498 

498 

497 

494 

494 

485 

478 

463 

450 

438 

422 

412 

391 

364 

353 

344 

332 

324 

311 

297 

280 

265 

245 

221 

192 

144 

Mean for weeks 

1-13 282 

14-52 448 

53-103 484 

12 

13 

16 

23 

25 

26 

29 

31 

35 

36 

40 

42 

45 

54 

54 

55 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

443 

456 

460 

468 

478 

476 

476 

486 

490 

493 

494 

493 

495 

495 

496 

494 

490 

488 

480 

474 

462 

447 

434 

421 

411 

390 

363 

354 

344 

334 

325 

312 

298 

283 

267 

245 

221 

193 

143 

481 

445 

283 

97 

99 

99 

100 

100 

99 

99 

100 

100 

99 

100 

99 

100 

99 

100 

99 

99 

99 

99 

99 

100 

99 

99 

100 

100 

100 

100 

100 

100 

100 

100 

101 

101 

101 

101 

100 

100 

100 

99 

99 

99 

100 

15 

17 

19 

22 

22 

25 

27 

28 

30 

34 

41 

44 

45 

53 

54 

54 

54 

54 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

55 

459 

477 

470 

475 

476 

480 

479 

485 

473 

490 

492 

494 

487 

493 

495 

491 

487 

488 

482 

476 

460 

448 

436 

421 

412 

391 

362 

352 

344 

333 

323 

310 

296 

282 

266 

245 

221 

192 

142 

483 

446 

282 

100 

100 

100 

100 

100 

100 

101 

100 

100 

100 

100 

98 

100 

104 

101 

102 

100 

100 

100 

,100 

97 

99 

100 

99 

99 

99 

99 

99 

99 

99 

99 

100 

99 

100 

99 

100 

100 

100 

99 

100 

100 

100 

11 

16 

19 

22 

25 

27 

29 

33 

37 

40 

41 

44 

53 

54 

54 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

9 

494 

494 

494 

494 

488 

485 

480 

473 

461 

449 

437 

422 

408 

391 

365 

352 

343 

333 

323 

309 

295 

281 

265 

244 

21 8 

190 

140 

469 

476 

481 

482 

477 

476 

477 

483 

488 

494 

500 

499 

a86 

445 

281 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

98 

99 

97 

100 

99 

101 

101 

100 

99 

99 

99 

99 

98 

99 

99 

100 

100 

100 

100 

99 

103 

104 

103 

103 

100 

99 

'100 

?9 

100 

99 

100 

56 

56 

56 , 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

21 

22 

22 - 

26 

30 ~ 

32 

35 

37 

39 

43 

46 

46 

48 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

a .Interim evaluation occurred during-week 66. 
Results 37 

Mean Body Weights and Survival of Female Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE 5 

on Av. Wt. No. of Av. Wt Wt. (% of No. of Av. Wt. Wt (% of No. of Av. Wt. Wt. (% of No. of 

Weeks 0 ppm LOO ppm 200 ppm 400 ppm 

Study (8) Survivors (8) controls) Survivors (8) controls) Survivors (9) controls) Survivors 

99 

97 

95 

93 

91 

89 

85 

81 

77 

73 

69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29 

25 

21 

17 

13 

12 

11 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

103 

101 

342 

339 

336 

338 

340 

339 

338 

339 

332 

332 

326 

327 

318 

311 

306 

300 

292 

288 

275 

263 

252 

241 

231 

226 

218 

212 

201 

198 

195 

191 

187 

182 

179 

171 

164 

156 

149 

140 

117 

Mean for weeks 

53-103 

14-52 

1-13 

327 

245 

172 

25 

29 

32 

32 

33 

34 

34 

36 

38 

39 

39 

41 

41 

50 

51 

51 

52 

53 

54 

55 

55 

55 

55 

55 

55 

55 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

351 

346 

346 

347 

350 

350 

349 

347 

342 

342 

336 

331 

324 

316 

312 

303 

294 

290 

279 

268 

254 

241 

233 

229 

221 

213 

204 

201 

196 

192 

188 

184 

180 

174 

168 

159 

151 

140 

115 

334 

248 

173 

103 

102 

103 

103 

103 

103 

103 

102 

103 

103 

103 

101 

102 

101 

102 

101 

101 

101 

101 

102 

101 

100 

101 

102 

101 

101 

102 

102 

101 

101 

100 

101 

101 

102 

102 

102 

102 

100 

99 

102 

101 

101 

21 

26 

29 

31 

33 

35 

36 

37 

43 

44 

44 

45 

46 

55 

55 

55 

55 

55 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

343 

347 

349 

344 

344 

343 

343 

345 

332 

336 

335 

326 

318 

314 

309 

301 

290 

287 

276 

263 

252 

241 

233 

229 

222 

213 

202 

199 

194 

190 

187 

182 

178 

172 

167 

157 

149 

138 

114 

33 1 

246 

171 

100 

100 

100 

101 

101 

100 

100 

99 

98 

100 

101 

100 

100 

100 

101 

101 

102 

101 

101 

100 

100 

99 

100 

102 

104 

102 

101 

101 

102 

102 

100 

101 

103 

100 

100 

101 

101 

101 

99 

101 

100 

99 

27 

28 

28 

30 

32 

33 

35 

36 

38 

39 

43 

44 

45 

54 

54 

54 

55 

55 

55 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

347 

346 

346 

343 

342 

345 

344 

343 

342 

339 

335 

330 

322 

317 

313 

305 

299 

293 

280 

270 

258 

246 

237 

230 

222 

215 

205 

199 

197 

191 

187 

182 

177 

173 

166 

156 

147 

137 

113 

333 

250 

172 

101 

102 

103 

101 

101 

102 

102 

101 

103 

102 

103 

101 

101 

102 

102 

102 

103 

102 

102 

103 

102 

102 

103 

102 

102 

102 

102 

101 

101 

100 

100 

100 

99 

101 

101 

100 

99 

98 

97 

102 

102 

100 

31 

32 

33 

34 

41 

41 

41 

42 

44 

45 

46 

46 

46 

54 

54 

55 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

56 

a Interim evaluation occurred during week 66. 
38 Acetonitrile, NTP TR 447 

and Appendix B for female rats. 

section are presented in Appendix A for male rats 

incidences for the neoplasms mentioned in this 

5% in at least, one animal group, and historical 

neoplasms that occurred with an incidence of at least 

tumor diagnoses, statistical analyses of primary 

plasms and nonneoplastic lesions, individual animal 

other organs. Summaries of the incidences of neononneoplastic lesions and neoplasms of the liver and 

biologically noteworthy changes in the incidences of 

This section describes the statistically significant or 

Pathology and Statistical Analyses 

those in the controls (Tables B1 and B4). 

lesions in exposed female groups were similar to 

controls (Tables 6 and A5). The incidences of liver 

marginally greater in 400 ppm males than those in 

incidences of eosinophilic and mixed cell foci were 

200 and 400 ppm males than that in controls, and the 

A4a). The incidence of basophilic foci was greater in 

range for inhalation study controls (Tables 6 and 

of males was only slightly greater than the historical 

ma or carcinoma (combined) in the 400 ppm group 

different, and the incidence of hepatocellular adenosure groups and controls were not significantly 

A3). However, painvise comparisons between expocally significant positive trend in males (Tables 6 and 

ma or carcinoma (combined) occurred with a statistihepatocellular carcinoma, and hepatocellular adenoLiver: The incidences of hepatocellular adenoma, 

historical control range (0%-8%, Table A4d). 

keratoacanthomas in 400 ppm males was within the 

observed in the control group, and the incidence of 

to chemical exposure. No keratoacanthomas were ' 

and A3), the increase was not considered to be due 

significant trend (0/48, 1/47, 0148, 4/48; Tables A1 

acanthoma in the skin occurred with a statistically 

groups. Although the increased incidence of keratohave been observed in certain historical control 

chance; in fact, higher incidences of these neoplasms 

increased incidences were considered to be due to 

not significantly increased. Therefore, these 

and A~c), and incidences in the 400 ppm group were 

when compared to historical controls (Tables A4b 

or carcinoma (combined) in the controls were low 

cytoma (combined) and of pancreatic islet adenoma 

the incidences of benign or malignant pheochromothe increases in neoplasm incidences were marginal; 

both the adrenal medulla and the pancreatic islets, 

4/47, 8/48, 2/48; Tables A1 and A3). However, in 

islets was also increased in the 200 ppm males (2/48, 

adenoma or carcinoma (combined) of the pancreatic 

Tables A1 and A3]. In addition, the incidence of 

chromocytoma (combined): 4/48, 15/46, 14/48, 8/48; 

12/48; 400 ppm, 7/48; benign or malignaIIL pheocytoma: 0 ppm, 4/48; 100 ppm, 14/46; 200 ppm, 

in 100 and 200 ppm male rats [benign pheochromonant pheochromocytoma (combined) were increased 

of benign pheochromocytoma and benign or maligOther organs: In the adrenal medulla, the incidences 
Results 39 

in the 2-Year Inhalation Study of Acetonitrile 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver of Male Rats 

TABLE 6 

Dose 0 PPm 100 ppm 200 ppm 400 ppm 

15-Month Interim Evaluation 

Number Examined 

Clear Cell Focus 

Basophilic Focusa 

Number Examined 

2-Year Study 

Mixed Cell Focus 

Eosinophilic Focus 

Clear Cell Focus 

Basophilic Focus 

Hepatocellular Adenomab 

Logistic regression testf 

First incidence (days) 

Terminal ratee 

Adjusted rated 

Overall rate' 

Hepatocellular Carcinomah 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

3 

6 

8 

0 

6 

8 

0 

7 

8 

2 

7 

8 

1 

3 

3 

15 

48 

1 

7 

1 

22 

47 

1 

5 

2 

25 * 

48 

5 

31** 

48 

5 

10 

P=O.O83 P=O.495 P=O.204 

2 623 727 

0/13 (0%) 2/17 (12%) 

0.0% 3.4% 4.8% 16.2% 

0/48 (0%) 1/47 (2%) 1/48 (2%) 3/48 (6%) 

0/11 (0%) 

P=O.492 

673 

OD (0%) 

1/48 (2%) 0/47 (0%) 0/48 (0%) 3/48 (6%) 

3.3% 0.0% 0.0% 14.5% 

P=O.121 P=0.504N P=0.500N P=O.374 

631 693 

on1 (0%) 0/13 (0%) OD (0%) 1/17 (6%) - - 

Hepatocellular Adenoma or Carcinoma' 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 1/48 (2%) 1/47 (2%) 1/48 (2%) 5/48 (10%) 

3.3% 3.4% 4.8% 25.2% 

P=O.O45 P=O.757 P=O.758 P=O.164 

637 623 673 693 

Oh1 (0%) 0/13 (0%) 0/9 (0%) 3/17 (18%) 

a Number of animals with lesion 

** PSO.01 

* Significantly different (PSO.05) from the control by the logistic regression test. 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Number of animals with neoplasm per number of animals with liver examined microscopically 

range, 0%-8% 

Historical incidence for 2-year inhalation studies with control groups (mean -C standard deviation): 11B98 (2.8% f 2.6%); 

lesions as nonfatal. A lower incidence in an exposure group is indicated by N. 

corresponding to the painvise comparisons between the controls and that exposed group. The logistic regression test regards these 

In the control column are the P values associated with the trend test. In the exposed group columns are the P values 

e Observed incidence in animals surviving until the end of the study 

g Not applicable; no neoplasms in animal group 

h Historical incidence: 4B98 (1.0% f 1.5%); range, 0%-4% ' Historical incidence: 15B98 (3.8% f 2.7%); range, 2%-8% 
40 Acetonitrile, NTP TR 447 

MICE 

females that survived were similar to those of the 

and body weight gains of all exposed groups of 

of the study (Table 7). The final mean body weights 

females receiving 800 ppm also died before the end 

one female receiving 400 ppm and one male and four 

died by week 3 of the study. Six additional animals, 

All male and female mice in the 1,600 ppm groups 

13-WEEK STUDY 

group. 

treatment-related findings were observed in any 

during the remainder of the study, and no other 

first week of the study. These findings did not recur 

were observed in 800 and 1,600 ppm mice during the 

controls. Hypoactivity and a bunched, rigid posture 

male groups were slightly lower than that of the 

controls. The final mean body weights of all exposed 

TABLE 7 

Survival and Body Weights of Mice in the 13-Week Inhalation Study of Acetonitrile 

Mean Body Weightb (9) 

Change Relative to Controls 

Final Weight 

Dose Survivala Initial Final' 

(PPm) ("/.I 

Male 

800 

400 

200 

100 

0 

1,600 

Female 

800 

400 

200 

100 

0 

1,600 

10/1 0 

10/10 

10/1 0 

9/10 

0/1 oe 

10/lOd 

10/10 

10/10 

0/10 

6/10; 

9/10 

'O/1Of 

25.1 f 0.2 

25.1 f 0.3 

24.8 f 0.2 

24.7 f 0.2 

25.0 f 0.4 

25.4 0.3 

20.5 f 0.2 

20.4 f 0.3 

20.2 f 0.3 

20.3 f 0.2 

21.0 f 0.4 

21.1 f 0.3 

32.5 +- 0.5** 

33.7 0.7 

34.2 ? 0.5 

32.9 +- 0.5 

35.1 f 0.5 

- 

29.4 f 0.9 

30.2 f 0.9 

29.0 ? 0.4 

29 9 k 0.8 

29.9 k 0.7 

- 

7.3 f 0.4* 

8.9 f 0.6 

9.5 f 0.5 

8.0 f 0.6 

9.7 f 0.5 

- 

8.3 f 0.7 

9.7 f 1.0 

8.6 f 0.5 

9.5 f 0.7 

8.9 f 0.6 

- 

92 

96 

97 

94 

103 

99 

102 

102 

a Number of animals surviving at 13 weekdnumber initially in group 

** P50.01 

* Significantly different (P<O.O5) from the control group by Williams' or Dunnett's test. 

Week of death: sometime during weeks 6-13 

Body weight taken during week 12. 

end of the study. No final mean body weights were calculated for groups with 100% mortality. 

Weights and weight changes are given as mean 1 standard error. Subsequent calculations are based on animals surviving to the 

Week of death: 1, 1, 1, 1, 3, 3, 3, 3, 3, 3 

g Week of death: 3, 3, 2 during weeks 6-13 

Week of death: 2 

e Week of death: 1, 1, 3, 3, 3, 3, 3, 3, 3, 3 
Results 41 

(Table F3). 

significantly greater than those of the controls 

of females exposed to 400 ppm and above were 

Relative liver weights of all exposed male groups and 

were significantly greater than those of the controls. 

tions of 200 ppm and above and of 800 ppm females 

Absolute liver weights of males exposed to concentrathese lesions corresponded to focal or multifocal 

200 ppm and above, respectively. Microscopically, 

exposed to concentrations of 400 ppm and above and 

anterior forestomach of male and female mice 

were consistently observed in the mucosa of the 

Focal or multifocal pale, dark brown or black lesions 

stomach, liver, and adrenal gland of mice (Table 8). 

Microscopic lesions were observed in the foreand female mice exposed to 400 ppm and 800 ppm. 

cellular cytoplasmic vacuolation occurred in the male 

exposed to 1,600 ppm. A high incidence of hepatoexposed to 200 ppm and one male and five females 

hyperplasia (Plates 5 and 6) occurred in one female 

Focal ulcers associated with areas of epithelial 

matory cell infiltrate in the adjacent submucosa. 

keratin layer (hyperkeratosis) and mixed inflamwas associated with mild thickening of the overlying 

thickening and folding (Plates 3 and 4). Hyperplasia 

epithelium (Plates 1 and 2) to marked epithelial 

Lesions varied from uniform, mildly thickened 

200 ppm in which the lesions were less prominent. 

groups, with the exception of female mice exposed to 

ity of these lesions was similar between exposure 

squamous epithelial hyperplasia. The average severof Acetonitrile 

Incidences of Selected Nonneoplastic Lesions in Mice in the 13-Week Inhalation Study 

TABLE 8 

0 ppm 100 ppm 200 ppm 400 ppm 800 ppm 1,600 ppm 

Male 

Forestomacha 

Ulcer 

Hyperplasiab 

Liver 

Cytoplasmic Vacuolation 

Female 

Ulcer 

Hyperplasia 

Forestomach 

Liver 

Cytoplasmic Vacuolation 

Fatty Degeneration 

Adrenal Gland, Cortex 

0 

0 

10 

0 

10 

0 

0 

10 

0 

10 

10 

0 

3 (1.7)d 

10 

8** (1.8) 

0 

0 

10 

0 

0 

10 10 

0 

7**(1.4) 8** (2.6) 

10 

1 (1.0) 

0 

10 - 

10 

0 

10 10 

7** (2.4) 

10 - 10 

9 (1.2) 10 (1.1) 6 (1.0) 

0 

6** (2.0) 

9 

7** (2.3) 

9 

0 

10 

7'* (2.1) 

10 

6** (3.0) 

10 

0** 

' 1 (2.0) 

1 (2.0) 

9e 

0 

10 

1 oe 

5' (2.0) 

5* (1.8) 

0 

10 

10 

0** 

* Significantly different (PSO.05) from the control group by the Fisher exact test 

Average severity grade of lesions in affected animals (1 = minimal; 2 = mild; 3 = moderate; 4 = marked) 

Animals in these groups not examined microscopically 

Number of animals with lesion 

a Number of animals with organ examined microscopically 

** (PSO.01) 

e All mice died by week 4. 
42 Acetonitrile, NTP TR 447 

may be an indication of stress- or exposure-related 

change in female mice exposed to 800 or 1,600 ppm 

exposed to 800 or 1,600 ppm, The absence of such a 

zone of the adrenal cortex, was absent in females 

normal age-related regression or involution of this 

exposed to 400 ppm. This change, which represents 

200 ppm and, to a lesser extent, in female mice 

female control mice and female mice exposed to 

observed in the X-zone of the adrenal cortex in 

hepatocyte glycogen stores. Fatty degeneration was 

that died may be indicative of increased utilization of 

of such changes in hepatocytes of 1,600 ppm mice 

to represent increased glycogen storage. The absence 

preexisting cytoplasmic clear spaces and is considered 

Vacuolation appeared to be a slight distension of 

bund animals. 

to be nonspecific changes typically observed in morimarrow, and pulmonary congestion, were considered 

lymphocytolysis in the thymus, spleen, and bone 

during the study, including lymphoid depletion and 

alterations that occurred only in mice that died 

acceleration of this normal process. Additional 

200 ppm. 

2-year inhalation study in mice were 50, 100, and 

acetonitrile exposure concentrations selected for the 

1,600 ppm males and females in the 13-week study, 

and gross and histopathologic lesions in 400,800, and 

Dose Selection Rationale: Based on reduced survival 
13 weeks. H&E: 45.5~ 

female B6C3F, mouse exposed to 400 ppm acetonitrile by inhalation for epithelium and mildly thickened keratin layer (hyperkeratosis). H&E; 87.5 X 

Mild focal hyperplasia of the squamous epithelium in the forestomach of a Higher magnification of Plate i. Note orderly maturation of the thickened 

PLATE 1 PLATE 2 

layer. HCE; 56x 

13 weeks. Note papillary-like folding of the epithelium and thickened keratin 

a female B6C3F, mouse exposed to 800 ppm acetonitrile by inhalation for 

Marked focal hyperplasia of the squamous epithelium in the forestomach of Higher magnification of Plate 3. H&E; 87.5 X 

PLATE 3 PLATE 4 
13 weeks. H&E; 35 X The keratin layer on the surface of the adjacent hyperplastic epithelium is 

of a male B6C3F, mouse exposed to 1,600 ppm acetonitrile by inhalation for inflammatory cell infiltrates (neutrophils) in the adjacent submucosal stroma. 

Ulcer within focal hyperplasia of the squamous epithelium in the forestomach Higher magnification of Plate 5. Note focal loss of the epithelium and 

PLATE 5 

slightly thickened. H&E; 115.5 X 

PLATE 6 
Results 43 

was significantly greater than that of controls. acetonitriIe. 

that of the controls. Survival of 200 ppm male mice were not considered related to exposure to 

exposed males and females was generally similar to (Figure 4, Tables 10 and 11). Clinical observations 

Kaplan-Meier survival curves in Figure 3. Survival of mean body weights of male and female mice 

and female mice are shown in Table 9 and in the 2 years had no effect on body weight gains or final 

Estimates of 2-year survival probabilities for male Exposure to acetonitrile by inhalation for up to 

Survival 

%YEAR STUDY 

Body Weights and Clinical Findings 

Survival of Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE 9 

50 PPm 100 ppm 200 ppm 

Male 

Animals initially in study 

Mean survival (days)c 

Percent probability of survival at end of studyb 

Animals surviving to study termination 

Moribund 

Natural deaths 

15-Month interim evaluationa 

Female 

Survival analysisd 

60 60 60 60 

64 

32 

13 

5 

10 

651 

64 

32 

14 

4 

10 

653 

64 

32 

16 

2 

10 

653 

6 

1 

10 

677 

86 

43 

P=0.013N P=l.OOON P = 1.000N P=0.017N 

Animals initially in study 

Mean survival (days) 

Percent probability of survival at end of study 

Animals surviving to study termination 

Moribund 

Natural deaths 

Accidental deatha 

15-Month interim evaluationa 

Survival analysis 

60 60 60 60 

642 

58 

28 

15 

6 

1 

10 

655 

67 

33e 

12 

5 

0 

10 

665 

58 

29 

13 

8 

0 

10 

629 

65 

32 

13 

5 

0 

10 

P=0.905N P=0.554N P=0.918N P=0.822N 

a Censored from survival analyses 

is indicated by N. 

comparisons (Cox, 1972) with the controls are in the exposed columns. A negative trend or lower mortality in an exposure group 

The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise 

Mean of all deaths (uncensored, censored, and terminal sacrifice) 

Kaplan-Meier determinations based on the number of animals alive on the first day of terminal sacrifice 

e Includes one animal that died the last week of the study 
44 Acetonitrile, NTP TR 447 

............. 

is 30 

-I 

............. 

.............. 

............... 

.............. 

i 

WEEKS 1 

............. 

............. 

............. 

............. 

t STUDY 

- 

- 

- 

i- 

..... 

t 

.... 

.... 

.... 

0.5 ! 75 0 15 so 45 60 

I 

WEEKS ON STUDY 

I 

90 105 

90 105 

0 

0 

by Inhalation for 2 Years 

Kaplan-Meier Survival Curves for Mice Administered Acetonitrile 

FIGURE 3 
Results 45 

a 30 60 90 

WEEKS ON STUDY 

IO5 

Growth Curves for Mice Administered Acetonitrile by Inhalation for 2 Years 

FIGURE 4
46 Acetonitrile, NTP TR 447 

Mean Body Weights and Survival of Male Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE 10 

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of 

Weeks 0 ppm 50 ppm 100 ppm 200 ppm 

Study (8) Survivors (8) controls) Survivors (8) controls) Survivors (g) controls) Survivors 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

* 11 

103 

101 

99 

97 

95 

93 

91 

89 

85 

81 

77 

73 

69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29 

25 

21 

17 

13 

12 

48.0 

48.0 

48.3 

47.9 

48.5 

48.4 

47.9 

47.7 

48.1 

47.6 

48.3 

48.4 

48.3 

48.0 

48.2 

48.0 

47.3 

47.8 

47.0 

45.8 

44.3 

43.2 

41.4 

40.4 

38.8 

37.7 

35.5 

34.6 

34.0 

33.8 

32.7 

32.2 

31.7 

31.5 

30.5 

29.3 

28.1 

27.2 

25.0 

Mean for weeks 

1-13 

53-103 48.1 

14-52 42.9 

31.2 

33 

35 

36 

36b 

38 

38 

40 

41 

44 

45 

48 

48 

48 

59 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

48.0 

47.2 

48.1 

47.4 

48.4 

48.7 

48.3 

49.0 

48.8 

47.9 

48.8 

48.9 

47.4 

47.8 

47.7 

47.3 

47.1 

46.9 

46.1 

45.2 

43.8 

42.4 

40.3 

38.6 

38.1 

35.9 

35.0 

34.5 

33.9 

33.4 

32.3 

31.6 

31.3 

30.8 

30.1 

29.1 

28.0 

27.0 

24.9 

48.1 

41.9 

30.9 

100 

98 

100 

99 

100 

101 

101 

103 

102 

101 

101 

101 

98 

100 

99 

99 

100 

98 

98 

99 

99 

98 

97 

96 

98 

95 

99 

100 

100 

99 

99 

98 

99 

98 

99 

99 

100 

99 

100 

100 

98 

99 

32 

34 

35 

36 

37 

40 

41 

43 

46 

48 

48 

48 

48 

58 

59 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

47.9 

47.8 

48.1 

48.6 

49.4 

49.0 

47.8 

48.5 

49.1 

49.3 

49.3 

49.2 

47.9 

48.2 

48.2 

47.5 

47.2 

47.3 

46.9 

45.8 

44.8 

43.2 

41.4 

39.5 

39.0 

37.3 

35.4 

34.7 

34.1 

33.4 

32.6 

32.0 

32.0 

31.3 

30.3 

29.1 

28.4 

27.3 

25.0 

48.4 

42.8 

31.2 

100 

100 

100 

102 

102 

101 

100 

102 

102 

104 

102 

102 

99 

100 

100 

99 

100 

99 

100 

100 

101 

100 

100 

98 

101 

99 

100 

100 

100 

99 

100 

99 

101 

99 

99 

99 

101 

100 

100 

101 

100 

100 

33 

34 

34 

35 

36 

39 

42 

43 

46 

48 

48 

49 

49 

59 

59 

59 

59 

59 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

47.9 

48.3 

48.7 

48.9 

49.4 

49.2 

49:0 

49.8 

49.9 

49.5 

50.0 

49.3 

48.7 

48.9 

48.6 

47.7 

47.3 

47.6 

47.4 

46.8 

44.8 

43.4 

41.4 

39.4 

38.2 

37.6 

35.7 

35.3 

35.0 

34.2 

33.1 

32.7 

32.1 

31.6 

30.5 

29.4 

28.4 

27.1 

24.6 

48.9 

43.0 

31.5 

100 

101 

101 

102 

102 

102 

102 

104 

104 

104 

104 

102 

101 

102 

101 

99 

100 

100 

101 

102 

101 

101 

100 

98 

99 

100 

101 

102 

103 

101 

101 

102 

101 

100 

100 

100 

101 

100 

98 

102 

100 

101 

44 

44 

45 

46 

47 

47 

48 

48 

48 

48 

49 

50 

50 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

a Interim evaluation occurred during week 66. 

The number of animals weighed for this week is fewer than the number of animals surviving. 
Results 

TABLE 11 

Mean Body Weights and Survival of Female Mice in the 2-Year Inhalation Study of Acetonitrile 

Study 

Weeks 0 ppm 50 ppm 100 pprn 200 ppm 

On Av. WL No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt Wt. (% of No. of 

(€9 Survivors (9) controls) Survivors (8) controls) Survivors (8) controls) Survivors 

9 

8 

7 

6 

5 

4 

3 

2 

1 

103 

101 

99 

97 

95 

93 

91 

89 

85 

81 

77 

73 

69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29 

25 

21 

17 

13 

12 

11 

10 

48.2 

47.2 

47.3 

46.9 

47.5 

48.0 

47.9 

48.0 

49.8 

48.4 

48.6 

47.7 

47.6 

47.8 

47.1 

46.1 

44.9 

44.5 

43.6 

41.7 

39.7 

37.3 

36.2 

34.1 

33.1 

31.8 

29.5 

28.8 

28.0 

27.5 

26.7 

26.4 

25.5 

25.5 

24.8 

23.9 

22.8 

22.2 

20.3 

Mean for weeks 

53-103 47.6 

14-52 

1-13 

38.0 

25.5 

28 

30 

35 

37 

38 

40 

41 

43 

44 

46 

46 

47 

47 

57 

57 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

59 

60 

47.7 

46.3 

47.0 

46.8 

47.3 

48.4 

48.9 

48.5 

50.7 

50.2 

49.6 

49.5 

47.6 

47.8 

47.2 

46.1 

44.9 

44.9 

43.8 

42.0 

39.8 

38.0 

36.2 

34.7 

33.4 

31.0 

29.9 

29.6 

28.3 

27.1 

26.8 

26.3 

25.4 

25.2 

24.3 

23.6 

22.5 

21.8 

20.0 

47.9 

38.2 

25.5 

100 

100 

101 

102 

101 

102 

104 

102 

104 

100 

100 

100 

100 

100 

101 

101 

101 

100 

102 

100 

102 

101 

98 

101 

103 

101 

101 

100 

100 

100 

99 

98 

99 

99 

98 

99 

99 

98 

99 

101 

101 

100 

33 

35 

35 

37 

38 

40 

42 

43 

46 

47 

48 

48 

48 

58 

59 

59 

59 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

47.4 

46.1 

46.3 

47.5 

47.8 

48.9 

48.2 

48.4 

50.2 

49.3 

49.0 

48.6 

47.9 

47.9 

47.0 

46.4 

45.7 

44.7 

44.0 

41.4 

40.5 

38.8 

37.0 

35.2 

34.0 

31.7 

29.4 

29.1 

28.4 

27.6 

26.9 

26.1 

25.5 

25.4 

24.6 

23.6 

22.7 

21.8 

20.0 

47.8 

38.6 

25.5 

102 

104 

102 

103 

103 

100 

100 

101 

101 

100 

101 

99 

100 

100 

99 

99 

100 

98 

99 

101 

101 

102 

101 

101 

101 

102 

101 

102 

101 

100 

100 

101 

102 

100 

101 

99 

98 

98 

98 

100 

102 

100 

31 

35 

37 

37 

43 

44 

46 

47 

47 

49 

50 

50 

50 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

45.7 

45.6 

46.2 

46.3 

47.1 

46.9 

45.5 

46.2 

46.8 

47.3 

48.1 

46.9 

46.4 

45.8 

45.7 

44.6 

43.8 

44.0 

43.2 

41.4 

38.0 

36.6 

34.9 

33.0 

31.7 

30.7 

28.5 

28.5 

28.3 

27.5 

26.9 

26.3 

26.0 

25.6 

24.5 

23.6 

23.1 

21.9 

19.8 

46.2 

37.1 

25.4 

95 

97 

98 

99 

99 

98 

95 

96 

94 

98 

99 

98 

98 

96 

97 

97 

98 

99 

99 

99 

96 

98 

96 

97 

96 

97 

97 

99 

101 

100 

101 

100 

102 

100 

99 

99 

101 

99 

98 

97 

98 

100 

32 

34 

34 

34 

35 

37 

39 

40 

40 

40 

41 

41 

45 

56 

57 

57 

57 

57 

58 

59 

59 

59 

59 

59 

59 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

60 

a Interim evaluation occurred during week 66. 
48 Acetonitrile, NTP TR 447 

that occurred with an incidence of at least 5% in at 

diagnoses, statistical analyses of primary neoplasms 

nonneoplastic lesions, individual animal tumor 

Summaries of the incidences of neoplasms and 

plasms of the lung, liver, and forestomach. 

nonneoplastic lesions of the forestomach, and neobiologically noteworthy changes in the incidences of 

This section describes the statistically significant or 

Pathology and Statistical Analyses 

for female mice. 

sented in Appendix C for male mice and Appendix D 

the neoplasms mentioned in this section are preleast one animal group, and historical incidences for 

males (Tables 12 and C3) and was at the upper limit 

incidence was significantly increased in 200 ppm 

bronchiolar adenoma in exposed male mice. The 

Lung There was an increased incidence of alveolar/ 

in the 2-Year Inhalation Study of Acetonitrile 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung of Mice 

TABLE 12 

Dose 0 PPm 50 PPm 100 ppm 200 ppm 

Male 

15-Month Interim Evaluation 

Number Examined 

2 

1 

10 

Alveolar/bronchiolar Carcinoma 

Alveolarbronchiolar Adenomaa 

&Year Study 

Lung 50 

Alveolarbronchiolar Adenoma' 

Alveolar Epithelium, Hyperplasia 4 (I$ 

Logistic regression testg 

First incidence (days) 

Terminal ratef 

Adjusted ratee 

overall rated 

733 (9 

6/32 (19%) 

18.8% 

6/50 (12%) 

P=O.OlO 

Alveolar/bronchiolar Carcinomah 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

P=0.450N 

729 

3/32 (9%) 

12.1% 

4/50 (8%) 

Alveolarbronchiolar Adenoma or Carcinoma' 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

P=O.O38 

I29 

9/32 (28%) 

30.3% 

10/50 (20%) 

(continued) 

1 

3 

10 

50 

5 (1.2) 

9/32 (28%) 

28.1% 

9/50 (18%) 

P=O.279 

733 (T) 

P=O.374 

583 

4/32 (13%) 

16.5% 

6/50 (12%) 

P=O.239 

583 

12/32 (38%) 

40.3% 

14/50 (28%) 

0 

2 

10 

48 

3 (1.3) 

P=O.375 

727 

7/32 (22%) 

24.2% 

8/48 (17%) 

P=O.367 

607 

3/32 (9%) 

16.4% 

6/48 (13%) 

P=O.234 

607 

1002 (31%) 

38.5% 

14/48 (29%) 

0 

3 

10 

50 

2 (1.5) 

P=O.O11 

554 

14/43 (33%) 

38.2% 

18/50 (36%) 

9.3% 

4/50 (8%) 

733 (T> 

4/43 (9%) 

P=0.491N 

P=O.O42 

554 

17/43 (40%) 

44.6% 

21/50 (42%) 
Results 49 

in the 2-Year Inhalation Study of Acetonitrile (continued) 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung of Mice 

TABLE 12 

Dose 0 PPm 50 PPm 100 ppm 200 ppm 

Female 

15-Month Interim Evaluation 

Number Examined 

Adenoma 

2-Year Study 

0 

10 

Number Examined 49 

Alveolar/bronchiolar Adenoma] 

Alveolar Epithelium, Hyperplasia 3 (1.0) 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

P=0.003N 

735 (T) 

7/28 (25%) 

25.0% 

7/49 (14%) 

Alveolarbronchiolar Carcinoma' 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

3.1% 

1/49 (2%) 

P=0.621N 

706 

0/28 (0%) 

Alveolarbronchiolar Adenoma or Carcinomam 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

P=0.007N 

706 

7/28 (25%) 

27.3% 

8/49 (16%) 

0 

10 

50 

4 (1.8) 

P=0.044N 

735 (T) 

2/33 (6%) 

6.1% 

2/50 (4%) 

P=0.753N 

735 (T) 

1/33 (3%) 

3.0% 

1/50 (2%) 

P=0.065N 

735 (T) 

9.1% 

3/50 (6%) 

3/33 (9%) 

2 

10 

50 

2 (1.5) 

P=0.067N 

735 (T) 

2/29 (7%) 

6.9% 

2/50 (4%) 

0/50 (0%) 

P=0.491N 

0/29 (0%) 

0.0% 

- 

2/29 (7%) 

6.9% 

2/50 (4%) 

P=0.032N 

735 (") 

1 

10 

49 

1 (2.0) 

0/32 (0%) 

0.0% 

0/49 (0%) 

P=0.005N 

- k 

3.1% 

1/49 (2%) 

735 (T> 

1/32 (3%) 

P=O.758 

P=0.012N 

735 (T) 

1/32 (3%) 

3.1% 

1/49 (2%) 

a Number of animals with lesion 

(")Terminal sacrifice 

Number of animals with neoplasm per number of animals with lung examined microscopically 

range, 6%-36% 

Historical incidence for 2-year inhalation studies with control groups (mean rC_ standard deviation): 113/673 (16.8% f 7.6%); 

Average severity of lesions in affected mice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

corresponding to the pairwise comparisons between the controls and that exposed group. The logistic regression test regards these 

Observed incidence in animals surviving until the end of the study 

Historical incidence: 58/659 (8.8% f 3.5%); range, 0%-15% 

Not applicable; no neoplasms in animal group 

Historical incidence: 150/673 (22.3% rf: 9.0%); range, 10%-42% 

lesions as nonfatal. A negative trend or lower incidence in an exposure group is indicated by W. 

g In the control column are the P values associated with the trend test. In the exposed group columns are the P values 

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

' Historical incidence: 19/659 (2.9% f 2.5%); range, 0%-6% 

1 Historical incidence: 40/659 (6.1% f 2.8%); range, 0%-10% 

h Historical incidence: 45/673 (6.7% f 5.6%); range, 0%-16% 

m 
50 Acetonitrile, NTP TR 447 

of the range o f historical controls (Ta 

cal controls (Tables 12 and D4a). 

controls were greater than the upper range of historiand D3); the incidences of these neoplasms in female 

exposure concentration in females (Tables 12 

or carcinoma (combined) were inversely related to 

dences of alveolar/bronchiolar adenoma and adenoma 

increased in exposed males. In contrast, the inciadenoma or carcinoma (combined) were similarly 

and C4a). The incidences of alveolar/bronchiolar 

tbles 12 

controls (Tables 13 and D3). 

or carcinoma (combined) were similar to those of 

hepatocellular adenoma and hepatocellular adenoma 

C3, and C4b). in exposed females, the incidences of 

exceeded the range of historical controls (Tables 13, 

significantly increased in 100 ppm ales and 

cellular adenoma or carcinoma (combined) was 

than that in the controls. The incidence of hepatocarcinoma in 100 ppm males was significantly greater 

Liver: At 2 years, the incidence of hepatocellular 

Incidences of Neoplasms of the Liver of Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE 13 

Dose 0 PPm 50 PPm 100 ppm 200 ppm 

Number Examined 

15-Month Interim Evaluation 

Male 

Hepatocellular Carcinoma 

Hepatocellular Adenomaa 

2-Year Study 

Hepatocellular Adenomab 

Logistic regression testf 

First incidence (days) 

Terminal ratee 

Adjusted rated 

Overall rate' 

Hepatocellular Carcinomag 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Hepatocellular Adenoma or Carcinomah 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

(continued) 

1 

2 

10 

P=0.293N 

563 

9/32 (28%) 

35.0% 

13/50 (26%) 

P=O.163 

437 

15.3% 

7/50 (14%) 

1/32 (3%) 

P=0.437N 

437 

10/32 (31%) 

44.5% 

19/50 (38%) 

3 

1 

10 

P=0.500N 

434 

8/32 (25%) 

31.7% 

12/50 (24%) 

P=O.128 

571 

24.6% 

11/50 (22%) 

1/32 (3%) 

P=O.394 

434 

9/32 (28%) 

46.7% 

21/50 (42%) 

0 

2 

10 

P=O.189 

595 

14/32 (44%) 

49.1% 

18/49 (37%) 

P=O.O38 

342 

4/32 (13%) 

30.3% 

13/49 (27%) 

P=O.O13 

342 

18/32 (56%) 

67.7% 

30/49 (61%) 

0 

1 

10 

P=0.249N 

701 

8/43 (19%) 

22.2% 

10/50 (20%) 

P =0.208 

532 

4/43 (9%) 

15.0% 

7/50 (14%) 

P=0.454N 

532 

11/43 (26%) 

31.8% 

15/50 (30%) 
Results 51 

Incidences of Neoplasms of the Liver of Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE 13 

Dose 0 PPrn 50 PPrn 100 pprn 200 pprn 

Female 

15-Month Interirn Evaluation 

Number Examined 

Hepatocellular Adenoma 

2-Year Study 

Hepatocellular Adenoma' 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Hepatocellular Carcinoma] 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Hepatocellular Adenoma or Carcinomak 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

0 

10 

2/28 (7%) 

4/49 (8%) 

P=O.390 

569 

12.0% 

P=0.340N 

706 

5/28 (18%) 

23.1% 

7/49 (14%) 

P=O.489 

569 

6/28 (21 %) 

28.0% 

9/49 (18%) 

2 

10 

P=O.191 

664 

6/33 (18%) 

22.4% 

8/50 (16%) 

P=0.475N 

595 

15.8% 

6/50 (12%) 

3/33 (9%) 

P=O.257 

595 

8/33 (24%) 

33.5% 

13/50 (26%) 

1 

10 

P=O.211 

708 

6/29 (21 %) 

25.6% 

8/50 (16%) 

P=0.457N 

570 

2/29 (7%) 

16.4% 

6/50 (12%) 

P=O.284 

570 

8/29 (28%) 

36.9% 

13/50 (26%) 

0 

10 

6/32 (19%) 

18.8% 

6/49 (12%) 

P =0.346 

735 (T) 

P=0.399N 

510 

3/32 (9%) 

13.6% 

5/49 (10%) 

P=O.466 

510 

8/32 (25%) 

28.5% 

10/49 (20%) 

a Number of animals with neoplasm per number of animals with liver examined microscopically 

(T)Terminal sacrifice 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Number of animals with neoplasm per number of animals examined microscopically 

range, 4%-38% 

Historical incidence for 2-year inhalation studies with control groups (mean f standard deviation): 120/673 (17.8% f 11.0%); 

Historical incidence: 136/673 (20.2% f 5.9%); range, 9%-29% 

lesions as nonfatal. A negative trend or lower incidence in an exposure group is indicated by N. 

corresponding to the pairwise comparisons between the controls and that exposed group. The logistic regression test regards these 

In the control column are the P values associated with the trend test. In the exposed group columns are the P values 

Historical incidence: 111/657 (16.9% f 8.7%); range, 3%-31% 

1 Historical incidence: 57/657 (8.7% 2 4.8%); range, 0%-16% 

Historical incidence: 56/657 (8.5% f 6.2%); range, 0%-22% 

h Historical incidence: 241/673 (35.8% f 12.1%); range, 11%-56% 

e Observed incidence in animals surviving until the end of the study 
52 Acetonitrile, NTP TR 447 

100 and 200 ppm females (Tables 14, C5, and D5). 

the increases were significant in 200 ppm males and 

increasing exposure concentration in exposed groups; 

females. At 2 years, the incidences increased with 

however, the increase was only significant in 200 ppm 

200 ppm was greater than that in the controls; 

stomach in males and females exposed to 50 or 

the incidence of squamous hyperplasia of the forecontrols (Tables 14, C~C, and D4c). At 15 months, 

equaled the highest values observed in the historical 

200 ppm (Tables 14, C1, and Dl) and these rates 

increased in male and female mice exposed to 

papilloma of the forestomach was marginally 

Forestomach: The incidence of squamous cell 

branched core of fibrous connective tissue stroma. 

hyperkeratotic, squamous epithelium supported by a 

like masses composed of hyperplastic, sometimes 

Papillomas were exophytic, pedunculated, and frondinflammation occurred in some severe lesions. 

the mucosal surface. Focal ulcers and suppurative 

lesions, folds of thickened epithelium projected above 

epithelial thickening and folding. In markedly severe 

Increasing severity was accompanied by progressive 

keratin layer and increased numbers of basal cells. 

accompanied by slight thickening of the overlying 

by slight thickening of the epithelium, frequently 

marked severity. Minimal lesions were characterized 

Hyperplasia was generally focal and of minimal to 

in the 2-Year Inhalation Study of Acetonitrile 

Incidences of Neoplasms and Nonneoplastic Lesions of the Forestomach of Mice 

TABLE 14 

0 PPm 50 PPm 100 ppm 200 ppm 

Male 

15-Month Interim Evaluation 

Number Examined 

Squamous Hyperplasiaa 

2-Year Study 

Number Examined 

Squamous Hyperplasia 

0 

10 

2 

10 

0 

10 

3 

10 

49 50 48 50 

3 (2.3)b 3 (3.3) 6 (2.5) 12' (1.9) 

Squamous cell PapilIoma' 0 0 1 2 

Female 

Number Examined 

15-Month Interim Evaluation 

Squamous Hyperplasia 

Number Examined 

2-Year Study 

Squamous Cell Papillomad 

Squamous Hyperplasia 

0 

10 

1 

10 

0 

10 10 

6* * 

49 50 50 

1 

9** (2.3) 19** (2.6) 

48 

2 (3.5) 

0 

I (2.6) 

1 3 

regression test (2-year study) 

** Significantly different (PsO.01) from the control group by the Fisher exact test (15-month interim evaluation) or the logistic 

* Significantly different (P50.05) from the control group by the logistic regression test 

Historical incidence: 8/661 (1.2% 2 2.0%); range, 0%-6% 

range, 0%-4% 

Historical incidence for 2-year inhalation studies with control groups (mean -t- standard deviation): W676 (0.7% 2 1.3%); 

Average severity grade of lesions in affected animals (1 = minimal; 2 = mild; 3 = moderate; 4 = marked) 

a Number of animals with lesion 
Results 53 

GENETIC TOXICOLOGY 

concluded to be equivocal. 

significant (P>O.O15) and the trial results were 

trial conducted with S9, the trend test was not 

increase in aberrations noted at the high dose in the 

highest dose tested (5,000 pLg/mL). Despite the 

both endpoints, the increases were noted at the 

presence of S9 (Galloway et al., 1987; Table E3); for 

1987; Table E2) and chromosomal aberrations in the 

atid exchanges in the absence of S9 (Galloway et al., 

cells, acetonitrile was a weak inducer of sister chromcytogenetic tests with cultured Chinese hamster ovary 

nation (Mortelmans et al., 1986; Table El). In 

activity was observed in any straidactivation combi1254-induced rat and hamster liver S9; no mutagenic 

TA1535, and TA1537, with and without Aroclor 

Salmonella typhimurium strains TA97, TA98, TA100, 

two laboratories for induction of mutations in 

Acetonitrile (100 to 10,000 /..@plate) was tested in 

observed in the 400 ppm group. 

micronucleated normochromatic erythrocytes was 

but in males, a small but significant increase in 

(Table E4). Results with female mice were negative 

acetonitrile (100 to 800 ppm) by inhalation 

male and female mice treated for 13 weeks with 

chromatic erythrocytes in peripheral blood samples of 

determining the frequency of micronucleated normodamage in mammalian cells in vivo was assessed by 

The ability of acetonitrile to induce chromosomal 

micronucleated normochromatic erythrocytes. 

observed in the form of increased frequencies of 

evidence for chromosomal damage in male mice was 

genic activity in cultured mammalian cells; in vivo, 

mutations in bacteria and showed only weak clastoIn conclusion, acetonitrile did not induce gene 
54 Acetonitrile, NTP TR 447 
55 

DISCUSSION AND CONCLUSIONS 

in which some animals died. 

resulted in death, or in survivors in exposure groups 

exposed to concentrations lower than those that 

with little evidence of adverse effects in animals 

dose response for acetonitrile toxicity is quite steep, 

on the toxicity information in the literature. The 

acetonitrile are consistent with those expected based 

The results of the 13-week inhalation studies of 

these concentrations. 

survivors were affected by acetonitrile exposure at 

800 or 1,600 ppm. This would suggest that he 

males exposed to 1,600 ppm and females exposed to 

edema. A mild nonresponsive anemia occurred in 

of organ weights, suggesting possible congestion and 

exposed to 1,600 ppm did show increases in a number 

(Way, 1981; Ballantyne, 1983). Surviving female rats 

organs, and gastric erosions have been reported 

often show congestion with hemorrhage in various 

produced in acute cyanide poisoning, but tissues 

There are no specific gross or histopathologic lesions 

current study only in those rats that died early. 

seen in debilitated animals and occurred in the 

tissues and corpora lutea of the ovary are frequently 

anoxia. Atrophy and cellular depletion of lymphoid 

1975b) and have been attributed to cyanide-induced 

reports (Pozzani et al., 1959a; Haguenoer et al., 

of hemorrhage in the brain are consistent with prior 

edema, and hemorrhage in the alveoli and evidence 

NTP studies. Pulmonary lesions of congestion, 

typically encountered in early death animals in other 

acetonitrile-induced toxicity, in addition to lesions 

lesions consistent with previous descriptions of 

Rats that died during the study showed a spectrum of 

there significant exposure-related clinical findings. 

exposure concentrations below 1,600 ppm, nor were 

lethality. Body weight gains were not affected at 

or a range of susceptibility to acetonitrile-induced 

tion of the remaining animals to continuing exposure 

the first week of exposure, suggesting either adapta800 ppm died. The deaths occurred primarily during 

exposed to 1,600 ppm and one male exposed to 

In the 13-week study, several male and female rats 

represent a true effect of acetonitrile exposure. 

with cyanide intoxication, as noted above, and may 

sure. However, gastric erosions have been observed 

other factor associated with the conditions of exporelated to acetonitrile exposure but rather to some 

suggesting that the effect may not be specifically 

other NTP inhalation studies (Melnick et al., 1990), 

forestomach lesions occasionally seen in mice in 

stomach. The character of the lesion was similar to 

occasional inflammation and ulceration in the foreThe most significant lesions were hyperplasia with 

the adrenal gland, the liver, and the forestomach. 

In mice, acetonitrile-related toxicity was observed in 

largely absent in mice dying during the first 3 weeks. 

surviving to the end of the study. These lesions were 

in mice, occurring primarily among those mice 

lesions other than lymphoid depletion were observed 

findings were not noted. In contrast to the rats, 

vors were not markedly affected and specific clinical 

group. As with the rats, body weight gains of surviand deaths of females extended down to the 400 ppm 

the 13-week mouse study was complete at 1,600 ppm, 

acetonitrile-induced mortality than rats. Mortality in 

Mice appeared somewhat more susceptible to 

were lethal in the 13-week studies. 

selected were within a factor of two of those that 

for ats. Thus, the high exposure concentrations 

highest exposure concentration for mice and 400 ppm 

effects in mice, led to the selection of 200 ppm as the 

400 and 800 ppm, coupled with the forestomach 

greater number of deaths of mice than rats at 

the mortality observed in the 13-week studies. The 

exposure concentrations for the 2-year studies was 

The primary factor influencing the selection of 

15 months, minimal anemia was noted in females 

age-related decline in acetonitrile metabolism. At 

1975a,b) and suggests that there is no significant 

does not accumulate in the body (Haguenoer et al., 

agrees with previous data indicating that acetonitrile 

survival were affected by acetonitrile exposure. This 

In the 2-year rat study, neither body weight gains nor 
56 Acetonitrile, NTP TR 447 

2 years. 

lesions in male or female rats at 15 months or 

dence of significant exposure-related nonneoplastic attribute to the chemical with any confidence. 

exposed to 400 ppm. However, there was no evi- magnitude of the neoplastic findings is insufficient to 

The incidence of alveolarbronchiolar adenoma in 

uncertain. 

exposure and liver neoplasia in male rats remains 

Overall, a causal relationship between acetonitrile 

that a liver tumor response is biologically plausible. 

acetonitrile-related effect on the liver and suggests 

hepatocellular foci does provide support for an 

evidcnce of chemical-related increased incidences of 

carcinogenic process (Harada et al., 1989). However, 

those considered more directly involved in the 

understood, these foci did not appear atypical, as with 

hepatocellular foci and neoplasia is not clearly 

While the specific relationship between various 

were marginally increased in the 400 ppm group. 

and the incidences of eosinophilic and mixed cell foci 

increased in the 200 and 400 ppm exposure groups, 

dences of basophilic foci were also significantly 

studies (NTP, 1989, 1991, 1993, 1994b). The incinoted in four previous control groups in dosed feed 

control group (8%). A 10% incidence has been 

upper ange of incidences observed in any one 

for inhalation studies and slightly higher than the 

higher than the historical control incidence of 3.8% 

adenoma or carcinoma (combined) (5/48, 10%) is 

400 ppm males, the incidence of hepatocellular 

versus the control incidence were not significant. In 

pairwise comparisons of the exposed group incidences 

achieved (P=O.O45) for a dose-related trend, but 

rats exposed to 400 ppm. Statistical significance was 

cellular adenoma or carcinoma (combined) in male 

was a marginal increase in the incidence of hepatoThe only partially positive neoplastic finding in rats 

nitrile exposure on the forestomach of mice, but the 

these findings establish an effect of prolonged acetowere within the ranges of historical controls. Thus, 

not statistically significant in males or females and 

female. The incidences of this benign tumor were 

to 100 or 200 ppm, and one occurred in a control 

squamous cell papillomas were noted in mice exposed 

and in females exposed to 100 or 200 ppm. A few 

squamous hyperplasia in males exposed to 200 ppm 

mice, with significant increases in the incidences of 

on the forestomach were again noted in exposed 

significantly greater than that of the controls. Effects 

fact, survival of male mice exposed to 200 ppm was 

weight gains and survival were not observed and, in 

In the 2-year study in mice, adverse effects on body 

attributed to acetonitrile exposure. 

bronchiolar adenoma in the 200 ppm males was not 

these reasons, the increased incidence of alveolar/ 

may have contributed to the apparent effect. For 

survival of this group at 2 years was quite high and 

control groups for inhalation studies, the 86% 

equal to the highest incidence seen in previous 

adenoma (18/50,36%) in the 200 ppm male group is 

study. Although the incidence of alveolar/bronchiolar 

three stages, which was not apparent in the current 

manifest as an increase in proliferative lesions of all 

incidence in this neoplasm type would be expected to 

the lesion. Thus, a true chemical-related increased 

based on the size and morphologic characteristics of 

tion between hyperplasia, adenoma, and carcinoma 

region were present as a continuum, with the distincProliferative lesions of the alveolarbronchiolar 

with a significant dose-related trend in females. 

incidence of alveolarbronchiolar adenoma decreased 

greater than that in the controls. Conversely, the 

male mice exposed to 200 ppm was significantly 

exposure. 

this is a sporadic finding unrelated to acetonitrile 

lower body weight of 200 ppm males, suggests that 

decrease the neoplasm response, such as a markedly 

confounding factors that would be expected to 

neoplasms in males coupled with an absence of 

evidence for a dose response in hepatocellular 

hepatocellular neoplasia in the females. The lack of 

indication of an effect of acetonitrile exposure on 

less than those in the controls, and there was no 

of hepatocellular neoplasms in 200 ppm males were 

increased in 100 ppm males. However, the incidences 

cellular adenoma or carcinoma (combined) was also 

100 ppm acetonitrile. The incidence of hepatosignificantly increased in male mice exposed to 

The incidence of hepatocellular carcinoma was 

Tennant, 1991). 

structural alerts for genotoxic activity (Ashby and 

Nitriles have not been specifically designated as 

chromatic erythrocytes in mice in the 13-week study. 

a marginal increase inmicronucleated normoeffects in cultured mammalian cells. It also induced 

with Salmonella and exhibited weak clastogenic 

Acetonitrile was found to be nonmutagenic in studies 
Discussion and Conclusions 57 

CONCLUSIONS evidence ofcarcinogenic activity of acetonitrile in male 

rats exposed to 100, 200, or 400 ppm. There was no stomach in male and female mice. 

carcinogenic activity of acetonitrile in female F344/N male rats and of squamous hyperplasia of the foreadenoma and carcinoma. There was no evidence of increased incidences of hepatic basophilic foci in 

marginally increased incidences of hepatocellular Exposure to acetonitrile by inhalation resulted in 

activity* of acetonitrile in male F344/N rats based on 

studies, there was equivocal evidence of carcinogenic 2oo 

Under the conditions of these 2-year inhalation Or B6C3F1 mice exposed to 50, loo Or 

Subcommittee comments and the public discussion on this Technical Report appears on page 11. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 9. A summay of the Technical Reports Review 
58 Acetonitrile, NTP TR 447 
59 

REFERENCES 

Lett. 12, 157-163. 

Comparative toxicities of aliphatic nitriles. Toxicol. 

Ahmed, A.E., and Farooqui, M.Y.H. (1982). 

70, 322-330. 

2-’4C-acetonitrile in mice. Pharmacol. Toxicol. 

distribution and macromolecular interaction of 

acetonitrile toxicity I: Whole body autoradiographic 

Hussein, G.I. (1992). Studies on the mechanism of 

Ahmed, A.E., Loh, J.-P., Ghanayem, B., and 

acetonitrile. J. Occup. Med. 1, 627-633. 

Amdur, M.L. (1959). Accidental group exposure to 

J. Appl. Toxicol. 3, 272-290. 

214 industrial chemicals in air and water dilution. 

with threshold limit values and volatilities for 

aid to chemical safety: Odor thresholds compared 

Amoore, J.E., and Hautala, E. (1983). Odor as an 

York. 

Research, pp. 362-365. John Wiley and Sons, New 

Armitage, P. (1971). Statistical Methods in Medical 

the U.S. NTP. Mutat. Res. 257, 229-306. 

genicity, and mutagenicity for 301 chemicals tested by 

relationships among chemical structure, carcinoAshby, J., and Tennant, R.W. (1991). Definitive 

Toxicol. 3, 400-408. 

toxicity by cyanides and cyanogens. Fundam. Appl. 

Ballantyne, B. (1983). Artifacts in the definition of 

9, 1349-1351. 

the lower troposphere. Geophys. Res. Lett. 

Becker, K.H., and Ionescu, A. (1982). Acetonitrile in 

rats. Toxicologist 2, 118. 

(1982). Teratogenic evaluation of aliphatic nitriles in 

Berteau, P.E., Levinskas, G.J., and Rodwell, D.E. 

Report (April 27, 1984). Project No. 11725. 

hamster ovary (CHO) cells on acetonitrile. Revised 

mutation assay (HGPRT locus) in cultured Chinese 

Bioassay Systems Corp. (1984). In vivo gene 

pp. 345-357. Noyes Publications, Park Ridge, NJ. 

Testing (H.A. Milman and E.K. Weisburger, Eds.), 

carcinogenicity studies. In Handbook of Carcinogen 

(1985). Quality assurance in pathology for rodent 

Wolfe, M.J., McConnell, E.E., and Hardisty, J.F. 

Boorman, G.A., Montgomery, C.A., Jr., Eustis, S.L., 

research laboratories. Chem. Eng. News 68, 15. 

Borman, S. (1990). Acetonitrile shortage hurts 

inhibition test. Water Res. 14, 231-241. 

algae, and protozoa in the cell multiplication 

the toxicity thresholds of water pollutants to bacteria, 

Bringmann, G., and Kuhn, R. (1980). Comparison of 

containing cosmetic. JAM 260, 3470-3473. 

cyanide intoxication and death from an acetonitrileCaravati, E.M., and Litovitz, T.L. (1988). Pediatric 

J. R. Stat. Soc. B34, 187-220. 

Cox, D.R. (1972). Regression models and life-tables. 

Code of Federal Regulations (CFR) 21, Part 58. 

Co., Inc., Princeton, NJ. 

Eds.), pp. 13-59. Princeton Scientific Publishing 

(M.A. Mehlman, W.G. Flamm, and R.J. Lorentzen, 

Toxicity of Chemical Carcinogens and Mutagens 

Modern Environmental Toxicology: Mechanisms and 

mutation model of carcinogenesis. In Advances in 

Crawford, B.D. (1985). Perspectives on the somatic 

in human lungs. Arch. Environ. Health 17, 746-748. 

(1968a). Retention of cigarette smoke components 

Dalhamn, T., Edfors, M.-L., and Rylander, R. 

cigarette smoke. Arch. Environ. Health 16, 831-835. 

(1968b). Mouth absorption of various compounds in 

Dalhamn, T., Edfors, M.-L., and Rylander, R. 

mortel. Eur. J. Toxicol. Environ. Hyg. 7, 91-97. 

Les intoxications par l’acktonitrile B propos d’un cas 

Scherpereel, P., Gosselin, B., and Ginestet, A. (1974). 

DeQuidt, J., Furon, D., Wattel, F., Haguenoer, J.M., 
60 Acetonitrile, NTP TR 447 

Toxicol. 6, 44-52. Margolin, B.H., Resnick, M.A., Anderson, B., and 

animal carcinogenicity experiments. Fundam. Appl. Nakamura, F., Ahmed, M., Duk, S., Rimpo, J., 

regression analysis of incidental-tumor data from Colman, S., Brown, B., Cannon, C., Bloom, A.D., 

Dime, G.E., and Haseman, J.K. (1986). Logistic Galloway, S.M., Armstrong, M.J., Reuben, C., 

32, 236-248. 

analysis of tumour prevalence data. Appl. statist. Cells: Evaluations Of 108 chemicals. Environ. Mol. 

Dinse, G.E., and Lagakos, S.W. (1983). Regression Sister chromatid exchanges in.Chinese hamster Ovary 

Zeiger, E. (1987). Chromosome aberrations and 

Mutagen. 10, 1-175. 

Hill Book Company, Inc., New York. 

to Statistical Analysis, 1st ed., pp. 145-147. McGrawDixon, W.J., and Massey, F.J., Jr. (1951). Introduction 

62, 957-974. 

tests for carcinogenicity. J. Natl. Cancer Inst. 

Statistical issues in interpretation of chronic bioassay 

Gart, J.J., Chu, KC., and Tarone, R.E. (1979). 

sums. Technometrics 6, 241-252. 

Dum, O.J. (1964). Multiple comparisons using rank 

control. J. Am. Stat. Assoc. 50, 1096-1121. 

procedure for comparing several treatments with a 

Dunnett, C.W. (1955). A multiple comparison 

John Wiley and Sons, New York. 

Industrial Hygene and Toxicology (F.A. Patty, Ed.). 

Fassett, D.W. (1963). Cyanide and nitriles. In Patty's 

18, 219-232. 

mutagenicity using the Ames' test. Toxicology 

(1980). Screening of tobacco smoke constituents for 

Florin, I., Rutberg, L., Curvall, M., and Enzell, C.R. 

J. Toxicol. Environ. Health 15, 609-621. 

potentiation of acute acetonitrile toxicity in rats. 

Freeman, J.J., and Hayes, E.P. (1985a). Acetone 

cyanide. Toxicologist 5, 246. 

acetone on microsomal metabolism of acetonitrile to 

Freeman, J.J., and Hayes, E.P. (1985b). Effects of 

hepatocytes. Fundam. Appl. Toxicol. 8, 263-271. 

olism of acetonitrile to cyanide by isolated rat 

Freeman, J.J., and Hayes, E.P. (1987). The metabEncyclopedia, Inc., New York. 

D.F. Othmer, Eds.), pp. 352-374. The Interscience 

Technology, 1st ed., Vol. 9. (R.E. Kirk and: 

isocyanides. In Kirk-Othmer Encyclopedia of Chemical 

Gergel, M.G., and Revelise, M. (1952). Nitriles and 

par voie intraperitonkale. Eur. J. Toxicol. 8, 94-101. 

tales par l'acktonitrile. lre note: Intoxications aigues 

Jacquemont, M.-C. (1975a). Intoxications expkrimenHaguenoer, J.-M., DeQuidt, J., and 

par voie pulmonaire. Eur. J. Toxicol. 8, 102-106. 

tales par l'acktonitrile. 2' note: Intoxications aigues 

Jacquemont, M.-C. (1975b). Intoxications expkrimenHaguenoer, J.-M., DeQuidt, J., and 

Pathol. 17, 690-708. 

two-year carcinogenicity studies in rats. Toxicol. 

hepatocellular foci in National Toxicology Program 

Boorman, G.A. (1989). Observations on altered 

Harada, T., Maronpot, R.R., Morris, R.W., and 

studies. Environ. Health Perspect. 58, 385-392. 

analysis and interpretation of animal carcinogenicity 

Haseman, J.K. (1984). Statistical issues in the design, 

studies in rodents. Toxicul. Pathol. 12, 126-135. 

Use of historical control data in carcinogenicity 

Haseman, J.K., Huff, J., and Boorman, G.A. (1984). 
References 61 

C3H/HeN)Fl (B6C3FJ mice. JNCI 75,975-984. 

control groups of F344/N rats and (C57BL/6N x 

Neoplasms observed in untreated and corn oil gavage 

Boorman, G.A., and McConnell, E.E. (1985). 

Haseman, J.K., Huff, J.E., Rao, G.N., Arnold, J.E., 

Wiley and Sons, New York. 

Nonparametric Statistical Methods, pp. 120-123. John 

Hollander, M., and Wolfe, D.A. (1973). 

(Abstr.). 

acetonitrile. Acta Pharmacol. Toxicol. 41, 340 

Mantz, J.M. (1977). Acute voluntary intoxication by 

Jaeger, A., Tempe, J.D., Porte, A., Stoeckel, L., and 

41, 133-145. 

k-sample test against ordered alternatives. Biometrika 

Jonckheere, A.R. (1954). A distribution-free 

raphy. J. Anal. Toxicol. 16, 104-106. 

specificity of analysis by headspace gas chromatogTwo fatalities from ingestion of acetonitrile: Limited 

Jones, A.W., Ufgren, A., and Eklund, A. (1992). 

Stat. Assoc. 53, 457-481. 

estimation from incomplete observations. J. Am. 

Kaplan, E.L., and Meier, P. (1958). Nonparametric 

19, 699-704. 

organic solvents. Toxicol. Appl. Pharmacol. 

Acute toxicity and limits of solvent residue for sixteen 

Kimura, E.T., Ebert, D.M., and Dodge, P.W. (1971). 

888. MacMillan Publishing Co., New York. 

In Casarett and Doull’s Toxicology, pp. 241, 372, and 

Klaassen, C.D., Amdur, M.O., and Doull, J. (1986). 

J. Biol. Chem. 260, 13,607-13,612. 

and acetol monooxygenase of rabbit microsomes. 

of ethanol-inducible P-450 isozyme 3a as the acetone 

Koop, D.R., and Casazza, J.P. (1985). Identification 

pyrolyzate. Agric. Biol. Chem. 47, 1979-1982. 

Mutagen from the gaseous phase of protein 

Kushi, A., Matsumoto, T., and Yoshida, D. (1983). 

lathyrogenic agents. J. Bp. Med. 114, 295-311. 

Levene, C.I. (1961). Structural requirements for 

Pediatrics 88, 337-340. 

Cyanide poisoning from a cosmetic nail remover. 

Losek, J.D., Rock, A.L., and Boldt, R.R. (1991). 

Fed 33, 492-505. 

by conventional processes. J. Water Pollut. Control 

(1961). Experimental treatment of organic cyanides 

Ludzack, F.J., Schaffer, R.B., and Bloomhuff, R.N. 

Methods. McGraw-Hill, Inc., New York. 

(1982). Handbook of Chemical Property Estimation 

Lyman, W.J., Reehl, W.F., and Rosenblatt, D.H. 

studies. JNCI 76, 283-289. 

neoplasms for evaluation of rodent carcinogenesis 

Boorman, G.A. (1986). Guidelines for combining 

McConnell, E.E., Solleveld, H.A., Swenberg, J.A., and 

33258 and pyronin Y. Mutat. Res. 120, 269-275. 

micronuclei and RNA in erythrocytes using Hoechst 

(1983). A simple fluorescent staining procedure for 

MacGregor, J.T., Wehr, C.M., and Langlois, R.G. 

toxicity studies. Fundam. Appl. Toxicol. 14, 513-522. 

increases assay efficiency and permits integration with 

micronucleus test: Measurement at steady state 

Shelby, M.D. (1990). The in vivo erythrocyte 

MacGregor, J.T., Wehr, C.M., Henika, P.R., and 

related to smoking. Public Health Rep. 77, 553-554. 

Gross, A.L. (1962). Acetonitrile in body fluids 

McKee, H.C., Rhoades, J.W., Campbell, J., and 

79, 639-648. 

carcinogenesis experiments. J. Am. Stat. Assoc. 

differences in tumor incidence based on animal 

McKnight, B., and Crowley, J. (1984). Tests for 

Laboratory Systems, Research Triangle Park, NC. 

and Analysis System, Version 1.4a. Integrated 

Appendix 2: Micronucleus Assay Data Management 

Anatysis Program for Micronucleus Assay Data. 

Tice, R.R. (1990). A General Purpose Statistical 

Margolin, B.H., Risko, K.J., Frome, E.L., and 

Salmonellalmicrosome test. Mutat. Res. 88, 343-350. 

(1981). Compatibility of organic solvents with the 

Maron, D., Katzenellenbogen, J., and Ames, B.N. 

safety assessment. Toxicol. Pathol. 10, 71-80. 

alterations and hepatocellular tumors in chemical 

pretation of rodent hepatocellular proliferative 

Maronpot, R.R., and Boorman, G.A. (1982). Inter- 
62 Acetonitrile, NTP TR 447 

Perspect. 86, 93-98. 

following two-week exposures. Environ. Health 

toxicology of isoprene in F344 rats and B6C3Fl mice 

Ragan, H.A., and Miller, R.A. (1990). Inhalation 

Melnick, R.L., Roycroft, J.H., Chou, B.J., 

p. 8. Merck and Company, Rahway, NJ. 

The Merck Index (1976). 9th ed. (W. Windholz, Ed.), 

p. 11. Merck and Company, Rahway, NJ. 

The Merck Index (1989). 11th ed. (S. Budavari, Ed.), 

Toxicol. 29, 447-458. 

case of suicidal oral acetonitrile ingestion. Clin. 

serum concentrations and cyanide blood levels in a 

Neurath, H., and Eggert, A. (1991). Acetonitrile 

Michaelis, H.C., Clemens, C., Kijewski, H., 

Spring Harbor, NY. 

pp. 605-627. Cold Spring Harbor Laboratory, Cold 

(H.H. Hiatt, J.D. Watson, and J.A. Winsten, Eds.), 

electrophiles. In Origins of Human Cancer 

chemical carcinogens as reactive mutagenic 

Miller, J.A., and Miller, E.C. (1977). Ultimate 

(Abstr.). 

vitro and in vivo treatment. Environ. Mutagen. 5, 482 

DNA synthesis (UDS) in hepatocytes following in 

and Spalding, J. (1983). Induction of unscheduled 

Steinmetz, K., Contreras, C., Austere, L., Martin, S., 

Mirsalis, J., Tyson, K., Beck, J., Loh, F., 

Toxicol. 11, 343-358. 

Toxicology Program 13-week studies. Fundam. Appl. 

reproductive organ weight data from National 

Evaluation of rodent sperm, vaginal cytology, and 

Ross, M.D., Teague, J.L., and Morris, R.W. (1988). 

Morrissey, R.E., Schwetz, B.A., Lamb, J.C., IV, 

270 chemicals. Environ. Mutagen. 8 (Suppl. 7), 1-119. 

mutagenicity tests: 11. Results from the testing of 

Tainer, B., and Zeiger, E. (1986). Salmonella 

Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., 

Bethesda, MD. 

Public Health Service, National Institutes of Health, 

U.S. Department of Health, Edication, and Welfare, 

Report Series No. 1. NIH Publication No. 76-801. 

for Carcinogen Bioassay in Small Rodents. Technical 

National Cancer Institute (NCI) (1976). Guidelines 

Cincinnati, OH. 

survey: Updated projection data collected 1972-1974. 

(NIOSH) (1979). National occupational hazards 

National Institute for Occupational Safety and Health 

data as of January 31, 1994. 

Survey (NOES) (1981-1983), unpublished provisional 

(NIOSH) (1994). National Occupational Exposure 

National Institute for Occupational Safety and Health 

Service, National Institutes of Health, Bethesda, MD. 

Health, Education, and Welfare, Public Health 

Specification NIH-11-1335. U.S. Department of , 

Formula Rat and Mouse Ration (NIH-07). 

National Institutes of Health (NIH) (1978). Open 

Health, Research Triangle Park, NC. 

Services, Public Health Service, National Institutes of 

No. 89-2808. U.S. Department of Health and Human 

Report Series No. 353. NIH Publication 

Rats and B6C3Fl Mice (Feed Studies). Technical 

2,CDichlorophenol (CAS No. 120-83-2) in F344/N 

Toxicology and Carcinogenesis Studies of 

National Toxicology Program (NTP) (1989). 

Health, Research Triangle Park, NC. 

Services, Public Health Service, National Institutes of 

No. 91-2842. U.S. Department of Health and Human 

Technical Report Series No. 387. NIH Publication 

F344/N Rats and B6C3Fl Mice (Feed Studies). , 

dl-Amphetamine Sulfate (CAS No. 60-13-9) in 

Toxicology and Carcinogenesis Studies of ' 

National Toxicology Program (NTP) (1991). ' 

Health, Research Triangle Park, NC. 

Services, Public Health Service, National Institutes of 

No. 93-3158. U.S. Department of Health and Human 

Report Series No. 427. NIH Publication ' 

Rats and B6C3Fl Mice (Feed Studies). Technical 

79%-85% Curcumin, CAS No. 458-37-7) in F344/N 

Oleoresin (CAS No. 8024-37-1) (Major Component 

Toxicology and Carcinogenesis Studies of Tumeric 

National Toxicology Program (NTP) (1993). 

Research Triangle Park, NC. 

Acetonitrile in Rats. National Institutes of Health, 

Inhalation Developmental Toxicology Studies: 

National Toxicology Program (NTP) (1994a). 
References 63 

Department of Ekdth and Fhrnan Services, Public (1985). Comparison of subchronic inhalation toxicity 

Series NO. 420. NIH Publication NO. 94-3151. U.S. Roloff, v., Short, R., Ribelin, w., and Dietrich, M. 

B6C3Fl Mice (Feed Studies). Technical Report 

Triamterene (CAS No. 396-01-0) in F344/N Rats and Pharmaceutical Press, London. 

Toxicology and Carcinogenesis Studies of Martindale: The Extra Pharmacopoaia, 28th ed. The 

National Toxicology Program (NTP) (1994b). Reynolds, J.E.F., and Prasad, AB., Eds. (1982). 

Research Triangle Park, NC. 

Health Service, National Institutes of Health, of five aliphatic nitriles in rats. Toxicolopt 5, 30. 

Health Studies Report Series). 

Tokyo, Environment Agency, pp. 492-493 (Office of 

annual report of chemical assessment [in Japanese]. 

Japan (1990). Chemicals in the environment. The 

Environmental Health, Environmental Agency, 

Office of Health Studies (OHS), Department of 

Biochem. Physiol. 2, 95-112. 

organonitriles in mice and houseflies. Pestic. 

liberation from various organothiocyanates and 

toxicological significance of hydrogen .cyanide 

Casida, J.E. (1972). Enzymatic mechanisms and 

Ohkawa, H., Ohkawa, R., Yamamoto, I., and 

259, 147-163. 

female Drosophila melanogaster. Mutat. Res. 

the FIX and ZESTE genetic test systems employing 

Aneuploidy in Drosophila, 11. Further validation of 

Osgood, C., Zimmering, S., and Mason, J.M. (1991a). 

Mutat. Res. 259, 165-176. 

studies on the induction of aneuploidy by nitriles. 

(1991b). Aneuploidy in Drosophila. IV. Inhalation 

Osgood, C., Bloomfield, M., and Zimmering, S. 

9154M. Sadtler Research Laboratories, Philadelphia. 

Sadtler Standard Spectra. IR No. 269; NMR No. 

autoinduction. J. Appl. Toxicol. 8, 201-209. 

three short term assays with special reference to 

Mutagenic activity of acetonitrile and fumaronitrile in 

Schlegelmilch, R., Krug, A., and Wolf, H.U. (1988). 

Vol. 4, pp. 31-53. Plenum Press, New York. 

and Methodsfor their Detection (A. Hollaender, Ed.), 

cytogenetic analysis. In Chemical Mutagens: Principles 

Schmid, W. (1976). The micronucleus test for 

a treatment. Biometrics 33, 386-389. 

Williams' test for contrasting increasing dose levels of 

Shirley, E. (1977). A non-parametric equivalent of 

46, 83-93. 

products of chlorine disinfection. Toxicology 

halogenated acetonitriles: Drinking water byand Stober, J.A. (1987). Developmental toxicity of 

Smith, M.K., George, E.L., Zenick, H., Manson, J.M., 

1, 634-642. 

and Nair, J.H., 111. (1959a). An investigation of the 

Pozzani, U.C., Carpenter, C.P., Palm, P.E., Weil, C.S., Straus, D.S. (1981). Somatic mutation, cellular 

mammalian toxicity of acetonitrile. J. Occup. &fed. differentiation, and ancer causation. JNcz 

67, 233-241. 

data. Ind. Hyg. J. 20, 364-369. 

between single dose inhalation and single dose oral Tarone, R.E. (1975). Tests for trend in life table 

mixtures by rats, with notes upon the relationship 

values: 5. The experimental inhalation of vapor TOxiCOl. 55, 47-54. 

(1959b). The toxicological basis of threshold limit mechanism Of acute toxicity Of nitriles in mice. Arch. 

Pozzani, u.c., Weil, c.S., and Carpenter, C.P. Tanii, H., and Hashimoto, K. (1984). Studies on the 

analysis. Biometrika 62, 679-682. 
64 Acetonitrile, NTP TR 447 

toxicity assays. Science 236, 933-941. acetonitrile in the Syrian golden hamster. Teratology 

carcinogenicity in rodents from in vitro genetic Willhite, C.C. (1983). Developmental toxicology of 

Minor, R. (1987). Prediction of chemical 

Resnick, M., Stasiewicz, S., Anderson, B., and 18, 991-1003. 

Zeiger, E., Haseman, J.K., Spalding, J., Caspary, W., exposure to aliphatic nitriles. Clin. Toxicol. 

Tennant, R.W., Margolin, B.H., Shelby, M.D., Willhite, C.C. (1981). Inhalation toxicology of acute 

27, 313-325. 

Med 9, 264-267. 

of acetonitrile-containing cosmetic. Am. J. Emerg. 

(1991). Severe cyanide poisoning from the ingestion 

Turchen, S.G., Manoguerra, A.S., and Whitney, C. 

nitriles. Toxicol. Appl. Phannacol. 59, 589-602. 

cyanide liberation in the acute toxicity of aliphatic 

Willhite, C.C., and Smith, R.P. (1981). The role of 

Emergency Response, Washington, DC. 

profile for acetonitrile. Office of Solid Waste and 

(USEPA) (1985). Health and environmental effects 

United States Environmental Protection Agency 

Washington, DC. 

Publication 2607. U.S. Government Printing Office, 

United States Production and Sales, 1991. USITC 

(USITC) (1993). Synthetic organic chemicals: 

United States International Trade Commission 

compared with azero dose control. Biomem'cs ' 

treatment means when several dose levels are 

Williams, D.A. (1971). A test for differences between 

27, 103-117. 

28, 519-531. 

dose levels with a zero dose control. Biometrics 

Williams, D.A. (1972). The comparison of several 

polyurethane foams. Br. Poiym. J. 4, 27-43. 

from the thermal decomposition of flexible 

Woolley, W.D. (1972). Nitrogen-containing products 

New York. 

and F. Wissing, Eds.), pp. 29-49. Academic Press, 

(B. Vennesland, E.E. Conn, C.J. Knowles, J. Westley, 

and its antagonism. In Qanide in Biology 

Way, J.L. (1981). Pharmacologic aspects of cyanide 

study. Mutat. Res. 224, 31-78. 

loss in Saccharomyces cerevisiae - an interlaboratory 

(1989). Detection of induced mitotic chromosome 

Piegorsch, W.W., Fogel, S., and Resnick, M.A. 

Whittaker, S.G., Zimmerman, F.K., Dicus, B., 

242, 231-258. 

and cyclophosphamide monohydrate. Mutat. Res. 

methanesulfonate, dimethyl sulfoxide, propionitrile 

carbamate, methyl methanesulfonate, ethyl 

cerevisiae: Effects of methyl benzimidazol-2-yl 

chromosome gain the yeast Saccharomyces 

(1990). The detection of mitotic and meiotic 

Piegorsch, W.W., Resnick, M.A., and Fogel, S. 

Whittaker, S.G., Moser, S.F., Maloney, D.H., 

Environ. Mol. Mutagen. 11 (Suppl. 12), 1-158. 

tests: IV. Results from the testing of 300 chemicals. 

Mortelmans, K. (1988). Salmonella mutagenicity 

Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and 

Mol. Muzagen. 16 (Suppl. 18), 1-14. 

earlier results with 41 additional chemicals. Environ. 

predicting rodent carcinogenicity: Confirmation of 

Evaluation of four in vitro genetic toxicity tests for 

Margolin, B.H., and Tennant, R.W. (1990). 

Zeiger, E., Haseman, J.K., Shelby, M.D., 

Saccharomyces cerevisiae. Mutat. Res. 149, 339-351. , 

solvents are strong inducers of aneuploidy in 

ethyl acetate, acetonitrile and other polar aprotic 

Resnick, M.A. (1985). Acetone, methyl ethyl ketone, 

Zimmerman, F.K., Mayer, V.W., Scheel, I., and ~ 

224, 287-303. 

organic solvents including neurotoxins. Mutat. Res. 

(1989). Induction of chromosome loss by mixtures of 

Zimmerman, F.K., Scheel, I., and Resnick, M.A. 
65 

APPENDIX A 

TABLE A1 

TABLE A2 

TABLE A3 

TABLE A4a 

TABLE A4b 

OF ACETONITRILE 

SUMMARY OF LESIONS IN MIALE RATS 

IN THE 2-YEAR INHALATION STUDY 

in the 2-Year Inhalation Study of Acetonitrile 98 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

Historical Incidence of Skin Keratoacanthomas in Untreated Male F344/N Rats 97 

Historical Incidence of Pancreatic Islet Neoplasms in Untreated Male F344/N Rats 97 

in Untreated Male F344/N Rats 96 

Historical Incidence of Adrenal Gland Pheochromocytomas 

in Untreated Male F344/N Rats 

Historical Incidence of Hepatocellular Neoplasms 

in the 2-Year Inhalation Study of Acetonitrile 

Statistical Analysis of Primary Neoplasms in Male Rats 

in the 2-Year Inhalation Study of Acetonitrile 

Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Inhalation Study of Acetonitrile 

Summary of the Incidence of Neoplasms in Male Rats 

............................... 

96 

90 

72 

67 

............................... 

............................... 

.......................................... 

.......................................... ... ..... 

............................... 
66 Acetonitrile, NTP TR 44: 
Lesions in Male Rats 67 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrilea 

TABLE A1 

0 PPm 100 ppm 200 ppm 400 ppm 

Early deaths 

IS-Month hain evalrcarion 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

Survivors 

Animals examined microscopically 

8 

56 

1 

36 

11 

56 

8 

55 

8 

26 

13 

55 

8 

56 

4 

35 

9 

56 

8 

56 

5 

26 

17 

56 

15-Month Interim Evaluation 

Adrenal medulla 

Endocrine System 

(8) 

C-cell, adenoma 

Pars intermedia, adenoma 

Pars distalis, adenoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Pituitary gland (8) 

1 (13%) 

2 (25%) 

Thyroid gland (8) 

Testes 

Genital System 

(8) (8) (8) (8) 

Interstitid cell, adenoma 

Bilateral, interstitial cell, adenoma 

3 (38%) 

3 (38%) 

7 (88%) 

1 (13%) 

s (63%) 

3 (38%) 8 (100%) 

Skin 

Integumentary System 

Keratoacanthoma 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Hematopoietic System 

General Body System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Neo~lasms Observed 
68 Acetonitrile, NTP TR 447 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

0 PPm 100 ppm 200 ppm 400 ppm 

Intestine small, jejunum 

Intestine large, colon 

Alimentary System 

2- Year Study 

(47) 

(48) 

(42) 

(42) 

(46) 

(46) 

(44) 

(45) 

Fibrosarcoma 

Carcinoma 1 (2%) 

1 (2%) 

Liver 

Intestine small, ileum 

(48) 

(47) 

(47) 

(42) 

(48) 

(46) 

(48) 

(43) 

Osteosarcoma, metastatic, bone 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 3 (6%) 

Mesentery 

Stomach, forestomach 

Pharynx 

Pancreas 

Tongue 

Stomach, glandular 

(4) 

(10) (9) 

(47) 

(1) 

(47) 

(2) 

(46) 

(48) 

(9) (11) 

(47) 

(1) 

(48) 

(1) 

(47) 

Squamous cell papilloma 

Adenoma 

1 (100%) 

1 (2%) 

1 (100%) 

(48) 

(48) 

(48) 

(48) 

Squamous cell carcinoma 1 (50%) 1 (100%) 

Heart 

Cardiovascular System 

Adrenal medulla 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Carcinoma 

Adenoma, multiple 

Adenoma 

Pars intermedia, adenoma 

Pars distalis, adenoma 

Pituitary gland 

C-cell, carcinoma 

C-cell, adenoma 

Bilateral, C-cell, carcinoma 

Bilateral, C-cell, adenoma 

Follicular cell, carcinoma 

Islets, pancreatic 

Thyroid gland 

' Follicular cell, adenoma 

2 (4%) 

25 (52%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

(4) 

(48) 

(48) 

3 (6%) 

6 (13%) 

(46) 

4 (9%) 

10 (22%) 

1 (2%) 

4 (9%) 

(47) 

(46) 

27 (59%) 

(47) 

1 (2%) 

1 (2%) 

5 (11%) 

(48) 

2 (4%) 

10 (21%) 

2 (4%) 

(48) 

23 (48%) 

3 (6%) 

4 (8%) 

1 (2%) 

(48) 

(48) 

1 (2%) 

6 (13%) 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

6 (13%) 

(48) 

(47) 

23 (49%) 

(48) 

1 (2%) 

6 (13%) 

1 (2%) 

None 

General Body System 
Lesions in Male Rats 69 

TABLE AI 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

0 PPm 100 ppm 200 ppm 400 ppm 

Preputial gland 

Epididymis 

Genital System 

2-Year study (continued) 

Carcinoma 

Adenoma 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

Osteosarcoma, metastatic, bone 

(47) 

(48) 

5 (11%) 

Testes (48) 

8 (17%) 

25 (52%) 

(47) 

(47) 

(47) 

(48) 

(48) 

(48) 

1 (2%) 

10 (21%) 

1 (2%) 

1 (2%) 2 (4%) 1 (2%) 

(47) (48) (48) 

6 (13%) 

29 (62%) 30 (63%) 

8 (17%) 

34 (71%) 

Lymph node 

Bone marrow 

Hematopoietic System 

Lymph node, bronchial 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Osteosarcoma, metastatic, bone 

Axillary, osteosarcoma, metastatic, bone 

Sarcoma 

Osteosarcoma, metastatic, bone 

Fibrosarcoma 

Thymoma NOS 

Thymus 

2 (4%) 

(45) (47) 

1 (2%) 

Integumentary System 

Fibroadenoma 

Carcinoma 

Mammary gland 

Sebaceous gland, carcinoma 

Trichoepithelioma 

Keratoacanthoma 

Hemangiopericytoma 

Fibrosarcoma 

Fibroma, multiple 

Fibroma 

Basal cell adenoma 

Skin 

(47) (47) (48) (48) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

(48) (47) (48) (48) 

3 (6%) 3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

4 (8%) 

Musculoskeletal System 

Rib, osteosarcoma 

Humerus, osteosarcoma 

Bone (48) (47) 

1 (2%) 
70 Acetonitrile, NTP TR 447 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE Al 

0 PPm 100 ppm 200 ppm 400 ppm 

Brain 

Nervous System 

2-YeUr study (continued) 

Meninges, meningioma benign 

Sarcoma, metastatic, spleen 

Oligodendroglioma NOS 

Glioma NOS 

Glioma malignant 

Astrocytoma NOS 

(48) 

1 (2%) 

(47) 

1 (2%) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

Lung 

Respiratory System 

(48) (47) (48) (48) 

Neoplasm nos, metastatic, uncertain primary 

Carcinoma, metastatic, thyroid gland 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma 

Pheochromocytoma malignant, metastatic, 

Osteosarcoma, metastatic, bone 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

site 

1 (2%) 

1 (2%) 

1 (2%) 

adrenal medulla 1 (2%) 

Carcinoma 

Zymbal’s gland 

Pinna, schwannoma benign 

Ear 

Special Senses System 

(2) 

1 (50%) 

(1) 

1 (100%) 

Urinary bladder 

Kidney 

Urinary System 

Transitional epithelium, carcinoma 

Renal tubule, adenoma 

(47) 

(47) 

(48) 

1 (2%) 

Multiple organsb 

Systemic Lesions 

Mesothelioma NOS 

Leukemia mononuclear 

(48) (47) (48) (48) 

3 (6%) 

29 (60%) 32 (68%) 

2 (4%) 

35 (73%) 32 (67%) 

5 (11%) 1 (2%) 
Lesions in Male Rats 71 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE A1 

~ 

100 ppm 200 ppm 400 ppm 

Total animals with primary neoplasms' 

Neoplasm Summary 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

uncertain primary site 

2-Year study 

2-Year study 

2-Year study 

2-Year study 

2-Year study 

benign or malignant 

2-Year study 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with malignant neoplasmsTotal metastatic neoplasms 

Total animals with metastatic neoplasms 

Total animals with uncertain neoplasmsTotal uncertain neoplasms 

48 

8 

41 

8 

48 

8 

48 

8 

122 

10 

141 

12 

143 

11 

142 

8 

45 

8 

45 

8 

45 

8 

41 

8 

76 

10 

95 

11 

93 

11 

94 

8 

35 

1 

36 38 34 

40 

1 

4 

3 

43 47 

1 

6 2 

2 

45 

1 

1 

1 

1 

3 6 

10 6 

3 

6 

3 

' Primaly neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 
72 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 0 ppm 

TABLE A2 

4823210496167729327611249 

9178912234668900233455577 

3444455555555566666666666 

6147792061517820343969271 

1330333135211352100100142 

0000000000000000000000000 

Number of Days on Study 

Carcass ID Number 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

......................... 

......................... 

......................... 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Liver 

Intestine small, ileum 

Salivary glands 

Pancreas 

Mesentery 

Fibrosarcoma 

Hepatocellular carcinoma 

......................... 

......................... 

......................... , 

......................... 

......................... 

X 

++ 

......................... 

......................... + ++ + 

Stomach, glandular 

Stomach, forestomach 

......................... 

......................... 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

......................... 

Mesothelioma NOS, metastatic, testes X 

Adrenal cortex 

Endocrine System 

......................... Adrenal medulla 

Adenoma 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pituitary gland 

Parathyroid gland 

Carcinoma 

Islets, pancreatic 

C-cell, carcinoma 

C-cell, adenoma 

Pars intermedia, adenoma 

Pars distalis, adenoma 

Thyroid gland 

......................... X 

......................... X 

x xx xx x xx x xxxx ......................... 

+++++M+++++++++++++++++++ 

X 

......................... X 

xx x 

X 

General Body System 

None 

Genital System 

Preputial gland 

Epididymis 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

M++++++++++++++++++++++++ 

......................... 

......................... 

......................... 

......................... 

X X 

X X X X 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma X X X xxxx X 

xx 

A Autolysis precludes examination 

+: Tissue examined microscopically 

I: Insufficient tissue 

M: Missing tissue 

Blank Not examined 

X Lesion present 
Lesions in Male Rats 73 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

94455639171933333333333 

78888899002233333333333 

66666666777777777777777 

57952612508384483368356 

42404200341201223444555 

00000000000000000000000 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Intestine large, colon 

Esophagus 

Alimentary System 

....................... 

....................... 48 

48 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Liver 

Intestine small, ileum 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

Fibrosarcoma 

Hepatocellular carcinoma 

+++++++++++A+++++++++++ 

+++++++++++A+++++++++++ 

+++++++++++A+++++++++++ 

+++++++++++A+++++++++++ 

....................... 

+++++++++++A+++++++++++ 

48 

48 

48 

48 

10 

1 

48 

47 

1 

47 

47 

47 

47 

X 

+ ++ + 

....................... 

....................... 

........................ 

....................... 

Cardiovascular System 

Heart 

Blood vessel 

....................... 

....................... 

1 

48 

48 

Mesothelioma NOS, metastatic, testes 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

....................... 

....................... 

3 

6 

48 

1 

25 

48 

47 

1 

1 

48 

2 

2 

48 

48 

Adenoma 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pituitary gland 

Parathyroid gland 

Carcinoma 

X 

X X 

Islets, pancreatic ....................... 

X 

xxx xxxxx X 

....................... 

....................... 

General Body System 

C-cell, carcinoma 

C-cell, adenoma 

Pars intermedia, adenoma 

Pars distalis, adenoma x xx 

Thyroid gland ....................... X xx 

X 

None 

Genital System 

Preputial gland 

Epididymis 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

....................... 

....................... 

8 

25 

48 

48 

48 

5 

47 

48 

X 

....................... 

....................... 

....................... 

x x x xxxxxx xx xxxxxx 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

xxx X 
74 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE A2 

3444455555555566666666666 

Number of Days on Study 91789122.34668900233455577 

4823210496167729327611249 

6147792061517820343969271 

1330333135211352100100142 

0000000000000000000000000 

Carcass ID Number 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow ......................... 

......................... 

......................... 

......................... 

......................... 

+++++++++++++M+++++++M+++ 

M++M+++++++++++++M+++++++ 

++ ++++ ++ + 

Skin 

Mammaxy gland 

Integumentary System 

......................... 

M++++++++++++++++++++++++ 

Fibroma X 

Musculoskeletal System 

Bone 

Rib, osteosarcoma 

......................... 

Nervous System 

Spinal cord 

Peripheral nerve 

Brain 

Glioma malignant 

......................... X 

+ 

+ 

Respiratory System 

Larynx ......................... Lung .......................... 

Neoplasm NOS, metastatic, uncertain 

Mesothelioma NOS, metastatic, testes 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma 

Osteosarcoma, metastatic, bone 

X 

X 

primary site X 

Trachea 

Nose 

......................... 

......................... 

Special Senses System 

Ear 

Pinna, schwannoma benign 

Eye 

X 

+ + 

Kidney 

Urinary System 

Renal tubule, adenoma 

Urinary bladder 

......................... 

......................... 

Systemic Lesions 

Multiple organs 

xx x xxxx x xxx xxx ......................... 

Mesothelioma NOS 

Leukemia mononuclear 

xx X 
Lesions in Male Rats 75 

Individual Animal Tumor Pathology of Male Rats in the %Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE A2 

Number of Days on Study 

94455639111933333353333 

I8888899002233333333333 

66666666111111117711711 

51952612508384483368356 

42404200341201223444555 

00000000000000000000000 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow ....................... 

....................... 

....................... 

....................... 

....................... 

M+++++++++++++M+++++M++ 

M++++++++++++M++M+++M++ 

48 

48 

48 

48 

43 

41 

12 

48 

++ + 

Skin 

Mammary gland 

Integumentary System 

Fibroma 

....................... 

....................... 

3 

48 

41 

X X 

Musculoskeletal System 

Bone 

X ....................... 1 

48 

Rib, osteosarcoma 

Nervous System 

Spinal cord 

Peripheral nerve 

Brain ....................... 

1 

1 

1 

48 

Glioma malignant 

Respiratory System 

Lung 

Larynx 

Neoplasm NOS, metastatic, uncertain 

Mesothelioma NOS, metastatic, testes 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma 

Osteosarcoma, metastatic, bone 

primary site 

Trachea 

Nose 

....................... 

....................... 

X 

1 

1 

1 

48 

48 

....................... 

....................... X 

48 

48 

1 

Special Senses System 

Ear 

Pinna, schwannoma benign 

Eye + 1 

1 

2 

Kidney 

Urinary System 

Renal tubule, adenoma 

Urinary bladder 

....................... 

48 

1 

48 

X 

....................... 

Systemic Lesions 

Multiple organs 

Mesothelioma NOS 

Leukemia mononuclear xx xxx xxx xx xx xx x ....................... 

3 

29 

48 
76 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm 

TABLE A2 

Number of Days on Study 

166150589014166805379235 

349123555666688899233456 

344555555555555555666666 

903131143358824995695121 

442533226162344533314165 

111111111111111111111111 

Carcass ID Number 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

PhaIynx 

Pancreas 

Mesentery 

Tongue 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Tooth 

Hepatocellular adenoma 

Squamous cell papilloma 

Squamous cell carcinoma 

........................ 

++AA+++++++++++A+A++++++ 

++A++++A+++++++A+A++++++ 

+++++++++++++++A++++++++ 

++A+++++M++++++A++++++++ 

+++++++++++++++A+A++++++ 

++A++++A+++++++A+A++++++ 

........................ 

X 

+ + ++ ........................ 

+ ...................... ........................ 

........................ 

Cardiovascular System 

Heart 

Blood vessel 

........................ 

++++++++++++++++M+++++++ 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

........................ 

........................ 

........................ 

X x xxx 

+++++++++++++++++M++++++ 

........................ 

X xx x xx X X xx 

+++++++++++++++++I++++++ 

X 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Islets, pancreatic 

Pituitary gland 

Parathyroid gland 

Adenoma 

Thyroid gland 

X 

X X 

Pars distalis, adenoma 

Follicular cell, adenoma 

C-cell, carcinoma 

C-cell, adenoma 

General Body System 

None 

Genital System 

Preputial gland 

Epididymis 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

........................ 

........................ 

........................ 

......................... 

........................ X 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma X xx xxx xx x 

'X xx X X 
Lesions in Male Rats 77 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

TABLE A2 

58931781323333333333333 

68890012233333333333333 

66667777777777777777777 

5.1653728284801294103467 

53414434551122223455556 

11111111111111111111111 

Number of Days on Study 

Tumors 

Tissues1 

Total 

Carcass ID Number 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

++A++++++++++++++++++++ 

++A++++++++++++++++++++ 

++A++++++++++++++++++++ 

++A++++++++++++++++++++ 

....................... 

45 

43 

44 

42 

47 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Pharynx 

Pancreas 

Mesentery 

Tongue 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Tooth 

....................... 

++A++++++++++++++++++++ 

++A++++++++++++++++++++ 

1 

2 

46 

47 

47 

1 

1 

47 

9 

1 

47 

42 

42 

Hepatocellular adenoma 

....................... 

+ +++ + 

X 

+ 

Squamous cell papilloma 

....................... 

....................... 

....................... 

X 

+ + 

Squamous cell carcinoma 

Cardiovascular System 

Heart 

Blood vessel 

....................... 

....................... 47 

46 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Islets, pancreatic 

Pituitary gland 

Parathyroid gland 

Adenoma 

Thyroid gland 

Pars distalis, adenoma 

Follicular cell, adenoma 

C-cell, carcinoma 

C-cell, adenoma 

....................... 

....................... 

X xxx X 

X X X X ....................... X X 

....................... 

x x xxxxx xx xxxx x xxx ....................... 

+++++++++++++++++++++M+ 

X X X X 

X 

X 1 

1 

5 

47 

27 

46 

46 

4 

47 

4 

10 

1 

46 

47 

General Body System 

None 

Genital System 

Preputial gland 

Epididymis 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

....................... 

....................... 

6 

29 

47 

46 

47 

1 

47 

47 

....................... 

+M+++f+++++++++++++++++ 

....................... 

x xxxx xxxxxxxxxxxxxxx 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

X 
78 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

TABLE A2 

166150589014766805379235 

349123555666688899233456 

344555555555555555666666 

Number of Days on Study 

Carcass ID Number 

903131143358824995695721 

442533226162344533314165 

111111111111111111111111 

Hematopoietic System 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood 

Thymus 

Sarcoma 

+ ........................ ++ ++ 

........................ 

........................ 

........................ 

++++++M++++++++++M++++++ 

++++++M++M++++++MMM+++++ 

X 

........................ 

Fibroadenoma 

Mammary gland 

Integumentary System 

Keratoacanthoma 

Fibrosarcoma 

Fibroma 

Basal cell adenoma 

Skin 

........................ 

........................ 

Musculoskeletal System 

Bone ........................ 

Nervous System 

Brain 

Sarcoma, metastatic, spleen 

Oligodendroglioma NOS 

........................ X 

Respiratory System 

Lung 

Larynx 

........................ 

++++++++I+++++++++++++++ 

Alveolaribronchiolar carcinoma 

Alveolaribronchiolar adenoma 

Trachea 

Nose 

........................ 

........................ 

Special Senses System 

None 

Urinary bladder 

Kidney 

Urinary System 

........................ 

........................ 

Systemic Lesions 

Multiple organs 

xx x xx xxxxxxxxxxx ........................ 

Mesothelioma NOS 

Leukemia mononuclear 

X 
Lesions in Male Rats 79 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the %Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

589317813233333333.33333 

68890012233333333333333 

66667777777777777777777 

51653728284801294103467 

53414434551122223455556 

11111111111111111111111 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Hematopoietic System 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood 

Thymus 

Sarcoma 

45 

2 

47 

46 

47 

43 

40 

7 

47 

1 

....................... + + + 

....................... 

+++++++++++++++++++++M+ 

....................... 

++++++++++++M+M++++++++ 

M+++++++++++++++++M++++ 

X 

+++++++++++++++++++M+M+ 

Fibroadenoma 

Mammaxy gland 

Integumentary System 

Keratoacanthoma 

Fibrosarcoma 

Fibroma 

Basal cell adenoma 

Skin 

....................... 

1 

1 

2 

1 

41 

2 

47 

X X 

....................... X 

X X 

X 

X 

Musculoskeletal System 

Bone ....................... 47 

Nervous System 

Brain ....................... 

1 

1 

47 

Sarcoma, metastatic, spleen 

Oligodendroglioma NOS 

X 

Respiratory System 

Larynx ....................... 

....................... 

47 

47 

1 

1 

47 

46 

Lung 

Alveoladbronchiolar carcinoma 

Alveolarbronchiolar adenoma X 

X 

Trachea 

Nose 

........................ 

....................... 

Special Senses System 

None 

Urinaxy bladder 

Kidney 

Urinary System 

....................... 

....................... 47 

47 

Systemic Lesions 

~~______ 

Multiple organs 

X X X 

xx xxxxxx x xx xx xxx ....................... 

5 

32 

47 

Mesothelioma NOS 

Leukemia mononuclear 

X 
80 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 200 ppm 

TABLE A2 

1520377590133737182972430 

7948890236678899012234456 

3344445555555555666666666 

2250913836762555915482494 

3736333263224654562656427 

2222222222222222222222222 

Number of Days on Study 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

......................... 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

......................... 

+++A+++++++++++++++++++++ 

......................... 

+++A+++++++++++++++++++++ 

......................... 

Osteosarcoma, metastatic, bone 

Hepatocellular adenoma 

X 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery + + 

......................... 

......................... 

......................... 

......................... I 

, 

Cardiovascular System 

Heart 

Blood vessel ......................... I 

......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Carcinoma 

Adenoma, multiple 

Adenoma 

Thyroid gland 

Pituitaly gland 

Parathyroid gland 

Islets, pancreatic 

Pars distalis, adenoma 

Follicular cell, carcinoma 

C-cell, adenoma 

Bilateral, C-cell, adenoma 

X 

......................... 

......................... 

X 

xx X X X 

X 

......................... X 

X X 

......................... 

xx xxx 

......................... 

+M+++++M+++++++++++++++++ 

xx xx X 

xx X 

General Body System 

None 

Genital System 

Epididymis 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

Adenoma 

Preputial gland 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

Osteosarcoma, metastatic, bone 

......................... 

......................... 

......................... 

......................... 

......................... 

xxx xxx 

X 

X xxx X xx 
Lesions in Male Rats 81 

563444180577’99333333333 

66778899000001333333333 

66666666171177717711171 

i1017607163248401688035 

51854753664553344446777 

22222222222222222222222 

Number of Days on Study 

Tumors 

Tissuest 

Total 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

....................... 

+++++++++A+++++++++++++ 

+++++++++A+++++++++++++ 

....................... 

+++++++++A+++++++++++++ 

+++++++++A+++++++++++++ 

+++++++++A+++++++++++++ 

....................... 

48 

48 

48 

48 

9 

1 

1 

48 

46 

46 

48 

46 

41 

46 

48 

Osteosarcoma, metastatic, bone 

Hepatocellular adenoma X 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

....................... 

....................... 

....................... 

....................... 

++ ++ +++ 

Cardiovascular System 

Heart 

Blood vessel 

....................... 

....................... 48 

48 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Carcinoma 

Adenoma, multiple 

Adenoma 

Thyroid gland 

Pituitaty gland 

Parathyroid gland 

Islets, pancreatic 

Pars distalis, adenoma 

Follicular cell, carcinoma 

C-cell, adenoma 

Bilateral, C-cell, adenoma 

....................... 

....................... 

xx xx X 

xx 

X X 

X ....................... 

....................... 

x xxx xx xxx xxx ....................... 

....................... 

X 

X 

X X X 

X 1 

6 

1 

48 

23 

48 

46 

3 

1 

4 

48 

2 

10 

2 

48 

48 

General Body System 

None 

Genital System 

Epididymis 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

Adenoma 

Preputial gland +++++++M+++++++++++++++ 

....................... 

....................... 

....................... 

....................... 

xx 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma xxxx x xxxxxxxxxx xxxxx 

Osteosarcoma, metastatic, bone 

10 

30 

1 

48 

48 

48 

2 

1 

41 

48 

X 

xx X 
82 Acetonitrile, NTP TR 441 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TABLE A2 

Number of Days on Study 

1520377590133737182912430 

7948890236678899012234456 

3344445555555555666666666 

2250913836762555915482494 

3736333263224654562656427 

2222222222222222222222222 

Carcass ID Number 

Hematopoietic System 

bone 

Lymph node 

Bone marrow ......................... + ++++ + + ++ 

Axillary, osteosarcoma, metastatic, 

X 

X 

Lymph node, bronchial 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Thymus 

MM+++M+++++++M+++++++++++ 

......................... 

......................... 

......................... 

+++++++++++++M+++M+++++++ 

Osteosarcoma, metastatic, bone 

I 

Thymoma NOS 

Osteosarcoma, metastatic, bone X 

......................... 

Carcinoma 

Mammary gland 

Integumentary System 

Trichoepithelioma 

Hemangiopericytoma 

Fibroma, multiple 

Skin 

......................... 

......................... 

Musculoskeletal System 

Bone ......................... Humerus, osteosarcoma X 

Nervous System 

Brain ......................... Meninges, meningioma benign 

Respiratory System 

Larynx 

X 

......................... 

......................... 

......................... 

......................... 

Lung 

Osteosarcoma, metastatic, bone 

Alveolar/bronchiolar carcinoma 

X 

Trachea 

Nose 

Special Senses System 

Eye + M 

Kidnev 

Urinary System 

......................... Urinaj bladder ......................... Transitional epithelium, carcinoma X 

Systemic Lesions 

Multiple organs 

xxxxxxxxxxx x xxxxx ......................... 

Mesothelioma NOS 

Leukemia mononuclear 
Lesions in Male Rats 83 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TABLE A2 

Number of Days on Study 

56344418057799333333333 

66778899000001333333333 

66666666777777777777777 

Carcass ID Number 

11077607763248401688035 

57854753664553344446777 

22222222222222222222222 

Tumors 

Tissues/ 

Total 

Hematopoietic System 

bone 

Lymph node 

Bone marrow 

Axillary, osteosarcoma, metastatic, 

Lymph node, bronchial 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Thymus 

Osteosarcoma, metastatic, bone 

Thymoma NOS 

Osteosarcoma, metastatic, bone 

....................... + ++ + 

+++++M+++++++M+++++++MM 

....................... 

....................... 

+++++++++M+++++++++++++ 

++M++++++++++++++++++++ 

+++++++++++++++++++++M+ 

X 

13 

48 

1 

47 

1 

48 

48 

47 

45 

1 

40 

1 

Carcinoma 

Mammary gland 

Integumentary System 

Trichoepithelioma 

Hemangiopericytoma 

Fibroma, multiple 

Skin 

....................... 

1 

1 

1 

48 

1 

48 

X 

X ....................... 

X 

X 

Musculoskeletal System 

Boric 

Humerus, osteosarcoma 

....................... 1 48 

Nervous System 

Brain ....................... 1 48 

Meninges, meningioma benign X 

Respiratory System 

Lung 

Larynx 

Osteosarcoma, metastatic, bone 

Alveolarbronchiolar carcinoma 

Trachea 

Nose 

....................... 

....................... 

48 

48 

1 

1 

48 

48 

....................... 

....................... 

Special Senses System 

Eye + 2 

Urinary bladder 

Kidney 

Urinary System 

....................... 

....................... 

Transitional epithelium, carcinoma 1 

48 

48 

Systemic Lesions 

Multiple organs 

Mesothelioma NOS 

Leukemia mononuclear 

....................... 

2 

35 

48 

xxxxxxxx x xxxx xx 

X X 
84 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm 

TABLE A2 

3570766887382190252299122 

8846678889223445567777999 

4455555555666666666666666 

Number of Days on Study 

Carcass ID Number 

7034579023100153684851690 

5777888498456664855878469 

3333333333333333333333333 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Liver 

Intestine small, ileum 

Carcinoma 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Mesentery 

Salivary glands 

Pharynx 

Pancreas 

Tongue 

Stomach, glandular 

Stomach, forestomach 

Tooth 

Squamous cell papilloma 

Adenoma 

Squamous cell carcinoma 

++++++A++++A++A+++++++++A 

++++++++++++++++++++++++A 

++A++++++++A++A+++++++++A 

++++++A++++A+++++++++++++ 

+++++++++++A++A+++++++++A 

......................... 

X 

......................... 

++A+++A++++A++A+++++++++A 

+ ++ + ......................... 

......................... 

......................... 

......................... 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Pheochromocytoma benign 

Pheochromocytoma malignant 

......................... 

......................... 

xxx xx X 

Adenoma 

Bilateral, pheochromocytoma benign 

Pituitary gland 

Parathyroid gland 

Carcinoma 

Thyroid gland 

Islets, pancreatic ........................... 

......................... xx ......................... 

........................ 

C-celi, adenoma 

Bilateral, C-cell, carcinoma 

Pars distalis, adenoma X x xx xx xxxx 

X X 

General Body System 

None 
Lesions in Male Rats 85 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

36377733333333333333333 

91222233333333333333333 

67777771177777777777777 

,64770479589392346812892 

74148833444556666671718 

33333333333333333333333 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

....................... 

....................... 

....................... 

....................... 

....................... 

47 

44 

46 

45 

48 

Intestine small, jejunum ....................... 44 

Carcinoma 

Liver 

Intestine small, ileum 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Mesentery 

Salivary glands 

Pharynx 

Pancreas 

Tongue 

Stomach, glandular 

Stomach, forestomach 

Tooth 

Squamous cell papilloma 

Adenoma 

Squamous cell carcinoma 

X X 

....................... 

....................... 

X 

X 

X X 

++ + ++ + + ....................... X 

+ 

A 

+++++++++++++M+++++++++ 

+++++++++++++M+++++++++ 

....................... 

X 

+ 

+ 1 

1 

1 

47 

41 

48 

1 

1 

1 

48 

11 

3 

1 

2 

48 

43 

1 

Cardiovascular System 

Heart 

Blood vessel 

....................... 

....................... 48 

48 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Adenoma 

Pituitary gland 

Parathyroid gland 

Carcinoma 

Thyroid gland 

Islets, pancreatic 

C-cell, adenoma 

Bilateral, C-cell, carcinoma 

Pars distalis, adenoma 

....................... 

....................... 

X 

X 

....................... X 

X 

....................... 

++++++++++++++++M++++++ 

X xxxx 

X 

x xxx xx ....................... 

X X X X 6 

1 

48 

23 

41 

45 

1 

1 

48 

1 

6 

1 

48 

48 

General Body System 

None 
86 Acetonitrile, NTP TR 441 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

TABLE A2 

Number of Days on Study 

3570766887382190252299122 

8846678889223445567777999 

4455555555666666666666666 

Carcass ID Number 

7034579023100153684851690 

5777888498456664855878469 

3333333333333333333333333 

Genital System 

Prostate 

Carcinoma 

Preputial gland 

Epididymis 

......................... 

......................... 

X 

......................... 

Testes 

Seminal vesicle ......................... 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma X xx .......................... xxx x xxxxx 

x xxx x X X 

Hematopoietic System 

Bone marrow 

Blood 

......................... 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node ++ ++ +++ ++ 

......................... 

......................... 

......................... 

......................... 

+++++M+++++M++++MM+++M+++ 

Fibrosarcoma 

Thymus ......................... 

Integumentary System 

Skin 

Fibroadenoma 

Carcinoma 

Mammary gland 

Sebaceous gland, carcinoma 

Keratoacanthoma 

Fibroma 

Basal cell adenoma 

.......................... 

......................... 

X X 

X 

Musculoskeletal System 

Bone .......................... 

Nervous System 

Brain 

X 

.......................... 

Glioma NOS 

Astrocytoma NOS. 

x 

Respiratory System 

Larynx ......................... 

......................... Lung 

metastatic, adrenal medulla. 

Pheochromocytoma malignant, 

Carcinoma, .metastatic, thyroid gland 

Trachea 

Nose 

............................. 

.......................... 
Lesions in Male Rats 87 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

36377733333333333333333 

91222233333333333333333 

67777777777777777777777 

64770479589392346812892 

74748833444556666677’778 

33333333333333333333333 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Genital System 

Preputial gland 

Epididymis 

....................... 

....................... 48 

48 

Carcinoma 

Prostate 48 

1 

....................... 

Testes 

Seminal vesicle 

Bilateral, interstitial cell, adenoma xxxxx xxxxxxxxxxxxxxxxx 

....................... 

....................... 

Interstitial cell, adenoma 8 

34 

48 

48 

X 

Hematopoietic System 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood 

Thymus 

Fibrosarcoma 

++ ....................... + + + 

++++++++++++++++++M++++ 

M+++++++++++M++++++++++ 

47 

41 

12 

48 

2 

....................... 

....................... 

....................... 

48 

1 

48 

48 

48 

X 

....................... 

Integumentary System 

Fibroadenoma 

Carcinoma 

Mammary gland 

Sebaceous gland, carcinoma 

Keratoacanthoma 

Fibroma 

Basal cell adenoma 

....................... 

1 

4 

3 

1 

48 

2 

1 

48 

X 

X X 

Skin ....................... 

X 

X 

X X xx 

Musculoskeletal System 

Bone ....................... 48 

Nervous System 

Brain ....................... 

1 

1 

48 

Glioma NOS 

Astrocytoma NOS 

Respiratory System 

Larynx ....................... 

....................... 

1 

48 

48 

Lung 

metastatic, adrenal medulla 

Pheochromocytoma malignant, 

Carcinoma, metastatic, thyroid gland X 

X 

48 

48 

1 

Trachea 

Nose 

....................... 

....................... 
88 Acetonitrile, NTP TR 447 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

Number of Days on Study 

3570766887382190252299122 

8846678889223445567777999 

4455555555666666666666666 

Carcass ID Number 

7034579023100153684851690 

5777888498456664855878469 

3333333333333333333333333 

Special Senses System 

Eye + 

Carcinoma 

Zymbal’s gland 

Lacrimal gland 

X 

+ 

Urinary bladder 

Kidney 

Urinary System 

.......................... 

......................... 

Systemic Lesions 

Multiple organs ......................... 

Mesothelioma NOS 

Leukemia mononuclear xx x xxx xx x xxxxxx 
Lesions in Male Rats 89 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

TABLE A2 

36317733333333333333333 

91222233333333333333333 

61171111111111171111111 

Number of Days on Study 

Carcass ID Number 

64110419589392346812892 

14148833444556666611118 

33333333333333333333333 

Tumors 

Tissues/ 

Total 

Special Senses System 

Carcinoma 

Zymbal’s gland 

Lacrimal gland 

Eye + 

+ 

Urinary bladder 

Kidney 

Urinary System 

....................... 

....................... 48 

48 

Systemic Lesions 

Multiple organs 

Mesothelioma NOS 

Leukemia mononuclear xxxxxxx xxxx x xxxxx ....................... 

1 

32 

48 

X 
90 Acetonitrile, NTP TR 447 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armita e testd 

Logistic regression testd 

Life table testd 

First incidence (days) 

Terminal rate' 

Adjusted rateb 

Overall ratea 

Adrenal Medulla: Benign Pheochromocytoma 

P=O.502 

P=0.480N 

P=0.335N 

646 

2/11 (18%) 

25.0% 

4/48 (8%) 

% 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Adrenal Medulla: Benign or Malignant Pheochromocytoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma or Carcinoma 

. . First incidence (days) 

P=O.410 

P=O.518 

P=0.396N 

646 

2/11 (18%) 

25.0% 

4/48 (8%) 

e 

0.0% 

0/48 (0%) 

0/11 (0%) 

- 

P=O.O55 

P=O.O83 

P=O.110 

3.3% 

1/48 (2%) 

P=O.O87 

P=O.121 

P=O.150 

637 

0/11 (0%) 

3.3% 

1/48 (2%) 

P=O.O26 

P=O.O45 

P=O.O67 

637 

on1 (0%) 

P=O.O02 

P=O.O25 

588 

3/13 (23%) 

56.0% 

14/46 (30%) 

P=O.O06 

P=O.O03 

P=O.O16 

559 

3/13 (23%) 

57.1% 

15/46 (33%) 

P=O.O03 

P=O.495 

P=O.480 

623 

0/13 (0%) 

3.4% 

1/47 (2%) 

P=O.495 

0/13 (0%) 

0.0% 

0/47 (0%) 

P=0.504N 

P=0.514N 

- 

P=0.505N 

P=O.757 

P=O.742 

623 

OD3 (0%) 

3.4% 

1/47 (2%) 

P=O.747 

P=O.O22 

P=O:O25 

507 

2/9 (22%) 

52.1% 

12/48 (25%) 

P=O.O26 

55.0% 

14/48 (29%) 

P =0.008 

P=O.OlO 

507 

2/9 (22%) 

P=O.O08 

4.8% 

1/48 (2%) 

P=O.492 

P=O.465 

673 

0/9 (0%) 

P=O.500 

0/9 (0%) 

0.0% 

0/48 (0%) 

P=0.500N 

P=0.514N 

- 

P =0.500N 

P=O.758 

P=O.733 

673 

0/9 (0%) 

4.8% 

1/48 (2%) 

P=0.753N 

P=O.244 

P=O.441 

632 

0/17 (0%) 

22.1% 

7/48 (15%) 

P=O.262 

P=O.173 

P=O.358 

632 

0/17 (0%) 

26.2% 

8/48 (17%) 

P=O.178 

P=O.204 

P=O.214 

727 

2/17 (12%) 

16.2% 

3/48 (6%) 

P=O.121 

P=O.374 

P=O.438 

693 

1/17 (6%) 

14.5% 

3/48 (6%) 

P=O.308 

P=O.164 

P=O.213 

693 

3/17 (18%) 

25.2% 

5/48 (10%) 

P=O.102 
Lesions in Male Rats 91 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pancreatic Islets: Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pancreatic Islets: Carcinoma 

Fisher exact test 

Cochran-Annitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pancreatic Islets: Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pituitary Gland (Pars Distalis): Adenoma 

0.0% 

0/48 (0%) 

0/11 (0%) - 

P=O.105 

P=O.178 

P=O.202 

3.6% 

1/48 (2%) 

P=0.476N 

P=0.438N 

P=0.357N 

651 

o/l1 (0%) 

5.9% 

1/48 (2%) 

P=O.472 

P=O.483 

P=O.555 

693 

Oh1 (0%) 

9.2% 

2/48 (4%) 

P=0.566N 

P=0.527N 

P=0.413N 

65 1 

0/11 (0%) 

P=0.308N 

P=0.284N 

P=0.073N 

394 

4/11 (36%) 

72.1% 

25/48 (52%) 

2/13 (15%) 

15.4% 

2/47 (4%) 

P=O.242 

P=O.273 

P=O.273 

733 (T> 

P=O.174 

P=O.196 

559 

1/13 (8%) 

18.9% 

4/47 (9%) 

P=O.174 

OD3 (0%) 

0.0% 

0/47 (0%) 

P=0.498N 

P=0.468N 

- 

P=0.505N 

P=O.330 

P =0.367 

559 

1/13 (8%) 

18.9% 

4/47 (9%) 

P=O.329 

P=O.335 

P=O.540 

331 

10/13 (77%) 

88.5% 

27/46 (59%) 

P=O.331 

4.5% 

1/48 (2%) 

P=O.493 

P=O.474 

666 

0/9 (0%) 

P=O.500 

22.8% 

5/48 (10%) 

P=O.O98 

P=O.O90 

539 

P=O.102 

0/9 (0%) 

9.8% 

3/48 (6%) 

P=O.305 

P=O.295 

560 

OD (0%) 

P=O.308 

30.4% 

8/48 (17%) 

P=O.O45 

P=O.O45 

539 

0/9 (0%) 

P=O.O45 

P=0.412N 

P=O.543 

371 

619 (67%) 

82.8% 

23/48 (48%) 

P=0.419N 

P = 0.204 

P=O.214 

727 

2/17 (12%) 

16.2% 

3/48 (6%) 

P=O.121 

1/17 (6%) 

5.9% 

1/48 (2%) 

P 30.7UN 

P=0.697N 

733 (T> 

P=0.753N 

733 (T) 

1/17 (6%) 

5.9% 

1/48 (2%) 

P=0.702N 

P=0.676N 

P=0.753N 

2/17 (12%) 

11.8% 

2/48 (4%) 

P=0.616N 

P=0.572N 

733 (T> 

P =0.692N 

P =0.487N 

P=0.129N 

485 

8/17 (47%) 

68.2% 

23/47 (49%) 

P=0.460N 
92 Acetonitrile, NTP TR 447 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE A3 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Fibroma 

Fisher exact test 

Cochran-Armitage test. 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Fibroma or Fibrosarcoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test. 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Keratoacanthoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Preputial Gland: Carcinoma 

Preputial Gland: Adenoma or Carcinoma 

0/11 (0%) 

5/47 (11%) 

15.8% 

P=0.089N 

P=0.113N 

P=0.075N 

472 

0/11 (0%) 

5/47 (11%) 

15.8% 

P=0.109N 

P=0.133N 

P=0.087N 

472 

17.3% 

3/48 (6%) 

P=O.572 

P=0.544N 

P=0.471N 

602 

1/11 (9%) 

17.3% 

3/48 (6%). 

P=0.461N' 

P=0.394N 

602 

1/11 (9%) 

P=0:540N 

0.0% 

0/48 (0%). 

0/11 (0%) - 

E=0.014 

P=O.O36 

P = 0.039 

P=0.089N 

P=0.112N 

567 

0/13 (0%) 

2.9% 

1/47 (2%) 

P=0.102N 

P=0.089N 

P=0.112N 

567 

0/13 (0%) 

2.9% 

1/47 (2%) 

P=0.102N 

P=0.497N 

P=0.448N 

701 

1/13 (8%) 

12.6% 

2/47 (4%) 

P=0.510N 

P=0.659N 

P=0.603N 

701 

2/13 (15%) 

19.8% 

3/47 (6%) 

P=O.651 

P=O.516 

P=O.553 

718 

0/13 (0%) 

5.9% 

1/47 (2%) 

P=O.495 

8.7% 

2/47 (4%) 

P=0.195N 

P=0.257N 

665 

0/9 (0%) 

P=0.217N 

P=0.345N 

P=0.413N 

665 

1/9 (11%) 

18.8% 

3/47 (6%) 

P=0.357N 

P=0.325N 

P =0.347N 

707 

0/9 (0%) 

7.7% 

1/48 (2%) 

P=0.308N 

P=0.325N 

P =0.347N 

707 

0/9 (0%) 

7.7% 

1/48 (2%) 

P=0.308N 

0/9 (0%) 

0.0% 

0/48 (0%) 

- 

- 

- 

- 

P=0.201N 

P=O.O81N 

485 

0/17 (0%) 

2.1% 

1/48 (2%) 

P=0.097N 

P=0.201N 

P=O.O8lN 

485 

0/17 (0%) 

2.1% 

1/48 (2%) 

P=0.097N 

P=0.642N 

P=0.527N 

560 

0/17 (0%) 

10.4% 

3/48 (6%) 

P=0.661N 

P=0.642N 

P=0.527N 

560 

0/17 (0%) 

10.4% 

3/48 (6%) 

P=0.661N 

4/17 (24%) 

23.5% 

4/48 (8%) 

P=O.O59 

P=O.122 

P=O.122 

733 (T> 
Lesions in Male Rats 93 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE A3 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Keratoacanthoma, Trichoepithelioma, or Basal Cell Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Testes: Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Carcinoma 

Fisher exact test 

Cochran-Annitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

0.0% 

0/48 (0%) 

on1 (0%) - 

P=O.O14 

P=O.O37 

P=O.O49 

P=O.O12 

P=O.O55 

P=0.408N 

492 

10/11 (91%) 

96.9% 

33/48 (69%) 

31.1% 

6/48 (13%) 

P=O.506 

P=0.497N 

P=0.382N 

646 

2/11 (18%) 

10.7% 

3/48 (6%) 

P-0.198N 

P=0.180N 

P=0.161N 

511 

0/11 (0%) 

38.4% 

9/48 (19%) 

P=0.400N 

P=0.291N 

P=0.204N 

511 

2/11 (18%) 

P=O.265 

P=O.289 

718 

1/13 (8%) 

13.1% 

2/47 (4%) 

P=O.242 

P=O.347 

P=0.492N 

446 

13/13 (100%) 

100.0% 

35/47 (74%) 

P=O.348 

P=0.504N 

P=0.432N 

525 

2/13 (15%) 

25.5% 

5/47 (11%) 

P=0.515N 

1/13 (8%) 

7.7% 

1/47 (2%) 

P=0.311N 

P=0.299N 

733 (T) 

P=0.316N 

P=0.292N 

P=0.244N 

525 

3/13 (23%) 

32.3% 

6/47 (13%) 

P=0.303N 

P=O.493 

P=O.474 

666 

019 (0%) 

4.5% 

1/48 (2%) 

P=O.500 

100.0% 

40/48 (83%) 

P=O.O25 

P=O.O86 

395 

9/9 (100%) 

P=O.O75 

35.8% 

7/48 (1 5%) 

P=O.471 

P=O.415 

573 

2/9 (22%) 

P=O.500 

0.0% 

0/48 (0%) 

0/9 (0%) - 

P=0.118N 

P=O.lSON 

P=0.121N 

35.8% 

7/48 (15%) 

P=0.414N 

P=0.496N 

573 

2/9 (22%) 

P=0.392N 

P=O.O77 

P=O.O82 

727 

4/17 (24%) 

27.4% 

5/48 (10%) 

P=O.O28 

P=O.120 

P=0.403N 

483 

17/17 (100%) 

100.0% 

42/48 (88%) 

P = 0.023 

P=0.474N 

P=0.379N 

672 

3/17 (18%) 

27.3% 

6/48 (13%) 

P=0.621N 

1/17 (6%) 

5.9% 

1/48 (2%) 

P=0.308N 

P=0.239N 

733 (T> 

P=0.308N 

P=0.270N 

P=0.184N 

672 

4/17 (24%) 

32.5% 

7/48 (15%) 

P=0.392N 
94 Acetonitrile, NTP TR 447 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE A3 

- 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Mononuclear Cell Leukemia 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Mesothelioma NOS 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted. rate 

Overall rate 

All Organs: Benign Neoplasms 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall'rate 

All Organs: Malignant Neoplasms 

P=O.320 

P=O.423 

P=0.214N 

418 

7/11 (64%) 

85.4% 

29/48 (60%) 

7.4% 

3/48 (6%) 

P=0.121N 

P =0.087N 

492 

0/11 (0%) 

P=0;134N 

100.0% 

45/48 (94%) 

P=O.260 

P =0.490 

P=O.lOlN 

394 

11/11 (100%) 

P =0.376N' 

P=0.327N 

P=0.084N 

418 

7/11 (64%) 

88.6% 

36/48 (75%) 

P=O.289 

P=O.485 

530 

8/13 (62%) 

85.4% 

32/47 (68%) 

P=O.286 

P=O.344 

P-0.384 

639 

2/13 (15%) 

26.8% 

5/47 (11%) 

P=O.345 

P=O.514 

P=0.411N 

331 

13/13 (100%) 

100.0% 

45/47 (96%) 

P=O.510 

P=0.564N 

P=0.402N 

530 

9/13 (69%) 

89.5% 

35/47 (74%) 

P=0.570N 

85.4% 

35/48 (73%) 

P=O.134 

P=O.142 

442 

419 (44%) 

P=O.139 

15.3% 

2/48 (4%) 

P=0.486N 

P=0.545N 

673 

1/9 (11%) 

P=0.500N 

100.0% 

45/48 (94%) 

P=O.606 

P = 0.329 

371 

919 (100%) 

P=0.661N 

86.7% 

38/48 (79%) 

P=O.423 

P=O.293 

371. 

4/9 (44%) 

P = 0.404 

P=O.493 

P=0.231N 

547 

12/17 (71%) 

85.6% 

32/48 (67%) 

P=O.336 

1/17 (6%) 

5.9% 

1/48 (2%) 

P=0.441N 

P=0.250N 

733 (T) 

P=0.308N 

P=O.675 

P=O.lOSN 

483 

17/17 (100%) 

lOO:O% 

47/48 (98%) 

P=O.308 

P-0.360N. 

P=0.076N 

485 

12/17 (71%) 

86.5% 

34/48, (71 %) 

P=.O.409N 
Lesions in Male Rats 95 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

48/48 (100%) 47/47 (100%) 48/48 (100%) 48/48 (100%) 

100.0% 100.0% 100.0% 100.0% 

Pf=0.071N P=0.373N P=O.333 P=0.069N 

394 331 371 483 

11/11 (100%) 13/13 (100%) 9/9 (100%) 17/17 (100%) 

P = 1.000N P=l.OOON P = 1.000N 

(T)Terminal sacrifice 

exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Annitage and Fisher 

to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

Observed incidence at terminal kill 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

liver, pancreatic islets, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

Value of statistic cannot be computed. 

e Not applicable; no neoplasms in animal group 
96 Acetonitrile, NTP TR 447 

Historical Incidence of Hepatocellular Neoplasms in Untreated Male F344/N Ratsa 

TABLE A4a 

Study Carcinoma 

Incidence in Controls 

Adenoma Adenoma. or Carcinoma 

Historical Incidence at Battelle Pacific Northwest Laboratories 

Hexachlorocyclopentadiene 

Chloroethane 

I-Epinephrine Hydrochloride 

2-Chloroacetophenone 

o-Chlorobenzalmalononitrile (CS2) 

Overall Historical Incidence 

1/50 

0/50 

1/50 

1/49 

2/50 

0/50 

1/50 

0150 

0149 

2/50 

1/50 

1/50 

1/50 

1/49 

4/50 

Range 

Standard deviation 

Total 

2.6% 

11/398 (2.8%) 

1.5% 

4/398 (1.0%) 

0%-8% 0%-4% 2%-8% 

15/398 (3.8%) 

2.7% I 

a Data as of 31 March 1993 

Historical Incidence of Adrenal Gland Pheochromocytomas in Untreated Male F344/N Ratsa 

TABLE A4b 

Study Malignant 

Incidence in Controls 

Benign .. 

Complex, or NOS 

Benign, Malignant, 

. -. 

Historical Incidence at Battelle Pacific Northwest Lahoratories 

Hexachlorocyclopentadiene 

Chloroethane 

I-Epinephrine Hydrochloride 

2-Chloroacetophenone 

o-Chlorobenzalmalononitrile (CS2) 

15/50 

8/36 

11/50 

14/46 

18/42 

2/50 

0/36 

015 0 

2/46 

4/42 

16/50 

8/36 

11/50 

15/46 

20/42 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

0%-43% 

12.2% 

921368 (25.0~0) 

0%-10% 

3.4% 

101368 (2.7%) 

14%-48% 

10.3% 

107/368 (29.1%) 

.. 

a Data as of 31 March 1993 
Lesions in Male Rats 97 

Historical Incidence of Pancreatic Islet Neoplasms in Untreated Male F344/N Ratsa 

TABLE A4c 

Study Carcinoma 

Incidence in Controls 

Adenoma Adenoma or Carcinoma 

Historical Incidence at Battelle Pacific Northwest Laboratories 

Hexachlorocyclopentadiene 

Chloroethane 

I-Epinephrine Hydrochloride 

2-Chloroacetophenone 

o-Chlorobenzalmalononitrile (CS2) 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

7/50 

6/48 

5/50 

3/47 

1/50 

2%-14% 

4.6% 

33/390 (8.5%) 

4/50 

0148 

1/50 

1/47 

0150 

0%-10% 

3.9% 

12/390 (3.1%) 

11/50 

6/48 

6/50 

4/47 

1/50 

2%-22% 

6.1% 

45/390 (1 1 .5%) 

a Data as of 31 March 1993 

Historical Incidence of Skin Keratoacanthomas in Untreated Male F344/N Ratsa 

TABLE A4d 

Study Incidence in Controls 

Historical Incidence at Battelle Pacific Northwest Laboratories 

Hexachlorocyclopentadiene 

Chloroethane 

2-Epinephrine Hydrochloride 

2-Chloroacetophenone 

o-Chlorobenzalmalononitrile (CS2) 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

0/50 

4/50 

2/50 

4/50 

0150 

0%-8% 

3.3% 

14/399 (3.5%) 

a Data as of 31 March 1993 
98 Acetonitrile, NTP TR 447 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Acetonitrilea 

TABLE A5 

0 PPm 100 ppm 200 ppm 400 ppm 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Early deaths 

Terminal sacrifice 

15-Month ht&n ~~olrtotion 

Survivors 

Animals examined microscopically 

8 

56 

1 

36 

11 

56 

8 

55 

8 

26 

13 

55 

8 

56 

4 

35 

9 

56 

8 

56 

5 

26 

17 

56 

15-Month Interim Evaluation 

Parasite metazoan 

Intestine small, jejunum 

Lumen, parasite metazoan 

Intestine large, rectum 

Alimentary System 

Portal, fibrosis 

Hepatodiaphragmatic nodule 

Degeneration, cystic 

Clear cell focus 

Basophilic focus 

Liver 

Acinus, atrophy 

Fat, necrosis 

Fat, inflammation, granulomatous 

Mesentery 

Pancreas 

(8) 

(8) 

(8) 

1 (13%) 

3 (38%) 

6 (75%) 

1 (13%) 

(8) 

3 (38%) 

(1) 

2 (25%) 

1 (100%) 

1 (100%) 

(8) (8) (8) 

5 (63%) 2 (25%) 

Heart 

Blood vessel 

Cardiovascular System 

Cardiomyopathy 

Mineralization 

Thyroid gland 

Pituitary gland 

Endocrine System 

Pars distalis, hyperplasia 

Follicle. dilatation 

C-cell, hyperplasia 

Ultimobranchial cyst 

(8) 

(8) 

(8) 

(7) 

(8) 

(8) 

(8) 

(7) 

2 (25%) 4 (57%) 

2 (25%) 

2 (25%) 

4 (50%) 2 (29%) 

1 (13%) 

1 (13%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Male Rats 99 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study ofAcetonitrile 

TABLE A5 

0 PPm 100 ppm 200 ppm 400 ppm 

Preputial gland 

Genital System 

15-Mortth Interim Evaluation (continued) 

Inflammation, suppurative 

Inflammation, chronic 

Hyperplasia 

Granuloma 

Ectasia 

Inflammation, suppurative 

Inflammation, suppurative 

(8) 

3 (38%) 

2 (25%) 

2 (25%) 

Testes 

Seminal vesicle 

Prostate 

(8) 

(8) 

(8) 

Interstitial cell, hyperplasia 

Germinal epithelium, atrophy 

5 (63%) 

(8) 

1 (13%) 

2 (25%) 

1 (13%) 

1 (13%) 

2 (25%) 

I (88%) 

(8) 

(8) 

(8) 

1 (13%) 

4 (50%) 

1 (13%) 

1 (13%) 

(8) 

(8) 

(8) 

(8) 

(8) 

(8) 

1 (13%) 

5 (63%) 

Hematopoietic System 

Renal, pigmentation 

Lumbar, pigmentation 

Lymph node 

Hyperplasia, RE cell 

Fibrosis 

Spleen 

(1) (2) (1) 

1 (100%) 

1 (50%) 

1 (50%) 1 (100%) 

(8) (8) (8) (8) 

1 (13%) 

2 (25%) 1 (13%) 

Skin 

Integumentary System 

(8) 

Inflammation, granulomatous 

Acanthosis 

Larynx 

Respiratory System 

Mineralization 

Inflammation, suppurative 

Foreign body 

Artery, inflammation, chronic active 

Alveolar epithelium, hyperplasia 

Infiltration cellular, histiocyte 

Hemorrhage 

Respiratory epithelium, hyperplasia 

Inflammation, chronic 

Hemorrhage 

Foreign body 

Peritracheal tissue, inflammation, chronic 

Peritracheal tissue, fibrosis 

Lung 

Nose 

Trachea 

active 

(8) (8) 

1 (13%) 

1 (13%) 

(8) (8) 

8 (100%) 

4 (50%) 

4 (50%) 

8 (100%) 

3 (38%) 

(8) (8) 

1 (13%) 

1 (13%) 

(8) 

8 (100%) 

1 (13%) 

1 (13%) 

1 (13%) 

(8) 

(8) 

1 (13%) 

3 (38%) 

3 (38%) 

(8) 

8 (100%) 

1 (13%) 

2 (25%) 

(8) 

1 (13%) 

1 (13%) 

1 (13%) 

1 (13%) 

(8) 

1 (13%) 
100 Acetonitrile, NTP TR 447 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study ofAcetonitrile 

TABLE A5 

1 

0 PPm 100 ppm 200 ppm 400 ppm 

Eye 

Special Senses System 

15-Month Interim Evaluation (continued) 

Cataract 

(1) 

1 (100%) 

Gdney 

Urinary System 

Nephropathy, chronic 

Nervous System 

Musculoskeletal System 

General Body System 

Systems Examined With No Lesions Observed 

Intestine large, colon 

Alimentary System 

2- Year Study 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Liver 

Parasite metazoan 

Diverticulum 

Lumen, parasite metazoan 

Lumen, parasite metazoan 

Lumen, parasite metazoan 

Hepatocyte, necrosis 

Bile duct, hyperplasia 

Vacuolization cytoplasmic 

Thrombosis 

Pigmentation, hemosiderin 

Mixed cell focus 

Inflammation, chronic 

Hepatodiaphragmatic nodule 

Hematopoietic cell proliferation 

Granuloma, multifocal 

Eosinophilic focus 

Degeneration, cystic 

Clear cell focus 

Basophilic focus 

Angiectasis 

Fat, necrosis 

Fat, inflammation, granulomatous 

Hemorrhage 

Mesentery 

(48) 

(47) 

(47) 

(47) 

(47) 

(48) 

4 (8%) 

4 (9%) 

4 (9%) 

1 (2%) 

3 (6%) 

15 (31%) 

3 (6%) 

15 (31%) 

1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 

7 (70%) 

3 (30%) 

7 (15%) 

11 (23%) 

2 (4%) 

(10) 

1 (10%) 

(47) 

(42) 

(45) 

(43) 

(44) 

(42) 

1 (2%) 

3 (7%) 

4 (9%) 

2 (5%) 

1 (2%) 

22 (47%) 

1 (2%) 

7 (15%) 

17 (36%). 

4 (9%) 

1 (2%) 

2 (4%) 

10 (21%) 

1 (2%) 

1 (2%) 

8 (17%): 

(9) 

1 (11%) 

7 (78%) 

(48) 

(46) 

(48) 

(46) 

(47) 

(46) 

4 (9%) 

6 (13%) 

1 (2%) 

5. (10%) 

2 (4%). 

1 (2%) 

8 (17%) 

25 (52%) 

4 (8%) 

1 (2%) 

1 (2%) 

3 (6%) 

12 (25%) 

1 (11%) 

3 (33%). 

1 (11%) 

(9) 

(44) 

(46) 

(45) 

4 (9%) 

1 (2%) 

4 (8%) 

31 (65%) 

(48) 

(44) 

(47) 

10 (21%). 

5 (10%) 

3 (6%) 

5 (10%). 

2 (4%) 

5 (10%) 

7 (64%) 

4 (36%) 

3 (6%). 

(11) 

1 (9%) 
Lesions in Male Rats 101 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE A5 

0 PPm 100 ppm 200 ppm 400 ppm 

StUdy (continued) 

Pancreas 

Alimentary System (continued) 

Duct, hyperplasia 

Duct, cyst 

Artery, inflammation 

Acinus, atrophy 

Hemorrhage 

Ulcer 

Necrosis 

Mineralization 

Inflammation, suppurative 

Hyperkeratosis 

Edema 

Diverticulum 

Acanthosis 

Stomach, forestomach 

Ulcer 

Necrosis 

Mineralization 

Inflammation, suppurative 

Hemorrhage 

Epithelium, hyperplasia 

Tongue 

Stomach, glandular 

(48) 

(48) 

1 (2%) 

21 (44%) 

4 (8%) 

1 (2%) 

5 (10%) 

4 (8%) 

(48) 

1 (2%) 

(47) 

30 (64%) 

1 (2%) 

1 (2%) 

1 (2%) 

4 (9%) 

(47) 

3 (6%) 

1 (2%) 

4 (9%) 

2 (4%) 

3 (6%) 

(46) 

1 (2%) 

1 (2%) 

(2) 

1 (50%) 

(48) 

1 (2%) 

2 (4%) 

10 (21%) 

7 (15%) 

7 (15%) 

1 (2%) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(48) 

2 (4%) 

2 (4%) 

29 (60%) 

(47) 

2 (4%) 

4 (9%) 

1 (2%) 

3 (6%) 

(47) 

1 (2%) 

Cardiovascular System 

Aorta, mineralization 

Aorta, inflammation 

Blood vessel 

Myocardium, necrosis 

Myocardium, mineralization 

Atrium, thrombosis 

Cardiomyopathy 

Heart 

(48) (46) (48) (48) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

(48) (47) (48) (48) 

8 (17%) 

9 (19%) 

3 (6%) 

7 (15%) 

3 (6%) 

7 (15%) 7 (15%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Endocrine System 

Vacuolization cytoplasmic 

Hyperplasia 

Hemorrhage 

Adrenal cortex 

Hyperplasia 

Parathyroid gland 

Hyperplasia 

Islets, pancreatic 

Hyperplasia 

Adrenal medulla 

20 (42%) 

7 (15%) 

(47) 

(48) 

(48) 

2 (4%) 

1 (2%) 

(47) 

1 (2%) 

16 (35%) 

8 (17%) 

(46) 

(47) 

(46) 

5 (11%) 

(48) (48) 

10 (21%) 

1 (2%) 

2 (4%) 

4 (9%) 

2 (4%) 

21 (44%) 16 (33%) 

7 (15%) 

(46) 

(48) 

(48) 

(45) 

(48) 

(48) 

2 (4%) 
102 Acetonitrile, NTP TR 447 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE A5 

0 PPm 100 ppm 200 ppm 400 porn 

Pituitaxy gland 

Endocrine System (continued) 

%Year Study (continued) 

Pars intermedia, hyperplasia 

Pars distalis, hyperplasia 

Pars distalis, hemorrhage 

Hemorrhage 

Follicular cell, hyperplasia 

C-cell, hyperplasia 

Cyst 

Thyroid gland 

(48) '(46) (48) (47) 

3 (6%) 7 (15%) 8 (17%) 4 (9%) 

1 (2%) 

1 (2%) 

1 (2%) 

9 (19%) 10 (21%) 

1 (2%) 

5 (11%) 

2 (4%) 

5 (10%) 

1 (2%) 

6 (13%) 8 (17%) 

(48) (47) (48) (48) 

7 (15%) 12 (25%) 

None 

General Body System 

Inflammation, suppurative 

Epididymis 

Genital System 

Prostate 

Inflammation, suppurative 

Inflammation, chronic 

Hyperplasia 

Ectasia 

Preputial gland 

Cyst 

Epithelium, hyperplasia 

Inflammation, suppurative 

Inflammation, suppurative 

Hyperplasia 

Seminal vesicle 

Interstitial cell, hyperplasia 

Germinal epithelium, atrophy 

Necrosis 

Inflammation, suppurative 

Hemorrhage 

Testes 

(47) 

(48) 

19 .(40%) 

5 (11%) 

.1 (2%) 

1 (2%) 

(48) 

7 (15%) 

1 (2%) 

(48) 

1 (2%) 

10 (21%) 

1 (2%) 

1 .(2%) 

1 (2%) 

(48) 

16 (33%) 

(47) 

(47) 

14 (30%) 

1 (2%) 

5 (11%) 

2 (4%) 

1 (2%) 

1 42%) 

1 (2%) 

1 (2%) 

9 (19%) 

(47) 

(46) 

(47) 

12 (26%) 

13 (28%) 

(47) 

(48) 

5 (11%) 

1 (2%) 

20 .(43%) 

1 (2%) 

' (48) 

2 (4%) 

1 (2%) 

1 (2%) 

9 (19%) 

(48) 

.(48) 

'9 (19%) 

16 .(33%) 

(48) 

(48) 

7 (15%) 

3 (6%) 

16 (33%) 

(48) 

12 (25%) 

1 (2%) 

2 (4%) 

(48) 

8 (17%) 

9 (19%) 

Hematopoietic 'System 

Myelofibrosis 

Hyperplasia, RE cell 

Bone marrow 

Renal, pigmentation 

-Renal,'hemorrhage 

Renal, ectasia 

Pancreatic, hyperplasia, lymphoid 

Lumbar, pigmentation 

Iliac, pigmentation 

Inflammation, granulomatous 

Lymph node 
Lesions in Male Rats 103 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE A5 

0 PPm 100 ppm 200 ppm 400 ppm 

Lymph node, mandibular 

Hematopoietic System (continued) 

%Year Study (continued) 

Hyperplasia, lymphoid 

Hemorrhage 

Inflammation, granulomatous 

Hyperplasia, lymphoid 

Hemorrhage 

Lymph node, mesenteric 

Pigmentation 

Hyperplasia, lymphoid 

Hemorrhage 

Fibrosis 

Lymph node, mediastinal 

Necrosis 

Hyperplasia, RE cell 

Hemorrhage 

Fibrosis 

Developmental malformation 

Congestion 

Hemorrhage 

(43) (43) (45) (47) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (5%) 

1 (2%) 

1 (2%) 

(48) (47) (47) (48) 

1 (2%) 1 (2%) 

1 (2%) 

(48) (46) (48) (48) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

5 (11%) 

1 (2%) 

1 (2%) 1 (2%) 

Spleen (48) (47) (48) (48) 

2 (4%) 

1 (2%) 

2 (4%) 2 (4%) 

4 (8%) 

1 (2%) 

7 (15%) 11 (23%) 18 (38%) 14 (29%) 

1 (2%) 

1 (2%) 

2 (4%) 

Thymus (48) (45) (47) (48) 

Integumentary System 

Epithelium, hyperplasia 

Inflammation, chronic 

Hemorrhage 

Galactocele 

Mammary gland 

Acanthosis 

Subcutaneous tissue, hemorrhage 

Ulcer 

Inflammation, suppurative 

Inflammation, granulomatous 

Inflammation, chronic 

Hyperkeratosis 

Cyst epithelial inclusion 

Skin 

Cyst 

(47) (47) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

(48) (47) 

2 (4%) 

4 (8%) 

2 (4%) 

1 (2%) 

(48) (48) 

3 (6%) 

1 (2%) 

3 (6%) 1 (2%) 

(48) (48) 

2 (4%) 

1 (2%) 

1 (2%) 1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Bone 

Musculoskeletal System 

(48) 

Fibrosis 

Developmental malformation 

(47) (48) 

.1 (2%) 

1 (2%) 
104 Acetonitrile, NTP TR 447 

TABLE A5 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the %Year Inhalation Study of Acetonitrile 

~~ ~~~~~ 

0 PPm 100 ppm 200 ppm 400 ppm 

Brain 

Nervous System 

%Year study (continued) 

Necrosis 

Hydrocephalus 

Hemorrhage 

Gliosis 

Demyelination 

(48) (47) (48) (48) 

1 (2%) 

5 (11%) 

1 (2%) 

7 (15%) 

3 (6%) 

1 (2%) 

7 (1.5%) 

6 (13%) 4 (8%) 

7 (15%) 2 (4%) 

Larynx 

Respiratory System 

(48) (46) (48) (48) 

Mineralization 

Metaplasia, squamous 

Inflammation, suppurative 

Hyperplasia 

Foreign body 

Pleura, fibrosis 

Alveolus, pigmentation 

Alveolus, mineralization 

Alveolus, inflammation, chronic 

Alveolus, fibrosis 

Alveolar epithelium, hyperplasia 

Thrombosis 

Metaplasia, osseous 

Infiltration cellular, histiocyte 

Infarct 

Hemorrhage 

Embolus 

Goblet cell, respiratory epithelium, 

Inflammation, suppurative 

Inflammation, chronic 

Hemorrhage 

Foreign body 

Respiratory epithelium, hyperplasia 

Olfactory epithelium, metaplasia 

Nasopharyngeal duct, hyperplasia 

Nasolacrimal duct, inflammation 

4 (8%) 

4 (8%) 

1 (2%) 

2 (4%) 

3 (7%) 

4 (9%) 

3 (7%) 

1 (2%) 

1 (2%) 

5 @O%) 

4 (8%) 

6 (13%) 

1 (2%) 

1 (2%) 

4 (8%) 5 (10%) 

Lung (48) (47) (48) (48) 

1 (2%) 

7 (15%) 

16 (33%) 

12 (26%) 

29 (62%) 

13 (27%) 

26 (54%) 

9 (19%) 

27 (56%) 

1 (2%) 

1 (2%) 

1 (2%) 

4 (8%) 

3 (6%) 3 (6%) 

7 (15%) 

3 (6%) 9 (19%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

10 (21%) 

1 (2%) 1 (2%) 

Nose (48) (47) (48) (48) 

8 (17%) 

8 (17%) 

6 (13%) 

3 (6%) 

7 (15%) 6 (13%) 

9 (19%) 

4 (8%) 

6 (13%) 

4 (9%) 

2 (4%) 

2 (4%) 

4 (9%) 

2 (4%) 

10 (21%) 

1 (2%) 

5 (10%) 

hypertrophy 3 (6%) 

1 (2%) 

1 (2%) 

5 (10%) 5 (11%) 

1 (2%) 

6 (13%) 7 (15%) 

Eye 

Special Senses System 

Cataract 

(2) 

2 (100%) 
Lesions in Male Rats 105 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE A5 

0 PPm 100 ppm 200 ppm 400 ppm 

Kidney 

Urinary System 

2-YefZr shdy (continued) 

Renal tubule, mineralization 

Renal tubule, hyperplasia 

Pelvis, transitional epithelium, hyperplasia 

Pelvis, dilatation 

Cortex, necrosis 

Thrombosis 

Nephropathy, chronic 

Transitional'epithelium, hyperplasia 

Ulcer 

Mineralization 

Metaplasia, squamous 

Inflammaiion, suppurative 

Hemorrhage 

Urinary bladder 

(48) 

1 (2%) 

1 (2%) 

45 (94%) 

3 (6%) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

2 (4%) 

(47) 

1 (2%) 

1 (2%) 

47 (100%) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(47) 

2 (4%) 

(48) (48) 

3 (6%) 

47 (98%) 48 (100%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(48) (48) 

2 (4%) 
106 Acetonitrile, NTP TR 447 
107 

OF ACETONITRILE 

SUMMARY OF LESIONS IN FEMALE RATS 

APPENDIX B 

IN THE 2-YEAR INHALATION STUDY 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE B3 Statistical Analysis of Primary Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE B2 Individual Animal Tumor Pathology of Female Rats 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE B1 Summary of the Incidence of Neoplasms in Female Rats 

............................... 

136 

132 

114 

109 

............................... 

............................... 

............................... 
Acetonitrile, NTP TR 447 
Lesions in Female Rats 109 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrilea 

TABLE Bl 

0 PPm 100 ppm 200 ppm 400 ppm 

Early deaths 

I5-Month intabn evalucyion 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Survivors 

Terminal sacrifice 

Animals examined microscopically 

8 

56 

24 

1 

23 

56 

8 

56 

2 

25 

21 

56 

8 

56 

1 

21 

26 

56 

8 

56 

5 

14 

29 

56 

15-Month Interim Evaluation 

Thyroid gland 

Pituitary gland 

Endocrine System 

(8) 

(8) 

C-cell. adenoma 

Pars distalis, adenoma 3 (38%) 

Carcinoma 

Clitoral gland 

Genital System 

Bilateral, polyp stromal 

Polyp stromal 

Uterus 

Fibroadenoma 

Mammary gland 

Integumentary System 

(8) (8) 

1 (13%) 2 (25%) 

Multiple organsb 

Systemic Lesions 

Leukemia mononuclear 

(8) 

1 (13%) 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Hematopoietic System 

General Body System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Neoplasms Observed 
110 Acetonitrile, NTP TR 447 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B1 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Alimentary System 

2- Year Study 

Hepatocellular, adenoma 

Fibrous histiocytoma, metastatic, skin. 

Lipoma 

metastatic, ovary 

Granulosa-theca tumor malignant, 

Mesentery 

Salivary glands 

Pharynx 

Pancreas 

Fibrous .histiocytoma, metastatic, skin 

Squamous cell papilloma 

(48) 

(47) 

(47) 

1 (2%) 

2 (4%) 

(9) 

1 (11%). 

1 (11%). 

(48) 

Heart: 

Blood.vessel 

Cardiovascular System 

Fibrous histiocytoma, metastatic, skin (48) 

1 (2%) 

(48). (47) 

(47) 

Adrenal cortex 

Endocrine System 

Granulosa-theca tumor; malignant, 

Neoplasm NOS 

Adrenal medulla. 

metastatic, ovary 

Adenoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Schwannoma malignant, metastatic, brain 

Carcinoma, metastatic, thyroid gland 

carcinoma 

Pars.intermedia, adenoma 

Pars distalis, adenoma 

Follicular cell, carcinoma 

Follicular cell, adenoma 

C-cell, carcinoma 

C-cell, adenoma. 

Islets, pancreatic 

Pituitary gland 

Parathyroid.gland 

Thyroid gland. 

1 (2%) 

(48) 

24 (50%) 

(48) 

3. (6%), 

1 (2%) 

26 (54%) 

1 (2%) 

(448). 

4 (8%) 

2 (4%) 

1 (2%) 

(47) 

2 (4%) 

2 (4%) 

1 (2%) 

(48), 

(47) 

(44) 

1 (2%) 

28 (60%) 29~ (64%). 

None 

General Body..System 
Lesions in Female Rats 111 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B1 

~~ ~~~ 

100 ppm 200 ppm 400 ppm 

Clitoral gland 

Genital System 

%Year Study (continued) 

Adenoma 

Carcinoma 

Granulosa-theca tumor malignant 

Carcinoma 

Bilateral, polyp stromal 

Polyp stromal 

Leiomyosarcoma 

Hemangioma 

Uterus 

Ovary 

1 (2%) 

7 (15%) 4 (8%) 

(47) (45) 

4 (9%) 

1 (2%) 

2 (4%) 

3 (7%) 

(48) (47) 

(47) 

1 (2%) 

1 (2%) 

7 (15%) 5 (11%) 

2 (4%) 

Lymph node 

Bone marrow 

Hematopoietic System 

Renal, granulosa-theca tumor malignant, 

metastatic, ovary 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Thymus 

Spleen 

Lymph node, mediastinal 

Fibrous histiocytoma, metastatic, skin 

Fibrous histiocytoma, metastatic, skin 

Fibrous histiocytoma, metastatic 

Thymoma NOS 

Integumentary System 

Fibrosarcoma 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammary gland 

Subcutaneous tissue, sarcoma 

Pinna, melanoma NOS 

Trichoepithelioma 

Squamous cell carcinoma 

Fibrous histiocytoma 

Fibroma, multiple 

Fibroma 

Basal cell carcinoma 

Skin 

(48) 

2 (4%) 

4 (8%) 

12 (25%) 

1 (2%) 

1 (2%) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

(48) 

6 

21 

4 

(48) (47) 

(44%) 

(8%) 

4 (8%) 

17 (35%) 

3 (6%) 

16 (34%) 

3 (6%) 

(13%) 7 (15%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 
1.12 Acetwitrile, NTP TR. 447 

Summary of the Incidence of Neoplasms in Female Rats in the. 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B1 

400 ppm 

Bone 

Musculoskeletal System 

Cranium, carcinoma, metastatic, Zymbal’s 

Schwannoma. malignant, metastatic, brain 

Skeletal muscle 

gland, 1 (2%) 

(1) 

Fibrous histiocytoma, metastatic, skin 1. (100%) 

(48) 

1 (2%) 

(47) 

Brain 

Nervous System 

(48) 

Cranial nerve, schwannoma malignant 

Glioma NOS 

Carcinoma, metastatic, Zymbal’s gland 

Astrocytoma NOS 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(47) 

Lung 

Respiratory System 

(48) 

Fibrous histiocytoma, metastatic, skin 

Carcinoma, metastatic, mammary gland 

Alveolar/bronchiolar adenoma 

2 (4%) 

1 (2%) 

(46) 

2 (4%) 

Carcinoma 

Zymbal’s gland 

Special Senses System 

(2) (1) 

2 (100%) 1 (100%) 

.. . . . . , .. 

Kidney 

Urinary System 

(48) (48) (48) (46) 

Papilloma 

Lipoma 

Granulosa-theca tumor malignant, 

metastatic, ovary 1 (2%) 

1 (2%) 

Urinary bladder (48) (48) (471, (45) 

1 (2%) 

Multiple organs 

Systemic Lesions 

Leukemia mononuclear 

(48) (48) 

18 (38%) 22 (46%) 

(48) 

20 (42%) 
Lesions in Female Rats 113 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B1 

0 PPm 100 PPm 200 ppm 400 ppm 

Total animals with primary neoplasms' 

Neoplasm Summary 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

benign or malignant 

2-Year study 

2-Year study 

2-Year study 

2-Year study 

2-Year study 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with uncertain neoplasmsTotal metastatic neoplasms 

Total animals with metastatic neoplasms 

Total uncertain neoplasms 

47 

3 

46 

2 

45 

5 

46 

5 

106 

2 

100 

5 

103 

9 

88 

3 

38 

3 

39 

1 

38 

5 

38 

5 

69 

5 

70 

8 

56 

3 

70 

1 

28 

1 

27 

1 

29 23 

31 

1 

35 

1 

29 

2 

1 

31 

2 

2 6 

16 

2 

1 2 

1 

1 2 

1 2 

' Primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 
114 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 0 ppm 

TABLE B2 

5010770508735725267411179 

01367272.38922366000122222 

1333344555566666777777777 

Number of Days on Study 

Carcass ID Number 

5793386815122299671549448 

7889750600770197951966087 

0000001011001100001000100 

Pharynx 

Pancreas 

Mesentery 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Squamous cell papilloma 

......................... 

++A++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

++A++++++++++++++M+++++++ 

++A++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

......................... 

......................... +++ + + + 

+++++++++++++++++++I+++++ 

......................... 

......................... 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

......................... 

......................... 

......................... 

++++M+++++++++++++M+M++++ 

......................... 

X X 

......................... x x xx xxx 

Pheochromocytoma benign 

Follicular cell, carcinoma 

C-cell, adenoma 

Pars distalis, adenoma 

X 

General Body System 

None 

Genital System 

Clitoral gland +++++++++ +++++++++++++++ 

Carcinoma 

Adenoma 

Uterus 

Ovary 

X X 

......................... 

......................... I 

Polyp stromal X 

A: Autolysis precludes examination 

+: Tissue examined microscopically 

I: Insufficient tissue 

M: Missing tissue 

Blank Not examined 

X Lesion present 
Lesions in Female Rats 115 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE B2 

77777777777777777777777 

Number of Days on Study 

44444444444444444444444 

33333333333333333333333 

Carcass ID Number 

01256706012680127037890 

66666677888889999000001 

00000000000000000111111 

Tumors 

Tissues/ 

Total 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

....................... 

....................... 

....................... 

....................... 

47 

47 

47 

48 

Intestine small, duodenum ....................... 46 

Pharynx 

Pancreas 

Mesentery 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Squamous cell papilloma 

....................... 

....................... 

....................... 

++ ++ 

....................... 

....................... 

....................... 

X 

+ 

....................... 

47 

48 

48 

1 

1 

48 

10 

48 

47 

47 

Cardiovascular System 

Heart 

Blood vessel 

....................... 

....................... 48 

48 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

....................... 48 

48 

........................ Pheochromocytoma benign 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

Follicular cell, carcinoma 

Ccell, adenoma 

Pars distalis, adenoma 

X 

1 

3 

48 

24 

48 

44 

48 

1 

....................... 

++M++++++++++++++++++++ 

....................... 

X 

xx xx xxx xxx xxx ....................... 

General Body System 

None 

~ 

Genital System 

Uterus 

Ovary 

Carcinoma 

Adenoma 

Clitoral gland 

Polyp stromal 

++++++++++++++++M++++++ i 

7 

48 

48 

4 

1 

46 

X 

X X 

....................... 

....................... 

x x xxx X 
116 Acetonitrile, NTP TR 447 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

5010770508735725267411179 

0136727238922366000122222 

1333344555566666777777777 

Number of Days on Study 

5793386815722299671549448 

7889750600770197951966087 

0000001011001100001000100 

Carcass ID Number 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood 

......................... +++ + ++ + 

......................... 

......................... 

++++++++++M++++++++++++++ 

Thymus 

Spleen 

Lymph node, mediastinal 

......................... 

......................... 

......................... 

Integumentary System 

Fibroadenoma. multiule 

Fibroadenoma 

Carcinoma 

Mammary gland ......................... 

X 

X 

xxx 

Fibrosarcoma X 

Skin ......................... 

Subcutaneous tissue, sarcoma 

Trichoepithelioma 

Squamous cell carcinoma 

Fibroma 

Basal cell carcinoma 

X 

X 

Musculoskeletal System 

Bone ......................... Cranium, carcinoma, metastatic, 

Zymbal’s gland X 

Nervous System 

Brain ......................... 

Carcinoma, metastatic, Zymbal’s gland 

Astrocytoma NOS 

Spinal cord 

X 

X 

+ 

Respiratory System 

LaNnX ++A++++++++++++++++++++++ 

Trachea 

Nose 

Lung 

......................... 

++A++++++++++++++++++++++ 

......................... 

Carcinoma 

Zymbal’s gland 

Special Senses System 

Urinary bladder 

Kidney 

Urinary System 

Eye 

X 

+ 

+ + 

X 

+ 

......................... 

......................... 

Papilloma X 

Systemic Lesions 

Multiple organs 

xxxxx x x xx x xx x ......................... Leukemia mononuclear 
Lesions in Female Rats 117 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE B2 

44444444444444444444444 

33333333333333333333333 

77777777777777777777777 

01256706012680127037890 

66666677888889999000001 

00000000000000000111111 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood + 

48 

48 

48 

46 

46 

43 

7 

48 

1 

....................... 

....................... 

....................... 

....................... 

++M++++++ +++++++++++++ 

+++++++++ +M+++++++++++ 

+++++++++ +++++M++MM+++ 

Integumentary System 

Fibrosarcoma 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammary gland 

Subcutaneous tissue, sarcoma 

Trichoepithelioma 

Squamous cell carcinoma 

Fibroma 

Basal cell carcinoma 

X xx 

X xxx X X 

X X 

....................... 

1 

1 

1 

1 

1 

48 

1 

4 

12 

2 

48 

xx 

Skin ....................... X 

X 

X 

Musculoskeletal System 

Bone +++++++++++I+++++++++++ 47 

Cranium, carcinoma, metastatic, 

Zymbal’s gland 1 

Nervous System 

Brain ....................... 

1 

1 

1 

48 

Carcinoma, metastatic, Zymbal’s gland 

Astrocytoma NOS 

Respiratory System 

Spinal cord 

Larynx 

....................... 

....................... 

....................... 

....................... 

48 

47 

48 

47 

Trachea 

Nose 

Lung 

Carcinoma 

Zymbal’s gland 

Special Senses System 

Eye + 

2 

2 

3 

Urinary bladder 

Kidney 

Urinary System 

....................... 

....................... 

1 

48 

48 

Papilloma 

Systemic Lesions 

Multiple organs 

X X 

....................... 18 

48 

Leukemia mononuclear xx X 
118 Acetonitrile, NTP TR 447. 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm 

TABLE B2 

Number of Days on Study 

8867803413371551933867338 

2701800122235667799900111 

3455566666666666666677777 

2302064374685635975181934 

8227081009079780181987612 

1221212221211112212111122 

Carcass ID Number 

Intestine large, colon 

Esophagus 

Alimentary System 

++++++++A++++++++++++++++ 

......................... 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Granulosa-theca tumor malignant, 

Hepatocellular adenoma 

Fibrous histiocytoma, metastatic, skin 

Mesentery 

metastatic, ovary 

......................... 

++++++++A++++++++++++++++ 

++++++++A++++++++++++++++ 

++++++++A++++++++++++++++ 

++++++++A++++++++++++++++ 

......................... 

+ 

X 

+ ++ 

X 

Salivary glands 

Pancreas 

Lipoma 

Stomach, glandular 

Stomach, forestomach 

+++++++++++++++M+++++++M+ 

......................... 

Fibrous histiocytoma, metastatic, skin X 

......................... 

......................... 

Cardiovascular System 

Heart 

Blood vessel ......................... Fibrous histiocytoma, metastatic, skin X 

......................... 

Adrenal cortex 

Endocrine System 

Granulosa-theca tumor malignant, 

metastatic, ovary 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Pars distalis, adenoma 

Carcinoma, metastatic, thyroid gland 

Carcinoma 

Pheochromocytoma benign 

Pars intermedia, adenoma 

Follicular cell, adenoma 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland 

......................... 

X 

......................... 

+++++++++++++++M+++++++++ 

......................... X 

xxx xx xx xx xxxx ......................... 

......................... 

X 
Lesions in Female Rats 119 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

TABLE B2 

9155555555555555555.5555 

12333333333333333333333 

71177771171117777111777 

Number of Days on Study 

Carcass ID Number 

72035045926789124801782 

71177888899999000011112 

12111111111111222222222 

Tumors 

Tissues/ 

Total 

Intestine large, colon 

Esophagus 

Alimentary System 

....................... 

....................... 47 

48 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

....................... 

....................... 

....................... 

....................... 

....................... 

47 

47 

47 

47 

48 

Liver ....................... 48 

Fibrous histiocytoma, metastatic, skin 

Lipoma 

Granulosa-theca tumor malignant, 

Hepatocellular adenoma 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

metastatic, ovary 

Fibrous histiocytoma, metastatic, skin 

X 

X 

+++ + + 

X 

....................... 

....................... 

....................... 

....................... 

9 

1 

2 

48 

48 

1 

46 

48 

1 

1 

Cardiovascular System ~~ 

Heart 

Blood vessel ....................... 

48 

1 

48 

Fibrous histiocytoma, metastatic, skin 

....................... 

Adrenal cortex 

Endocrine System 

....................... 48 

2 

48 

1 

Granulosa-theca tumor malignant, 

metastatic, ovary 

Adrenal medulla 

X X 

....................... Pheochromocytoma benign 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Pars distalis, adenoma 

Carcinoma, metastatic, thyroid gland 

Carcinoma 

Pars intermedia, adenoma 

Follicular cell, adenoma 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland 

....................... 

++++++MM++++++++++++++M 

....................... 

2 

1 

4 

48 

1 

26 

48 

1 

44 

1 

48 

'X 

xxxxxx xxxx x xx 

X 

....................... X X X X 

X 

X 
120 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

TABLE B2 

8867803413371551933867338 

2701800122235667799900111 

3455566666666666666677777 

2302064374685635975181934 

8227081009079780181987612 

1221212221211112212111122 

Number of Days on Study 

Carcass ID Number 

General Body System 

None 

Genital System 

Carcinoma 

Adenoma 

Clitoral gland ++++++MM+++MM+++++++M++M+ 

X 

X 

Uterus 

Ovary ......................... 

Polyp stromal 

Leiomyosarcoma 

Granulosa-theca tumor malignant X X 

......................... 

X X 

Hematopoietic System 

Lymph node 

Bone marrow 

Blood 

Renal, granulosa-theca tumor 

......................... + + ++ 

malignant, metastatic, ovary X 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

+++++++++++++++M+++++++M+ 

++M++M++++++MM+++M+++++++ 

Fibrous histiocytoma, metastatic, skin 

Fibrous histiocytoma, metastatic, skin 

X 

X 

+++++++++M+++++++++++++++ 

Thymus 

Spleen 

Lymph node, mediastinal 

Fibrous histiocytoma, metastatic 

+++++++++++++++++++++++M+ 

X 

......................... 

......................... , 

Integumentary System 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammary gland 

Fibrous histiocytoma 

Fibroma 

......................... 

xx x xxx xx X xx xx 

......................... xx x 

X X 

Skin 

X 

Musculoskeletal System 

Skeletal muscle 

Bone 

Fibrous histiocytoma, metastatic, skin 

......................... 

X 

+ 

Nervous System 

Brain ......................... Glioma NOS X 
Lesions in Female Rats 121 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

TABLE B2 

Number of Days on Study 

91555555555555555555555 

12333333333333333333333 

77777777177777777’177777 

7203.5045926789124801782 

~1777888899999000011112 

12111111111111222222222 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

General Body System 

None 

Genital System 

Carcinoma 

Adenoma 

Clitoral gland 

Polyp stromal 

Leiomyosarcoma 

Granulosa-theca tumor malignant 

Uterus 

Ovary 

....................... 

48 

1 

1 

42 

....................... L 

....................... 

4 

1 

48 

X 

X X 

Hematopoietic System 

Lymph node 

Bone marrow 

Blood 

malignant, metastatic, ovary 

Renal, granulosa-theca tumor 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Thymus 

Spleen 

Lymph node, mediastinal 

Fibrous histiocytoma, metastatic, skin 

Fibrous histiocytoma, metastatic, skin 

Fibrous histiocytoma, metastatic 

+ ....................... 

+ 

++++++++M++M+++MM++++++ 

....................... 

++++++++++++M++++++++++ 

....................... 

....................... 

....................... 

5 

48 

1 

48 

48 

1 

46 

47 

1 

46 

1 

39 

1 

Integumentary System 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammary gland 

Fibrous histiocytoma 

Fibroma 

Skin 

....................... X 

X xxx x x xx 

X X 

....................... xx 

x 

X 2 

1 

48 

6 

21 

4 

48 

Musculoskeletal System 

Skeletal muscle 

Bone ....................... 

Fibrous histiocytoma, metastatic, skin 1 

1 

48 

Nervous System 

Brain 

Glioma NOS 

....................... 1 

48 
122 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

TABLE B2 

8867803413371551933867338 

27018001'22235667799900111 

3455566666666666666677777 

Number of Days on Study 

2302064374685635975181934 

8227081009079780181987612 

1221212221211112212111122 

Carcass ID Number 

Respiratory System 

Larynx ......................... 

......................... Lung 

Fibrous histiocytoma, metastatic, skin 

Carcinoma, metastatic, mammary gland 

X 

Trachea 

Nose 

......................... 

+++++++++++ +++++++++++++ 

Special Senses System 

Carcinoma 

Zymbal's gland 

Harderian gland 

Eye + 

X 

+ 

Kidney 

Urinary System 

......................... Granulosa-theca tumor malignant, 

metastatic, ovary X 

Urinary bladder ......................... 

Systemic Lesions 

Multiple organs 

X 

......................... Leukemia mononuclear x x x x xxxx xx x 
Lesions in Female Rats 123 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

Number of Days on Study 

9155555555555555555'5555 

12333333333333333333333 

77777777777777777777777 

72035045926789124801782 

71777888899999000011112 

12111111111111222222222~ 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Respiratory System 

Larynx 

....................... 

....................... 

48 

47 

2 

1 

48 

48 

Lung 

Fibrous histiocytoma, metastatic, skin 

Carcinoma, metastatic, mammary gland X 

X 

Trachea 

Nose 

....................... 

....................... 

Special Senses System 

Carcinoma 

Zymbal's gland 

Harderian gland 

Eye + 

Kidney 

Urinary System 

....................... 

....................... ' 48 

1 

48 

Granulosa-theca tumor malignant, 

metastatic, ovary 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

....................... 

x xx x xx xx xx 22 

48 
124 Acetonitrile, NTP TR 447 

Number of Days on Study 

2695456678735115115397555 

9768356668922556778912333 

2344555555566666666677777 

8755943655840574968303126 

1081099203889923912138888 

3323322333222233233332222 

Carcass ID Number 

Mesentery 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Lipoma 

......................... 

+++A+A+++++++++++++++++++ 

......................... 

......................... 

+++++A+++++++++++++++++++ 

......................... 

......................... 

......................... 

+ + 

I 

......................... 

++++++++I +++++++++.+++++++ ......................... 

......................... 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Adenoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Parathyroid gland 

Islets, pancreatic 

M++++++++++++++++++++++++ 

......................... 

X 

X 

......................... 

+M++M+++M++++++++++++++++ 

Pituitary gland +++++I+++++++++++++++++++ 

C-cell, adenoma 

Pars distalis, adenoma 

Schwannoma malignant, metastatic, brain X 

xxx x x xx xxxxx 

Thyroid gland ......................... 

General Body System 

None 

Genital System 

Ovary 

Carcinoma 

Adenoma 

Clitoral gland 

UkNS 

Bilateral, polyp stromal 

Polyp stromal 

+++++++++++++++++M+++++++ 

......................... 

......................... 

X X 
Lesions in Female Rats 125 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

Number of Days on Study 

55555555555555555555555 

33333333333333333333333 

11111111111111111111111 

Carcass ID Number 

19268123480419023459126 

88999000001i11222222333 

22222333333333333333333 

Tumors 

Tissues/ 

Total 

Mesentery 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Lipoma 

....................... 

....................... 

....................... 

....................... 

....................... 

....................... 

....................... 

....................... 

+ + ++ 

X 

+ + 

....................... 

....................... 

....................... 

....................... 

48 

41 

48 

48 

1 

8 

48 

46 

48 

48 

41 

48 

48 

48 

Cardiovascular System 

Heart 

Blood vessel 

....................... 

....................... 48 

48 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Islets, pancreatic 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Pituitary gland 

Parathyroid gland 

Adenoma 

C-cell, adenoma 

Pars distalis, adenoma 

Schwannoma malignant, metastatic, brain 

Thyroid gland 

...................... 

...................... 

., 

A 

...................... xx 

...................... 

++++++++++++++++++M+++ 

X 

...................... 

xxxxxx x xxxx 

xxx 

+ 

+ 

4 

48 

28 

1 

41 

44 

2 

48 

2 

1 

41 

48 

+ 

+ 

+ 

+ 

General Body System 

None 

Genital System 

Ovary 

Carcinoma 

Adenoma 

Clitoral gland 

Uterus 

Bilateral, polyp stromal 

Polyp stromal 

....................... 

....................... 

X 

....................... 

2 

I 

48 

48 

4 

1 

41 

X X X X 

X x xxx 

X X 
126 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TABLE B2 

269545661813511511539755' 

976835666.8922556778912333 

2344555555566666666671171 

Number of Days on Study 

3323322333222233233332222 

Carcass ID Number 

8755943655840574968303126 

1081099203889923912138888 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow ......................... 

+++++++++++++++++M+++++++ 

+++++++++++++++++M++++M++ 

+++M++++M++M'+++++M+M+++++ 

+ + ++ + + 

Lymph node, mediastinal +++++++++++++++++++++M+Mt 

Thymus 

Spleen 

......................... 

......................... 

Integumentary System 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammary gland .......................... X 

X xx xx x xx 

Skin ......................... Pinna, melanoma NOS 

Musculoskeletal.System 

Bone ......................... 

Schwannoma malignant, metastatic, brain X 

Nervous System 

Brain ......................... Glioma NOS X 

Cranial erve, schwannoma malignant X 

Respiratory System 

Trachea 

Nose 

Lung 

Larynx 

......................... 

......................... 

......................... 

......................... 

Special Senses System 

Lacrimal gland 

Ear 

Eye 

+ 

+ 

Kidney 

Urinary System 

......................... Lipoma 

Urinary bladder ......................... 

Multiple organs 

Systemic Lesions, 

xxx xxx xxx .......................... Leukemia mononuclear X 
Lesions in Female Rats 127 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TmLE B2 

5555555555555555555-5555 

33333333333333333333333 

77777777777777777777777 

79268123480479023459126 

88999000001111222222333 

22222333333333333333333 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow ....................... 

....................... 

....................... 

M++++++++++++++++++++++ 

....................... 

++M++++++++++++++++++++ 

++++M+++++++++M++++++++ 

48 

48 

45 

47 

45 

41 

8 

48 

+ + 

Integumentary System 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammary gland 

Pinna, melanoma NOS 

Skin 

X X X 

X xxxx x 

xx ....................... 

1 

48 

4 

17 

3 

48 

X 

X 

....................... X 

Musculoskeletal System 

Bone ....................... 1 48 

Schwannoma malignant, metastatic, brain 

Nervous System 

Brain ....................... 

1 

1 

48 

Cranial nerve, schwannoma malignant 

Glioma NOS 

Respiratory System 

Larynx 

....................... 

....................... 

....................... 

....................... 

48 

48 

48 

48 

Trachea 

Nose 

Lung 

Special Senses System 

Lacrimal gland 

Ear 

Eye + 

+ 

1 

2 

1 

Urinary bladder 

Kidney 

Urinary System 

Lipoma 

....................... 

41 

1 

48 

X 

....................... 

Systemic Lesions 

Multiple organs 

xxx x - X x xxxx ....................... 20 

48 

Leukemia mononuclear 
128 Acetonitrile, NTP TR 447 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm 

4782989602299937139444444 

9251912677717190222333333 

3455566666666661111777711 

0956192441337816825368069 

3291493090301322002999000 

4434434434444444444333444 

Number of Days on Study 

Carcass ID Number 

Intestine large, colon 

Esophagus 

Alimentary System 

+++A++A+A++++++++++++++++ 

++++++A++++++++++++++++++ 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

+++A++A++++++++++++++++++ 

+++AA+A++++++++++++++++++ 

++AA++A++++++++++++++++++ 

+++A++A++++++++++++++++++ 

+++A++A+A++++++++++++++-++ 

+++A++A++++++++++++++++++ 

+++A++A+++++++++++++-+++++ 

+++A++A++++++++++++++++++ 

++++++A++++++++++++++++++ 

+++A++A++++++++++++++++++ 

+ + ++ + 

Cardiovascular System 

Heart 

Blood vessel ++++++A++++++++++++++++++ 

++++++A++++++++++++++++++ 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Pheochromocytoma benign 

Neoplasm NOS 

Parathyroid gland 

Carcinoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

C-cell, adenoma 

Pars .distalis, adenoma 

+++A'++M++++++++++++++++++ 

++++++M++++++++++++++++++ 

X 

X X 

+++A++A++++++++++++++++++ 

X 

+++A++A++++++++++++++++++ 

X xxxx xxx X xxx 

++IA++A++++++++++++++++++ 

+++A++A++++++++++++++.++++ 

X xx 

General Body System 

None 

Genital System 

Adenoma 

Clitoral gland 

X X 

++++++A+++M++++++++++++++ 

Carcinoma 

ovary 

Uterus 

Polyp stromal 

Hemangioma 

Carcinoma 

++++++A++++++++++++++++++ 

++++++A++++++++++++++++++ 

X 

X 
Lesions in Female Rats 129 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

TABLE B2 

44444444444444444444444 

33333333333333333333333 

77777777777777777777777 

12345892347146702345678 

11111112222333344444444 

44444444444444444444444 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Alimentary System 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

....................... 

....................... 

....................... 

....................... 

+ ++ ....................... 

....................... 

....................... 

....................... 

....................... 

....................... 

....................... 

....................... 

46 

46 

47 

46 

9 

46 

45 

45 

46 

45 

45 

45 

47 

+ 

Cardiovascular System 

Heart 

Blood vessel 

....................... 

....................... 47 47 

Adrenal cortex 

Endocrine System 

....................... 46 

Adrenal medulla 

Pheochromocytoma benign 

Neoplasm NOS 

Parathyroid gland 

Carcinoma 

Islets, pancreatic 

Thyroid gland 

PituitaIy gland 

C-cell, adenoma 

Pars distalis, adenoma 

....................... 

4 

46 

29 

45 

45 

1 

46 

2 

1 

47 

....................... 

....................... xxxxxxxx x xxxx xx x ....................... 

+++++++++++M+++++++++++ 

X 

General Body System 

None 

Genital System 

Carcinoma 

Adenoma 

Clitoral gland ++++++++++++++++++++M++ 

5 

1 

1 

47 

47 

2 

3 

45 

X 

X X 

Uterus 

Ovary 

....................... 

....................... 

Polyp stromal 

Hemangioma 

Carcinoma 

X 

X X X X 
130 Acetonitrile, NTP TR '447 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

TABLE B2 

Number of Days on Study 

4782989602299937139444444 

9257912677777790222333333 

3455566666666667777777777 

Carcass ID Number 

0956192441337816825368069 

3291493090301322002999000 

4434434434444444444333444 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Thymoma NOS 

+++A++A++++++++++++++++++ 

+ ++ + ++ 

++++++A++++++++++++++++++ 

+++A++A++++++++++++++++++ 

......................... 

......................... 

++++++A++M++++f++++++++++ 

++MM++A+M+++M++++++++M+++ 

X 

Integumentary System 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammaly gland 

Fibroma, multiple 

X 

++++++A++++++++++++++++++ 

X X 

xx xx X 

X 

X 

X X xx 

Skin ......................... 

Musculoskeletal System 

Bone ++++++A++++++++++++++++++ 

Nervous System 

Brain ......................... 

Respiratory System 

Larynx 

Trachea 

Nose 

Lung 

Alveolar/bronchiolar adenoma 

+++A++A++++++++++++++++++ 

+++A++A++++++++++++++++++ 

X 

+++A++A++++++++++++++++++ 

+++A++A++++++++++++++++++ 

Harderian gland 

Special Senses System 

Eye + + 

+ 

Urinary bladder 

Kidney 

Urinary System 

+++A++A+++++++++++++++++I 

+++A++A++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

xx xx xxxx xxxx xx x ......................... Leukemia mononuclear 
Lesions in Female Rats 131 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Acetonitrile: 400 ppm (continued) 

TABLE B2 

17117777717177777777171 

Number of Days on Study 33333333333333333333.333 

44444444444444444444444 

Carcass ID Number 

12345892347146702345678 

11111112222333344444444 

44444444444444444444444 

Tumors 

Tissues1 

Total 

Hematopoietic System 

Bone marrow ....................... 46 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Thymoma NOS 

+ + 

1 

47 

45 

46 

41 

42 

40 

8 

....................... 

+ ..................... +++++++++++++++++++M+++ 

....................... 

+++++M+++++++M+++++++MM 

+++M+++++M+++++++++++++ 

Integumentary System 

Fibroadenoma, multiple 

Fibroadenoma 

Carcinoma 

Mammary gland 

Fibroma, multiple 

....................... 

1 

48 

7 

16 

3 

47 

xx xxxx xx X 

....................... 

X 

X 

X X 

Skin 

Musculoskeletal System 

Bone 

~~~~~ ~ 

....................... 47 

Nervous System 

Brain ....................... 47 

Respiratory System 

Larynx 

Trachea 

Nose 

Lung 

Alveolarbronchiolar adenoma 

....................... 

....................... 

X 

++++++++++++++M++++++++ 

....................... 45 

46 

2 

46 

46 

Harderian gland 

Special Senses System 

Eye + 

1 

3 

Urinary bladder 

Kidney 

Urinary System 

....................... 

....................... 45 

46 

Systemic Lesions 

Multiple organs ....................... 24 48 

Leukemia mononuclear X xxxx xx x 
132 Acetonitrile, NTP TR 447 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrile 

TABLE B3 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armita e testd P=O.394 

Logistic ion testd P=O.356 

Life testd P=O.498 

First ence 734 (T) 

Terminal 1/23 (4%) 

Adjusted rateb 4.3% 

Overall ratea 1/48 (2%) 

Adrenal Medulla: Benign or Malignant Pheochromocytoma 

% 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Clitoral Gland: Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Clitoral Gland: Carcinoma 

4.5% 

1/46 (2%) 

734 (T) 

1/22 (5%) 

P=O.166 

P=O.196 

P=O.231 

14.7% 

4/46 (9%) 

P=0.392N 

P=0.340N 

P=0.286N 

623 

2/22 (9%) 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Clitoral Gland: Adenoma or Carcinoma 

P=O.421 

P=O.492 

P=0.561N 

623 

3/22 (14%) 

19.0% 

5/46 (11%) 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma 

P=O.224 

P=O.333 

P=0.459N525 

11/23 (48%) 

55.9% 

16/48 (33%) 

8.9% 

2/48 (4%) 

P=O.470 

P=O.456 

719 

1/21 (5%) 

P=O.500 

2.3% 

1/42 (2%) 

P=O.745 

P=0.749N 

600 

on1 (0%) 

P=O.730 

2.3% 

1/42 (2%) 

P=0.196N 

P=0.192N 

588 

on1 (0%) 

P=0.210N 

4.5% 

2/42 (5%) 

P=0.242N 

P=0.224N 

588 

on1 (0%) 

P=0.256N 

72.7% 

27/48 (56%) 

P = 0.030 ' 

P=O.O22 

478 

12/21 (57%) 

P=O.O20 

P = 0.230 

P = 0.346 

376 

1/26 (4%) 

8.0% 

3/47 (6%) 

P=O.301 

1/26 (4%) 

3.8% 

1/47 (2%) 

P=0.725N 

P=0.725N 

734 (T) 

P=0.747N 

4/26 (15%) 

15.4% 

4/47 (9%) 

P=0.632N 

P=0.576N 

734 (T) 

P=0.631N 

5/47 (11%) 

P=0.621N 

P=0.547N 

5/26 (19%) 

19.2% 

734 (T) 

P=0.616N 

P=O.225 

P=O.318 

469 

13/26 (50%) 

60.0% 

21/48 (44%) 

P=O.201 

P=O.538 

P=O.576 

672 

1/29 (3%) 

5.9% 

2/47 (4%) 

P=O.492 

3/28 (11%) 

3/45 (7%) 

10.7% 

P=O.393 

P =0.393 

734 (T) 

P=O.300 

7.1% 

2/45 (4%) 

734 (T) 

2/28 (7%) 

P =0.288N 

P=0.250N 

P=0.349N 

734 (T) 

5/28 (18%) 

5/45 (11%) 

17.9% 

P=0.553N 

P=0.488N 

P=O.616 

P=O.199 

P=O.344 

394 

15/29 (52%) 

61.2% 

23/48 (48%) 

P=O.106 
Lesions in Female Rats 133 

Statistical Analysis of Primaly Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B3 

0 PPm 100 ppm 200 pprn 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pituitary Gland (Pars Distalis): Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Fibroma, Sarcoma, or Fibrous Histiocytoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Adenoma 

P=O.500 

P=O.566 

P=0.536N 

734 (T) 

2/23 (9%) 

8.7% 

2/48 (4%) 

P=O.263 

P=O.402 

P=0.388N 

525 

12/23 (52%) 

59.6% 

1 7/48 (35 %) 

P=O.O84 

P=O.253 

P=0.438N 

530 

13/23 (57%) 

69.7% 

24/48 (50%) 

P=0.242N 

P=0.272N 

P=0.199N 

310 

0/23 (0%) 

4.8% 

2/48 (4%) 

P=O.420 

P=O.547 

P=0.542N 

702 

1/23 (4%) 

10.5% 

3/48 (6%) 

13.4% 

4/48 (8%) 

P=O.354 

P=O.319 

67 1 

1/21 (5%) 

P=O.339 

P=O.O12 

P=O.OlO 

478 

13/21 (62%) 

77.2% 

30/48 (63%) 

P=O.O07 

600 

73.0% 

26/48 (54%) 

12/21 (57%) 

P=O.543 

P=O.327 

P=O.419 

12.8% 

3/48 (6%) 

P=O.451 

P=O.479 

706 

2/21 (10%) 

P=O.500 

P=O.479 

P=O.429 

721 

3/21 (14%) 

18.2% 

4/48 (8%) 

P=O.500 

P=O.557 

P=O.557 

734 (T) 

3/26 (12%) 

11.5% 

3/48 (6%) 

P=O.500 

P=O.168 

P=O.262 

469 

15/26 (58%) 

66.2% 

23/48 (48%) 

P=O.150 

P=O.288 

P=O.422 

534 

17/26 (65%) 

75.2% 

28/47 (60%) 

P=O.232 

0/26 (0%) 

0.0% 

0/48 (0%) 

P = 0.326N 

P=0.240N 

e - 

P=O.Z47N 

4/26 (15%) 

15.4% 

4/48 (8%) 

P=O.494 

P=O.535 

734 (T) 

P=O.500 

P=O.559 

P=O.589 

672 

1/29 (3%) 

8.8% 

3/48 (6%) 

P=O.500 

P=O.211 

P=O.360 

394 

16/29 (55%) 

64.0% 

24/48 (50%) 

P=O.l08 

P=O.341 

P=0.554N 

599 

21/29 (72%) 

77.9% 

29/45 (64%) 

P=O.116 

1/29 (3%) 

3.4% 

1/48 (2%) 

P=0.659N 

P=0.453N 

734 (T) 

P=0.500N 

13.3% 

4/46 (9%) 

P=O.572 

P=O.597 

729 

3/29 (10%) 

P=O.476 
134 Acetonitrile, NTP TR 447 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B3 

100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

10.5% 

3/48 (6%) 

P=O.473 

P=0.534N 

P=0.478N 

702 

1/23 (4%) 

P=O.325 

P=O.291 

721 

4/21 (19%) 

22.7% 

5/48 (10%) 

4/26 (1 5%) 

15.4% 

4/48 (8%) 

P=O.494 

P=O.535 

734 (T) 

13.3% 

4/46 (9%) 

P=O.572 

P=O.597 

729 

3/29 (10%) 

P=O.357 P=O.500 P=O.476 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Uterus: Stromal Polyp 

P=0.457N 

P=0.397N 

P=0.273N 

331 

6/23 (26%) 

27.7% 

7/48 (15%) 

14.3% 

4/48 (8%) 

P=0.243N 

P=0.291N 

621 

2/21 (10%) 

P=O.412 

P=O.484 

485 

7/26 (27%) 

30.5% 

9/48 (19%) 

P=0.317N 

P=0.236N 

723 

4/29 (14%) 

16.6% 

5/48 (10%) 

P = 0.262N P=O.392 P=0.379N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Mononuclear Cell Leukemia 

46.4% 

18/48 (38%) 

P=O.163 

P=O.158 

P=O.491 

331 

5/23 (22%) 

P = 0.227 

P=O.261 

328 

9/21 (43%) 

62.3% 

22/48 (46%) 

P=O.403 

P=O.513 

534 

11/26 (42%) 

55.6% 

20/48 (42%) 

P=O.101 

P=O.417 

427 

12/29 (41%) 

57.9% 

24/48 (50%) 

P=O.267 P=O.417 P=O.152 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign Neoplasms 

100.0% 

38/48 (79%) 

P=0.512N 

P=0.188N 

P=0.069N 

331 

23/23 (100%) 

P=0.405N 

P=O.350 

478 

18/21 (86%). 

92.7% 

39/48 (81%) 

P=0.422N 

P=0.371N 

469 

22/26 (85%) 

90.3% 

38/48 (79%) 

92.6% 

38/48 (79%) 

P=0.190N 

P=0.115N 

394 

26/29 (90%) 

P=O.500 P=0.599N P=0.599N 

Fisher exact test. 

Cochran-Armitage test 

Logistic regression test 

Life table test 

Fiist .incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Neoplasms 

P=0.338N 

P=0.384N 

P=0.116N 

310 

10/23.(43%) 

67.8% 

29/48 (60%) 

74.0% 

28/48 (58%) 

P=0.542Nc 

P=O.545 

328- 

12/21 (57%) 

P=0.173N 

P=0.154N 

292 

12/26 (46%) 

60.3% 

23/48 (48%) 

P.=0.499N, 

P=0.182N 

427 

14/29 (48%), 

63.8% 

27/48 (56%) 

P=0.500N P=0.153N' P.=0.418N 
Lesions in Female Rats 135 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

0 PPm 100 ppm 200 ppm 400 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

P=0.404N 

P=0.339N P=0.313N P=0.248N P=O.24ON 

P=0.060N P=O.489 P=0.274N P=0.090N 

310 328 292 4 

23/23 (100%) 23/26 (88%) 28/29 (97%)

100.0% 97.9% 93.8% 97.9% 

47/48 (98%) 46/48(96%) 45/48(94%) 46/48(96%) 

P=0.500N P=0.308N P=0.500N 

2oi21 (95%) 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

(T)Terminal sacrifice 

exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher 

to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

Observed incidence at terminal kill 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

clitoral gland, pituitaxy gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

e Not applicable; no neoplasms in animal group 
136 Acetonitrile, NTP TR 447 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study ofAcetonitrilea 

TABLE B4 

0 PPm 100 ppm 200 ppm 400 ppm 

Early deaths 

IS" intabn me 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

SuIvivors 

8 

56 

8 

56 

8 

56 

8 

56 

24 

1 

2 

25 

1 

21 

5 

14 

23 21 26 29 

Animals examined microscopically 56 56 56 56 

Esophagus 

Alimentary System 

15-Month Interim Evaluation 

Ulcer 

Intestine large, rectum 

Inflammation, suppurative 

Intestine large, colon 

Mediastinum, cyst 

Serosa, hemorrhage 

Hepatodiaphragmatic nodule 

Granuloma, multifocal 

Eosinophilic focus 

Clear cell focus 

Basophilic focus 

Angiectasis 

Liver 

Fat, inflammation, granulomatous 

Hyperplasia, squamous 

Acinus, atrophy 

Fat, necrosis 

Mesentery 

Stomach, forestomach 

Pancreas 

1 (13%) 

6 (75%) 7 (88%) 5 (63%) 

1 (13%) 

1 (13%) 

1 (13%). 

1 (13%) 

1 (13%) 

2 (25%) 1 (13%) 

1 (13%) 

(2) 

2 (100%) 

2 (25%) 

1 (50%) 

(8) 

(8) 

Heart 

Cardiovascular System 

Cardiomyopathy 

Pituitary gland 

Endocrine System 

Pars distalis, hyperplasia 

Pars distalis, cyst 

Cyst 

C-cell, hyperplasia 

Dilatation 

Thyroid gland 

(8) 

1 (13%) 

1 (13%) 

(8) 

1 (13%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Female Rats 137 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B4 

0 PPm 100 ppm 200 ppm 400 ppm 

Clitoral gland 

Genital System 

IS-Month Interim Evaluation (continued) 

(8) (8) 

Inflammation, suppurative 

Ectasia 

Uterus 

Ovary 

(8) 

(8) 

(8) 

(8) 

Cyst 2 (25%) 

Endometrium, hyperplasia 

Hematopoietic System 

Pancreatic, pigmentation 

Pancreatic, hemorrhage 

Lymph node 

Lymph node, mesenteric 

Hyperplasia, lymphoid 

Lymph node, mandibular 

Hyperplasia, lymphoid 

Hemorrhage 

Hyperplasia, lymphoid 

Hemorrhage 

Lymph node, mediastinal 

Hyperplasia, lymphoid 

Thymus 

Skin 

Integumentary System 

(8) (8) 

Subcutaneous tissue, hemorrhage 

Ulcer 

Inflammation, granulomatous 

Acanthosis 

(8) (8) 

1 (13%) 

1 (13%) 

1 (13%) 

1 (13%) 

Bone 

Musculoskeletal System 

(8) (8) 

Periosteum, cranium, hemorrhage 

(8) 

1 (13%) 

Larynx 

Respiratory System 

Mineralization 

Metaplasia, squamous 

Inflammation, suppurative 

Hyperplasia 

Foreign body 

Pleura, fibrosis 

Infiltration cellular, histiocyte 

Hemorrhage 

Lung 

(8) (8) (8) (8) 

2 (25%) 

2 (25%) 

1 (13%) 

1 (13%) 

2 (25%) 

2 (25%) 

2 (25%) 

1 (13%) 

8 (100%) 

1 (13%) 

1 (13%) 

(8) (8) (8) (8) 

1 (13%) 

1 (13%) 

8 (100%) 

1 (13%) 

8 (100%) 

1 (13%) 

8 (100%) 
138 Acetonitrile, NTP TR 447 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B4 

0 PPm 100 ppm 200 ppm 400 ppm 

15-Month Interim Evaluation (continued) 

Eye 

Special Senses System 

Hemorrhage 

Cataract 

(1) (1) (2) (1) 

1 (100%) 

1 (100%) 

1 (100%) 1 (100%) 1 (50%) 

Kidney 

Urinary System 

Renal tubule, mineralization 

Nephropathy, chronic 

(8) (8) (8) (8) 

5 (63%) 

3 (38%) 

2 (25%) 

1 (13%) 

3 (38%) 

1 (13%) 

4 (50%) 

Nervous System 

General Body System 

Systems Examined With No Lesions Observed 

2-Year Study 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

2 (4%) 

(47) . (48) 

2 (4%) 

(47) (47) 

(47) (47) 

(47) (47) 

(47) (47) 

(48) (48) (48) 

(46) 

(48) 

(47) 

(48) 

(48) 

Atrophy 

Inflammation, granulomatous 

Inflammation, granulomatous 

Lumen, parasite metazoan 

Lumen, parasite metazoan 

Lumen, parasite metazoan 

Serosa, hemorrhage 

Serosa, cyst 

Portal, fibrosis 

Hepatocyte, necrosis 

Hepatocyte, hyperplasia 

Hepatocyte, degeneration, cystic 

Bile duct, hyperplasia 

Vacuolization cytoplasmic 

Mixed cell focus 

Infarct 

Hepatodiaphragmatic nodule 

Hematopoietic cell proliferation 

Granuloma, multifocal 

Degeneration, cystic 

Clear cell focus 

Basophilic focus 

6 (13%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

2 (4%) 

34 (71%) 34 (71%) 33 (69%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

6 (13%) 7 (15%) 

11 (23%) 14 (29%) 12 (25%) 

2 (4%) 

1 (2%) 1 (2%) 

7 (15%) 9 (19%) 

3 (6%) 

4 (8%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(45) 

(45) 

(45) 

3 (7%) 

1 (2%) 

36 (78%) 

2 (4%) 

1 (2%) 

8 (17%) 

20 (43%) 

2 (4%) 

10 (22%) 

3 (7%) 
Lesions in Female Rats 139 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B4 

0 PPm 100 ppm 200 ppm 400 ppm 

study (continued) 

Mesentery 

Alimentary System (continued) 

Fat, necrosis 

Fat, inflammation, granulomatous 

Artery, inflammation 

Hemorrhage 

Duct, mineralization 

Salivary glands 

Artery, inflammation 

Acinus, atrophy 

Ulcer 

Necrosis 

Inflammation, suppurative 

Hyperplasia, squamous 

Hyperplasia, basal cell 

Edema 

Diverticulum 

Stomach, forestomach 

Necrosis 

Mineralization 

Stomach, glandular 

Pancreas 

(10) 

1 (10%) 

1 (10%) 

14 (29%) 

7 (70%) 

(48) 

(48) 

(48) 

1 (2%) 

2 (4%) 

3 (6%) 

2 (4%) 

2 (4%) 

(47) 

1 (2%) 

(9) 

2 (22%) 

1 (11%) 

1 (11%) 

6 (67%) 

(48) 

19 (40%) 

3 (6%) 

6 (13%) 

5 (10%) 

3 (6%) 

(48) 

(8) 

4 (50%) 

4 (50%) 

1 (13%) 

2 (25%) 

(48) 

23 (48%) 

1 (2%) 

(47) 

(48) 

5 (11%) 

1 (2%) 

4 (9%) 

7 (15%) 

1 (2%) 

5 (11%) 

2 (4%) 

(48) 

(9) 

1 (11%) 

8 (89%) 

5 (56%) 

(46) 

1 (2%) 

25 (54%) 

(46) 

(47) 

1 (2%) 

5 (11%) 

3 (7%) 

2 (4%) 

7 (15%) 

(46) 

Blood vessel 

Cardiovascular System 

Aorta, mineralization 

Aorta, inflammation 

Endocardium, hyperplasia 

Thrombosis 

Cardiomyopathy 

Heart 

(48) (48) (48) (47) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

(48) (48) (48) (47) 

1 (2%) 

1 (2%) 

2 (4%) 2 (4%) 

1 (2%) 

Endocrine System 

Vacuolization cytoplasmic 

Mineralization 

Hyperplasia 

Hemorrhage 

Adrenal cortex 

Hyperplasia 

Angiectasis 

Adrenal medulla 

Pituitary gland 

Hyperplasia 

Parathyroid gland 

Pars distalis, hyperplasia 

Pars distalis, hemorrhage 

Pars distalis, cyst 

Cyst 

(48) 

1 (2%) 

1 (2%) 

6 (13%) 

(48) 

5 (10%) 

(48) 

(44) 

11 (23%) 

1 (2%) 

9 (19%) 

2 (4%) 

11 (23%) 

(48) 

6 (13%) 

(48) 

(44) 

15 (31%) 

3 (6%) 

1 (2%) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

5 (10%) 

(47) 

3 (6%) 

(47) 

(44) 

3 (7%) 

14 (30%) 

3 (6%) 

1 (2%) 

8 (17%) 

(47) 

1 (2%) 

1 (2%) 

(45) 

(45) 

14 (31%) 

3 (7%) 
140 Acetonitrile, NTP TR 44'7 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B4 

0 PPm 100 ppm 200 ppm 400 ppm 

Thyroid gland 

Endocrine System (continued) 

%-Year Study (continued) 

(48) (48) (48) (46) 

C-cell, hyperplasia 

Ultimobranchial cyst 1 (2%) 

26 (54%) 38 (79%) 36 (75%) 38 (83%) 

None 

General Body System 

Genital System 

Ovary 

Inflammation, suppurative 

Hyperplasia 

Ectasia 

Clitoral gland 

Cyst 

Inflammation, granulomatous 

Endometrium, hyperplasia 

Cervix, endometrium, hyperplasia 

Thrombosis 

Hemorrhage 

Uterus 

(46) 

11 (24%) 

2 (4%) 

4 (9%) 

2 (4%) 

(48) 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

(42) 

3 (7%) 

4 (10%) 

4 (10%) 

(48) 

3 (6%) 

1 (2%) 

(48) 

1 (2%) 

(47) (45) 

9 (19%) 

6 (13%) 

14 (31%) 

1 (2%) 

1 (2%) 

4 (9%) 3 (7%) 

(48) (47) 

5 (11%) 

(48) (47) 

1 (2%) 

1 (2%) 

Hematopoietic System 

Myelofibrosis 

Hyperplasia, RE cell 

Bone marrow 

Renal, hemorrhage 

Lumbar, pigmentation 

Iliac, pigmentation 

Axillary, hyperplasia, lymphoid 

Inflammation, suppurative 

Hemorrhage 

Lymph node, bronchial 

Hyperplasia, lymphoid 

Hemorrhage 

Lymph node, mandibular 

Inflammation, granulomatous 

Hyperplasia, lymphoid 

Hemorrhage 

Lymph node, mesenteric 

Pigmentation 

Hemorrhage 

'Lymph node, mediastinal 

Lymph node 

1 (14%) 

(43) 

2 (4%) 

1 (2%) 

1 (2%) 

4 (9%) 

(46) 

(46) 

2 .(4%) 

(48) 

11 (23%) 

(48) 

3 (6%) 

2 (4%) 

(5) 

1 (3%) 

1 (20%) 

439) 

2 (4%) 

1 (2%) 

(47) 

1 (2%) 

(46) 

6 (13%) 

(48) 

2 (4%) 

(8) 

1 (13%) 

1 (13%) 

1 (13%) 

(41) 

(453 

2 (4%) 

(47) 

(453 

3 (7%) 

(47) 

2 (4%) 

.(46) 

1 (2%) 

11 (24%) 
Lesions in Female Rats 141 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE B4 

0 PPm 100 ppm 200 ppm 400 ppm 

Spleen 

Hematopoietic System (continued) 

%Year Study (continued) 

(48) (48) 

Necrosis 

Hyperplasia, RE cell 

Hemorrhage 

Hematopoietic cell proliferation 

Fibrosis 

Developmental malformation 

Hemorrhage 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

2 (4%) 

3 (6%) 

1 (2%) 

1 (2%) 

Thymus (48) (48) 

(48) (45) 

1 (2%) 

3 (7%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

(48) (47) 

Integumentary System 

Epithelium, hyperplasia 

Galactocele 

Mammary gland 

Subcutaneous tissue, necrosis 

Ulcer 

Inflammation, suppurative 

Inflammation, granulomatous 

Acanthosis 

(48) 

Skin (48) 

1 (2%) 

(48) (48) (47) 

2 (4%) 

1 (2%) 

2 (4%) 

(48) (48) (48) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

Bone 

Musculoskeletal System 

Vertebra, fracture 

Fibrosis 

Nervous System 

Necrosis 

Hydrocephalus 

Hemorrhage 

Brain 

Hemorrhage 

Spinal cord 

(48) (48) (48) (47) 

4 (8%) 

4 (8%) 5 (10%) 

3 (6%) 

8 (17%) 

5 (11%) 

2 (4%) 

6 (13%) 

1 (100%) 

1 (2%) 

(1) 

Larynx 

Respiratory System 

Metaplasia, squamous 

Inflammation, suppurative 

Hyperplasia 

Foreign body 

(47) (48) (48) (46) 

7 (15%) 5 (10%) 

3 (6%) 

5 (11%) 1 (2%) 

6 (13%) 2 (4%) 

1 (2%) 

2 (4%) 

3 (7%) 

3 (7%) 

3 (7%) 

3 (7%) 

6 (13%) 

4 (8%) 
142 Acetonitrile, NTP TR 447 

TABLE B4 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Acetonitrile (continued) 

0 PPm 100 ppm 200 ppm 400 ppm 

Lung 

Respiratory System (continued) 

2-YeUr study (continued) 

(48) (48) (48) (46) 

Pleura, fibrosis 

Alveolus, inflammation, suppurative 

Alveolus, inflammation, chronic 

Alveolus, fibrosis 

Alveolar epithelium, hyperplasia 

Metaplasia, osseous 

Infiltration cellular, histiocyte 

Hemorrhage 

Goblet cell, respiratory epithelium, 

Inflammation, suppurative 

Inflammation, chronic 

Hemorrhage 

Foreign body 

Respiratory epithelium, metaplasia, squamous 

Respiratory epithelium, hyperplasia 

Olfactory epithelium, metaplasia 

Nasolacrimal duct, inflammation, suppurative 

Epithelium, hyperplasia 

10 (21%) 

35 (73%) 

16 (33%) 

29 (60%) 

16 (33%) 

32 (67%) 

12 (26%) 

30 (65%) 

1 (2%) 

2 (4%) 

1 (2%) 

3 (6%) 

6 (13%) 

1 (2%) 

1 (2%) 

5 (10%) 

5 (11%) 

2 (4%) 

2 (4%) 

2 (4%) 

1 (2%) 

3 (7%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1. (2%) 

2 (4%) 

4 (9%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

Nose (47) (47) (48) (46) 

3 (6%) 

hypertrophy 3 (6%) 2 (4%) 

3 (6%) 

2 (4%) 2 (4%) 

1 (2%) 

2 (4%) 

Trachea (48) (48) (48) (45) 

Eye 

Special Senses System 

Retina, atrophy 

Hemorrhage 

Cataract 

(3) (1) 

1 (33%) 

1 (33%) 

(2) (3) 

2 (100%) 

1. (50%) 

1 (50%) 

Kidney 

Urinary System. 

Cortex, necrosis 

Nephropathy, chronic 

Infarct 

5 (10%) 

37 (77%) 

.. 

Renal tubule, pigmentation, hemosiderin. 

Renal tubule, mineralization 

Renal tubule, inflammation, suppurative 

Pelvis, transitional epithelium, hyperplasia. 

Transitional epithelium, hyperplasia 

39 (81%) 

Urinary bladder (48) 

(48) (48) (46) 

1 (25%) 

1 (2%) 

2, (4%) 

37 (77%) 

1 (2%) 

1 (2%). 

1 (2%) 

5 (10%) 

37 (77%). 40 (87%) 

5 (11%) 

40 (83%) 45 (94%) 37 (80%) 

(48) (47) (45) 
143 

APPENDIX C 

SUMMARY OF LESIONS IN MALE MICE 

IN THE 2-YEAR INHALATION STUDY 

OF ACETONITRILE 

TABLE C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice 

TABLE C4c Historical Incidence of Forestomach Squamous Cell Papilloma 

TABLE C4b Historical Incidence of Hepatocellular Neoplasms 

TABLE C4a Historical Incidence of Alveolar/bronchiolar Neoplasms 

TABLE C3 Statistical Analysis of Primary Neoplasms in Male Mice 

TABLE C2 Individual Animal Tumor Pathology of Male Mice 

TABLE C1 Summary of the Incidence of Neoplasms in Male Mice 

in the 2-Year Inhalation Study of Acetonitrile 

in Untreated Male B6C3Fl Mice 

in Untreated Male B6C3Fl Mice 

in Untreated Male B6C3Fl Mice 

in the 2-Year Inhalation Study of Acetonitrile 

in the 2-Year Inhalation Study of Acetonitrile 

in the 2-Year Inhalation Study of Acetonitrile ............................... 

170 

169 

168 

168 

164 

148 

144 

............................... 

............................... 

......................................... 

......................................... 

......................................... 

............................... 
144 Acetonitrile, NTP TR 447 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Acetonitrilea 

TABLE C1 

0 PPm 50 PPm 100 ppm 200 ppm 

Early deaths 

I5-Mod1 irrtaim evaluation 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

Survivors 

10 

60 

5 

13 

32 

10 ' 

60 

4 

14 

32 

10 

60 

2 

16 

32 

10 

60 

1 

6 

43 

Animals examined microscopically 60 60 60 60 

Liver 

Alimentary System 

15-Month Interim Evaluation 

(10) (10) (10). (10) 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

1 (10%) 

1 (10%) 1 (10%) 

1 (10%) 3 (30%) 

2 (20%) 1 (10%) 

Thyroid gland 

Endocrine System 

Follicular cell, adenoma 

Lung 

Respiratory System 

(10) (10) (10) (10) 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple 

Alveolar/bronchiolar adenoma 

2 (20%) 1 (10%) 

1 (10%) 

1 (10%) 2 (20%) 2 (20%) 3 (30%) 

Urinary System 

Special Senses System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

Genital System 

General Body System 

Cardiovascular System 

Systems Examined With No Neoplasms Observed 

/ 

Intestine large, rectum 

Alimentary System 

2-Year Study 

Adenoma 

Intestine small, duodenum 

Anus, leiomyosarcoma 
Lesions in Male Mice 145 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE C1 

Intestine small, jejunum 

Alimentary System (continued) 

&Year shdy (continued) 

Liver 

Carcinoma 

Bile duct, carcinoma 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Hemangiosarcoma 

Hemangioma 

Pancreas 

Fat, hemangioma 

Sarcoma, metastatic, skin 

Stomach, forestomach 

Salivary glands 

Adenocarcinoma 

Stomach, glandular 

Mesentery 

Carcinoma, metastatic, pancreas 

Squamous cell papilloma 

10 (20%) 

4 (8%) 

1 (2%) 

3 (6%) 

3 (6%) 

(3) 

1 (33%) 

(49) 

(50) 

3 (6%) 

8 (16%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

10 (20%) 

(47) 

(49) 

1 (2%) 

5 (10%) 

13 (27%) 

6 (12%) 

7 (14%) 

(50) 

(49) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

9 (18%) 

1 (2%) 

1 (2%) 

6 (12%) 

(3) 

None 

Cardiovascular System 

Endocrine System 

Capsule, adenoma 

Adenoma 

Adrenal cortex 

Pheochromocytoma benign 

Adrenal medulla 

Pituitary gland 

Adenoma 

Islets, pancreatic 

Pars intermedia, adenoma 

Follicular cell, adenoma 

C-cell, adenoma 

Thyroid gland 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

(46) 

(47) 

(49) 

2 (4%) 

1 (2%) 

1 (2%) 

None 

General Body System 

Testes 

Genital System 

Interstitial cell, adenoma 
146 Acetonitrile, NTP TR 447 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE C1 

0, PPm 50 ppm 100 ppm. 200 ppm 

Bone marrow 

Hematopoietic System 

2-YeW study (continued) 

Hemangioma 

Lymph node, bronchial 

Lymph node 

Hemangiosarcoma 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Carcinoma, metastatic, pancreas 

Hemangiosarcoma 

Sarcoma, metastatic, skin 

Hemangiosarcoma 

Carcinoma, metastatic, pancreas 

Hemangiosarcoma 

Thymus 

Spleen 

1 (2%) 

(44) 

(49) 

(35) 

Hemangiosarcoma 

Carcinoma, metastatic, pancreas 1 (2%) 

1 (2%) 

Integumentary System 

Subcutaneous tissue, sarcoma 

Prepuce, squamous. cell carcinoma. 

Basal cell carcinoma 

Skin (50) (49) 

1 (2%) 

1 (2%) 

None 

Musculoskeletal System 

None 

Nervous System 

Lung 

Respiratory System 

(50) 

Mediastinum, hemangioma 

Sarcoma, metastatic, skin 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 4 (8%) 

Hepatocellular carcinoma 

Hemangiosarcoma 

Carcinoma, metastatic, pancreas 

Alveolarbronchiolar carcinoma, multiple 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple 

Alveolarbronchiolar adenoma 

4 (8%) 

4 (8%) 

2 (4%) 

1 (2%). 

1 (2%) 

(501 (48) (50) 

6 (12%) 

9 (18%) 

4 (8%) 

6 (13%) 

4 (8%) 

17 (34%) 

2 (4%) 

2 (4%) 1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

5 (10%). 
Lesions in Male Mice 147 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE C1 

0 PPm 50 PPm 100 ppm 200 ppm 

shdy (continued) 

Harderian gland 

Special Senses System 

Adenoma (5) (5) (1) (2) 

5 (100%) 4 (80%) 1 (100%) 2 (100%) 

Kidney 

Urinary System 

Pelvis, hemangioma 

Multiple organsb 

Systemic Lesions 

(50) (50) (50) (50) 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Histiocytic sarcoma 1 (2%) 

2 (4%) 

1 (2%) 1 (2%) 

3 (6%) 3 (6%) 2 (4%) 

Total animals with primary neoplasms' 

Neoplasm Summary 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

2-Year study 

2-Year study 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total metastatic neoplasms 

Total animals with metastatic neoplasms 

32 

4 

38 

6 

31 

3 

35 

3 

54 

6 

61 

10 

55 

4 

58 

4 

24 

2 

25 

4 

25 

3 

28 

3 

33 

3 

33 

6 

30 

4 

31 

4 

18 

3 

23 

4 

19 18 

21 

3 

28 

4 

25 21 

5 3 

5 

5 

7 I 

' Primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 
148 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm 

TABLE C2 

7768315583705884693333333 

3544689992357791123333333 

4455555556666667777777777 

9306128974785017662458123. 

5214252412232400230000111 

0000000000000000000000000 

Number of Days on Study 

Carcass ID Number 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Intestine small, ileum 

Intestine small, jejunum 

Carcinoma 

Adenoma 

++++A+++A+++A++++++++++++ 

++++++++A+++M+++A++++++++ 

++++++++A+++A++++++++++++ 

......................... 

++M+M+++A+M+A++++++++++++ 

......................... 

++++A+++M+++A++++A+++++++ 

++++A+++M+++A++M+++++++++ 

Liver ......................... 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple x xx 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

X X X 

X X X 

X 

X 

X 

X 

Salivary glands 

Pancreas 

Mesentery 

Sarcoma, metastatic, skin X 

+ + 

......................... 

++++++++A++++++++++++++++ 

Tooth 

Stomach, glandular 

Stomach, forestomach 

++++++++A++++++++++++++++ 

++++++++A++++++++++++++++ 

++ + 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

++++++++++++M++++++++++++ 

Adrenal cortex 

Endocrine System 

+++++ ++A++++++++++++++++ 

Pheochromocytoma benign 

Capsule, adenoma 

Adenoma 

Adrenahmedulla 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

Follicular cell, adenoma 

C-cell, adenoma 

Pars intermedia, adenoma 

++++++++A++++++++++++++++ 

X 

++++++++++++M++++++++++++ 

M+MMM+MMM+++M+++M++++++++ 

++++++++A++M+++++++++++++ 

++++++++A++++++++++++++++ 

X 

General Body System 

Genital' System 

Tissue NOS 

Testes 

Seminal vesicle. 

Prostate 

Preputial gland 

Epididymis 

......................... 

++++++++A++++++++++++++++ 

+++M+++++++++++++++++M++S 

+M+++++++++++++++++++++++ 

......................... 

A. Autolysis precludes examination 

+: Tissue examined microscopically 

I: Insufficient tissue 

M: Missing tissue 

Blank: Not examined 

X: Lesion present 
Lesions in Male Mice 149 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

3333333333333333333333333 

3333333333333333333333333 

7177777771771777717777777 

0000000000000000000000000 Total 

,1111223333333444445555556 Tissues/ 

4569020234579145781345780 Tumors 

Number of Days on Study 

Carcass ID Number 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

......................... 50 

......................... 45 

........................ 46 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Carcinoma 

Adenoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Sarcoma, metastatic, skin 

Mesentery 

......................... 48 

......................... 47 

......................... 47 

1 

3 

3 

X 10 

4 

3 

1 

......................... 50 

.......................... 46 

1 

++++++++++++++++M++++++++ 45 

X 1 

X 

X 

X X xx 

+ 

X X 

Pancreas ......................... 49 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands ......................... 50 

......................... 49 

......................... 49 

3 

Cardiovascular System 

Heart 

Blood vessel +++++++++M+++++++++++++++ 48 

......................... 50 

Endocrine System 

Capsule, adenoma 

Adenoma 

Adrenal cortex 

Pheochromocytoma benign 

Islets, pancreatic 

Adrenal medulla 

1 

1 

......................... 48 

+++M+++++++++++++++++++++ 47 

1 

......................... 49 

X 

X 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

2 

1 

......................... 49 

1 

++++++++++++++++M++++IM++ 46 

+++++M+++++++++++++M+++++ 39 

Follicular cell, adenoma 

C-cell, adenoma 

Pars intermedia, adenoma X 

xx 

General Body System 

Genital System 

Tissue NOS + 1 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Epididymis ......................... 50 

......................... 49 

+M++++++M++++++++++++M+++ 45 

......................... 49 

......................... 50 
150 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE C2 

Number of Days on Study 

I768315583105884693333333 

3544689992351791123333333 

44555555.56666661777777771 

Carcass ID Number 

9306128914185011662458123 

5214252412232400230000111 

0000000000000000000000000 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Thymus 

Spleen 

Lymph node, mediastinal 

Sarcoma, metastatic, skin 

Hemangiosarcoma 

Hemangiosarcoma 

++++++++A++++++++++++++++ 

+ 

++++++++A+++++++++++++M++ 

+++MMM++M+++M+++++++++++M 

MMM+M+M+M++MMM+++++++++++ 

X 

++MM++++MM+++++++M+++++++ 

X 

+'M++++M+AM++++++++++M++++ 

++++++++A++++++++++++++++ 

X 

Skin 

Mammary gland 

Integumentary System 

......................... 

MMMMMMMMMMMMMMMMMMMMMMM.MM 

Subcutaneous tissue, sarcoma 

Prepuce, squamous cell carcinoma 

X 

Musculoskeletal System 

Bone ......................... 

Nervous System 

Brain .......................... 

Respiratory System 

Lung 

Larynx 

......................... 

++++++++A++++++++++++++++ 

Alveolarhronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Alveolarhronchiolar adenoma, multiple 

'X X 

Sarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, liver xx 

Hemangiosarcoma X 

X 

X 

Trachea 

Nose 

++++++++A++++++++++++++++ 

........................... 

.. . 

Harderian gland 

Special Senses System 

+ + + 

Adenoma X x X .. 

Urinary bladder 

Kidney 

Urinary System 

Pelvis, hemangioma 

++++++++A++++++++++++.++++ 

++++++++A++++++++++++++++ 

Systemic Lesions 

Multiple organs ............................ 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic X 
Lesions in Male Mice 151 

TABLE c2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

3333333333333333333333333 

33333333333333333333.33333 

7117777777111711711717117 

Number of Days on Study 

Carcass ID Number 

0000000000000000000000000 Total 

1111223333333444445555556 Tissues/ 

4569020234579145781345780 Tumors 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Spleen 

Lymph node, mediastinal 

Thymus 

Sarcoma, metastatic, skin 

Hemangiosarcoma 

Hemangiosarcoma 1 

......................... 44 

......................... 49 

1 

M+++++++++M++++++++++++I+ 42 

1 

......................... 48 

+++++M+MM+M++M++M+++MMM++ 35 

+++++++++MM+++++++++M++++ 38 

3 

......................... 49 

+ + 

Skin 

Mammary glandIntegumentary System MMMMMMMMMMMMMMMMMMMMMMMMM 

1 

1 

......................... 50 

Subcutaneous tissue, sarcoma 

Prepuce, squamous cell carcinoma X 

Musculoskeletal System 

Bone ......................... 50 

Nervous System 

Brain ......................... 50 

Respiratory System 

Larynx 

......................... 49 

......................... 50 

1 

4 

1 

4 

X 2 

xx X 4 

......................... 50 

......................... 49 

Lung 

Sarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, liver 

Hemangiosarcoma 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple X 

Alveolarbronchiolar adenoma X 

X X 

X 

Trachea 

Nose 

Harderian gland 

Special Senses System 

+ + 

X X 5 

5 

Adenoma 

Urinary bladder 

Kidney 

Urinary System 

~ ~~ ~ 

.......................... 49 

1 

......................... 49 

Pelvis, hemangioma X 

Systemic Lesions 

Multiple organs 

2 

1 

......................... 50 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

X X 
152 Acetonitrile, NTP.TR 447 

1111111111111111111111111 

64135,52368844420144444444 

4455556666666667777777777 

7517398049734801321245679 

33746.72354747386732222222 

Number of Days on Study 2378990.234566790113333333 

Carcass ID Number 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

+A+++++++++++++++++++++++ 

+A+M++M+++++++++A++++++++ 

......................... 

Intestine large, rectum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Anus, leiomyosarcoma 

......................... 

......................... 

+A++++++++++++++A++++++++ 

+A+++++++++++++++++++++++ 

Intestine small, ileum 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Hemangiosarcoma 

Liver ......................... 

+A++++++++++++++.+++++++++ 

xx 

X xxx x x X X 

xx X 

X 

X 

Pancreas 

Mesentery + ......................... 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Adenocarcinoma 

Carcinoma, metastatic, pancreas 

X 

.......................... 

.......................... 

......................... 

X 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

++++++M++++++++++++++++++ 

Endocrine System 

~~~~ 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Thyroid gland 

Pituitary gland 

Parathyroid, gland 

Adenoma 

Capsule, adenoma 

......................... X 

......................... 

......................... 

......................... 

++++M++++++++++++++.++++++ 

+++-k++M++++++++++M+M+++++ 

X 

General Body System 

None 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Epididymis 

Interstitial cell, adenoma 

......................... 

......................... 

+++++++++IM++++++++++++++ 

........................... 

......................... 

X 
Lesions in Male Mice 153 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm (continued) 

TABLE C2 

4444444444444444444444444 

3333333333333333333333333 

7777777777777777777777777 

1111111111111111111111111 Total 

3333444445555555566666777 Tissues/ 

1469045680123678925678568 Tumors 

Number of Days on Study 

Carcass ID Number 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Anus, leiomyosarcoma 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Hemangiosarcoma 

Pancreas 

Mesentely 

Adenocarcinoma 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Carcinoma, metastatic, pancreas 

......................... 50 

......................... 46 

......................... 49 

......................... 50 

......................... 50 

......................... 49 

......................... 48 

......................... 48 

......................... 1 

1 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

2 

1 

2 

10 

3 

8 

X 2 

1 

X 

49 \ 

X 

X 

x xxx xx X 

X 

X 

+ 

Cardiovascular System 

Heart 

Blood vessel ......................... 49 

......................... 50 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Adenoma 

......................... 50 

......................... 50 

X 4 

......................... 50 

......................... 50 

I++++++++++++++++++++++++ 48 

+++MMMM+MIM+++M++M++M++M+ 36 

1 

Capsule, adenoma xx 

General Body System 

None 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Epididymis .. 

Interstitial cell, adenoma 

......................... 50 

......................... 50 

+++MM++++++++++++M+++++++ 45 

......................... 50 

......................... 50 

1 
154 Acetonitrile, NTP TR 447 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm (continued) 

641.3552368844420144444444 

2318990234566190113333333 

4455556666666661111717171 

7511398049134801321245619 

3374612354141386132222222 

1111111111111111111111111 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Hemangioma 

Lymph node, bronchial 

Lymph node 

Hemangiosarcoma 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Spleen 

Carcinoma, metastatic, pancreas 

Carcinoma, metastatic, pancreas 

Thymus 

Carcinoma, metastatic, pancreas 

......................... 

X 

++ 

++++M++++++++++++++++++++ 

M+++MM+++++M+M+M+M++MMM++ 

++++M+M+++++++++MM+++++++ 

X 

++M++++++++++++++++MM++M+ 

X 

+++MM+M+++++++++MM+++++++ 

......................... 

X 

Skin 

Mammary gland 

Integumentary System 

+++++++++++++M+++++++++++ 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Musculoskeletal System 

Skeletal muscle. 

Bone ......................... 

Nervous System 

Brain ......................... 

Respiratory System 

Lung 

Lalynx ......................... 

xx x 

X 

......................... 

Mediastinum, hemangioma 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular carcinoma 

Carcinoma, metastatic, pancreas 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma 

X 

X 

X 

X 

X X 

Trachea 

Nose 

......................... 

......................... 

Harderian gland 

Ear 

Special Senses System 

Adenoma 

+ 

X 

++ 

Urinary bladder 

Kidney 

Urinary System 

+++++++++++++++++M+++++++ 

......................... 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 

Histiocytic sarcoma 

......................... 

xx 
TaBLB c2 

hdhidpna! himd Tumor $athology Sf Male Mice in the %year hhakstion Study of Acetonitrile: 58 ppm (continued) 

4444444444444444444444444 

3333333333333333333333333 

7777777777777777777777777 

1111111111111111111111111 Total 

3333444445555555566666777 Tissues/ 

1469045680123678925678568 Tumors 

Number of Days on Study 

Carcass BD Number 

Hemangioma 

Wematopoietic System 

Bone marrow ......................... 50 

X 1 

Lymph node, bronchial 

Lymph node 

Hemangiosarcoma 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Spleen 

Carcinoma, metastatic, pancreas 

Carcinoma, metastatic, pancreas 

Thymus 

Carcinoma, metastatic, pancreas 1 

......................... 45 

......................... 50 

1 

++IMM+++++MM++M+++++++MM+ 38 

......................... 49 

MM+++M++++M+++M+++++M+M++ 33 

1 

M+++M+++MM++++++++MMMMM++ 37 

2 

1 

......................... 49 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Muscunoskenetaa system 

Skeletal muscle 

Bone 

1 

......................... 50 

+ 

......................... 50 

!hSpilL%tQlFj' BgrStetZl 

Lalynx 

Lung 

Mediastinum, hemangioma 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular carcinoma 

Carcinoma, metastatic, pancreas 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma 

Trachea 

Nose 

......................... 50 

......................... 50 

1 

1 

2 

1 

1 

6 

xx xx x X 9 

......................... 50 

......................... 50 

x X 

x 

x 

x 

Harderian gland 

Ear 

Special Senses System 

Adenoma xx 

++ 

X 

+ 

4 

5 

1 

Urinaly bladder 

Kidney 

Urinary System 

......................... 50 

......................... 49 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 

Histiocytic sarcoma 

3 

1 

......................... 50 

X 

X 
156 Acetonitrile, NTP TR 447 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the &Year Inhalation Study of Acetonitrile: 100 ppm 

2773516664088452074444444 

4361901334555669223333333 

3555566666666666777777777 

1840500539476783482346781 

4779907478557858864444445 

2222232222222222222222222 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

+A+++++++++++++++++++++++ 

......................... 

AA++++M++++++++++++++++++ 

......................... 

......................... 

......................... 

......................... 

AM+++++++"++++++++++++++ 

+A++++++++++++++++M++++++ 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

Fat, hemangioma 

X 

X 

X 

X X X X 

xx xx X 

X xx 

X 

X 

MesenteIy 

Stomach, forestomach 

Salivary glands 

Pancreas 

Stomach, glandular- 

+ + 

AA+++++++++++++.++++++.++++ 

AA+++++++++++++++++++++++ 

......................... 

.......................... 

Squamous cell papilloma 

Cardiovascular System 

Heart 

. . Blood vessel 

+A+++++++++++++++++++++++ 

.......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

MMM+++++++++++++++++++?++ 

........................ 

Pheochromocytoma benign 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

......................... 

AM++M+M++++++++++++++++++ 

+A+M+-+M+++++.++++++M++M+++ 

......................... 

General Body System 

Genital System 

Tissue NOS + 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Epididymis 

MAM,+f+M+++++++++++M+-+++++ 

AMM+++++++++++++++++M++++ 

AA+++++++++++++++++++++++ 

AAfM++++++++f++++++++++++ 

+A,++++++++++++++++++++++-+ 
IIndividanaP AmimaU TUW~SB- Pathology of Male Mice in the %-Year InhaPation Study of Acetonitrile: 100 ppm (continued) 

TABLE ea: 

4444444444444444444444444 

3333333333333333333333333 

7717771771777711777717717 

2222222222222222222222222 Total 

5556666666677788888999999 Tissues/ 

2390123467925901568234689 Tumors 

Numbe~ of Days on Study 

Carcass ID Number 

In'testine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus ......................... 48 

......................... 46 

......................... 48 

......................... 48 

Intestine large, cecum ........................... 48 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

Fat, hemangioma 

Mesentery 

Stomach, forestomach 

Salivary glands 

Pancreas 

Stomach, glandular 

Squamous cell papilloma 

......................... 48 

.......................... 47 

......................... 48 

......................... 49 

1 

3 

5 

13 

6 

7 

1 

......................... 48 

......................... 48 

......................... 48 

......................... 48 

1 

X 

X X 

xx X x x xx x 

X X X X 

X 

+ 

X 

CaPdiQVa5cUbP SyS(leE3l 

Heart 

Blood vessel ......................... 48 

......................... 49 

~~dQcI&lC? sgrS&eHaa 

Adrenal medulla 

Adrenal cortex 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Pheochromocytoma benign 

......................... 46 

+MM++M+++++'++++++M+++M+++ 40 

......................... 48 

1 

......................... 47 

......................... 48 

x 

General Body System 

Thyroid gland ......................... 48 

Genital System 

Tissue NOS 1 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Epididymis ......................... 48 

......................... 46 

++I++M+M+++++++++++++++++ 42 

......................... 47 

......................... 49 
158 Acetonitrile, NTP TR 447 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

2773576664088452074444444 

4367901334555669223333333 

3555566666666666777777777 

1840500539476783482346181 

4119901418551858864444445 

2222232222222222222222222 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Lymph node 

Bone marrow ......................... 

++ 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

AA++++MMMM++++++++I+M++++ 

......................... 

AA+++++MM+++M+M++++++++++ 

AA++++++M++++++++++++++++ 

MM+MM+M+M+MMM++M+++MMM+++ 

+M++MM++M+++M++++++M++M++ 

Skin 

Mammary gland 

Integumentary System 

+A+++++++++++++.++++++++++ 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Basal cell carcinoma X 

Musculoskeletal. System 

Bone +A+++++++++++++++++++++++ 

Nervous System. 

Brain .......................... 

Respiratory System 

Larynx .......................... 

AA++++++++++++++.+++++++++ 

Lung 

Hepatocellular carcinoma, metastatic, liver 

Alveolar/bronchiolar ,carcinoma, multiple 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple 

Alveolarbronchiolar adenoma . X 

xx 

X 

xx xx 

Trachea 

Nose 

~ 

......................... 

......................... 

Harderian glandSpecial Senses System 

~~~ ~ 

x 

+ 

Adenoma 

Urinary bladder 

Kidney 

Urinary System 

+A+++++++++++++++++++++++ 

AA+++++++++++++++++++++++ 

Systemic hsions 

Multiple organs 

Lymphoma malignant mixed. 

Lymphoma-malignant lymphocytic 

+ ....................... 

xx X 
Lesions in Male Mice 159 

TABLE c2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 

4144444444444444444444444 

3333333333333333333333333 

2222222222222222222222222 Total 

Carcass ID Number 5556666666677788888999999 Tissues1 

2390123467925901568234689 Tumors 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

+++M+M+M+++++++++++++M+++ 38 

......................... 48 

+M+MM+IM+++M++++M+++MM+++ 35 

......................... 47 

M+++M++M+++++++M++M++M+MM 29 

++++++++M+M+++MI++++M++++ 38 

2 

......................... 48 

Skin 

Mammary gland 

Integumentary System 

Basal cell carcinoma 1 

......................... 49 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Musculoskeletal System 

Bone ......................... 49 

Nervous System 

Brain ......................... 48 

Respiratory System 

Larynx 

Lung 

Hepatocellular carcinoma, metastatic, liver 

Alveolaribronchiolar carcinoma, multiple 

Alveolaribronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple 

Alveolarbronchiolar adenoma 

Trachea 

Nose 

......................... 48 

2 

6 

......................... 48 

5 

2 

X 4 

......................... 48 

......................... 48 

xx X X X 

X X 

X 

X 

X 

Special Senses System 

Harderian gland 

Adenoma 1 

1 

Urinary bladder 

Kidney 

Urinary System 

......................... 48 

......................... 49 

Systemic Lesions 

Multiple organs 

3 

1 

.......................... 49 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic X 
160 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 200 ppm 

TABLE C2 

2435217333333333333333333 

35469023333333333,33333333 

5566677777777.777777777777 

0681735235623480123456890 

7066197666677778888888889 

3433433333333333333333333 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Liver 

Intestine small, ileum 

Carcinoma 

++++++A++++++++++++++++++ 

++++++.A++++++++M+++++++++ 

++++++I,+M++++++++++++++++ 

......................... 

+++++++++++++++M+++++++++ 

MM++++A+M++++++++++++++++ 

......................... 

......................... 

++++++A++++++++++++++++++ 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Hemangiosarcoma 

Hemangioma 

xx 

X xx 

X X 

X 

X 

X X 

X 

X 

Fat, hemangioma 

Bile duct, carcinoma 

Hepatocellular adenoma,. multiple 

X 

Pancreas 

Mesentery 

......................... 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivaly glands 

Squamous cell papilloma 

......................... 

......................... 

......................... 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

......................... 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Adenoma 

Thyroid gland 

......................... Capsule, adenoma X 

++++++++M++++++++++++++++ 

+M++M+MM++M+M++++++++++MM 

......................... 

......................... 

X 

Pars intermedia, adenoma 

......................... 

General Body System 

None 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Epididymis 

.......................... 

.......................... 

+++++++++++M++++++++M++++ 

......................... 

......................... 
Lesions in Male Mice 161 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TABLE C2 

Number of Days on Study 

3333333333333333333333333 

3333333333333333333333333 

1117111111111111111711111 

3333333444444444444444444 Total 

9999999000000001111111112 Tissues/ 

1245679013451890123456890 Tumors 

Carcass ID Number 

Gallbladder 

Esophagus 

Alimentary System 

......................... 50 

++++++++++M++++++++++++++ 45 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Liver 

Intestine small, ileum 

Carcinoma 

Bile duct, carcinoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Hemangiosarcoma 

Hemangioma 

Fat, hemangioma 

Mesentery 

Stomach, forestomach 

Salivary glands 

Pancreas 

Tooth 

Stomach, glandular 

Squamous cell papilloma 

......................... 

......................... 

......................... 

++++++++++++++M++++++++++ 

......................... 

X 

......................... 

......................... 

X 

2 

1 

1 

50 

49 

1 

49 

48 

48 

49 

49 

X X X 

X 

9 

1 

6 

1 

......................... 50 

X X 2 

......................... 50 

......................... 50 

......................... 50 

X 1 

+ 3 

1 

X 1 

xx 

++ 

+ 

Cardiovascular System 

Heart 

Blood vessel ++++++++++++++++++++M++++ 49 

......................... 50 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Adenoma 

Thyroid gland 

Capsule, adenoma 

Pars intermedia, adenoma 

......................... 50 

1 

......................... 49 

++MMI+MMMMM+MMM+M++M+++MM 21 

1 

......................... 50 

......................... 50 

1 

......................... 50 

X 

General Body System 

None 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Epididymis ......................... 50 

......................... 50 

+++++++++M++++M+M++++++++ 45 

......................... 50 

......................... 50 
162 Acetonitrile, NTP TR 447 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

5566671777777717777711777 

Number of Days on Study 

2435217333333333333333333 

35469023.33333333333333333 

0681135235623480123456890 

7066197666677778888888889 

3433433333333333333333333 

Carcass ID Number 

Bone marrow 

Hematopoietic System 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Hemangiosarcoma 

Spleen 

......................... 

+M+MM+MI++MM+++M+M+M+++M+ 

......................... 

M+MM+++++MM++I+M++++++M+M 

+++M+++MMM+++++++++++++++ 

X 

Hemangiosarcoma 

Hemangiosarcoma 

++M+M++++++++++++++++++++ 

X 

......................... 

Thymus 

Skin 

Mammary gland 

Integumentary System 

......................... 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Musculoskeletal System 

Bone ......................... 

Nervous System 

Brain ......................... 

Respiratory System 

Larynx 

xx xxx x xx x X xxx ......................... 

......................... 

......................... 

.......................... X 

Lung 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple 

Alveolarbronchiolar adenoma 

X 

X 

Trachea 

Nose 

Harderian gland 

Special Senses System 

Adenoma X 

+ 

X 

+ 

Urinary Bladder 

Kidney 

Urinary System 

......................... + + + + + + +++++ + ~~ ~~ ~~~ 

Systemic Lesions 

~~ 

Multiple organs ......................... Lymphoma malignant mixed X X 
Lesions in Male Mice 163 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the &Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

3333333333333333333.333333 

3333333333333333333333333 

7171717117711111111171777 

3333333444444444444444444 Total 

9999999000000001111111112 Tissues/ 

1245679013451890123456890 Tumors 

Number of Days on Study 

Carcass ID Number 

Bone marrow 

Hematopoietic System 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Hemangiosarcoma 

Spleen ......................... 50 

1 

+++++MM+++++++++I+++++MMI 33 

+++++++++++++++++M+++++++ 49 

M+++++++M+M++M++++M++++++ 36 

+M++++++M+++M+M+++++++++M 41 

1 

......................... 50 

Hemangiosarcoma X 

Hemangiosarcoma 

Thymus ++++++++M++++++++++++++++ 47 

1 

Skin 

Mammary gland 

Integumentary System 

......................... 50 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Musculoskeletal System 

Bone ......................... 50 

Nervous System 

Brain ......................... 50 

Respiratory System 

Lalynx ......................... 50 

......................... 50 

xx 11 

......................... 50 

......................... 50 

4 

1 

Lung 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple 

Alveolarbronchiolar adenoma xx 

X X 

Trachea 

Nose 

Harderian gland 

Special Senses System 

Adenoma 2 

2 

Urinary bladder 

Kidney 

Urinary System 

......................... 50 

......................... 50 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 2 

......................... 50 
164 Acetonitrile, NTP TR 447 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE C3 

0 PPm 50 PPm 100 ppm 200 ppm 

Fisher exact test 

Cochran-Armita e testd 

Logistic regression testd 

Life table testd 

First incidence (days) 

Terminal rate' 

Adjusted rateb 

Overall ratea 

Adrenal Cortex: Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Harderian Gland: Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hemangiosarcoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Carcinoma 

% P=0.197N 

P=0.130N 

P=0.130N 

733 (T) 

2/32 (6%) 

6.3% 

2/49 (4%) 

P=0.114N 

P=0.150N 

P=0.073N 

437 

2/32 (6%) 

12.2% 

5/50 (10%) 

3.0% 

1/50 (2%) 

P=O.312 

P=O.321 

P=O.431 

729 

0/32 (0%) 

P=0.345N 

P=0.293N 

P=0.114N 

563 

9/32 (28%) 

35.0% 

13/50 (26%) 

P=0.478N 

P=O.163 

P=0.304N 

437 

1/32 (3%) 

15.3% 

7/50 (14%) 

P=O.335 

P=O.335 

733 (T) 

4/32 (13%) 

12.5% 

4/50 (8%) 

P=O.349 

P=0.514N 

P=0.492N 

602 

3/32 (9%) 

11.4% 

4/50 (8%) 

P=0.500N 

3/32 (9%) 

9.4% 

3/50 (6%) 

P=O.295 

P=O.304 

733 (T> 

P=O.309 

P=0.500N 

P=0.491N 

434 

8/32 (25%) 

31.7% 

12/50 (24%) 

P=0.500N 

P=O.128 

P=O.255 

571 

1/32 (3%) 

24.6% 

11/50 (22%) 

P=O.218 

0/32 (0%) 

0.0% 

0/48 (0%) 

P=0.238N 

P=0.238N 

e - 

P=0.253N 

3.1% 

1/50 (2%) 

P=0.102N 

P=0.106N 

733 (T) 

1/32 (3%) 

P=0.102N 

P=O.763 

P=O.760 

720 

0/32 (0%) 

2.9% 

1/49 (2%) 

P=O.747 

P=O.189 

P=O.208 

595 

14/32 (44%) 

49.1% 

18/49 (37%) 

P=O.175 

P=O.O38 

P=O.139 

342 

4/32 (1 3%) 

30.3% 

13/49 (27%) 

P=O.O96 

P=0.397N 

P=0.397N 

733 (T) 

1/43 (2%) 

2.3% 

1/50 (2%) 

P=0.492N 

P=0.418N 

P=0.156N 

727 

1/43 (2%) 

4.5% 

2/50 (4%) 

P=0.218N 

P=O.221 

P=O.387 

554 

0/43 (0%) 

6.4% 

3/50 (6%) 

P=O.309 

P=0.249N 

P=0.123N 

701 

8/43 (19%) 

22.2% 

10/50 (20%) 

P=0.318N 

15.0% 

7/50 (14%) 

P=O.208 

P=0.475N 

532 

4/43 (9%) 

, 

P=0.613N 
Lesions in Male Mice 165 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE C3 

0 PPm 50 PPm 100 ppm 200 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma or Carcinoma 

P=0.260N 

P=0.437N 

P=0.070N 

437 

10/32 (31%) 

44.5% 

19/50 (38%) 

P=O.394 

P=O.447 

434 

9/32 (28%) 

46.7% 

21/50 (42%) 

P=O.O13 

P=O.O54 

342 

18/32 (56%) 

67.7% 

30/49 (61%) 

P=0.454N 

P=0.094N 

532 

11/43 (26%) 

31.8% 

15/50 (30%) 

P=O.419 P=O.O17 P=0.263N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Adenoma 

6/32 (19%) 

18.8% 

6/50 (12%) 

P=O.O02 

P=O.OlO 

P=O.O24 

733 (T> 

9/32 (28%) 

28.1% 

9/50 (18%) 

P=O.279 

P = 0.279 

733 (T> 

P=O.375 

P=O.384 

727 

7/32 (22%) 

24.2% 

8/48 (17%) 

P=O.O11 

P=O.O37 

554 

14/43 (33%) 

38.2% 

18/50 (36%) 

P=O.288 P=O.355 P = 0.005 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Carcinoma 

P=0.503N 

P=0.450N 

P=0.315N 

729 

3/32 (9%) 

12.1% 

4/50 (8%) 

P=O.374 

P=O.377 

583 

4/32 (13%) 

16.5% 

6/50 (12%) 

P=O.367 

P=O.375 

607 

3/32 (9%) 

16.4% 

6/48 (13%) 

733 (T> 

4/43 (9%) 

9.3% 

4/50 (8%) 

P=0.491N 

P =0.477N 

P=O.370 P=O.344 P=0.643N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

P=O.O11 

P=O.O38 

P=O.119 

729 

9/32 (28%) 

30.3% 

lobo (20%) 

P=O.239 

P=O.239 

583 

12/32 (38%) 

40.3% 

14/50 (28%) 

P=O.234 

P=O.245 

607 

10/32 (31%) 

38.5% 

14/48 (29%) 

P=O.O42 

P=O.113 

554 

17/43 (40%) 

44.6% 

21/50 (42%) 

P=O.241 P=O.206 P=O.O15 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Hemangiosarcoma 

5.9% 

2/50 (4%) 

P=O.210 

P=O.208 

P=O.339 

716 

OB2 (0%) 

P=O.340 

P =0.338 

583 

3/32 (9%) 

11.3% 

4/50 (8%) 

2.9% 

1/50 (2%) 

P 30.499N 

P=O.SOON 

720 

OB2 (0%) 

P=O.189 

P=O.331 

554 

2/43 (5%) 

10.7% 

5/50 (10%) 

P=O.339 P=0.500N P=O.218 
166 Acetonitrile, NTP TR 447 

0 PPm 50 PPm 100 ppm 200 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Hemangioma or Hemangiosarcoma 

Fisher exact test 

Cochran-Armitage test P=0.420N 

Logistic  P=0.373N 

Life P=0.292N 

First incidence s) 714 

Terminal rate 2/32 (6%) 

Adjusted rate 8.9% 

Overall rate 3/50 (6%) 

All Organs: Malignant Lymphoma (Lymphocytic or Mixed) 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign Neoplasms 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Neoplasms 

8.8% 

3/50 (6%) 

P=O.179 

P=O.216 

P=O.340 

716 

1/32 (3%) 

P=O.308 

P = 0.432 

P=0.207N 

437 

18/32 (56%) 

63.1% 

25/50 (50%) 

P=0.255N 

P=0.427N 

P=0.076N 

437 

10/32 (31%) 

46.2% 

20/50 (40%) 

P=O.246 

P=O.245 

583 

5/32 (16%) 

17.4% 

6/50 (12%) 

P=O.243 

8.7% 

3/50 (6%) 

P=0.661N 

P=O.653 

700 

1/32 (3%) 

P=0.661N 

P=O.497 

P=O.505 

434 

20/32 (63%) 

67.6% 

26/50 (52%) 

P=O.500 

P=O.257 

P=O.319 

571 

12/32 (38%) 

53.7% 

24/50 (48%) 

P=O.273 

1/32 (3%) 

2/50 (4%) 

6.0% 

P=0.454N 

P=0.500N 

720 

P=0.500N 

10.6% 

4/50 (8%) 

P=O.504 

P=O.504 

644 

1/32 (3%) 

P=O.500 

P=O.514 

P=O.506 

537 

20/32 (63%) 

67.8% 

26/50 (52%) 

P=O.500 

P=0.526N 

P=0.486N 

342 

8/32 (25%) 

43.4% 

19/50 (38%) 

P=0.500N 

15.1% 

7/50 (14%) 

P=O.165 

P=O.290 

554 

4/43 (9%) 

P=O.159 

2/43 (5%) 

4.7% 

2/50 (4%) 

P=0.500N 

P=0.401N 

P=0.374N 

733 (T) 

P=O.389 

P=0.291N 

554 

24/43 (56%) 

59.5% 

28/50 (56%) 

P =0.344 

P=O.491 

P=0.164N 

532 

13/43 (30%) 

37.4% 

18/50 (36%) 

P=0.418N 
Lesions in Male Mice 167 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

P=O.532 

P=O.482 P-0.251 P=O.247 P=O.414 

P=0.061N P=O.326 P=O.335 P=0.123N 

437 434 342 

22/32 (69%) 23/32 (72%) 29/43 (67%) 

76.7% 82.3% 80.7% 71.4% 

34/50 (68%) 3/50(76%) 38/50 (76%) 35/50 (70%) 

P=O.252 P=O.252 P=O.500 

24/32 (75%) 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

(VTerminal sacrifice 

exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher 

to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

Observed incidence at terminal kill 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

liver, and lung; for other tissues, denominator is number of animals necropsied. 

e Not applicable; no neoplasms in animal group 
168 Acetonitrile, NTP TR 447 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Untreated Male B6C3F, Micea 

TABLE C4a 

Study Carcinoma 

Incidence in Controls 

Adenoma Adenoma or Carcinoma 

Historical Incidence at Battelle Pacific Northwest Laboratories 

o-Chlorobenzalmalononitrile (CS2) 

Hexachlorocyclopentadiene 

Chloroethane 

I-Epinephrine Hydrochloride 

2-Chloroacetophenone 

Allyl Glycidyl Ether 

1,3-Butadiene 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

7/49 

11/49 

3/50 

11/50 

7/50 

7/50 

18/50 

7/49 

0/49 

2/50 

5/50 

6/50 

0/50 

5/50 

6%-36% 

7.6% 

113/673 (16.8%) 

0%-16% 

5.6% 

45/673 (6.7%) 

14/49 

11/49 

5/50 

15/50 

11/50 

7/50 

21/50 

10%-42% 

9.0% 

150/673 (22.3%) 

~~ ~~ 

a Data as of 31 March 1993 

Historical Incidence of Hepatocellular Neoplasms in Untreated Male B6C3F, Micea 

TABLE C4b 

Study Carcinoma 

Incidence in Controls 

Adenoma Adenoma or Carcinoma 

Historical Incidence at Battelle Pacific Northwest Laboratories 

o-Chlorobenzalmalononitrile (CS2) 

Hexachlorocyclopentadiene 

Chloroethane 

I-Epinephrine Hydrochloride 

2-Chloroacetophenone 

Allyl Glycidyl Ether 

1,3-Butadiene 

4/49 

19/50 

6/5 0 

10/50 

5/50 

15/49 

13/50 

14/49 

7/50 

9/50 

12/50 

11/50 

10/49 

11/50 

18/49 

24/50 

15/50 

20/50 

16/50 

23/49 

21/50 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

4%-38% 

11.0% 

120/673 (17.8%) 

9%-29% 

5.9% 

136/673 (20.2%) 

11%-56% 

12.1% 

241/673 (35.8%) 

a Data as of 31 March 1993 
Lesions in Male Mice 169 

Study 

Historical Incidence of Forestomach Squamous Cell Papilloma in Untreated Male B6C3Fl Micea 

TABLE C4c 

Incidence in Controls 

Historical Incidence at Battelle Pacific Northwest Laboratories 

o-Chlorobenzalmalononitrile (CS2) 

Hexachlorocyclopentadiene 

Chloroethane 

[-Epinephrine Hydrochloride 

2-Chloroacetophenone 

Allyl Glycidyl Ether 

1,3-Butadiene 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

0150 

0150 

0/50 

0/50 

2/50 

1/50 

1/50 

0%-4% 

1.3% 

5/676 (0.7%) 

~ ~~~ ~ ~~ 

a Data as of 31 March 1993 
170 Acetonitrile, NTP TR 447 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Acetonitrilea 

TABLE C5 

~ ~~~ 

0 PPm 50 PPm 100 ppm 200 ppm 

Early deaths 

15-Monfh intainr cvlrcuation 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

Animals examined microscopically 

10 

60 

5 

13 

32 

60 

10 

60 

4 

14 

32 

60 

10 

60 

2 

16 

32 

60 

10 

60 

1 

6 

43 

60 

Gallbladder 

Alimentary System 

15-Month Interim Evaluation 

Liver 

Inflammation, suppurative 

Eosinophilic focus 

Degeneration, fatty 

Basophilic focus 

Angiectasis 

Hyperplasia, squamous 

Fat, necrosis 

Stomach, forestomach 

Mesentery 

5 (50%) 

2 (20%) 

1 (100%) 

1 (10%) 

(10) 

(1) 

Heart 

Cardiovascular System 

Cardiomyopathy 

Angiectasis 

Endocrine System 

Hypertrophy 

Hyperplasia 

Adrenal cortex 

Pituitary gland 

Hyperplasia 

Islets, pancreatic 

Pars distalis, cyst 

Preputial gland 

Epididymis 

Genital System 

(10) 

(10) 

(10) 

(10) 

Inflammation, chronic active 

Granuloma sperm 

1 (10%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Male Mice 171 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE C5 

0 PPm 50 PPm 100 ppm 200 ppm 

Lymph node, mesenteric 

Hematopoietic System 

15-Month Interim Evaluation (continued) 

(8) (10) (10) 

Hyperplasia 

Congestion 1 (10%) 

2 (25%) 

Skin 

Integumentary System 

(10) (10) (10) (10) Subcutaneous tissue, inflammation, chronic 1 (10%) 1 (10%) 

Bone 

Musculoskeletal System 

Inflammation, suppurative 

Mineralization 

Brain 

Nervous System 

Respiratory System 

Larynx 

(10) 

(10) 

(10) 

(10) 

(10) 

(10)- 

(10) 

(10) 

Inflammation, suppurative 

Perivascular, infiltration cellular, 

Alveolar epithelium, hyperplasia 

Hemorrhage 

1 (10%) 

Lung 

1 (10%) 1 (10%) 

1 (10%) 

2 (20%) 

mononuclear cell 

Urinary System 

Renal tubule, hyperplasia 

Nephropathy 

Hydronephrosis 

Kidney (10) (10) (10) (10) 

1. (10%) 

2 (20%) 

4 (40%) 2 (20%) 4 (40%) 1 (10%) 

Special Senses System 

General Body System 

Systems Examined With No Leswm Observed 
172 Acetonitrile, NTP TR 447 

Gallbladder 

Alimentary System 

2- Year Study 

(45) (46) (46) (45) 

Cyst 

3 (7%) 

1 (2%) 

1 (2%) 

Inflammation, suppurative 

Hyperplasia 

Degeneration 

Angiectasis 

Peyer’s patch, hyperplasia 

Peyer’s patch, hyperplasia 

Peyer’s patch, hyperplasia 

Necrosis 

Kupffer cell, pigmentation 

Centrilobular, necrosis 

Bile duct, hyperplasia 

Bile duct, degeneration 

Bile duct, cyst 

Vacuolization cytoplasmic 

Necrosis 

Mixed cell focus 

Mitotic alteration 

Karyomegaly 

Infarct 

Hemorrhage 

Hematopoietic cell proliferation 

Focal cellular change 

Eosinophilic focus 

Degeneration, fatty 

Cytomegaly 

Clear cell focus 

Basophilic focus 

Fat, necrosis 

Inflammation, chronic 

Angiectasis 

Duct. ectasia 

Inflammation, chronic 

Focal cellular change 

Atrophy 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 1 

2 (4%) 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

(50) 

(46) 

(45) 

(47) 

(47) 

(50) 

(49) 

(48) 

(48) 

(49) 

(49) 

(48) 

(47) 

(48) 

(48) 

(50) 

(49) 

(49) 

(48) 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

5 (10%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

2 (4%) 

1 (2%) 

5 (10%) 

2 (4%) 

1 (2%) 

4 (8%) 1 (2%) 

1 (2%) 

2 (4%) 

3 (6%) ~ 

1 (2%) 

1 (2%) 

3 (6%) 

1 (2%) ~ 

2 (4%) 1 

3 (6%) 

4 (8%) 

3 (6%) 

4 (8%) 

4 (8%) 7 (14%) 

5 (10%) 4 (8%) 

4 (8%) 

2 (4%) 

1 (2%) 

2 (4%) 

2 (4%) 

4 (8%) 

4 (8%) 

6 (12%) 

3 (6%) 

3 (6%) 

3 (6%) 

3 (6%) 

3 (6%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

Mesentery (3) (2) (3) (3) 

1 (50%) 

1 (50%) 

2 (67%) 

3 (6%) 

2 (67%) 2 (67%) , 

Pancreas (49) (50) (48) (50) 

1 (2%) 

2 (4%) 

1 (2%) 

3 (6%) 1 (2%) 

1 (2%) 

2 (4%) 1 

Salivary glands (50) 

Inflammation, suppurative 

Inflammation, chronic 

Cyst 
Lesions in Male Mice 173 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE C5 

0 PPm 50 PPm 100 ppm 200 ppm 

study (continued) 

Stomach, forestomach 

Alimentary System (continued) 

Ulcer 

Inflammation, suppurative 

Hyperplasia, squamous 

Hyperkeratosis 

Pigmentation, hemosiderin 

Inflammation, suppurative 

Hyperplasia 

Erosion 

Ectopic tissue 

Degeneration 

Stomach, glandular 

Inflammation 

Dysplasia 

Tooth 

(49) 

1 (2%) 

2 (4%) 

1 (2%) 

3 (6%) 

(49) 

2 (4%) 

1 (2%) 

2 (4%) 

(3) 

2 (67%) 

1 (33%) 

(50) (48) 

4 (8%) 

6 (13%) 

3 (6%) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) (48) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

2 (4%) 

2 (4%) 

12 (24%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(1) 

1 (100%) 

Heart 

Cardiovascular System 

(50) (50) (49) (50) 

Arteriole, inflammation, chronic 

Necrosis 

Cardiomyopathy 40 (80%) 41 (82%) 46 (94%) 44 (88%) 

2 (4%) 

1 (2%) 1 (2%) 

Adrenal cortex 

Endocrine System 

Accessory adrenal cortical nodule 

Capsule, hyperplasia 

Hypertrophy 

Hyperplasia 

Hyperplasia 

Parathyroid gland 

Hyperplasia 

Islets, pancreatic 

Hyperplasia 

Adrenal medulla 

Pars distalis, hyperplasia 

Pars distalis, cyst 

Pituitary gland 

Follicular cell, hyperplasia 

Follicle, cyst 

Thyroid gland 

Cyst 

25 (52%) 

7 (15%) 

4 (9%) 

1 (3%) 

4 (9%) 

2 (4%) 

1 (2%) 

1 (2%) 

(46) 

(39) 

(47) 

(49) 

(49) 

7 (14%) 

(50) 

1 (2%) 

1 (2%) 

30 (60%) 

9 (18%) 

(48) 

1 (2%) 

3 (6%) 

(50) 

5 (10%) 

1 (2%) 

(48) 

19 (40%) 

9 (19%) 

1 (2%) 

1 (2%) 

(46) 

2 (4%) 

2 (4%) 

2 (4%) 

1 (2%) 

(48) 

(50) 

1 (2%) 

33 (66%) 

9 (18%) 

(49) 

1 (2%) 

(50) 

1 (2%) 

None 

General Body System 
174 Acetonitrile, NTP TR 447 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE C5 

0 PPm 50 PPm 100 ppm 200 ppm 

Epididymis 

Genital System 

2-kW study (continued) 

Inflammation, suppurative 

Inflammation, chronic active 

Hyperplasia 

Granuloma sperm 

Inflammation, chronic active 

Ectasia 

Atrophy 

Inflammation, suppurative 

Inflammation, chronic active 

Inflammation, suppurative 

Interstitial cell, hyperplasia 

Mineralization 

Atrophy 

Preputial gland 

Seminal vesicle 

Prostate 

Testes 

13 (27%) 

(49) 

(45) 

1 (2%) 

1 (2%) 

(50) 

3 (6%) 

12 (24%) 

16 (32%) 

(50) 

(45) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

(48) (50) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(46) (50) 

4 (9%) 

1 (2%) 

1 (2%) 

7 (15%) 

(47) 

(42) 

(49) 

1 (2%) 

1 (2%) 

2 (4%) 

17 (34%) 

(50) 

(45) 

2 (4%) 

I 

2 (4%) 

1 (2%) 

(50) 

Hematopoietic System 

Thrombosis 

Hyperplasia, neutrophil 

Hyperplasia, megakaryocyte 

Fibrosis 

Angiectasis 

Bone marrow 

Renal, hyperplasia 

Pancreatic, hyperplasia 

Iliac, hyperplasia 

Hyperplasia 

Lymph node, mandibular 

Hyperplasia 

Lymph node, bronchial 

Hyperplasia 

Hematopoietic cell proliferation 

Congestion 

Angiectasis 

Lymph node, mesenteric 

Hyperplasia 

Hyperplasia, mononuclear cell 

Hyperplasia, lymphoid 

Hematopoietic cell proliferation 

Angiectasis 

Amyloid deposition 

(49) (50) (48) 

~ 

(50) 

1 (2%) 

5 (10%) 

2 (4%) 

1 (2%) 1 (2%) 

1 (2%) 

7 (14%) 9 (19%) 11 (22%) 

Lymph node (3) (2) (2) 

1 (50%) 

1 (50%) 

1 (50%) 

(48) 

(35) 

(38) 

(49) 

(33) 

(37) 

(47) 

(29) 

(38) 

(49) 

(36) 

(41) 

1 (3%) 

3 (9%) 

2 (5%) 

2 (6%) 1 (3%) 2 (6%) 

, 

2 (4%) 

1 (2%) 

2 (5%) 

10 (20%) 

1 (2%) 5 (11%) 1 (2%) I 

8 (17%) 

3 (6%) 

3 (6%) 3 (6%) 

Spleen 

Lymph node, mediastinal 

(49) 

(42) 

(50) 

(38) 

(48) 

(35) 

(50) 

(33) 

1 (2%) 

2 (5%) 1 (3%) , 

1 (2%) 

12 (24%) 15 (30%) 16 (33%) 

8 (16%) 

13 (26%) 

4 (8%) 2 (4%) 4 (8%) 

1 (2%) 
Lesions in Male Mice 175 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE C5 

0 PPm 50 PPm 100 ppm . 200 ppm 

Thymus 

Hematopoietic System (continued) 

2-YeUt study (continued) 

(44) (45) (38) (47) 

Epithelial cell, hyperplasia 

Necrosis 

Hyperplasia, lymphoid 

Atrophy 

1 (2%) 

1 (2%) 1 (2%) 

4 (9%) 2 (4%) 

1 (2%) 

1 (3%) 1 (2%) 

1 (3%) 1 (2%) 

Integumentary System 

Subcutaneous tissue, inflammation, chronic 5 (10%) 

Pinna, granuloma 

Inflammation, suppurative 

Cyst epithelial inclusion 

Skin (50) (49) (49) (50) 

10 (20%) 10 (20%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

7 (14%) 

Fibrous osteodystrophy 

Bone 

Musculoskeletal System 

(50) (50) 

Nervous System 

Thrombosis 

Mineralization 

Brain (50) (50) (48) (50) 

20 (40%) 21 (42%) 13 (27%) 18 (36%) 

1 (2%) 

Lung 

Respiratory System 

Inflammation, chronic, focal 

Hemorrhage 

(50) 

5 (10%) 

Alveolar epithelium, hyperplasia 

Inflammation, granulomatous 

Respiratory epithelium, metaplasia, squamous 1 (2%) 

Respiratory epithelium, hyperplasia 

Olfactory epithelium, metaplasia 3 (6%) 

Olfactory epithelium, atrophy 4 (8%) 

Nasolacrimal duct, inflammation, suppurative 4 (8%) 

Inflammation, suppurative 3 (6%) 

Exudate 

Metaplasia, squamous 

4 (8%) 

Nose (50) 

1 (2%) 

Trachea (49) 

(50) 

3 (6%) 

1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 

3 (6%) 

(48) 

3 (6%) 

2 (4%) 

2 (4%) 

(48) 

1 (2%) 

(50) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

(501 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

None 

Special Senses System 

. 
176 Acetonitrile, NTP TR 447 

TABLE C5 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Acetonitrile 

0 PPm 50 PPm 100 ppm 200 ppm 

Kidney 

Urinary System 

%Year Study (continued) 

Arteriole, inflammation, chronic 

Nephropathy 

Metaplasia, osseous 

Inflammation, suppurative 

Hyperplasia, cystic 

Hydronephrosis 

Amyloid deposition 

Renal tubule, mineralization 

Transitional epithelium, hyperplasia 

Ulcer 

Pigmentation, melanin 

Inflammation, suppurative 

Inflammation, chronic 

Hemorrhage 

Dilatation 

Urinaly bladder 

cortex, cyst 

(49) 

1 (2%) 

2 (4%) 

1 (2%) 

3 (6%) 

37 (76%) 

1 (2%) 

5 (10%) 

1 (2%) 

(49) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

5 (10%) 

2 (4%) 

39 (78%) 

3 (6%) 

(49) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

(48) 

2 (4%) 

35 (73%) 

1 (2%) 

3 (6%) 

3 (6%) 

(49) 

2 (4%) 

3 (6%) 

1 (2%) 

1 (2%) 

6 (12%) 

2 (4%) 

2 (4%) 

42 (84%) 

1 (2%) 

(50) 

3 (6%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 
177 

OF ACETONITRILE 

SUMMARY OF LESIONS IN FEMALE MICE 

APPENDIX D 

IN THE 2-YEAR INHALATION STUDY 

TABLE D4a Historical Incidence of Alveolar/bronchiolar Neoplasms 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE D3 Statistical Analysis of Primary Neoplasms in Female Mice 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE D2 Individual Animal Tumor Pathology of Female Mice 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE Dl Summary of the Incidence of Neoplasms in Female Mice 

in Untreated Female B6C3Fl Mice 

TABLE D4b Historical Incidence of Hepatocellular Neoplasms 

in Untreated Female B6C3Fl Mice 

in the 2-Year Inhalation Study of Acetonitrile 

TABLE D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice 

in Untreated Female B6C3Fl Mice 

TABLE D4c Historical Incidence of Forestomach Squamous Cell Papilloma 

............................... 

211 

210 

209 

209 

204 

184 

179 

............................... 

............................... 

........................................ 

........................................ 

........................................ 

............................... 
178 Acetonitrile, NTP TR 447 
Lesions in Female Mice 179 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrilea 

TABLE Dl 

0 PPm 50 PPm 100 ppm 200 ppm 

Early deaths 

1SMd intsbn cvaliration 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

Died last week of study 

Survivors 

10 

60 

6 

15 

1 

28 

10 

60 

5 

12 

32 

1 

10 

60 

8 

13 

29 

10 

60 

5 

13 

32 

Animals examined microscopically 60 60 60 60 

Liver 

Alimentary System 

15-Month Interim Evaluation 

Salivary glands 

Mesentexy 

(10) 

(2) 

(10) 

Hepatocellular adenoma 

Lung 

Respiratory System 

(10) (10) 

Alveolarbronchiolar adenoma 

Multiple organsb 

Systemic Lesions 

(10) (10) 

Lymphoma malignant lymphocytic 1 (10%) 

Urinary System 

Special Senses System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Systems Examined With No Neoplasms Observed 

Intestine large, cecum 

Gallbladder 

Alimentary System 

2-Year Study 

Intestine small, duodenum 

Leiomyosarcoma 
180 Acetonitrile, NTP TR 447 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE Dl 

0 PPm 50 PPm 100 ppm 200 ppm 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Alimentary System (continued) 

%Year study (continued) 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Hemangiosarcoma 

Fat, histiocytic sarcoma 

Fat, hemangioma 

Fat, carcinoma, metastatic, pancreas 

Sarcoma stromal, metastatic, uterus 

Carcinoma, metastatic, ovary 

Sarcoma stromal, metastatic, uterus 

Fibrosarcoma 

Adenocarcinoma 

Mesentery 

Pancreas 

Stomach, forestomach 

Salivary glands 

Squamous cell papilloma 

Sarcoma stromal, metastatic, uterus 

Carcinoma, metastatic, pancreas 

Sarcoma stromal, metastatic, uterus 

Carcinoma, metastatic, pancreas 

Stomach, glandular 

(49) 

(45) 

(44) 

2 (4%) 

1 (2%) 

2 (4%) 

5 (10%) 

3 (6%) 

(8) 

(49) 

(49) 

(49) 

1 (2%) 

(49) 

(50) 

(47) 

(46) 

1 (2%) 

1 (2%) 

5 (10%) 

7 (14%) 

(16) 

1 (6%) 

1 (6%) 

1 (6%) 

1 (6%) 

(49) 

1 (2%) 

(50) 

(50) 

1 (2%) 

(48) 

1 (2%) 

(50) 

(45) 

(46) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

6 (12%) 

4 (8%) 

(6) 

1 (17%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

(49) 

(47) 

(47) 

1 (2%) 

4 (8%) 

4 (8%) 

1 (2%) 

2 (4%) 

1 (2%) 

(5) 

(48) 

(48) 

(49) 

3 (6%) 

(48) 

Heart 

Cardiovascular System 

Histiocytic sarcoma 

Adrenal cortex 

Endocrine System 

Capsule, adenoma 

Histiocytic sarcoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Histiocytic sarcoma 

Pituitary gland 

Adenoma 

Islets, pancreatic 

Follicular cell, adenoma 

Bilateral, follicular cell, adenoma 

Pars intermedia, adenoma 

Pars distalis, adenoma 

Adrenal medulla 

Thyroid gland 

(49) 

1 (2%) 

(48) 

2 (4%) 

1 (2%) 

1 (2%) 

(48) 

(49) 

14 (29%) 

(49) 

2 (4%) 

(49) 

1 (2%) 

(48) 

3 (6%) 

(50) 

(50) 

2 (4%) 

15 (30%) 

(50) 

1 (2%) 

1 (2%) 

(49) 

(49) 

(48) 

(48) 

2 (4%j 

6 (13%) 

(49) 

1 (2%) 
Lesions in Female Mice 181 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE Dl 

Tissue NOS 

General Body System 

%Yf?Uf &Udy (continued) 

Sarcoma 

(2) 

1 (50%) 

Genital System 

ovary 

Yolk sac carcinoma 

Sarcoma stromal, metastatic, uterus 

Luteoma 

Histiocytic sarcoma 

Hemangioma 

Cystadenoma 

Arrhenoblastoma NOS 

Cervix, fibroma 

Sarcoma stromal 

Polyp stromal 

Leiomyosarcoma 

Leiomyoma 

Hemangiosarcoma 

Hemangioma 

Uterus 

(4) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

2 (4%) 

1 (2%) 

2 (4%) 

(47) (48) (48) 

2 (4%) 1 (2%) 1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

(49) (50) (49) 

3 (6%) 3 (6%) 

1 (2%) 

Bone marrow 

Hematopoietic System 

Osteosarcoma, metastatic, bone 

Histiocytic sarcoma 

Hemangiosarcoma 

Renal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Histiocytic sarcoma 

Carcinoma, metastatic, pancreas 

Lymph node, bronchial 

Lymph node, mesenteric 

Histiocytic sarcoma 

Lymph node, mandibular 

Sarcoma stromal, metastatic, uterus 

Histiocytic sarcoma 

Carcinoma, metastatic, pancreas 

Sarcoma stromal, metastatic, uterus 

Histiocytic sarcoma 

Lymph node, mediastinal 

Sarcoma stromal, metastatic 

Histiocytic sarcoma 

Hemangiosarcoma 

Lymph node 

Spleen 

Thymus 

(49) 

2 (4%) 

1 (2%) 

(7) 

1 (14%) 

1 (14%) 

(38) 

1 (3%) 

2 (5%) 

(44) 

(35) 

2 (5%) 

(36) 

2 (6%) 

(49) 

2 (4%) 

2 (4%) 

(44) 

(49) 

(44) 

(44) 

(39) 

1 (2%) 

1 (2%) 

(35) 

1 (3%) 

(49) 

(4) 

1 (2%) 

1 (2%) 

(46) 

(49) 

1 (2%) 

(1) 

(43) 

(33) 

(34) 
182 Acetonitrile, NTP TR 447 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE Dl 

0 PPm 50 PPm 100 ppm 200 ppm 

study (continued) I 

Mammary gland 

Integumentary System 

(49) (47) 

Adenocarcinoma 

Adenoacanthoma 

3 (6%) 

Skin (49) (50) 

Subcutaneous tissue, sarcoma 

Subcutaneous tissue, hemangiosarcoma 

(50) (48) 

1 (2%) 

3 (6%) 

1 (2%) 

1 (2%) 

(50) (49) 

2 (4%) 

Subcutaneous tissue, sarcoma stromal, 

metastatic, uterus 1 (2%) 

Bone 

Musculoskeletal System 

Osteosarcoma 

(50) 

1 (2%) 

Brain 

Nervous System 

Lung 

Respiratory System 

(49) (50) 

Pheochromocytoma malignant, metastatic, 

Osteosarcoma, metastatic, bone 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 1 (2%) 

Carcinoma, metastatic, pancreas 

Alveolarbronchiolar carcinoma, multiple 

Alveolarbronchiolar carcinoma 

Alveolaribronchiolar adenoma, multiple 

Alveolaribronchiolar adenoma 

Adenocarcinoma, metastatic, mammary gland 1 (2%) 

Adenocarcinoma, metastatic, harderian gland 

Sarcoma stromal, metastatic, uterus 

6 (12%) 2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

adrenal medulla 1 (2%) 

1 (2%) 

Nose (49) (50) 

(50) (49) 

2 (4%) 

1 (2%) 1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Harderian gland 

Special Senses System 

Adenoma 

Adenocarcinoma 

(3) (1) (2) (1) 

1 (50%) 

1 (50%) 1 (100%) , 

3 (100%) 1 (100%) 

Kidney 

Urinary System 

Renal tubule, carcinoma 

Renal tubule, adenoma 

Histiocytic sarcoma 

Urinary bladder (47) 
Lesions in Female Mice 183 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE Dl 

0 PPm 50 PPm 100 ppm 200 ppm 

StUdy (continued) 

Multiple organs 

Systemic Lesions 

(50) (50) (50) (50) 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Lymphoma malignant 

Histiocytic sarcoma 

5 (10%) 

5 (10%) 10 (20%) 

1 (2%) 

2 (4%) 1 (2%) 1 (2%) 1 (2%) 

7 (14%) 

9 (18%) 3 (6%) 

7 (14%) 11 (22%) 

Total animals with primary neoplasms' 

Neoplasm Summary 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

benign or malignant 

2-Year study 

2-Year study 

2-Year strldy 

2-Year study 

2-Year study 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with uncertain neoplasmsTotal metastatic neoplasms 

Total animals with metastatic neoplasms 

Total uncertain neoplasms 

36 

2 

37 

3 

35 

1 

32 

1 

73 

1 

64 

2 

68 

3 

51 

1 

25 

2 

24 

3 

18 

1 

29 

36 

2 

35 

3 

23 

1 

40 

23 

1 

23 27 24 

32 

1 

3 

28 33 

3 

28 

3 3 

3 12 9 4 

1 

1 

~ ~~ 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 
184 Acetonitrile, NTP TR 447 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm 

5667912351848114906600555 

0221682234567889900012333 

0445556666666666677777777 

0372570137696140899001256 

1619808109960697008298666 

1010010110001000110100000 

Number of Days on Study 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System I 

........................ 

+++++A+++A++A+A+++++++++ 

+++A++A++A++A+A+++++++++ 

+++A+A+++A++AMA++++++++M 

+++++AM++A++A+++++++++++ 

+++++++++++++++M++++++++ 

+++++++++A++++A+++M+++++ 

+++A+AA++A++A+++++++++++ 

......................... 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma xx 

X 

X 

X 

X 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

........................ 

......................... 

........................ 

+ + + + + , 

I 

Squamous cell papilloma 

........................ 

Cardiovascular System 

Heart 

Blood vessel 

Histiocytic sarcoma 

........................ 

....................... 

X ~~~ ~~ ~ ~~ 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Histiocytic sarcoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Histiocytic sarcoma 

Pituitaly gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

Follicular cell, adenoma 

Pars distalis, adenoma 

........................ X 

+++++++++++++++++++++M++ .J 

A 

X 

........................ 

++M+++++MMM+MMM+++M+++M+ 

........................ 

........................ X x xx 

X 

X xx 

General Body System 

Sarcoma 

Tissue NOS 

X 

+ + 

A: Autolysis precludes examination 

+: Tissue examined microscopically 

I: Insufficient tissue 

M: Missing tissue 

Blank: Not examined 

X Lesion present 
Lesions in Female Mice 185 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE D2 

5555555555555555555555555 

3333333333333333333333333 

11111111111111111?7111117 

0000000000000000111111111 Total 

~6171111188889999000111111 Tissues/ 

8345678923671389014345689 Tumors 

Number of Days on Study 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

~~ ~ ~~ ~ 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Stomach, forestomach 

Salivaxy glands 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Squamous cell papilloma 

+++++++++++M+++++++++++++ 48 

++++++++++M+++++++++M++++ 42 

......................... 46 

......................... 48 

......................... 45 

......................... 42 

......................... 44 

......................... 45 

......................... 49 

8 

2 

1 

3 

2 

xx 5 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

1 

X 

X 

X 

X 

X 

+ + + 

X 

Cardiovascular System 

Heart 

Blood vessel 

1 

......................... 49 

M+M++++++M++++++++++M+M++ 42 

Histiocytic sarcoma 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Histiocytic sarcoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Histiocytic sarcoma 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

Follicular cell, adenoma 

Pars distalis, adenoma 

2 

......................... 49 

14 

+++++++++++++++++++++M+++ 48 

MMMMMM+++MMMM+MM+M+++MM+M 24 

......................... 49 

2 

1 

1 

......................... 48 

1 

......................... 49 

X 

X 

X xxx X xx 

X 

General Body System 

Sarcoma 

Tissue NOS 

1 

2 
186 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE D2 

5667912351848114906600555 

0221682234567889900012333 

0445556666666666677777117 

Number of Days on Study 

Carcass ID Number 

0312510131696140899001256 

1619808109960697008298666 

1010010110001000110100000 

Ovary 

Clitoral gland 

Genital System 

Luteoma 

Histiocytic sarcoma 

Hemangioma 

Cystadenoma 

khenoblastoma NOS 

Polyp stromal 

Hemangiosarcoma 

Hemangioma 

Uterus 

........................ 

++++M+++M+++++++++M++MM+ 

X 

X 

........................ 

X X 

X 

Hematopoietic System 

Bone marrow ........................ 

Renal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Histiocytic sarcoma 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node, mediastinal 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, bronchial 

Histiocytic sarcoma 

Hemangiosarcoma 

Spleen 

Lymph node 

Thymus 

X 

X 

++ 

X 

+ + , 

I 

+MM+++++M+M++++++M+M++.++ 

X 

........................ 

+++M+M++++M++++++++M++MM 

+++++++++M++++++++M+++M+ 

++MM+M++++++++ M++++++M+ 

X 

X 

X 

X 

X 

++M+++MM++++++++++M+++++ 

Skin 

Mammaxy gland 

Integumentary System 

Adenocarcinoma 

........................ 

........................ X X X 

Musculoskeletal System 

Bone ........................ 

Nervous System 

Brain ........................ 
Lesions in Female Mice 187 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE D2 

5555555555555555555555555 

3333333333333333333333333 

7177777717717177771777717 

Number of Days on Study 

Carcass ID Number 

0000000000000000111111111 Total 

6717177188889999000111111 Tissues/ 

8345678923611389014345689 Tumors 

Ovary 

Clitoral gland 

Genital System 

Luteoma 

Histiocytic sarcoma 

Hemangioma 

Cystadenoma 

Arrhenoblastoma NOS 

Polyp stromal 

Hemangiosarcoma 

Hemangioma 

Uterus 

1 

1 

1 

1 

48 

MM+++M++MM++++MM+MMM+M+M+ 32 

X 1 

......................... 49 

X 

++++++++++++++++++++M++++ 

X 

X 

X 2 

2 

1 

Renal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Histiocytic sarcoma 

Hemangiosarcoma 

Hematopoietic System 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, bronchial 

Bone marrow 

2 

1 

......................... 49 

1 

1 

+ I 

+M+++M+++M+M+++M+M+++MM++ 35 

2 

++M++++M++++++M++M++++M++ 38 

X 

X 

Lymph node + + 

X 

Histiocytic sarcoma 

Lymph node, mediastinal 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Hemangiosarcoma 

Spleen 

++++++++M++++++++++++++++ 44 

2 

2 

......................... 49 

2 

+I+++M+M+MM+++++M+++++++M 36 

2 

++++++++M+++M++++++++++++ 44 

1 

X 

X 

X 

X 

Skin 

Mammary gland 

Integumentary System 

Adenocarcinoma 

......................... 49 

3 

......................... 49 

Musculoskeletal System 

Bone ......................... 49 

Nervous System 

Brain ......................... 49 
188 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

TABLE D2 

Number of Days on Study 

5667912351848114906600555 

0221682234567889900012333 

0445556666666666677777777 

0372570137696140899001256 

1619808109960697008298666 

1010010110001000110100000 

Carcass ID Number 

Respiratory System 

Lung 

Larynx 

........................ 

........................ 

Adenocarcinoma, metastatic, mammary 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar adenoma 

gland 

X 

X 

X 

Pheochromocytoma malignant, 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

metastatic, adrenal medulla 

X 

Trachea 

Nose 

........................ 

........................ 

Harderian gland 

Special Senses System 

Adenoma X 

+ 

X 

+ 

Kidney 

Urinary System 

........................ 

Renal tubule, carcinoma 

Renal tubule, adenoma 

Histiocytic sarcoma X 

X 

X 

I 

Urinary bladder ++++++++++M+++++++++++++ 

Systemic Lesions 

Multiple organs ........................ 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Lymphoma malignant 

Histiocytic sarcoma X 

X 

xxx 

X xx , 
Lesions in Female Mice 189 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 0 ppm (continued) 

5555555555555555555555555 

3333333333333333333333333 

7777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

0000000000000000111111111 Total 

6777777788889999000111111 Tissues/ 

8345678923611389014345689 Tumors 

Respiratory System 

J-alynx 

Lung 

Adenocarcinoma, metastatic, mammary 

Pheochromocytoma malignant, 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Alveolarhronchiolar carcinoma 

Alveolarbronchiolar adenoma, multiple 

Alveolarbronchiolar adenoma 

metastatic, adrenal medulla 

gland 

Trachea 

Nose 

......................... 49 

......................... 49 

X X 

2 

1 

1 

1 

6 

1 

X xx 

X 

X 

X 

X 

......................... 49 

......................... 49 

1 

Harderian gland 

Special Senses System 

Adenoma 

+ 3 

X 3 

Kidney 

Urinary System 

~ ~~ ~ 

Renal tubule, carcinoma 

Renal tubule, adenoma 

Histiocytic sarcoma 

Urinary bladder ......................... 48 

1 

1 

2 

......................... 49 

X 

Systemic Lesions 

Multiple organs 

1 

2 

......................... 49 

5 

X 5 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Lymphoma malignant 

Histiocytic sarcoma X 

X 

X X 
190 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm 

TABLE D2 

Number of Days on Study 

958952129.6445671766666666 

6341902234666881133333333 

3455566666666667777777777 

7694319482349278568901234 

3198213191030883388899999 

2211222212222112211111111 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Fat, histiocytic sarcoma 

Fat, hemangioma 

Sarcoma stromal, metastatic, uterus 

Carcinoma, metastatic, ovary 

Sarcoma stromal, metastatic, uterus 

Mesentery 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Sarcoma stromal, metastatic, uterus 

Sarcoma stromal, metastatic, uterus 

......................... 

A++++A+++++++++++++++++++ 

A++++A++++++++++A++++++++ 

A++++A++++++++++++++++++M 

A++++A++A++++++++++++++++ 

A++++A+++++++++++++++++++ 

A++++A++++M+++++M++++++++ 

A++++A++++++++++A++++++++ 

++++++++++++M++++++++++++ 

X 

X X xx 

X X 

X 

X 

+ + ++ +++ ++++ + 

X 

X 

X 

A++++++++++++++++++++++++ 

X 

......................... 

......................... 

X 

......................... 

X 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

M+M+++++M++++++++++M+++++ 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Thyroid gland 

Capsule, adenoma 

Follicular cell, adenoma 

Pars distalis, adenoma 

A++++++++++++++++++++++++ 

X 

MM+MM++++M++M+M++MM+M++MM 

A++++++++++++++++++++++++ 

A+++++++++++++]++++++++++ 

A++++++++++++++++++++++++ 

xx X X xx 

A+++++++++++M++++++++++++ 

X 

General Body System 

None 
Lesions in Female Mice 191 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm (continued) 

TABLE D2 

6666666666666666666666666 

3333333333333333333333333 

7777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

1112222222222222222222222 Total 

9990000000111222222223333 Tissues/ 

5671245678057012457890123 Tumors 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Fat, histiocytic sarcoma 

Fat, hemangioma 

Sarcoma stromal, metastatic, uterus 

Carcinoma, metastatic, ovary 

Sarcoma stromal, metastatic, uterus 

Mesentery 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Sarcoma stromal, metastatic, uterus 

Sarcoma stromal, metastatic, uterus 

......................... 49 

++++++++A+++++I++++++++++ 45 

++++++++A++++++++++++++++ 45 

......................... 48 

++++++++A++++++++++++++++ 46 

+++++M+MA++++++++++++++++ 44 

++++++++A++++++++++++++++ 46 

++++++++A++++++++++++++++ 47 

......................... 50 

......................... 48 

1 

......................... 50 

......................... 50 

1 

......................... 49 

1 

1 

1 

1 

+ 16 

1 

X 7 

1 

X X 5 

X X 

X 

+ + + 

1 

Cardiovascular System 

Heart 

Blood vessel ++++M++MM++++++++++++++++ 43 

......................... 50 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Thyroid gland 

Capsule, adenoma 

Follicular cell, adenoma 

Pars distalis, adenoma 

X 3 

......................... 48 

xxx X 14 

.......................... 49 

+M++M+MI ++++M++MMMM+M+I M+ 26 

......................... 49 

......................... 48 

1 

......................... 49 

X 

X 

X X X 

General Body System 

None 
192 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm (continued) 

TABLE D2 

9589521296445671166666666 

6347902234666861133333333 

3455566666666661111711111 

Number of Days on Study 

Carcass ID Number 

I694319482349218568901234 

3198213191030883388899999 

2211222212222112211111111 

@sly 

Genital System 

Clitoral gland 

A++++AM++++++++++++++++++ 

M+MM++++MM+++M++++M++M+MM 

Yolk sac carcinoma 

Sarcoma stromal, metastatic, uterus 

Cystadenoma 

Leiomyoma 

Hemangioma 

X 

X 

Uterus ......................... 

Sarcoma stromal 

Polyp stromal 

Leiomyosarcoma X 

X 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Sarcoma stromal, metastatic, uterus 

Sarcoma stromal, metastatic, uterus 

Histiocytic sarcoma 

......................... 

A+++++++++++M+++++++++++M 

++++++++I +++I ++M++M+M++M+ 

A+M+++I+++++M++M+++++++++ 

++ ++ + 

, 

X 

X 

X 

Spleen 

Lymph node, mediastinal A++++MM++++++++++M++MM+MM 

A++++M+++++++++++++++++++ 

A++++++++++++++++++++++++ I 

Thymus 

Sarcoma stromal, metastatic X 

I 

Skin 

Mammary gland 

Integumentary System 

......................... 

M+M++++M+++++++++++++++++ 

I 

Subcutaneous tissue, sarcoma stromal, 

metastatic, uterus X , 

Musculoskeletal System 

Skeletal muscle 

Bone ......................... 

Nervous System 

~~~ 

Brain ......................... 

Respiratory System 

Larynx ......................... 

A++++++++++++++++++++++++ 

Lung 

Trachea 

Nose 

Sarcoma stromal, metastatic, uterus 

Hepatocellular carcinoma, metastatic, liver 

Alveolarbronchiolar carcinoma, multiple 

Alveolarbronchiolar adenoma X 

X 

X 

A++++++++++++++++++++++++ 

......................... 
Lesions in Female Mice 193 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm (continued) 

TABLE D2 

Number of Days on Study 

6666666666666666666666666 

3333333333333333333333333 

7777777777777777777777777 

1112222222222222222222222 Total 

9990000000111222222223333 Tissues/ 

5671245678057012457890123 Tumors 

Carcass ID Number 

Ovary 

Clitoral gland 

Genital System 

2 

......................... 47 

+M+++M+M+++M+++M+M+++++++ 34 

Cystadenoma X X 

Yolk sac carcinoma 

Sarcoma stromal, metastatic, uterus 

Sarcoma stromal 

Polyp stromal 

Leiomyosarcoma 

Leiomyoma 

Hemangioma 

Uterus 

1 

X 2 

1 

1 

1 

......................... 49 

1 

1 

X 

X 

X 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Sarcoma stromal, metastatic, uterus 

Sarcoma stromal, metastatic, uterus 

Histiocytic sarcoma 

Thymus 

Spleen 

Lymph node, mediastinal 

Sarcoma stromal, metastatic 1 

......................... 48 

......................... 49 

1 

M+++++++++MMM++M++M+++++M 35 

1 

1 

+M++++++++++++M+++M++++++ 44 

M++++++++M++M+++++++++MM+ 39 

++++++++M++++++++++++++++ 44 

+ 9 

......................... 49 

+ + + 

Skin 

Mammary gland 

Integumentary System 

......................... 47 

.......................... 50 

Subcutaneous tissue, sarcoma stromal, 

metastatic, uterus 1 

Musculoskeletal System 

Skeletal muscle 

Bone 

1 

......................... 50 

+ 

Nervous System 

Brain ......................... 50 

Respiratory System 

Larynx 

Lung 

Trachea 

Nose 

Sarcoma stromal, metastatic, uterus 

Hepatocellular carcinoma, metastatic, liver 

Alveolaribronchiolar carcinoma, multiple 

Alveolaribronchiolar adenoma 

......................... 49 

......................... 50 

1 

1 

1 

X 2 

......................... 50 

......................... 49 

X 
194 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm (continued) 

TABLE D2 

9589521296445671766666666 

6347902234666881133333333 

3455566666666667717717711 

Number of Days on Study 

Carcass ID Number 

7694319482349218568901234 

3198213191030883388899999 

2211222212222112211111111 

Harderian gland 

Special Senses System 

X 

+ I 

Adenoma 

Urinary bladder 

Kidney 

Urinary System 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs ......................... 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Hisiiocytic sarcoma X 

X X X xx x 

X X X 
Lesions in Female Mice 195 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 50 ppm (continued) 

TABLE D2 

6666666666666666666666666 

3333333333333333333333333 

1111111117117111111111117 

1112222222222222222222222 Total 

9990000000111222222223333 Tissues/ 

5611245618057012451890123 Tumors 

Number of Days on Study 

Carcass ID Number 

Harderian gland 

Special Senses System 

Adenoma 1 

1 

Urinary bladder 

Kidney 

Urinary System 

......................... 49 

......................... 48 

Systemic Lesions 

Multiple organs 

7 

X X X 10 

1 

......................... 50 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Histiocytic sarcoma 

X 

X xx X 
196 Acetonitrile, NTP TR 447 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 100 ppm 

2035303458001046829146666 

5183456666117000011233333 

5556666666666111111111111 

3236355923100091954124618 

3503421535134610205200000 

3333333333333333333333333 

Number of Days on Study 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Adenocarcinoma 

Fat, carcinoma, metastatic, pancreas 

Stomach, forestomach 

Salivary glands 

Fibrosarcoma 

Pancreas 

MesenteIy 

Carcinoma, metastatic, pancreas 

Squamous cell papilloma 

Carcinoma, metastatic, pancreas 

Stomach, glandular 

......................... 

M++A++A++++A++A++++++++++ 

++++A+A++++A++A++++++++++ 

+++MA+A++++A++A++++++++++ 

......................... 

......................... 

++++++A++++A++A++++++++++ 

+++AA+A++++A++A+++I++++++ 

......................... 

L 

X X 

X X 

X 

X 

X 

X 

+ 

X 

X 

++ 

......................... 

......................... 

......................... 

X 

......................... X 

Cardiovascular System 

Heart 

Blood vessel 

+M+++++++++++++++++++++++ 

+++++M+++++++++++++++++M+ 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Adenoma 

Thyroid gland 

......................... 

......................... 

......................... 

......................... X X X X 

......................... 

M++M++++++MMM+++MM+MMMMMM 

! 

Follicular cell, adenoma 

Bilateral, follicular cell, adenoma 

Pars distalis, adenoma 

X 

X 

General Body System 

ovary 

Genital System 

Tissue NOS I 

Clitoral gland +++MM+++M+++++++M+++++M++ 

......................... 

++++++++++++.+++++M++++++I 

Polyp stromal 

Cystadenoma X 

Uterus 

X 
Lesions in Female Mice 197 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the &Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

6666666666666666666666666 

3333333333333333333333333 

7777777717117777777177777 

3333333333333333333333333 Total 

1111112222333344444445555 Tissues/ 

0234670467147812457890167 Tumors 

Number of Days on Study 

Carcass ID Number 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

+++++++++++++++++++++M+++ 49 

++++I+++++++M+M++++++++++ 41 

......................... 47 

Intestine large, rectum ......................... 48 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma, multiple 

Mesentery 

......................... 48 

++++I++++++++++++++++++++ 44 

......................... 46 

......................... 45 

......................... 50 

X 1 

X 

X 

X X X 

X 

++ + 

X 

Adenocarcinoma 

Fat, carcinoma, metastatic, pancreas 

Stomach, forestomach 

Salivary glands 

Fibrosarcoma 

1 

......................... 50 

X 1 

1 

......................... 50 

......................... 50 

X 1 

1 

......................... 50 

1 

Pancreas 

Carcinoma, metastatic, pancreas 

Squamous cell papilloma 

Carcinoma, metastatic, pancreas 

Stomach, glandular 

Cardiovascular System 

Heart 

Blood vessel ++++++M++++++++++++++++++ 47 

......................... 49 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Adenoma 

Thyroid gland 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

++MMM++MMMM+MMM+M+MMMM+++ 22 

2 

1 

1 

......................... 50 

xx xx x x xx X X 15 

X X 

Follicular cell, adenoma 

Bilateral, follicular cell, adenoma 

Pars distalis, adenoma 

X 

General Body System 

Tissue NOS + 1 

Ovary 

Clitoral gland 

Genital System 

Uterus 

3 

......................... 50 

1 

......................... 48 

+M+++M++M+MM++++++++M++++ 39 

Polyp stromal 

Cystadenoma 

X X 
198 Acetonitrile, NTP TR 447 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

2035303458001046829746666 

518345666~6111000011233333 

5556666666666111111111111 

3236355923100091954124618 

3503421535134610205200000 

3333333333333333333333333 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow ......................... Histiocytic sarcoma X I 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Carcinoma, metastatic, pancreas 

Carcinoma, metastatic, pancreas 

Histiocytic sarcoma 

Thymus 

+ +++ ++ 

......................... 

+++++M+MM+++M+M++++MM++++ 

......................... 

+++M++++M+++++M++++++++M+ 

+++++++++++++M++++++++M+M I 

X I 

X 

X I 

........................ 

Mammary gland 

Integumentary System 

......................... 

Subcutaneous tissue, sarcoma 

Adenocarcinoma 

Adenoacanthoma 

X X X 

Skin 

X 

......................... X 

Musculoskeletal System 

Skeletal muscle 

Bone ......................... 

Nervous System 

Brain ......................... 

Respiratory System 

Larynx ......................... 

......................... Lung 

Adenocarcinoma, metastatic, 

Hepatocellular carcinoma, metastatic, liver 

Carcinoma, metastatic, pancreas 

Alveolar/bronchiolar adenoma 

harderian gland 

X 

X 

Trachea 

Nose 

......................... 

......................... 

Harderian gland 

Special Senses System 

Adenoma 

Adenocarcinoma 

+ 

X 

Urinaly bladder 

Kidney 

Urinary System 

......................... 

......................... 

Systemic Lesions 

Multiple organs ......................... 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic X xxx 

Histiocytic sarcoma X 

X xx 

X 
Lesions in Female Mice 199 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 100 ppm (continued) 

TABLE D2 

6666666666666666666666666 

3333333333333333333333333 

1117171111111111111111117 

3333333333333333333333333 Total 

1111112222333344444445555 Tissues/ 

0234610461147812457890161 Tumors 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

1 

......................... 50 

Histiocytic sarcoma 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Carcinoma, metastatic, pancreas 

+ ++ 

+++++I+++M+++++++++++++++ 41 

++++++M++++++++++++++M+++ 44 

1 

+M+++M++M++++M+++M+++++++ 42 

9 

Carcinoma, metastatic, pancreas 

......................... 50 

+++M+++++++++M+++++M+++++ 40 

1 

+++M+++++++++M+++++++++++ 46 

1 

Thymus 

Spleen 

Lymph node, mediastinal 

Histiocytic sarcoma 

Mammary gland 

Integumentary System 

3 

1 

......................... 50 

2 

......................... 50 

Subcutaneous tissue, sarcoma 

Adenocarcinoma 

Adenoacanthoma X 

Skin 

Musculoskeletal System 

Skeletal muscle 

Bone 

1 

......................... 50 

+ 

Nervous System 

Brain ......................... 50 

Respiratory System 

Lung 

Larynx ......................... 50 

......................... 50 

Adenocarcinoma, metastatic, 

Hepatocellular carcinoma, metastatic, liver 

Carcinoma, metastatic, pancreas 

Alveolarlbronchiolar adenoma 

harderian gland 

2 

1 

2 

X 1 

......................... 50 

......................... 49 

X X 

'X 

Trachea 

Nose 

Harderian gland 

Special Senses System 

1 

X 1 

+ 2 

Adenoma 

Adenocarcinoma 

Urinary bladder 

Kidney 

Urinary System 

......................... 50 

......................... 50 

Systemic Lesions 

Multiple organs 

xx I 

X 9 

1 

......................... 50 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Histiocytic sarcoma 

X 

xx X X 
200 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 200 ppm 

TABLE D2 

7494219006040448905555555 

2924790116345667123333333 

1234445555666666777777777 

2828471220834754971368901 

6376535247433674742222233 

4444444444444444444444444 

Number of Days on Study 

Carcass ID Number 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

......................... 

+AAAA++++++++++++++++++++ 

++A++++++++++++++++++++++ 

Intestine large, cecum 

Intestine large, rectum 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Leiomyosarcoma 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Squamous cell papilloma 

+AA+A+++++++++M++++++++++ 

+AA++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

+AA+A++++++++++++++++++++ 

+AA+A++++++++++++++++++++ 

+AA+I+++++++++++M++++++++ 

X 

xx X 

X 

X 

+ + + I 

++A+++++++++++++ ++++++++ 

++A++++++++++++++++++++++ 

+AA++++++++++++++++++++++ 

X 

+ 

......................... , 

Cardiovascular System 

Heart 

Blood vessel 

......................... 

++MM++++++++++++++M++++++ 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Follicular cell, adenoma 

Pars intermedia, adenoma 

Pars distalis, adenoma 

Thyroid gland 

++A+++M++++++++++++++++++ 

M+MM++M+++++++++++M+MM+M+ 

+AA++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

X X 

X 

X 

++A++++++++++++++++++++++ 

General Body System 

None 

Ovary 

Clitoral gland 

Genital System 

Uterus 

Cervix, fibroma 

Polyp stromal 

Cystadenoma 

++A++++++++++++++++++++++ 

M+A+++MM+M+++++M+++++++++ 

++A++++++++++++++++++++++ 
Lesions in Female Mice 201 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TABLE D2 

5555555555555555555555555 

333333.3333333333333333333 

1111111111111111117111111 

4444444444444444444444444 Total 

3334444455555566666611178 Tissues/ 

5691356923618901456934180 Tumors 

Number of Days on Study 

Carcass ID Number 

Gallbladder 

Esophagus 

Alimentary System 

......................... 49 

......................... 46 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Leiomyosarcoma 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Squamous cell papilloma 

......................... 46 

++++++++++++M++++++++++++ 41 

......................... 48 

......................... 49 

......................... 41 

......................... 41 

+++++++++++++M+++++++++++ 45 

1 

1 

......................... 48 

X 3 

......................... 48 

......................... 49 

......................... 48 

+ 5 

1 

2 

X 4 

1 

X 4 

1 

X 

X X 

X 

X 

X 

+ 

X 

Cardiovascular System 

Heart 

Blood vessel +++++M++++++M++++++++++++ 45 

......................... 50 

~ ~ 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Follicular cell, adenoma 

Pars intermedia, adenoma 

Pars distalis, adenoma 

Thyroid gland 

......................... 

M++MMM+MMMMM+++M++++MMMMM 

......................... 

......................... 

......................... 

X xx X 

X ......................... 1 

49 

2 

6 

48 

21 

48 

49 

49 

General Body System 

None 

Ovary 

Clitoral gland 

Genital System 

Uterus 

Cervix, fibroma 

Polyp stromal 

Cystadenoma 

1 

3 

......................... 49 

1 

++++++++++++++M++++++++++ 48 

M+M++M+M++M++++++++M+M+++ 31 

X 

X xx 

X 
202 Acetonitrile, NTP TR 447 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TABLE D2 

7494219006040448905555555 

2924790116345667123333333 

1234445555666666777777777 

2828471220834754971368901 

6376535247433674742222233 

4444444444444444444444444 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

++A++++++++++++++++++++++ 

Osteosarcoma, metastatic, bone X 

+AA++++++++++++++++++++++ 

++A++MMM++M+++MMMM+I+M+++ 

+MA+++M++++++M+++++++M+++ 

++M+M+M++M++MM+++M+M+M+MM 

M+M+M++++M++++++M+MM+++MM 

Hemangiosarcoma X 

Thymus ++A+++++MI +++I +M+M++++MM+ 

Skin 

Mammary gland 

Integumentary System 

++A++++++++++++++++++++++ 

++A+++M++++++++++++++++++ 

Subcutaneous tissue, hemangiosarcoma 

Adenocarcinoma 

Skeletal muscle 

Osteosarcoma 

Bone 

Musculoskeletal System 

......................... X 

+ 

Nervous System 

Brain ++A++++++++++++++++++++++ 

Respiratory System 

Larynx 

Lung 

++A++++++++++++++++++++++' 

++A++++++++++++++++++++++ 

Adenocarcinoma, metastatic, 

Osteosarcoma, metastatic, bone 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

&eolar/bronchiolar carcinoma 

harderian gland 

X 

X 

Trachea 

Nose ++A++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

Harderian gland 

Special Senses System 

Eye 1 

Adenocarcinoma 

Urinary bladder 

Kidney 

Urinary System 

+AA++++++++++++++++++++++ 

+AA++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Histiocytic sarcoma 

......................... 

X 

X 

X X X 
Lesions in Female Mice 203 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Acetonitrile: 200 ppm (continued) 

TABLE D2 

55555555555555555555.55555 

3333333333333333333333333 

1711111171111111177711111 

Number of Days on Study 

Carcass ID Number 

4444444444444444444444444 Total 

3334444455555566666611118 Tissues/ 

5691356923618901456934180 Tumors 

Hematopoietic System 

Bone marrow 

1 

......................... 49 

Osteosarcoma, metastatic, bone 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Hemangiosarcoma 

Thymus 

+ 

......................... 48 

M++M++++++++++++MM+M+++++ 34 

++++++++++M++++++++++M+++ 43 

+MMM+++MM++++M+++++++++++ 33 

++++++M++++++MMM+++++M+++ 36 

1 

+M+++++++++++++++++++++++ 41 

1 

Skin 

Mammary gland 

Integumentary System 

1 

......................... 49 

X 1 

......................... 48 

Subcutaneous tissue, hemangiosarcoma 

Adenocarcinoma 

X 

Musculoskeletal System 

Bone ......................... 50 

Skeletal muscle 

Osteosarcoma 

1 

1 

Nervous System 

Brain ......................... 49 

Respiratory System 

Larynx ......................... 49 

......................... 49 

1 

1 

1 

1 

1 

.......................... 49 

......................... 49 

Lung 

Adenocarcinoma, metastatic, 

Osteosarcoma, metastatic, bone 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver X 

Alveolarbronchiolar carcinoma 

harderian gland X 

X 

Trachea 

Nose 

Harderian gland 

Special Senses System 

Eye 

X 

+ 

+ 

1 

1 

1 

Adenocarcinoma 

Urinary bladder 

Kidney 

Urinary System 

......................... 48 

++++++++.+++++++++++++++M+ 41 

Systemic Lesions 

Multiple organs 

11 

3 

1 

......................... 50 

Lymphoma malignant mixed 

Lymphoma malignant lymphocytic 

Histiocytic sarcoma 

xx 

X xxx x xxx 
204 Acetonitrile, NTP TR 447 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE D3 

0 PPm 50 PPm 100 ppm 200 ppm 

Fisher exact test 

Cochran-Armita e testd P=0.045N 

Logistic sion testd P=0.029N 

Life P=0.044N 

First 426 

Terminal rate' 

Adjusted rateb 9.3% 

Overall ratea 3/48 (6%) 

Adrenal Medulla: Benign or Malignant Pheochromocytoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Harderian Gland: Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Harderian Gland Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma 

Fisher exact test 

Cochran-Annitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Carcinoma 

2n7 (7%) 

% 

8.3% 

3/50 (6%) 

P=0.073N 

P=0.074N 

P=0.071N 

623 

1/28 (4%) 

8.3% 

3/50 (6%) 

P=0.279N 

P=0.289N 

P=0.266N 

623 

1/23 (4%) 

12.0% 

4/49 (8%) 

P=O.411 

P=O.390 

P=O.466 

569 

2/28 (7%) 

P =0.332N 

P=0.340N 

P=0.309N 

706 

5/28 (18%) 

23.1% 

7/49 (14%) 

0/33 (0%) 

0.0% 

0/48 (0%) 

P=0.121N 

P=0.141N 

P=O.lOON 

e - 

1/50 (2%) 

P=0.301N 

P=0.283N 

1/33 (3%) 

3.0% 

735 (T> 

P=0.309N 

1/33 (3%) 

3.0% 

1/50 (2%) 

P=0.309N 

P=0.301N 

P = 0.283N 

735 (T> 

8/50 (16%) 

P=O.191 

P=O.248 

664 

6/33 (18%) 

22.4% 

P=O.188 

15.8% 

6/50 (12%) 

P=0.475N 

P=0.397N 

595 

3/33 (9%) 

P=0.484N 

0.0% 

0/50 (0%) 

on9 (0%) - 

P=0.165N 

P=0.113N 

P=0.114N 

3.4% 

1/50 (2%) 

735 (T) 

in9 (3%) 

P=0.303N 

P=0.296N 

P = 0.309N 

2/29 (7%) 

6.9% 

2/50 (4%) 

P=0.500N 

P=0.487N 

P=0.485N 

735 (T> 

P=O.211 

P=O.209 

708 

6/29 (21 %) 

25.6% 

8/50 (16%) 

P=O.188 

P=0.457N 

P=0.456N 

570 

2/29 (7%) 

16.4% 

6/50 (12%) 

P=0.484N 

0.0% 

0/49 (0%) 

OB2 (0%) - 

P=0.117N 

P=0.091N 

P=0.106N 

1 

0.0% 

0/50 (0%) 

0/32 (0%) - 

P=0.122N 

P=0.125N 

P=0.121N 

3.1% 

1/50 (2%) 

735 (T> 

1/32 (3%) 

P=0.309N 

P=0.313N 

P=0.298N 

6/32 (19%) 

18.8% 

6/49 (12%) 

P=O.346 

P =0.433 

735 (T> 

P=O.370 

P =0.399N 

P=0.323N 

510 

3/32 (9%) 

13.6% 

5/49 (10%) 

P=0.380N 
Lesions in Female' Mice 205 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

TABLE D3 

100 ppm 200 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pituitary Gland (Pars Distalis): Adenoma 

P=O.529 

P=O.489 

P =0.503N 

569 

6/23 (21%) 

28.0% 

9/49 (18%) 

7/28 (25%) 

25.0% 

7/49 (14%) 

P=O.OOSN 

P=0.003N 

P=0.003N 

735 (T) 

P=0.009N 

P=0.007N 

P=0.007N 

706 

7/28 (25%) 

27.3% 

8/49 (16%) 

8.9% 

3/50 (6%) 

P=0.373N 

P=0.379N 

P=0.358N 

641 

1/23 (4%) 

41.8% 

14/48 (29%) 

P=0.030N 

P=0.033N 

P =0.023N 

622 

9/27 (33%) 

P=O.257 

P=O.359 

595 

8/33 (24%) 

33.5% 

13/50 (26%) 

P=O.251 

2/50 (4%) 

P=0.075N 

P =0.044N 

P=0.044N 

2/33 (6%) 

6.1% 

735 (T) 

9.1% 

3/50 (6%) 

735 (T) 

3/33 (9%) 

P=0.094N 

P=0.065N 

P=0.057N 

0/50 (0%) 

P=0.118N 

P=0.109N 

0/33 (0%) 

0.0% 

- 

P=0.121N 

P=0.516N 

P =0.406N 

646 

11/33 (33%) 

38.3% 

14/49 (29%) 

P=0.563N 

P=O.284 

P=O.290 

570 

8/29 (28%) 

36.9% 

13/50 (26%) 

P=O.251 

2/29 (7%) 

6.9% 

2/50 (4%) 

P=0.075N 

P =0.067N 

P=0.067N 

735 (T) 

6.9% 

2/50 (4%) 

735 (T) 

2/29 (7%) 

P=O.O32N 

P=0.039N 

P=0.043N 

P=O.663 

P=0.612N 

650 

On9 (0%) 

7.8% 

3/50 (6%) 

P=0.661N 

P=0.572N 

P=0.582N 

583 

10/29 (34%) 

42.2% 

15/50 (30%) 

P=O.552 

P=O.466 

P=O.585 

510 

8/32 (25%) 

28.5% 

10/49 (20%) 

P=O.500 

0.0% 

0/49 (0%) 

0/32 (0%) - 

P=0.006N 

P=0.005N 

P =O.005N 

3.1% 

1/49 (2%) 

735 (T) 

1/32 (3%) 

P=0.015N 

P=0.012N 

P=O.OlON 

3.1% 

1/50 (2%) 

735 (T) 

1/32 (3%) 

P=0.316N 

P=0.283N 

P=0.309N 

P=0.041N 

P=0.027N 

664 

4/32 (13%) 

17.4% 

6/48 (13%) 

P=0.038N 
206 Acetonitrile, NTP TR 447 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Stomach (Forestomach): Squamous Cell Papilloma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (Follicular Cell): Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Uterus: Stromal Polyp 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Uterus: Stromal Polyp or Stromal Sarcoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Hemangiosarcoma 

3.6% 

1/50 (2%) 

P=O.O97 

P=O.107 

P=O.108 

735 (T) 

1/28 (4%) 

7.1% 

2/49 (4%) 

735 (T) 

2/28 (7%) 

P=0.309N 

P=0.320N 

P=0.287N 

6.9% 

2/50 (4%) 

P=O.371 

P=O.365 

P=O.404 

720 

1/28 (4%) 

6.9% 

2/50 (4%) 

P=O.438 

P=O.419 

P=O.468 

720 

1/28 (4%) 

8.9% 

3/50 (6%) 

P = 0.423 

P=O.424 

P=O.438 

622 

1/28 (4%) 

0/33 (0%) 

0.0% 

0/50 (0%) 

P=0.500N 

P=0.467N 

P=0.467N 

- 

P=O.508 

P=O.562 

639 

2/33 (6%) 

8.3% 

3/48 (6%) 

P=O.490 

2/33 (6%) 

6.1% 

2/50 (4%) 

P=0.662N 

P=0.637N 

735 (T) 

P=0.691N 

P=O.515 

P=O.558 

639 

2/33 (6%) 

8.3% 

3/50 (6%) 

P=O.500 

0/33 (0%) 

0.0% 

0/50 (0%) 

P=0.121N 

P=0.119N 

P=0.107N 

- 

1/29 (3%) 

3.4% 

1/50 (2%) 

P=0.754N 

P=0.754N 

735 (T) 

P=0.753N 

P=0.667N 

P =0.673N 

712 

li29 (3%) 

6.4% 

2/50 (4%) 

P=0.684N 

9.1% 

3/50 (6%) 

P=O.533 

P=O.524 

664 

2/29 (7%) 

P=O.500 

P=O.533 

P=O.524 

664 

2/29 (7%) 

9.1% 

3/50 (6%) 

P=O.500 

1/29 (3%) 

3.4% 

1/50 (2%) 

P=0.294N 

P=0.288N 

735 (T) 

P=0.309N 

3/32 (9%) 

9.4% 

3/50 (6%) 

P=O.309 

P=O.353 

P=O.353 

735 (T) 

3.1% 

1/49 (2%) 

735 (T) 

1/32 (3%) 

P=0.500N 

P=0.453N 

P=0.453N 

3/32 (9%) 

9.4% 

3/50 (6%) 

P=O.530 

P-0.560 

735 (T) 

P=O.500 

3/32 (9%) 

9.4% 

3/50 (6%) 

P=O.500 

P=O.530 

P=O.560 

735 (T) 

P = 0.662 

P=0.632N 

509 

2/32 (6%) 

8.4% 

3/50 (6%) 

P=0.661N 
Lesions in Female Mice 207 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

0 PPm 50 PPm 100 ppm 200 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Hemangioma or Hemangiosarcoma 

5/50 (10%) 2/50 (4%) 

15.7% 6.1% 

P=0.319N 

P=0.329N P=0.201N 

P=0.295N P=0.168N 

622 

3/28 (11%) 2/33 (6%) 

735 (T> 

P=0.218N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Lymphoma (Lymphocytic, Mixed, or Undifferentiated Cell Type) 

P=O.396 

P =0.365 P=O.138 

P=O.456 P=O.247 

581 369 

6/28 (21%) 9/33 (27%) 

32.1% 40.6% 

11/50 (22%) 17/50 (34%) 

P=O.133 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign Neoplasms 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Neoplasms 

18/28 (64%) 20/33 (61%) 

73.5% 65.4% 

29/50 (58%) 25/50 (50%) 

P-0.031N 

P=O.O42N P=0.219N 

P50.022N P=0.125N 

639 42p 

P=0.274N 

24/50 (48%) 23/50 (46%) 

P=0.506N 

P=0.340N 

369 

12/33 (36%) 

59.2% 51.5% 

P=O.481 

P=O.475 

P=0.519N 

426 

12/28 (43%) 

P=0.500N 

1/29 (3%) 

3.4% 

1/50 (2%) 

P=0.090N 

P=0.095N 

735 (T> 

P=0.102N 

P=O.211 

P=O.256 

552 

8/29 (28%) 

40.9% 

16/50 (32%) 

P=O.184 

P=0.187N 

P=0.229N 

583 

17/29 (59%) 

66.6% 

25/50 (50%) 

P=0.274N 

P=O.364 

P=O.473 

552 

12/29 (41%) 

60.4% 

27/50 (54%) 

P=O.345 

P=0.366N 

P=0.317N 

509 

2/32 (6%) 

8.4% 

3/50 (6%) 

P=0.357N 

P=O.281 

P=O.424 

444 

12/32 (38%) 

40.6% 

14/50 (28%) 

P=O.322 

P=0.047N 

P=0.019N 

644 

16/32 (50%) 

54.0% 

19/50 (38%) 

P=0.036N 

P=0.548 

P=0.453N 

444 

17/32 (53%) 

60.5% 

24/50 (48%) 

P=0.579N 
208 Acetonitrile, NTP TR 447 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Acetonitrile (continued) 

0 PPm 50 PPm 100 ppm 200 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

P=0.289N 

P=0.345N P=0.548N P = 0.497 

P=0.263N P=0.314N P=0.558N 

426 369 552 

2OR8 (71%) 23/33 (70%) 21/29 (72%) 

81.6% 78.0% 82.2% 

36/50 (72%) 36/50 (72%) 38/50 (76%) 

P=0.588N P=O.410 

P=0.405N 

P=0.234N 

444 

23/32 (72%) 

78.2% 

33/50 (66%) 

P=0.333N 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

(T)Terminal sacrifice 

exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated WIN. 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher 

to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

Observed incidence at terminal kill 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

liver, lung, pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

e Not applicable; no neoplasms in animal group 
Lesions in Female Mice 209 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Untreated Female B6C3Fl Micea 

TABLE D4a 

Study Carcinoma 

Incidence in Controls 

Adenoma Adenoma or Carcinoma 

Historical Incidence at Battelle Pacific Northwest Laboratories 

o-Chlorobenzalmalononitrile (CS2) 

Hexachlorocyclopentadiene 

Chloroethane 

I-Epinephrine Hydrochloride 

2-Chloroacetophenone 

Allyl Glycidyl Ether 

1,3-Butadiene 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

4/50 

4/48 

2/49 

3/50 

4/50 

0/50 

4/50 

0%-10% 

2.8% 

40/659 (6.1%) 

1/50 

3/48 

3/49 

2/50 

3/50 

0/50 

0/50 

0%-6% 

2.5% 

191659 (2.9%) 

5/50 

7/48 

5/49 

5/50 

6/50 

0/50 

4/50 

0%-15% 

3.5% 

581659 (8.8%) 

a Data as of 27 April 1993 

Historical Incidence of Hepatocellular Neoplasms in Untreated Female B6C3Fl Micea 

TABLE D4b 

Study Carcinoma 

Incidence in Controls 

Adenoma Adenoma or Carcinoma 

Historical Incidence at Battelle Pacific Northwest Laboratories 

o-Chlorobenzalmalononitrile (CS2) 

Hexachlorocyclopentadiene 

Chloroethane 

/-Epinephrine Hydrochloride 

2-Chloroacetophenone 

Allyl Glycidyl Ether 

1,3-Butadiene 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

4/50 

5/49 

0/49 

2/50 

4/50 

1/50 

11/49 

0%-22% 

6.2% 

56/657 (8.5%) 

7/50 

4/49 

3/49 

1/50 

8/50 

5/50 

4/49 

0%-16% 

4.8% 

57/657 (8.7%) 

11/50 

9/49 

3/49 

3/50 

12/50 

6/50 

15/49 

3%-31% 

8.7% 

111/657 (16.9%) 

a Data as of 27 April 1993 
210 Acetonitrile, NTP TR 447 

Study 

Historical Incidence of Forestomach Squamous Cell Papilloma in Untreated Female B6C3Fl Micea 

TABLE D4c 

Incidence in Controls 

Historical Incidence at Battelle Pacific Northwest Laboratories 

o-Chlorobenzalmalononitrile (CS2) 

Hexachlorocyclopentadiene 

Chloroethane 

I-Epinephrine Hydrochloride 

2-Chloroacetophenone 

Allyl Glycidyl Ether 

1,3-Butadiene 

2/50 

0/49 

0/49 

0/50 

0150 

1/50 

0/50, 

Overall Historical Incidence 

Range 

Standard deviation 

Total 

0%-6% 

2.0% 

8/661 (12%). 

a Data as of 27 April 1993 
Lesions in Female Mice 211 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Acetonitrilea 

TABLE D5 

0 PPm 50 PPm 100 ppm 200 ppm 

Early deaths 

zs-luon#l inraim evaluplion 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

Died last week of study 

Survivors 

10 

60 

10 

60 

10 

60 

10 

60 

6 

15 

1 

5 

12 

8 

13 

5 

13 

32 

1 

28 29 

60 

32 

Animals examined microscopically 60 60 60 

Gallbladder 

Alimentary System 

15-Month Interim Evaluation 

Liver 

Inflammation, suppurative 

Bile duct, cyst 

Necrosis 

Eosinophilic focus 

Basophilic focus 

Fat, necrosis 

Mesentery 

Duct, ectasia 

Inflammation, chronic 

Pancreas 

Inflammation, chronic 

Salivary glands 

Hyperplasia, squamous 

Diverticulum 

Stomach, forestomach 

(10) 

1 (10%) 

1 (10%) 

1 (10%) 

(10) 

(2) 

1 (50%) 

1 (10%) 

1 (10%) 

1 (10%) 

(10) 

(10) 

1 (10%) 

Heart 

Cardiovascular System 

Cardiomyopathy 

Endocrine System 

Pituitary gland 

Hyperplasia 

Adrenal cortex 

Pars distalis, hyperplasia 

Pars distalis, cyst 

Ovary 

Genital System 

Atrophy 

Cyst 

Uterus 

Hyperplasia, cystic 

Angiectasis 

(10) (10) (10) (9) 

1 (10%) 

3 (30%) 4 (40%) 2 (20%) 

8 (80%) 

1 (10%) 

1 (11%) 

(10) (10) (10) (10) 

9 (90%) 9 (90%) 9 (90%) 

~~ 

a Number of animals examined microscopically at the site and the number of animals with lesion 
212 Acetonitrile, NTP TR 447 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE D5 

0 PPm 50 PPm 100 ppm 200 ppm 

15-Month Interim Evaluation (continued) 

Lymph node, mesenteric 

Lymph node, bronchial 

Hematopoietic System 

Hyperplasia, lymphoid 

Hyperplasia 

Angiectasis 

Hyperplasia 

Spleen 

Fibrous osteodystrophy 

Bone 

Musculoskeletal System 

Mineralization 

Brain 

Nervous System 

Lung 

Respiratory System 

(10) (10) (10) 

Perivascular, infiltration cellular, 

Hemorrhage 

Embolus 

1 (10%) 

1 (10%) 

3 (30%) 

mononuclear cell 1 (10%) 

Inflammation, chronic 

Urinary bladder 

.Nephropathy 

Kidney 

Urinary System 

Special Senses System 

Integumentary System 

General Body System 

Systems Examined With No Lesions Observed 

Gallbladder 

Alimentary System 

2-Year Study 

Necrosis 

Hemorrhage 

Necrosis 

Hemorrhage 

Inflammation, suppurative 

Hyperplasia 

Intestine large, colon 

Intestine large, cecum 
Lesions in Female Mice 213 

(continued) 

Summary of the'Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE D5 

0 PPm 50 PPm 100 ppm 200 ppm 

Intestine small, duodenum 

Alimentary System (continued) 

2-Year Study (continued) 

Amyloid deposition 

Intestine small, ileum 

Peyer's patch, hyperplasia 

Intestine small, jejunum 

Inflammation, suppurative 

Clear cell focus 

Basophilic focus 

Angiectasis 

Liver 

Serosa, inflammation, suppurative 

Centrilobular, necrosis 

Bile duct, degeneration 

Bile duct, cyst 

Regeneration 

Pigmentation, hemosiderin 

Necrosis 

Mixed cell focus 

Mitotic alteration 

Karyomegaly 

Infiltration cellular, mononuclear c 

Infarct 

Hyperplasia, lymphoid 

Hematopoietic cell proliferation 

Focal cellular change 

Eosinophilic focus 

Degeneration, fatty 

Degeneration, cystic 

Cytomegaly 

Fat, necrosis 

Inflammation, chronic 

Angiectasis 

Duct, inflammation, chronic 

Duct, ectasia 

Inflammation, chronic 

Hyperplasia, lymphoid 

Hyperplasia 

Focal cellular change 

Atrophy 

Amyloid deposition 

Inflammation, suppurative 

Salivary glands 

Epithelium, hyperplasia 

Ulcer 

Necrosis 

Inflammation, suppurative 

Hyperplasia, squamous 

Hyperkeratosis 

Angiectasis 

Stomach, forestomach 

erst 

Mesentery 

Pancreas 

(49) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

5 (10%) 

1 (2%) 

8 (16%) 

e11 

4 (8%) 

4 (8%) 

2 (4%) 

1 (2%) 

1 (2%) 

(8) 

6 (75%) 

1 (13%) 

2 (25%) 

(49) 

2 (4%) 

2 (4%) 

2 (4%) 

2 (4%) 

2 (4%) 

1 (2%) 

(49) 

(49) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

3 (6%) 

2 (4%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

4 (8%) 

4 (8%) 

9 (56%) 

(49) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

3 (6%) 

7 (14%) 

2 (4%) 

3 (6%) 

4 (8%) 

(50) 

(45) 

(46) 

(44) 

(49) 

(47) 

(47) 

(45) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

3 (6%) 

8 (16%) 

1 (2%) 

2 (4%) 

1 (2%) 

(6) 

2 (33%) 

(50) 

2 (4%) 

1 (2%) 

1 (2%) 

(50) 

(50) 

(48) 

(49) 

5 (10%) 

4 (8%) 

9 (18%) 

3 (6%) 

19 (40%) 

2 (4%) 

1 (2%) 

5 (10%) 

1 (2%) 

3 (6%) 

(5) 

4 (80%) 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 
214 Acetonitrile, NTP TR 447 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE D5 

0 PPm 50 PPm 100 ppm 200 ppm 

Stomach, glandular 

Alimentary System (continued) 

&Year study (continued) 

(49) (48) (50) 

Mineralization 

Inflammation, suppurative 

Hyperplasia 

Dysplasia 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

Tooth (1) 

1 (100%) 

Blood vesselCardiovascular System 

Thrombosis 

Mineralization 

Inflammation, suppurative 

Hyperplasia, atypical 

Cardiomyopathy 

Angiectasis 

Heart 

(42) (43) (47) (45) 

1 (2%). 

1 (2%) 1 (2%) 

(49) (50) (49) (50) 

1 (2%). 

22 (45%) 

1 (2%) 

16 (33%) 19 (38%), 15 (30%) 

1 (2%). 

1' (2%) 

Adrenal cortex 

Endocrine System 

Capsule, hyperplasia 

Hypertrophy 

Hyperplasia 

Hematopoietic cell.proliferation 

Cyst 

Hyperplasia 

Islets, .pancreatic 

Hyperplasia 

Adrenal medulla 

Pars intermedia, hyperplasia 

Pars distalis, inflammation, chronic 

Pars distalis, hyperplasia 

Pars distalis, cyst 

PituitaIy gland 

Follicular cell;. hyperplasia. 

Follicle, cyst 

Inflammation,.suppurative 

Inflammation 

Thyroid gland 

(49) 

1 (2%) 

3 (6%) 

3 (6%). 

2 (4%): 

2 (4%) 

2 (4%) 

(48) 

(49) 

(48) 

17 (35%) 

3 (6%) 

(49) 

12 (24%) 

1 (2%) 

(49) 

3 (6%) 

4 (8%) 

2 (4%) 

2 (4%). 

(49) 

(49) 

(48) 

5 (10%) 

2 (4%) 

16 (33%) 

3 (6%) 

6 (13%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

7 (14%). 

1 (2%) 

(!O) 

9 (18%) 

(49) 

1 (2%) 

3 (6%) 

1 (2%). 

1. (2%) 

1 (2%) 

2. ($%) 

1 (2%) 

20 (42%) 

(48) 

(,448) 

(49) 

(.49) 

1 (2%) 

3. (6%). 

6 (12%).. 

None 

General. Body System 
Lesions in Female Mice 215 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE D5 

0 PPm 50 PPm 100 ppm 200 ppm 

Clitoral gland 

Genital System 

%Year Study (continued) 

Atrophy 

Atrophy 

Angiectasis 

Amyloid deposition 

Abscess 

Interstitium, hyperplasia 

Germinal epithelium, hyperplasia 

Mineralization 

Myometrium, hyperplasia 

Artery, inflammation, chronic 

Arteriole, hypertrophy 

Thrombosis 

Inflammation, suppurative 

Hyperplasia, cystic 

Dilatation 

Angiectasis 

Amyloid deposition 

(48) 

(32) 

(47) 

(34) 

(48) 

(39) 

(48) 

(37) 

1 (3%) 

ovary 

4 (8%) 

16 (33%) 

17 (35%) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

14 (30%) 

18 (38%) 

16 (33%) 

20 (42%) 13 (27%) 

Cyst 

1 (2%) 

1 (2%) 

12 (25%) 

1 (2%) 

Uterus (49) (49) (50) (49) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 1 (2%) 

44 (90%) 

2 (4%) 

48 (98%) 46 (92%) 44 (90%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Bone marrow 

Hematopoietic System 

(49) (49) (50) (49) 

Hyperplasia, neutrophil 

Hyperplasia, megakaryocyte 

Renal, congestion 

Renal, angiectasis 

Popliteal, hyperplasia 

Iliac, hyperplasia 

Iliac, hemorrhage 

Axillary, hyperplasia 

Hyperplasia 

Hematopoietic cell proliferation 

Erythrophagocytosis 

Lymph node, bronchial 

Hyperplasia 

Lymph node, mandibular 

Inflammation, chronic 

Hyperplasia, histiocytic 

Hyperplasia 

Hemorrhage 

Hematopoietic cell proliferation 

Congestion 

Angiectasis 

Amyloid deposition 

Lymph node, mesenteric 

1 (2%) 

6 (12%) 1 (2%) 3 (6%) 3 (6%) 

Lymph node (7) (9) (9) (1) 

1 (11%) 

1 (11%) 

1 (11%) 

1 (11%) 1 (11%) 

1 (11%) 

2 (29%) 

1 (11%) 

10 (26%) 3 (7%) 

2 (5%) 

1 (2%) 

(38) (44) (42) 

7 (17%) 

(44) 

(35) 

(44) 

(39) 

(47) 

(44) 

5 (14%) 

1 (2%) 

1 (2%) 

4 (9%) 

1 (2%) 

2 (5%) 

1 (2%) 

1 (2%) 

1 (2%) 

3 (7%) 

1 (2%) 

1 (2%) 

7 (15%) 

1 (2%) 

5 (11%) 4 (9%) 

5 (15%) 

4 (11%) 

(43) 

(33) 
216 Acetonitrile, NTP TR 447 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE DS 

0 PPm 50 PPm 100 ppm 200 ppm 

Lymph node, mediastinal 

Hematopoietic System (continued) 

2-YeUr study (continued) 

Inflammation, suppurative 

Hyperplasia 

Hematopoietic cell proliferation 

Erythrophagocytosis 

Capsule, fibrosis 

Hyperplasia, lymphoid 

Hemorrhage 

Hematopoietic cell proliferation 

Fibrosis 

Congestion 

Amyloid deposition 

Epithelial. cell, hyperplasia 

Necrosis 

Hyperplasia, lymphoid 

Developmental malformation 

Atrophy 

Angiectasis 

Spleen 

Thymus 

2 (6%) 

6 (17%) 

2 (6%) 

(49). 

1 (2%). 

12’ (24%) 

1 (2%) 

14 (29%) 

5 (11%) 

6 (14%) 

(35) 

1 (3%) 

1 (3%) 

1 (3%) 

(49) 

12 (24%). 

1 (2%) 

14 (29%) 

(48) 

1 (2%): 

5 (10%). 

1 (2%) 

2 (4%) 

1 (2%) 

10 (25%) 

(50) 

1 (2%) 

13 (26%) 

15. (30%) 

1 (2%) 

2. (4%) 

1 (2%) 

(46) 

2 (4%). 

12 (26%) 

(34) 

5 (15%) 

(48) 

1 (2%) 

11 (23%) 

5 610%) 

(41) 

5 (12%) 

1 (2%) 

8 (20%) 

1 (2%) 

1 (2%) 

Hyperplasia 

Mammaly gland 

Integumentary System 

Subcutaneous tissue, inflammation, chronic 1 (2%) 

Sebaceous gland, hyperplasia. 

Inflammation,. suppurative 

Skin (49) 

(49) 

(50). 

(47) 

(50) 

(50) 

(49) 

(48) 

T (2%) 

1 (2%) 

2 (4%) 

1 (2%). 

1 (2%) 

2 (4%) 

1 (2%). 

2 (4%) 

1 (2%) 

Musculoskeletal. System 

Inflammation, chronic 

Fracture 

Fibrous osteodystrophy 

Bone 

Inflammation 

Hemorrhage 

Skeletal muscle 

(49) (50) (50) (50) 

26 (53%) 18 (36%) 16 (32%) 18, (36%.) 

1 (100%) 

1. (loo,%) 1 (100%) 

1 (2%). 

1 (2%) 

(1). (1) (1) 

1 (100%) 

Nervous System 

Pigmentation, hemosiderin 

Necrosis 

Mineralization 

Inflammation, chronic 

Inflammation 

Hemorrhage 

Brain (49) (50) (50) (49) 

1 (2%) 

1 (2%) 

27 (55%) 

1 (2%) 

1. (2%) 

1 (2%) 

20 (41%) 

3 (6%) 

28 (56%). 27 (54%). 

1 (2%) 
Lesions in Female Mice 217 

(continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Acetonitrile 

TABLE D5 

0 PPm 50 PPm 100 ppm 200 ppm 

study (continued) 

Lung 

Respiratory System 

Mediastinum, hemorrhage 

Artery, mediastinum, inflammation 

Arteriole, inflammation 

Alveolus, infiltration cellular, histiocyte 

Alveolar epithelium, hyperplasia 

Thrombosis 

Inflammation, suppurative 

Inflammation, acute 

Infiltration cellular, mononuclear cell 

Hemorrhage 

(49) 

4 (8%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

3 (6%) 

Perivascular, infiltration cellular, 

Mediastinum, inflammation, suppurative 

5 (10%) 

1 (2%) 

1 (2%) 

1 (2%) 

4 (8%) 

mononuclear cell 

Nose (49) 

Respiratory epithelium, necrosis 

Respiratory epithelium, metaplasia, squamous 

Respiratory epithelium, hyperplasia 

Olfactory epithelium, necrosis 

Olfactory epithelium, metaplasia 

Olfactory epithelium, atrophy 

Nasolacrimal duct, inflammation, suppurative 1 (2%) 

Inflammation, suppurative 

Metaplasia, squamous 

Trachea (49) 

(50) 

1 (2%) 

4 (8%) 

3 (6%) 

1 (2%) 

4 (8%) 

2 (4%) 

3 (6%) 

1 (2%) 

(49) 

(50) 

2 (4%) 

2 (4%) 

2 (4%) 

2 (4%) 

3 (6%) 

(50) 

2 (4%) 

5 (10%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

(49) 

1 (2%) 

4 (8%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

(49) 

(49) 

None 

Special Senses System 

Kidney 

Urinary System 

Arteriole, inflammation, chronic 

Nephropathy 

Metaplasia, osseous 

Inflammation, suppurative 

Infiltration cellular, mononuclear cell 

Infarct 

Hyperplasia, atypical 

Hydronephrosis 

Amyloid deposition 

Renal tubule, necrosis 

Renal tubule, mineralization 

Glomerulus, embolus 

Cortex, pigmentation, hemosiderin 

Transitional epithelium, hyperplasia 

Arteriole, inflammation 

Ulcer 

Inflammation, chronic 

cortex, cyst 

Urinary bladder 

(49) 

1 (2%) 

1 (2%) 

1 (2%) 

23 (47%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) (50) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

31 (63%) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

26 (52%) 

(50) 

1 (2%) 

23 (48%) 

3 (6%) 

2 (4%) 

(47) 
218 Acetonitrile, .NTP 'TR 447 
219 

GENETIC TOXICOLOGY 

APPENDIX E 

SALMONELLA MUTAGENICITY TEST PROTOCOL . . . . . , . . . , . . . . . . , . . . . . . . , . , . . . . . . . . . . . . . . 220 

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . 220 

TABLE E2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

TABLE El Mutagenicity of Acetonitrile in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . 223 

RESULTS ..................................................................... 222 

Following Treatment with Acetonitrile by Inhalation for 13 Weeks . ., . . . . . . . . . . . . . . . 227 

TABLE E4 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes 

byAcetonitrile ........................................................ 226 

TABLE E3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

byAcetonitrile ........................................................ 225 

MOUSEPERIPHERALBLOOD MICRONUCLEUSTESTPROTOCOL ........ ................. ..... 221 
220 Acetonitrile, NTP TR 447 

GENETIC TOXICOLOGY 

for 2 days at 37" C. All tests were repeated using either the same or different S9 concentrations. 

plates. Histidine-independent mutant colonies arising on these plates were counted following incubation 

added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar 

Syrian hamster liver) for 20 minutes at 37" C. Top agar supplemented with 1-histidine and d-biotin was 

mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or 

Salmonella typhimurium tester strains (TA97, TA98, TA100, TA1.53.5, and TA1.537) either in buffer or S9 

laboratories as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the 

Testing was performed as reported by Mortelmans et al. (1986). Acetonitrile was sent to two testing 

SALMO~VELLA MUTAGENICITY TEST PROTOCOL 

repeated. 

of acetonitrile. In the absence of toxicity, 10,000 ,ug/plate was selected as the high dose. All assays were 

Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses 

increase required for a chemical to be judged positive or weakly positive. 

in revertant colonies is observed following chemical treatment. There was no minimum percentage or fold 

magnitude to support a determination of mutagenicity. A negative response is obtained when no increase 

defined as an increase in revertants that is not dose related, not reproducible, or is of insufficient 

independent (revertant) colonies in any one strain/activation combination. An equivocal response is 

In this assay, a positive response is defined as a reproducible, dose-related increase in histidineyielding equivocal or positive results were repeated. 

absence of toxicity, 5,000 ,ug/mL was selected as the high dose. A single flask per dose was used, and tests 

consisted of concurrent solvent and positive controls and of at least three doses of acetonitrile. In the 

handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test 

and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were 

induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs), both in the presence 

coded aliquot by Radian Corporation. It was tested in cultured Chinese hamster ovary (CHO) cells for 

Testing was performed as reported by Galloway et al. (1987). Acetonitrile was sent to the laboratory as a 

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS 

SCEs/cell from each dose level. 

were' read by the same person. Fifty second-division metaphase cells were scored for frequency of 

were the same as for cells treated without S9. All. slides were scored blind and' those from a. single test 

proceeded for an additional 26 hours, with Colcemid present for the final 2, hours.. . Harvesting and staining 

removed and replaced with medium containing serum and BrdU and no acetonitrile, and incubation 

S9, cells were incubated with acetonitrile, serum-free medium, and S9 for 2 hours. The medium was then 

harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with 

fresh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then 

culture initiation. After 26 hours, the medium containing acetonitrile was removed and replaced with 

acetonitrile in supplemented McCoy's 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after 

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 26.hours with 

by chance at one dose point is less than. 0.01'; the probability for such a chance occurrence at two dose 

chosen as a statistically conservative positive response. The probability of this level of difference occurring 

points (Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control. value was 

Statist.ica1 analyses were conducted on the slopes of the dose-response curves and the individual dose 
Genetic Toxicology 221 

led to a call of equivocal. 

statistically significant trend (P<O.O05) in the absence of any responses reaching 20% above background 

of activity; increases at two or more doses resulted in a determination that the trial was positive. A 

points is less than 0.001. An increase of 20% or greater at any single dose was considered weak evidence 

harvested in the same manner as for the treatment without S9. 

were incubated for 12 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were 

treated with acetonitrile and S9 for 2 hours, after which the treatment medium was removed and the cells 

then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were 

with acetonitrile for 12 hours; Colcemid was added and incubation continued for 2 hours. The cells were 

Chromosomal Aberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium 

(pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). 

included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other 

person. One hundred first-division metaphase cells were scored at each dose level. Classes of aberrations 

(21 & 2 chromosomes). All slides were scored blind and those from a single test were read by the same 

Cells were selected for scoring on the basis of good morphology and completeness of karyotype 

the reviewers. 

were based on a consideration of the statistical analyses as well as the biological information available to 

increase at any one dose resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls 

more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant 

trend (PsO.015) were considered weak evidence for a positive response; significant differences for two or 

points. For a single trial, a statistically significant (PsO.05) difference for one dose point and a significant 

statistical call for a trial, analyses were conducted on both the dose response curve and individual dose 

Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a 

and the magnitudes of those effects. 

scientific staff after considering the results of statistical analyses, reproducibility of any effects observed, 

based on reproducibly positive trials (as noted previously). Ultimately, the final call is determined by the 

N = the number of exposure groups. A final call of positive for micronucleus induction is preferably 

1) the trend test P value is s 0.025 or 2) the P value for any single exposure group is s O.O25/N where 

proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if 

a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in 

and the control group (Margolin et al., 1990). In the presence of excess binomial variation, as detected by 

one-tailed Cochran-Armitage trend test, followed by painvise comparisons between each exposure group 

was analyzed by a statistical software package that tested for increasing trend over exposure groups using a 

group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs 

cell. The results were tabulated as the mean of the pooled results from all animals within a treatment 

510 nm illumination); the minimum size limit was approximately one-twentieth the diameter of the NCE 

micronuclei exhibit the characteristic fluorescent emissions of DNA (blue with 360 nm and orange with 

criteria of Schmid (1976) were used to define micronuclei, with the additional requirement that the 

micronuclei in 10,000 normochromatic erythrocytes (NCEs) in each of 10 animals per dose group. The 

1,000 times magnification using a semi-automated image analysis system to determine the frequency of 

mixture of Hoechst 332Wpyronin Y (MacGregor et al., 1983), and coded. Slides were scanned at 630 or 

immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye 

were obtained from male and female B6C3Fl mice at the end of the 13-week toxicity study. Smears were 

A detailed discussion of this assay can be found in MacGregor et al. (1990). Peripheral blood samples 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL 
222 Acetonitrile, NTP TR 447 

trend test was not significant (P>O.O15) and the trial results were concluded to be equivocal. 

(5,000 pglmL). Despite the increase in Abs noted at the high dose in the trial conducted with S9, the 

et af., 1987; Table E3); for both endpoints, the increases were noted at the highest dose tested 

of SCEs in the absence of S9 (Galloway et aZ., 1987; Table E2) and Abs in the presence of S9 (Galloway 

(Mortelmans et aZ., 1986; Table El). In cytogenetic tests with CHO cells, acetonitrile was a weak inducer 

rat and hamster liver S9; no mutagenic activity was obseried in any straidactivation combination 

S. typhimurium strains TA97, TA98, TA100, TA1535, and TA1537, with and without Aroclor 1254-induced 

Acetonitrile (100 to 10,000 pg/plate) was tested in two laboratories for induction of mutations in 

RESULTS 

the 400 ppm group. 

mice were negative, but in males, a small but significant increase in micronucleated NCEs was observed in 

treated for 13 weeks with acetonitrile (100 to 800 ppm) by inhalation (Table E4). Results with female ~ 

determining the frequency of micronucleated NCEs in peripheral blood samples of male and female mice 

The ability of acetonitrile to induce chromosomal damage in mammalian cells in vivo was assessed by 

in the form of increased frequencies of micronucleated NCEs. 

activity in cultured mammalian cells; in vivo, evidence for chromosomal damage in male mice was observed 

In conclusion, acetonitrile did not induce gene mutations in bacteria and showed only weak clastogenic 
Genetic Toxicology 223 

Mutagenicity of Acetonitrile in Salmonella typhimurium" 

TABLE El 

Revertants/plate b 

Strain Dose - s9 +lo% hamster S9 +lo% rat S9 

(/&Plate) 

Study performed at SRI, International 

TAlOO 

333 

100 

0 

10,000 

3,333 

1,000 

178 f 2.7 

159 f 7.4 

174 f 3.5 

159 f 8.5 

157 f 7.9 

171 f 6.0 

Positive control' 

Trial summary 

471 f 17.5 

Negative 

TA1535 

333 

100 

0 

10.000 

3,333 

1,000 

25 f 2.6 

24 f 2.0 

18 f 1.2 

25 f 3.5 

23 f 4.8 

32 f 3.0 

Positive control 

Trial summary 

359 f 12.5 

Negative 

TA1537 

333 

100 

0 

10.000 

3,333 

1,000 

10 f 1.7 

11 f 1.7 

10 f 2.1 

8 f 2.6 

12 f 1.7 

13 f 0.7 

Positive control 

Trial summary 

215 f 8.4 

Negative 

TA98 

333 

100 

0 

10.000 

3,333 

1,000 

Positive control 

Trial summary 

30 f 1.5 

35 f 5.2 

32 f 3.8 

28 f 7.5 

32 f 2.5 

30 f 3.2 

815 f 4.9 

Negative 

172 f 7.5 

174 f 9.0 

155 f 2.9 

162 f 7.0 

166 f 9.2 

171 f 9.2 

1.584 f 55.1 

Negative 

22 f 2.2 

15 f 0.7 

13 f 4.1 

17 f 0.3 

17 f 2.3 

15 f 3.2 

505 f 13.2 

Negative 

11 f 1.2 

21 f 6.4 

18 f 1.8 

14 f 3.3 

21 f 4.5 

18 f 1.0 

55.9 f 5.5 

Negative 

44 f 2.0 

44 f 3.7 

47 f 4.4 

47 f 0.7 

44 f 3.2 

42 f 1.7 

Negative 

1,393 f 120.7 

126 f 5.5 

136 f 2.9 

135 f 2.6 

140 f 12.1 

134 f 10.6 

158 f 4.1 

1,262 f 54.6 

Negative 

15 f 3.5 

13 2 0.9 

14 f 4.0 

15 f 0.9 

14 2 1.2 

16 +. 1.5 

438 f 30.3 

Negative 

16 f 2.7 

14 f 1.3 

12 f 1.5 

15 f 2.0 

16 f 2.0 

16 f 1.9 

491 f 60.8 

Negative 

34 f 1.2 

38 f 1.0 

38 f 3.8 

43 f 5.8 

34 f 2.9 

44 f 3.7 

Negative 

1,031 f 178.1 
224 ,Acetonitrile, NT,P TR 447 

Mutagenicity ,of Acetonitrile .in Salmoneh typhimurium (continued) 

TABLE El 

'Revertaritsjplate 

Strain .Dose 49 +hamster S9 +rat S9 

OLg/Plate) 10% 30% 10% 30% 

Study performed at Microbiological Associates, Inc. 

333 

100 

TAM0 0 

10,000 

3,333 

1,000 

'81 f:6.2 

74 f 5.6 

74 f 6.2 

84 f 4.5 

73 f 5:2 

204 f 3.0 

.Negative 

83 f .6.4 

Positive control 

Trial summary 

333 

100 

TA1535 0 

10,000 

3,333 

1,000 

13 f 0.9 

15 f 1.0 

14 f 3.3 

14 f 3.0 

14 f 0.0 

16 f 0.6 

89 f 2.9 

Negative 

Rositive control 

Trial summary 

333 

100 

TA97 0 

10,000 

3,333 

1,000 

80 -+ 4.8 

89 f 2.3 

91 f 14.2 

96 f 3.8 

108 f 13.4 

94 f 3.9 

420 f .26.1 

Negative 

Positive control 

Trial summaty 

333 

100 

TA98 0 

10,000 

3,333 

1,000 

22 f 5.7 

15 f 2.0 

20 f 0.9 

23 f 1.2 

21 f 3.1 

21 f 1.9 

113 f 5.8 

Negative 

Positive control 

Trial summary 

94 f 3.7 

87 f 8.8 

80 't 0.6 

91 f 4.3 

97 f 4.'0 

110 f 1.5 

404 f 21.5 

Negative 

93 f 8.0 

90 f 3.5 

92 k 6.6 

102 f 4.3 

103 2 10.5 

108 f 4.2 

285 +- 21.2 

Negative 

30 f 2.3 

27 f 4.1 

'36 f 4.1 

27 f 2.4 

24 f 2.1 

31 f 1.7 

187 f 10.1 

Negative 

,79 2 4.3 

99 & 4.8 

88 +. 4.6 

80 f 2.7 

87 f '6:O 

91 % 1.3 

,285 f 15.0 

Negative 

10 f 1.0 

9 f 1.3 

12 f '0.9 

8 f 0.7 

8 f 1.2 

12 f l:o 

69 f 6.2 

Negative 

182 +. 14.7 

160 f 3.8 

165 f 6.8 

148 f 3:0 

167 r .9.3 

160 f 4.3 

291 f 4.9 

Negative 

26 f 3.6 

33 f 1.7 

30 f 3.0 

.31 f 5.3 

34 f 1.2 

35 f l:o 

117 f 9.5 

Negative 

-92 f 6.8 

76 f 10.0 

85 f 2.4 

80 f 4.1 

82 & 7.9 

88 f '5.2 

282 f 44.3 

Negative 

10 2 1.8 

7 f 0:6 

9 f'0.9 

9 f 2.3 

'8 f 1.3 

12 f 119 

119 f 10.5 

Negative 

121 f 10.0 

126 f 12;o 

120 f 1.0 

141 f 6.4 

130 f 3.3 

140 f 3.7 

723 f 1.5.7 

Negative 

31 f 2.4 

23 f 1.2 

22 f 2.7 

24 f 2.3 

21 f 4.0 

25 -+ 0.7 

160 f 12.9 

Negative 

84 f '4.4 

87 f 7.5 

'84 f .2.9 

84 rt 4.0 

76 f 10.0 

90 f 2.7 

700 f .23.7 

Negative 

9 2 2.9 

10 a 1.8 

12 a 1.8 

.9 f 3.6 

8 f 0.3 

7 +. 2.3 

109 'f 2.0 

Negative 

157 f 10.9 

168 f 14.2 

161 f 32.5 

166 f 6.9 

172 -t 14.4 

I95 f 6.2 

,403.f 10.1 

.Negative 

-37 f 1.2 

34 f 2.3 

31 f 3.5 

29 f 1.8 

40 d 3.7 

32 f 3.0 

211 f 20.8 

-Negative 

a The detailed protocol and these data are presented in Mortelmans et al. (1986). 0 Bg/plate dose is the solvent control. 

2-aminoanthracene. 

TA1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 

The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA97 and 

Revertants are presented as mean f the standard error from 3 plates. 
Genetic Toxicology 225 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Acetonitrilea 

TABLE E2 

Compound (FglmL) Cells somes SCEs some Cell in BrdU Chromosomeb 

Dose Total Chromo- No. of Chromo- SCEs/ HIS Change of SCEs/ 

No. of SCEs/ Relative 

(%) 

Acetonitrile 

Triethylenemelamine 

Distilled water 

Summary: Weak positive 

-s9 

Acetonitrile 

Cyclophosphamide 

Distilled water 

Summary: Negative 

+ s9 

50 

0.015 50 

160 50 

500 50 

1,600 50 

5,000 50 

1.042 383 0.36 7.7 26.0 

1,050 1,442 1.37 28.8 26.0 

1,049 440 0.41 8.8 26.0 

1,043 428 0.41 8.6 26.0 

1,050 459 0.43 9.2 26.0 

1,049 469 0.44 9.4 26.0 

P=0.003c 

273.63 

21.64: 

18.93 

11.64 

14.11 

50 1,042 408 0.39 8.2 26.0 

1.000 50 1,046 910 0.86 18.2 26.0 122.19 

500 50 1,039 431 0.41 8.6 26.0 

1,600 50 1,039 392 0.37 7.8 26.0 

5,000 50 1,037 414 0.39 8.3 26.0 1.96 

-3.65 

5.94 

P=O.568 

* Positive (PcO.01) a Study performed at Columbia University. A detailed description of the protocol and these data are presented in Galloway er al. 

Significance of relative SCEs/chromosome tested by the linear regression trend test vs. log of the dose 

SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells. 

(1987). SCE=sister chromatid exchange; BrdU=bromodeoxyuridine 
226 Acetonitrile, NTP TR 447 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Acetonitrilea 

TABLE E3 

Dose Total No. of Abs/ Cells with 

- s9 + s9 

Dose Total No. of Abs/ Cells with 

(&mL) Cells Abs Cell Abs (%) @g/mL) Cells Abs Cell Abs (%) 

Distilled water 

Summary: Negative 

.Halvest time: 14.0 

100 2 

Triethylenemelamine 

0.15 50 14 

Acetonitrile 

500 100 2 

1,600 100 9 

5,000 100 5 

Distilled water 

Summary: Equivocal 

Harvest time: 14.0 

0.02 2.0 100 1 0.01 1.0 

Cyclophosphamide 

0.28 20.0 15.0 50 27 0.54 44.0 

Acetonitrile 

0.02 2.0 

0.09 7.0 

0.05 5.0 

500 100 3 0.03 3.0 

1,600 100 2 0.02 

5,000 100 8 0.08 7.0 

P=O.O16 

2.0* 

P=0.054b 

* Positive (PcO.05) 

Significance of percent cells with aberrations tested by the linear regression trend test vs. log of the dose 

Abs=aberrations. 

a Study performed at Columbia University. The detailed protocol and these data are presented in Galloway et al. (1987). 
Genetic Toxicology 227 

with Acetonitrile by Inhalation for 13 Weeksa 

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Foltowing Treatment 

TABLE E4 

Dose 

(PPm) Erythrocytes/1,000 Cellsb 

Normochromatic 

Micronucleated 

of Mice 

Number 

Male 

800 

400 

200 

100 

0 

Trend test 

Female 

800 

400 

200 

100 

0 

Trend test 

2.06 f 0.28 

2.36 ? 0.29* 

1.71 f 0.10 

1.91 2 0.11 

1.42 k 0.13 

P=O.O07' 

1.57 k 0.15 

1.71 ? 0.15 

1.61 2 0.16 

1.16 2 0.16 

1.33 f 0.12 

P=O.O3 

10 

10 

10 

10 

10 

10 

10 

10 

6 

9 

* Po$tive (PcO.006) by pailwise comparison to the control group with a t-test. 

__- -a Teripheral blood samples obtained at termination of the 13-week toxicity study. A detailed protom; is presented in MacGregor 

Significance of 

Data presented as mean f standard error of the mean. NCE = normochromatic e@rrJb 

et al. (1990); a minimum of 10,000 NCEs were scored per animal. 

tested by a one-taki &- trend test. 

I 

/ 

- 

.-, /-- - _/' 

i 
228 

Acetonitrile, NTP TR 447 

. 
229 

ORGAN-WEXGHT-TO-BODY-WEXGHT RATIOS 

ORGAN WEIGHTS AND 

APPENDIX F 

TABLE F1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

TABLE F2 Organ Weights and Organ-Weight-to-Bodyweight Ratios for Rats at the 15-Month 

TABLE F3 Organ Weights and Organ-Weight-to-Bodyweight Ratios for Mice 

TABLE F4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the 15-Month 

in the 13-Week Inhalation Study of Acetonitrile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 

Interim Evaluation in the 2-Year Inhalation Study of Acetonitrile . . . . . . . . . . . . . . . . 232 

in the 13-Week Inhalation Study of Acetonitrile . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 233 

Interim Evaluation in the 2-Year Inhalation Study of Acetonitrile . . . . . . . . . . . . . . . . 235 
230 Acetonitrile, NTP TR 447 

Organ Weights and Organ-Weight-to-Bodyweight Ratios for Rats in the 13-Week Inhalation Study of Acetonitrilea 

TABLE F1 

.. 

0 PPm 100 ppm 200 ppm 400 ppm 800 ppm 1,600 ppm 

.. .. 

Male 

n 10 10 10 10 9 4 

Necropsy bpdy wt 345 f 4 354 * 5 364 f 6 348,f 5 349 * 7 285 +- 12** 

Brain 

Absolute , 

Relative, 

Absolute 

Absolute 

L. and R. Kidney.. 

Relative 

Relative 

Absolute 

Relative . 

Absolute 

Relative ,:. 

Absolute . 

. Relative 

Absolute , 

Relative. , 

Heart 

Liver 

Lungs 

R. Testis . 

~yrqus 

5.48 f 0.09 

1.886 f 0.014 

34.23 f 0.39 

11.800 k 0.195. 

6.62 2 0.07 

2.279 f 0.016, 

2.67 f 0.04 ,~ 

0.920 f 0.018: 

1.937 f 0.048 I 

3.86,f 0.09 ;, , 

1.331 f 0.035 -. 

5.62 f 0.12. 

0.81.. f 0.010 :: 

0.81 0.03. ,. 

2.90 g .0.22 

1.022 f 0.070 

5.38 f 0.06 

1.898 2 0.011 

12.565 $ 0.454 

6.67 2.0.09 

2.357 f 0.049 

0.26l,<.f, 0.022 . 

3.86 0.07 ~ 

1.364,~ 0.022 

5.59 f 0.19 ,. 

1.983 &, 0.089 

35.57 r 1.33 . . 

0.74 0;06.; 

5.29 f 0.08 

1.923 f 0.013 

0.969, 0.015 

2.65 * 0.02 

6.72 f 0.09, . 

2.446 5 0.039. 

2.200, f 0.094 

35.10 f 0.45 

12.775 f 0.238 

6.0.4, f 0.23 

1.355. f 0.021 . 

0.79 -f 0.04,; 

0.287: 0.017 

3.73 ; 0.06 

5.38 f 0.10 

1.867 f 0.021 

2.73 f 0.04 

0.948 f 0.013. 

34.52 f 0.28 

12.011 f 0.214 

6.65 f 0.07 

2.312 f 0.028 

1.972 f 0.086. . 5.67 $ 0.24 

0.79 f,O.03 . ,. 

0.274 2 0.012, , 

3.72 f 0.05 ., 

1.291 $ 0.017. ;, 

.. . 

1.876 f 0.013 

5.39 f 0.10 

2.77 i 0.04 

0.963 f 0.020 

.. 

1.998,.+ 0.083, 

35.74 f 0.57 

12.468 .r?l 0.345 

6.87 '+ 0.08. 

2.396 f 0.060 ,. 

5.73 f. 9.22, .. . 

1.283,.$ 0.018 , 

0.65.:,? 0.03** 

0.2@,.% 0.014* 

3.69 ,g 0.06 , . .., 

1.793 r 0.017** : 

6.32 1?1 OX** ' 

0.940 rt q.049 

3.30 f 0.10** , 

2.303 rt 0.146 

8.06 +- 0.20** , 

11.803 f.0.758 

1.800 ,rt 0.158 . 

41.41 rt 1,76** 

6.30 f 0.36 ., ~ 

., . 

1.063 f 0.028** ~ 

3.75 ' & 0.20 

. .. 

0.130 ; 0:008** . , 

0.46 .+. 0.03.* *: ,. 
Organ Weight Analyses 231 

(continued) 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Inhalation Study of Acetonitrile 

TABLE. F1 

0 PPm 100 ppm 200 ppm 400 ppm 800 PPm 1,600 ppm 

Brain 

Necropsy body wt 

n 

Female 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

L. and R. Kidney 

Relative 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

Liver 

Lungs 

Thymus 

10 

201 f 4 

8.77 f 0.16 

1.755 f 0.011 

3.05 f 0.06 

0.613 f 0.015 

1.370 f 0.031 

31.53 & 1.00 

6.313 f 0.173 

6.82 f 0.08 

1.17 & 0.03 

0.235 f 0.007 

6.11 f 0.06 

1.227 f 0.023 

10 

194 rt 4 

9.12 f 0.20 

1.758 f 0.014 

1.269 rt 0.025 

32.78 f 0.72 

6.328 rt 0.116 

7.22 f 0.12* 

1.395 f 0.028 

3.23 f 0.05 

0.625 f 0.014 

1.10 f 0.02 

0.212 f 0.006 

6.56 f 0.10* 

10 

206 f 3 

1.10 f 0.03 

0.226 f 0.008 

6.25 f 0.12 

1.284 f 0.032 

32.19 rt 0.96 

6.618 f 0.235 

7.06 f 0.09 

1.449 f 0.024 

3.32 f 0.07 

0.681 f 0.012. 

8.56 f 0.09 

1.756 f 0.014 

10 10 7 

214 f 4 206f3 183 & 4** 

1.745 f 0.020 1.742 f 0.008 1.703 f 0.011 

8.17 f 0.10. 8.46 e 0.11 9.35 f 0.19. 

0.663 f 0.011. 0671 f 0.022* 0.769 f 0.026** 

3.10 & 0.04 3.25 f 0.07 4.22 f O.W* 

1.496 f 0.030** 1.486 & 0.025** 1.514 f 0.039** 

7.00 f 0.08 7.21 f 0.07** 8.30 f 0.14** 

6.861 f 0.228 6.930 f 0.151* 7.709 f 0.371** 

32.04 f 0.73 33.64 f 0.77 42.44 f 2.56** 

1.362 & 0.043* 1.272 f 0.037 1.189 f 0.024 

6.36 f 0.14 6.16 f 0.14 6.52 f 0.14 

0.243 f 0.007 0.203 f 0.006** 0.118 f 0.010** 

1.13 f 0.02 0.98 f 0.03** 0.64 f 0.04** 

~ ~~ ~~ ~ 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

** P50.01 

* Significantly different (P50.05) from the control group by Williams’ or Dunnett’s test 

(mean f standard error). 
'232 

in the 2-Year Inhalation Study of Acetonitrilea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 15-Month Interim Evaluation 

TABLE F2 

0 PPm 100 ppm 200 pmm 400 ppm 

n 

Male 

R. Kidney 

Necropsy body wt 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Liver 

Lungs 

Female 

Necropsy body wt 

R. ,Kidney 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Liver 

Lungs 

8 

507 f 11 

4.35 f 0.13 

2.203 f 0.066 

33.10 f 0.72 

16.805 f 0.639 

3.24 f 0.06 

1.640 f 0.035 

307 f 13 

5.14 f 0.12 

1.569 f 0.047 

29.47 f 0.68 

9.026 f 0.380 

3.24 f 0.11 

0.989 f 0.031 

8 

500 2 7 

4.63 f 0.12 

2.314 f 0.065 

31.68 f 0.63 

15.845 f 0.349 

3.18 f 0.06 

1.590 f 0.035 

311 f 7 

3.21 f 0.13 

0.996 f 0.040 

9.601 f 0.359 

1.701 f 0.144 

30.98 f 1.36 

5.49 f 0.50 

8 

489 f 5 

4.65 f 0.28 

2.281 .f 0.152 

32.60 f 0.47 

15:961 f 0.310 

3.26 f 0.05 

1.595 r 0.628 

308 k 7 

5.41 f 0.26 

1.665 f 0.075 

30.66 f 0.77 

9.464 f 0.380 

3.26 f 0.07 

1.003 f 0.017 

8 

482 f 7 

3.47 f '0.07 

2.150 f 0.032 

32.31 2 035 

15.573 f 0.321 

3.25 f 0.06 

1.565 f 0.030 

320 f8 

4.83 f 0.ld 

1.543 f 0.042 

30.02 f 0.67 

9.630 f 0.364 

3.24 f 0.12 

1.033 f 0.028 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg dqan weight/g body weight 

(mean f standard error). 
Organ Weight Analyses 233 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Inhalation Study of Acetonitrilea 

TABLE F3 

0 PPm 100 ppm 200 ppm 400 PPm 800 ppm 1,600 ppm 

Brain 

Necropsy body wt 

n 

Male 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

L. and R. Kidney 

Relative 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

Liver 

Lungs 

R. Testis 

Thymus 

35.6 f 0.6 

10 

0.85 f 0.06 

0.030 f 0.002 

3.35 f 0.10 

0.119 f 0.002 

6.65 f 0.23 

0.236 f 0.007 

46.79 f 0.76 

1.665 f 0.044 

16.87 f 0.46 

0.599 f 0.017 

4.49 f 0.11 

0.160 f 0.006 

13.00 f 0.25 

0.461 f 0.005 

10 10 10 9 

34.1 f 0.6 34.8 f 0.5 34.3 f 0.8 33.7 f 0.3 b 

0 

- 

0.459 f 0.005 0.475 f 0.004 0.469 f 0.002 0.456 ? 0.003 

13.51 f 0.27 13.67 f 0.19 13.71 f 0.28 13.52 f 0.11 

- 

- 

0.156 f 0.004 .156 f 0.003 .155 f 0.005 .156 f 0.006 

4.59 f 0.12 4.49 f 0.09 4.52 f 0.13 4.62 f 0.19 

- 

- 

0.634 f 0.010 0.646 f 0.007* 0.636 f 0.014 0.599 f 0.014 

18.64 f 0.35*' 18.58 f 0.25** 18.53 f 0.17** 17.76 f 0.37 

1.758 f 0.034 1.822 f 0.030* 1.945 f 0.059*' 2.111 f 0.047.' 

51.62 f .0.61" 52.37 f 0.75** 56.56 f 0.60** 62.59 f 1.17** 

- 

- 

- 

- 

0.243 f 0.005 .257 f 0.006 .247 f 0.007 .249 f 0.006 - 

7.13 f 0.07. 7.38 f 0.10'' 7.20 f 0.15.' 7.38 f 0.14** - 

0.120 f 0.001 0.122 f 0.002 .118 f 0.002 .115 f 0.002 

3.52 f 0.06 3.51 f 0.05 3.45 f 0.08 3.40 f 0.06 

- 

- 

0.034 f 0.002 .035 f 0.001 .032 f 0.002 0.028 f 0.003 - 

1.01 f 0.04 0.99 f 0.03 0.94 f 0.05 0.83 f 0.08 - 
234 Acetonitrile, NTP TR 447 

(continued) 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Inhalation Study of Acetonitrile 

TABLE F3 

0 PPm 100 ppm 200 ppm 400 ppm 800 ppm 1,600 ppm 

Brain 

Necropsy body wt 

n 

Female 

Relative 

Absolute 

Heart 

Relative 

Absolute 

L. and R. Kidney 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Liver 

Lungs 

Thymus 

30.5 & 0.9 

10 

15.84 +- 0.44 

0.479 f 0.004 

0.143 k 0.003 

1.51 f 0.09 

0.046 f 0.002 

7.88 f 0.11 

0.240 f 0.007 

51.95 r 1.95 

1.582 r 0.074 

14.23 f 0.29 

0.432 f 0.011 

4.73 0.15 

30.6 f 0.9 

10 

15.82 f 0.38 

0.481 f 0.005 

13.93 f 0.31 

0.424 f 0.007 

4.67 f 0.12 

0.142 f 0.002 

52.97 f 0.92 

1.616 f 0.037 

0.244 f 0.007 

8.00 ? 0.19 

1.65 f 0.06 

0.050 f 0.002 

29.1 f 0.6 

10 

0.424 f 0.008 

4.75 f 0.10 

0.138 f 0.003 

16.67 f 0.29 

0.484 f 0.005 

14.57 f 0.16 

0.239 f 0.006 

55.00 f 0.52 

1.601 f 0.032 

8.21 f 0.15 

1.58 f 0.09 

0.046 f 0.003 

9 

29.4 f 0.6 

8.16 f 0.20 

0.240 f 0.007 

58.65 f 0.85** 

1.726 f 0.035 

14.53 f 0.27 

0.427 f 0.002 

4.69 f 0.11 

0.138 f 0.003 

16.36 f 0.35 

0.480 f 0.002 

0.044 f 0.003 

1.51 f 0.11 

31.5 +- 1.1 

6 

1.48 f 0.11 

0.046 f 0.003 

7.57 f 0.20 

0.238 r 0.011 

65.31 f 0.95** 

2.057 f 0.080** 

14.31 f 0.13 

0.450 & 0.014 

4.62 f 0.14 

0.145 +- 0.004 

15.18 f 0.54 

0.475 f 0.004 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

** PSO.01 

* Significantly different (PSO.05) from the control group by Williams’ or Dunnett’s test 

No data presented due to 100% mortaliiy in this group 

(mean f standard error). 
Organ Weight Analyses 235 

in the 2-Year Inhalation Study of Acetonitrilea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the 15-Month Interim Evaluation 

TABLE F4 

0 PPm 50 PPm 100 ppm 200 ppm 

n 

Male 

10 10 9 10 

Necropsy body wt 47.7 f 1.6 47.4 f 1.3 49.0 f 1.1 49.2 f 1.2 

R. Kidney 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Liver 

Lungs 

0.433 f 0.009 0.427 f 0.009 0.434 f 0.016 0.420 f 0.013 

9.19 f 0.40 9.06 f 0.30 8.85 f 0.21 8.55 f 0.18 

2.287 f 0.183 2.515 f 0.260 2.188 f 0.135 2.229 f 0.097 

48.34 f 4.34 54.53 f 7.72 44.51 f 2.60 45.23 f 1.18 

0.253 f 0.008 0.258 & 0.007 0.269 f 0.010 0.258 f 0.011 

5.33 f 0.16 5.48 f 0.21 5.50 f 0.23 5.25 f 0.19 

Female 

n 10 10 10 10 

Necropsy body wt 49.7 f 1.4 47.4 ? 2.5 51.5 f 2.6 48.3 f 2.3 

R. Kidney 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Liver 

Lungs 

0.300 f 0.008 0.271 f 0.004. 0.302 f 0.008 0.291 f 0.007 

6.07 f 0.17 5.84 f 0.28 5.93 f 0.17 6.11 f 0.23 

1.985 f 0.052 1.864 f 0.036 2.095 f 0.069 2.109 f 0.045 

40.18 f 1.23 40.05 f 1.58 41.13 f 1.26 44.31 f 1.62 

0.257 f 0.006 0.238 f 0.007 0.252 +. 0.006 0.267 f 0.010 

5.21 f 0.18 5.13 f 0.26 4.98 f 0.21 5.65 f 0.34 

* Significantly different (PSO.05) from the control group by Williams’ or Dunnett’s test a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean & standard error). 
236 Acetonitrile, NTP TR 447 
237 

THYROID HORMONE ASSAY RESULTS 

HEMATOLOGY AID 

APPENDIX G 

TABLE 62 Hematology Data for Rats at the 15-Month Interim Evaluation 

TABLE GI Hematology and Thyroid Hormone Assay Data for Rats 

in the 13-Week Inhalation Study of Acetonitrile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 

in the %Yeas Inhalation Study of Acetonitrile . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . 240 
P 

B 

+I +I +I +I +I 

.. 

2 

s: 

2 

* 

r?*oi+ rem 

* *- 1. 

00000 

mNOm00000 

ai 
Hematology and Thyroid Hormone Assays 239 

Hematology and Thyroid Hormone Assay Data for Rats in the 13-Week Inhalation Study of Acetonitrile (continued) 

TABLE G1 

~ 

0 PPm 100 ppm 200 ppm 400 ppm 800 ppm 1,600 ppm 

n 

Female (continued) 

10 10 10 10 10 7 

Thyroid Hormone Assays 

Thyroid-stimulating 

Thyroxine (bg/dL) 4f0 4kO 4f0 4f0 3f0 

Triiodothyronine (ng/dL) 126 f 7 114 f llb 112 f 6 122 f 5 104 f 6 80 f 3** 

hormone (ng/mL) If0 If0 1-0 1fO 1fO 

4 f ob 

1 f ob 

a Mean f standard error 

** PSO.01 

l Significantly different (PSO.05) from the control group by Dunn’s or Shirley’s test 

n=9 
240 Acetonitrile, NTP TR447 

Hematology Data for Rats at the 15-Month Interim Evaluation in the 2-Year Inhalation Study of Acetonitrilea 

TABLE 62 

0 PPm 100 ppm 200 ppm 400 ppm 

Male 

n 8 

Nucleated erythrocytes (103/bL) 

Eosinophils (103/bL) 

Monocytes (103/bL) 

Lymphocytes (103/bL) 

Segmented neutrophils (103/bL) 

Leukocytes (103/a) 

Reticulocytes (106/bL) 

Platelets (IO~/~L) 

Mean cell hemoglobin concentration (g/dL) 

Mean cell hemoglobin (pg) 

Mean cell volume (fL) 

Erythrocytes (106/bL) 

Hemoglobin (g/dL) 

Manual hematocrit (%) 

Hematocrit (%) 

Female 

34.5 f 0.2 

16.9 f 0.1 

49.1 f 0.4 

8.94 f 0.10 

15.1 f 0.2 

46.1 f 0.5 

43.8 f 0.5 

0.22 f O.OSb 

0.04 f 0.02b 

0.06 f 0.05b 

3.58 f 0.49b 

1.84 f 0.34b 

5.54 f 0.33b 

0.4 f 0.0 

540.3 f 30.6 

n 8 

Nucleated erythrocytes (103/bL> 

Eosinophils (103/bL) 

Monocytes (103/bL) 

Lymphocytes (103/bL) 

Segmented neutrophils (103/bL> 

Leukocytes (103/bL) 

Reticulocytes (106/bL) 

Platelets (103/~~) 

Mean cell hemoglobin concentration (g/dL) 

Mean cell hemoglobin (pg) 

Mean cell volume (E) 

Elythrocytes (106/bL) 

Hemoglobin (g/dL) 

Manual hematocrit (%) 

Hematocrit (%) 

34.8 f '0.1 

18.5 f 0.1 

53.1 f 0.2 

8.40 f 0.06 

15.5 f 0.1 

46.6 f 0.4 

44.6 f 0.4 

0.07 f 0.02 

0.03 f 0.01 

0.10 f 0.02 

2.15 f 0.15 

0.70 f 0.09 

2.99 f 0.16 

0.2 f 0.0 

483.9 f 18.5 

8 

34.5 f 0.1 

16.7 f 0.1 

48.5 f 0.4 

9.14 f 0.06 

15.3 f 0.1 

46.8 f 0.3 

44.3 f 0.3 

0.12 f 0.05 

0.09 f 0.02 

0.10 f 0.03 

3.77 f 0.19 

1.50 f 0.15 

5.49 f 0.25 

0.4 f 0.0 

519.9 f 8.0 

7 

34.7 f 0.1 

18.1 f 0.2 

52.1 f 0.7 

8.27 f 0.12 

15.0 f .0.4 

45.1 f 1.0 

43.2 f 1.1 

0.12 f 0.03 

0.05 f 0.02 

0.14 f 0.06 

2.61 f 0.19 

0.80 f 0.14 

3.60 f 0.28 

0.2 f 0.0 

431.6 f 20.7 

8 

34.6 f 0.2 

16.9 f 0:2 

48.6 f 0.6 

9.08 f 0.15 

15.3 f 0.1 

46.9 f 0.5 

44.2 f 0.4 

0.15 f 0.05 

0.04 f 0.02 

0.06 f 0.03 

3.75 f 0.29 

1.73 f 0.34 

5.60 f 0.58 

0.4 f 0.0 

518.1 f 19.3 

8 

34.9 f 0.2 

18.3 f 0.1 

52.6 f 0.3 

8.26 f 0.08 

15.2 f 0.1 

45.6 f 0.4 

43.5 f 0.5 

0.2 f 0.0 

464.5 f 22.0 

0.13 f 0.03 

0.05 f ,0:01 

0.18 f 0.06 

2.38 f 0.17 

0.91 f 0.14 

3.54 0.31 

8 

34.6 f 0.2 

16.3 f 0.1** 

47.0 f 0.2** 

9.40 f 0.10** 

15.3 f 0.1 

47.6 f 0.6 

44.3 f 0.4 

0.16 f 0.04 

0.06 f 0.02 

0.04 f 0.02 

3.88 f 0.23 

1.72 f 0.19 

5.70 .f '0.27 

0.3 f 0.0 

504.4 f 12.2 

8 

34.8 f 0.1 

18.0 f 0.1** 

51.5 f 0.3** 

8.15 f 0.08 

14.7 f '0.2** 

44.4 f 0.5** 

42.2 f 0.5** 

0.13 f 0.04 

0.05 f 0.02 

0.08 f 0.02 

2.81 f 0.25 

1.05 f 0.18 

3.99 f 0.29* 

0.2 f 0.0 

445.4 f 14.6 

a Mean f standard error 

** PSO.01 

* Significantly different (PSO.05) from the control group by Dunn's or Shirley's test 

n=7 
241 

GENERATION OF CHAMBER CONCENTRATIONS 

CHEMICAL CHARACTERIZATION AND 

APPENDIX H 

PROCUREMENT AD CHARACTERIZATION OF ACETONITRILE 

TABLE H1 Summary of Chamber Concentrations in the 13-Week Inhalation Studies 

FIGURE H5 Acetonitrile Exposure Suite 

FIGURE H4b Acetonitrile Inhalation Exposure Chamber for the 2-Year Studies 

FIGURE H4a Acetonitrile Inhalation Exposure Chamber for the 13-Week Studies 

FIGURE H3b Acetonitrile Vapor Generation and Delivery System for the 2-Year Studies 

FIGURE H3a Acetonitrile Liquid Vapor Generator 

FIGURE H2 Nuclear Magnetic Resonance Spectrum of Acetonitrile 

FIGURE H1 Infrared Absorption Spectrum of Acetonitrile 

in the 200 ppm Acetonitrile Mouse Exposure Chamber for the 2-Year Study 

FIGURE Hll Monthly Mean Concentration and Standard Deviation 

in the 100 ppm Acetonitrile Mouse Exposure Chamber for the 2-Year Study 

FIGURE H10 Monthly Mean Concentration and Standard Deviation 

in the 50 ppm Acetonitrile Mouse Exposure Chamber for the 2-Year Study 

FIGURE H9 Monthly Mean Concentration and Standard Deviation 

in the 400 ppm Acetonitrile Rat Exposure Chamber for the 2-Year Study 

FIGURE H8 Monthly Mean Concentration and Standard Deviation 

in the 200 ppm Acetonitrile Rat Exposure Chamber for the 2-Year Study 

FIGURE H7 Monthly Mean Concentration and Standard Deviation 

in the 100 ppm Acetonitrile Rat Exposure Chamber for the 2-Year Study 

FIGURE H6 Monthly Mean Concentration and Standard Deviation 

of Acetonitrile 

TABLE H2 Summary of Chamber Concentrations in the 2-Year Inhalation Studies 

of Acetonitrile 

................................. 242 

251 

250 

249 

248 

247 

246 

245 

258 

257 

256 

255 

254 

253 

252 

252 

GENERATION AND MONITORING OF CHAMBER CONCENTRATIONS ............................. 243 

................................. .......................... ....................................... ........... ................ 

................. .............................................. 

......................................................... 

........................................................ 

............ 

............ 

............ 

.......... 

......... 

......... 
242 Acetonitrile, NTP TR 447 

GENERATION OF CHAMBER CONCENTRATIONS 

CHEMICAL CHARACTERIZATION AND 

acetonitrile studies are on file at the National Institute of Environmental Health Sciences (NIEHS). 

Midwest Research Institute (Kansas City, MO). Reports on analyses performed in support of the 

studies. Identity, purity, and stability analyses were conducted by the analytical chemistry laboratory, 

VO41381, obtained from Vistron Corporation (Cleveland, OH), and were used for a portion of the 2-year 

and for the majority of the 2-year studies. Lots B082889, obtained from J.T. Baker (Phillipsburg, NJ), and 

Dupont deNemours and Company, Inc. (Wilmington, DE), and was used throughout the 13-week studies 

Acetonitrile was obtained in three lots (2485, B082889, and VO41381). Lot 2485 was obtained from E.I. 

PROCUREMENT AND CHARACTERIZATION OF ACETONITRILE 

Figures H1 and H2. 

Standard Spectra) of acetonitrile. The infrared and nuclear magnetic resonance spectra are presented in 

nuclear magnetic resonance spectroscopy. All spectra were consistent with the literature spectra (Sudtler 

The chemical, a clear, colorless liquid, was identified as acetonitrile by infrared, ultraviolethsible, and 

nitrogen carrier gas at a flow rate of 70 mL/minute. Two systems were used: 

3 M potassium chloride. Gas chromatography was performed using a flame ionization detector and a 

with 0.01 N sodium hydroxide, and monitored potentiometrically with an electrode filled with 

titration, and gas chromatography. For free acid titration, the samples were dissolved in water, titrated 

The purity of each lot was determined by elemental analyses, Karl Fischer water analysis, free acid 

A) Porapak QS on 100/120 mesh, with an oven temperature program of 50" C for 5 minutes, 

then 50" to 230" C at 10" C per minute, 

B) 10% Carbowax 20M-TPA on SO/lOO Chromosorb W (AW), with an oven temperature' 

program of 60" C for 6 minutes, then 60" to 200" C at 10" C per minute. 

analyzed using system A, except with an isothermal oven temperature of 145" C. 

Samples of 0.5% acetonitrile in methanol containing 0.7% isopropanol as the internal standard were , 

Concomitant analyses of each lot with an analytical chemistry laboratory sample were also performed. 

100.0% & 0.3% (lot VO41381). The overall purity for all lots was determined to be at least 99%. 

concomitant analyses indicated purities of 99.4% -C 0.2% (lot 2485), 100.4% & 0.3% (lot B082889), and 

one major peak and no impurities with areas greater than 0.1% relative to the major peak. The 

major peak and one impurity peak with an area of 0.14% relative to the major peak. System B indicated 

peak for lots 2485 and B082889. For lot VO41381, gas chromatography using system A indicated one 

system indicated one major peak and no impurities with areas greater than 0.1% relative to the major 

(lot 2485), 29 & 5 ppm (lot B082889), and 35 f 4 ppm (lot VO41381). Gas chromatography by each 

(lot B082889), and 0.19% & 0.04% (lot VO41381) water. Free acid titration indicated 33 2 1 ppm 

acetonitrile. Karl Fischer water analysis indicated 0.114% f 0.003% (lot 2485), 0.11% f 0.07% 

Elemental analyses for carbon, hydrogen, and nitrogen were in agreement with the theoretical values for 

degradation of the bulk chemical was observed. 

using gas chromatography and free acid titration methods similar to those previously described. No 

original containers. The stability of the bulk chemical was monitored periodically by the study laboratory 

temperatures up to 60" C. To ensure stability, the bulk chemical was stored at approximately 22" C in the 

that acetonitrile was stable as a bulk chemical for at least 2 weeks when stored protected from light, at 

performed using system A, except with an isothermal oven temperature of 145" C. These studies indicated 

Stability studies were performed by the analytical chemistry laboratory. Gas chromatography was 
Chemical Characterization and Generation of Chamber Concentrations 243 

diagram of the exposure suite is shown in Figure H5. 

chamber when the catchpans are in place. Total active mixing volume of each chamber is 1.7 m3. A 

MD) (Figures H4a and H4b), so that uniform vapor concentrations can be maintained throughout the 

exposure chamber, which was manufactured by Hartford System Division of Lab Products, Inc. (Aberdeen, 

inadvertently produced during generation of the atmosphere. The study laboratory designed the inhalation 

start and again during the study with animals in chambers to check all chambers for any aerosol 

Gardener Type CN Small Particle Detector (Gardner Associates, Schenectady, W) was used prior to study 

exposure chambers once the concentrations in the distribution system had stabilized (Figure H3b). A 

desired concentrations by adjusting the compressed air pressure to the vacuum pumps, and delivered to the 

HEPA-filtered air. The mixture was drawn into a stainless steel distribution manifold, diluted to the 

studies) with a glass fiber wick (Figure H3a). Acetonitrile vapor was mixed with charcoal-filtered and 

stainless steel cylinder heated to approximately 177" F f 8" F (13-week studies) or 200" F f 5" F (2-year 

(13-week studies) each week. Liquid was pumped from the reservoir to a vaporizer that consisted of a 

acetonitrile and in the reservoir. The reservoir was refilled approximately once (2-year studies) or twice 

5.6 L stainless steel reservoir. A nitrogen cover was maintained at all times while transferring the 

Vupor Generation System. Liquid acetonitrile was transferred from the original shipping container to a 

GENERATION AND MONITORING OFCHAMBER CONCENTRATIONS 

nitrogen. 

chromatograph was monitored throughout exposure days using an on-line standard of acetonitrile in 

flow assured the delivery of fresh sample to the stream select valve. Drift of the on-line gas 

lined stainless steel lines and the exhaust port of the %port or 12-port stream select valve. This constant 

standards using an off-line gas chromatograph. Samples were constantly drawn by vacuum through Tefloncalibrated critical-orifice sampling system. These samples were analyzed against gravimetrically prepared 

samples from each exposure chamber using dimethylformamide-filled, fritted-glass bubblers and a 

twice hourly during the 13-week and 2-year studies. Calibration was accomplished by acquiring grab 

(13-week studies) or 12-port (2-year studies) sampling valve. Each chamber was sampled approximately 

130" C. The monitor was coupled with the inhalation chambers using an automated, multiplexed, $-port 

80/100 Porapak Q. The nitrogen carrier gas flow rate was 30 mL/rninute and the column temperature was 

gas chromatograph equipped with a flame ionization detector and an OD nickel column packed with 

Vapor Concennution Monitoring. Chamber concentrations were monitored with a single on-line HP-5840 

studies. 

13-week studies and 7 to 12 minutes without animals or 14 to 17 minutes with animals in the 2-year 

after vapor generation was terminated (TI,,) ranged from 12 to 14 minutes with or without animals in the 

remainder of the study. The time for chamber concentration to decay to 10% of the target concentration 

12 minutes of concentration buildup. This change in the operating procedure was followed throughout the 

were reduced to the initial values by increasing air pressure to the delivery pumps during the initial 10 to 

was adopted for all studies. When longer buildup times were noted in the 2-year studies, the T, times 

15 air changes per hour, the theoretical value for T, is approximately 12.5..minutes. A T, of 12 minutes 

17 minutes in the 13-week studies and 10 to 15 minutes in the 2-year studies. At a chamber airflow rate of 

10 minutes for the 2-year studies. The Tw in chambers with animals was determined to be 15 to 

the start of vapor generation (T,) without animals was 9 to 12 minutes for the 13-week studies and 9 to 

with and without animals present in the chambers. The time to achieve 90% of target concentration after 

Chamber Concennution Monitoring. Buildup and decay rates for chamber concentrations were determined 

throughout the 13-week and 2-year studies. 

a single line. Chamber atmosphere uniformity (5% relative standard deviation) was maintained 

on-line gas chromatograph with the multiport sample valve disabled to allow continuous monitoring from 

approximately every 90 days during the 2-year studies. Vapor concentration was determined using the 

the 13-week studies, once during the 13-week studies, prior to the start of the 2-year studies, and 

Uniformity of vapor concentration in the inhalation exposure chambers was evaluated prior to the start of 
244 Acetonitrile, NTP TR 447 

counts above the minimum resolvable level were measured in any chamber. 

generation. The minimum resolution of the Gardner counter is approximately 200 particles/cm3. No 

Schenectady, NY) was used to check all chambers for any aerosol inadvertently produced during 

study with animals in the chambers, a Gardner Qpe CN Small Particle Detector (Gardner Associates, 

Prior to the start of the 2-year studies, before animals were placed in the chambers and again during the 

At least 99% of all individual concentration measurements were within 10% of the target concentrations. 

all chambers for the entire 2-year studies were 100% of the target with relative standard deviations of 3%. 

concentration measurements were within 10% of the target concentrations. The means of concentration in 

and 100% of the target, with relative standard deviations of 3%. .At least 98% of all individual 

The means of concentratims in all chambers for the 15-month interim evaluation ranged between 99% 

H6-Hll. 

The monthly mean exposure concentrations in the chambers for the 2-year studies are presented in Figures 

Summaries of the chamber concentrations for the 13-week and 2-year studies are in Tables H1 and H2.' 

generation. 

chemical degradation occurred in the chambers or in the vapor distribution line as a result of test chemical 

alcohol, and acrylonitrile using gas chromatography. The results of these analyses indicated that no test 

and dimethylformamide and the desorbed samples were analyzed for acetonitrile, propionitrile, allyl 

the vapor distribution line using charcoal-filled adsorbent tubes. The tubes were desorbed with methanol 

the vapor distribution line. Samples were obtained from occupied and unoccupied exposure chambers and 

the 100 and 1,600 ppm chambers, and during the 2-year studies in the 50 and 400 ppm chambers and in 

Acetonitrile Degradation. Studies of acetonitrile degradation were conducted during the 13-week studies in 

locations. 

distribution line, and.their relative amounts were substantially less than 1% 'by weight at all sampling 

indicated that allyl alcohol and acrylonitrile were below the detection limit in the chambers and vapor 

in generated test atmospheres ranged from ~0.1% to 0.2% by weight relative to acetonitrile. Analyses, 

observed in generated atmospheres of acetonitrile. However, the measured concentration of propionitrile 

the test material, although the concentrations were very low. Of these impurities, only propionitrile was 

Sample analysis indicated that propionitrile, acrylonitrile, and allyl alcohol were present as impurities in 

maintained. 

acetonitrile samples from the generator .reservoir indicated that the purity of the test chemical .was 

days 7, 14, and 21 during the 13-week studies, and after day 6 of.the 2-year studies. Analysis of 

Samples of acetonitrile were obtained from the generator reservoir when the reservoir was filled, after 
CBnemnical Characterization and Generation of Chamber Concentrations 245 

Infrared Absorption Spectrum of Acetonitrile 

FIGURE Hl 
A 

4 

h 

t 

2. 

E. 

B 

cc 

I-- t2 

W 
Chemical Characterization and Generation of Chamber Concentrations 241 

TUQL 

LIOUlb 

DlSTRlOUTlON 

SCRtW 

SLCURlrn 

CCNCRATOR 

Acetonitrile Liquid Vapor Generator 

FIGURE H3a 
M3lA 301s M3lh lNOtld 

t 

250 Acetonitrile, NTP 'TR 447 

Acetonitrile Inhalation Exposure Chamber for the 2-Year Studies 

FIGURE H4b 
Chemical Characterization and Generation of Chamber Concentrations 251 

'r 

ROOtl 

STORAGE 

CHEMICAL 

x' 

W 

P 

u 

D 

NOT USED 

ROOfl 323 

CHRONIC 

ACETONITRILE 

ROOM 418 

ROOM 420 

1 

ROOM 424 

Acetonitrile Exposure Suite 

FIGURE H5 
252 Acetonitrile, NTP TR 447 

Summary of Chamber Concentrations in the 13-Week Inhalation Studies of Acetonitrile 

TABLE H1 

Target Concentration Total Number of Readings 

(PPm) (PP@ 

Average Concentrationa 

Rat Chambers 

800 

400 

200 

100 

1.600 

Mouse Chambers 

800 

400 

200 

100 

1,600 

819 

818 

823 

823 

778 

836 

835 

840 

840 

793 

799 f 42 

397 f 25 

199 f 14 

101 f 5.5 

1,590 f 76 

798 f 49 

397 f 27 

,199 f 14 

101 f 6.1 

'1,590 f 88 

a ,Mean f standard deviation 

Summary of Chamber Concentrations in the 2-Year Inhalation Studies .of Acetonitrile 

TABLE H2 

Target Concentration Total Number of Readings 

(PPm) 

Average Concentrationa 

(PPm) 

Rat Chambers 

400 

200 

100 

'Mouse Chambers 

200 

1'00 

50 

:6,020 

6,242 

6,262 

6,371 

6,262 

.6,244 

100.k 2.9 

400 k 11:4 

,199 f'6:O 

200 & .6.0 

100 * 3.4 

49.8 k 6.6 

a Mean * standard deviation 
ficetonitrile - Chronic 

From 31 Mar 1988 through 23 Mar 1990 

Monthly Mean b Standard Davlation 

18F3 ppm - Rats 

1 

1, 

I t T 

I t I 

1 1 

I t t t 1 

1 t I 

1 I 

I I 

1 I 

I 
254 Acetonitrile, NTP TR 447 

in the '200 ppm ,Acetonitrile Rat .Exposure Chamber ,for ',the 2-Year 'Study 

Monthly Mean Concentration .and 'Standard Deviation 

FIGURE H7 
Chemical Characterization and Generation of Chamber Concentrations 255 

in the 400 ppm Acetonitrile Rat Exposure Chamber for the 2-Year Study 

Monthly Mean Concentration and Standard Deviation 

FIGURE HS 
n 

3' 3 

33 

DO 

5pl I 

03 I 

9sz 
L 

"-1 

3- 

_c_ 

-c 

3.- 

4- 

-+- 

3- 

+ 

4- 

+ 

-+ 

+ 

+ 

us 

r, 

33 

b (D 

DY 0 

-D 

or]' I -7pl 

LSZ 
258 .Acetoditrile, .NW TR 447 

- .rd 

dt 

>E 

c 

CE 

+, 

't 1 00 

in the 200 ppm Acetonitrile Mouse Exposure Chamber for the 2-Year Study 

Monthly Mean Concentration and Standard Deviation 

FIGURE H11 
259 

AND CONTAMINANT LEVELS 

INGREDIENTS, NUTRIENT COMPOSITION, 

APPENDIX I 

IN NIH-07 RAT AND MOUSE RATION 

TABLE I1 Ingredients of NIH-07 Rat and Mouse Ration 

TABLE I2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration 

TABLE I3 Nutrient Composition of NIH-07 Rat and Mouse Ration 

TABLE I4 Contaminant Levels in NIH-07 Rat and Mouse Ration 

............................... 

262 

261 

260 

260 

...................... 

....................... 

........................ 
260 Acetonitrile, NTP TR 447 

Ingredients of NIH-07 Rat and Mouse Rationa 

TABLE I1 

Ingredientsb Percent by Weight 

Premixes (vitamin and mineral) 

Salt 

Ground limestone 

Dicalcium phosphate 

Dry molasses 

Dried brewer's yeast 

Soy oil 

Corn gluten meal (60% protein) 

Alfalfa meal (dehydrated, 17% protein) 

Dried skim milk 

Wheat middlings 

Fish meal (60% protein) 

Soybean meal (49% protein) 

Ground hard winter wheat 

Ground #2 yellow shelled corn 

10.00 

10.00 

12.00 

23.00 

24.50 

0.25 

0.50 

050 

1.25 

1.50 

2.00 

2.50 

3.00 

4.00 

5.00 

a NCI, 1976; NIH, 1978 

Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed: 

Vitamins and Minerals in NIH-07 Rat and Mouse Rationa 

TABLE I2 

Amount Source 

Vitamins 

A 

K3 

D3 

Thiamine 

Riboflavin' 

&Pantothenic acid: 

Niacin 

Folic acid 

Choline 

d-a-Tocopheryl. acetate 

Biotin 

Pyridoxine 

B12 

Minerals. 

Cobalt: 

Iodine, 

Cbpper 

Zinc 

Manganese. 

Iron 

18.0 g 

30.0 g 

2.2-g 

560.0 g 

20,000.1u 

2.8 g 

4,600,000 IU 

5;5oo,ooo .IU 

10.0 g 

3.4 g4,000 fig 

140.0:mg 

1.7 g 

Menadione 

D-activated.anirna1 sterol 

Stabilized vitamin A>palmitate or acetate. 

Choline chloride 

&Calcium pantothenate 

Thiamine mononitrate 

&Biotin. 

Pyridoxine:liydrochloride 

120.0 'g 

Cobalt carbonate 

Calcium. iodate 

Copper sulfate. 

Zinc oxide. 

Manganous oxide 

Ironwlfate' 

0.4 g. 

1.4-g: 

16.0 g 

60.0 g, 

4;0 g 

a Per ton'(2;000~16), of finished product. 
Feed Analyses 261 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

TABLE I3 

Nutrient Deviation 

Mean 2 Standard 

Range Number of Samples 

Ash (% by weight) 

Crude fiber (% by weight) 

Crude fat (% by weight) 

Protein (% by weight) 

Amino Acids (% of total diet) 

Valine 

Qrosine 

Tryptophan 

Threonine 

Phenylalanine 

Methionine 

Lysine 

Leucine 

Isoleucine 

Histidine 

Glycine 

Cystine 

Arginine 

Essential Fatty Acids (% of total diet) 

Linolenic 

Linoleic 

Vitamins 

Choline (ppm) 

Vitamin B12 (ppb) 

Biotin (ppm) 

Folic acid (ppm) 

Pyndoxine (ppm) 

Pantothenic acid (ppm) 

Niacin (ppm) 

Riboflavin (ppm) 

Thiamine (ppm) 

a-Tocopherol (ppm) 

Vitamin D (IU/kg) 

Vitamin A (IU/kg) 

Minerals 

Sulfur (%) 

Magnesium (%) 

Sodium (%) 

Chloride (%) 

Potassium (%) 

Phosphorus (%) 

Calcium (%) 

Zinc (ppm) 

Manganese (ppm) 

Cobalt (ppm) 

Chromium (ppm) 

Iodine (ppm) 

Iron (PPm) 

Copper (PPm) 

6.63 f 0.29 

3.61 f 0.39 

5.39 k 0.34 

22.93 f 0.76 

1.089 k 0.057 

0.677 f 0.105 

0.223 f 0.160 

0.896 f 0.055 

0.994 & 0.125 

0.437 k 0.115 

1.273 f 0.051 

1.972 f 0.052 

0.917 f 0.034 

0.580 f 0.024 

1.160 f 0.050 

0.306 k 0.075 

1.287 k 0.084 

0.277 k 0.036 

2.389 f 0.233 

18.69 f 2.53 

36.92 k 9.32 

4,450 f 1,382 

6,532 f 1,894 

3,068 2 314 

40.14 & 20.04 

0.267 f 0.049 

2.51 f 0.64 

9.25 k 2.62 

30.30 f 3.60 

100.95 f 25.92 

7.92 f 0.93 

11.50 f 2.40 

58.14 k 9.91 

92.24 k 5.35 

356.2 f 90.0 

0.274 f 0.063 

0.168 k 0.008 

0.315 f 0.344 

0.526 f 0.092 

0.887 f 0.067 

0.95 f 0.03 

1.24 f 0.11 

3.70 & 1.14 

1.71 f 0.45 

0.797 f 0.23 

6.11 - 7.30 

2.80 - 4.30 

4.60 - 5.90 

21.70 - 24.20 

0.962 - 1.170 

0.564 - 0.794 

0.107 - 0.671 

0.824 - 0.985 

0.665 - 1.110 

0.306 - 0.699 

1.200 - 1.370 

1.850 - 2.040 

0.867 - 0.965 

0.531 - 0.608 

1.060 - 1.220 

0.181 - 0.400 

1.100 - 1.390 

0.210 - 0.320 

1.830 - 2.570 

2,400 - 3,430 

10.6 - 65.0 

0.19 - 0.35 

1.80 - 3.70 

5.60 - 14.0 

23.0 - 34.6 

65.0 - 150.0 

6.10 - 9.00 

14.0 - 28.0 

22.5 - 48.9 

3,000 - 6,300 

4,180 - 12,140 

8.090 - 15.39 

46.10 - 81.60 

81.70 - 99.40 

255.0 - 523.0 

0.208 - 0.420 

0.151 - 0.180 

0.258 - 0.370 

0.380 - 0.635 

0.772 - 0.971 

0.90 - 1.00 

1.00 - 1.54 

0.490 - 1.150 

0.85 - 2.09 

1.52 - 5.83 

26 

26 

26 

26 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

9 

9 

9 

10 

10 

10 

10 

10 

9 

10 

26 

9 

4 

26 

10 

10 

10 

10 

10 

10 

10 

10 

8 

8 

26 

26 

6 

9 
262 Acetonitrile, NTP TR 447 

Contaminant Levels in NIH-07 Rat and Mouse Ration 

TABLE I4 

Deviationa 

Mean f Standard 

Range Number of Samples 

Cadmium (ppm) 

Arsenic (ppm) 

Contaminants 

Nitrite nitrogen (ppm)d 

Nitrate nitrogen (ppm)d 

Aflatoxins (ppb)' 

Selenium-(ppm) 

Mercury (ppm)b 

Lead (PPm) 

BHT (PPm)" 

BHA (PPm)' 

N-Nitrosopyrrolidine (ppb) 

NLNitrosodimethylamine (p b)k 

Total nitrosoamines (ppb)k 

Escherichia coli (MPN/g)l 

Coliform (MPN/g)' . 

Coliform .(MPN/g)Fh 

Aerobic plate count (CFU/g)f 

R 

Pesticides (ppm) 

6-BHC 

y-BHC 

13-BHC 

,a-BHCI 

':Endosulfan sulfate 

'Endosulfan I1 

Endosulfan I 

'Malathion 

Bthyl :parathion 

Methyl :parathion 

iDiazinon 

Trithion 

(Ethion 

,Ronnel 

%Estimated PCBs 

Toxaphene 

Chlordane 

Telodrin 

Endrin 

Dieldrin 

'Methoxydhlor 

Mirex 

HCB 

DDT 

DDD 

DDE 

'Heptachlor epoxide 

Aldrin 

Heptachlor 

0.41 f 0.24 

0.25 2 0.17 

0.22 f 0.16 

<5.0 

<0.20 

0.05 ? 0:02 

1.27 rt .0.59 

1.58 * 0.63 

0.19 2 0.15 

15.86 ? 3.93 

47.15 2 215 

5.04 ? .5.37 

65,142 2 70,838 

2.28 f 1.38 

6.53 +- 3.22 

8.81 2 3.93 

3.00 ? 0.20 

< 0.1 

<0:05 

< 0:Ol 

<001 

co.01 

~0.05 

40.01 

-<0.01 

<0:01 

10.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

< 0.02 

<0.01 

co.02 

-iE 0.02 

< 0.1 

<0.05 

<0:02 

co:o1 

<0.2 

~0:03 

c0:01 

<0.01 

0.23 k '0.23 

0.05 - 0.60 

0.16 - 1.21 

0.02 - 0.11 

0.10 - 1.00 

6,700 - 320,000 

<0.10 - 3.00 

CO.10 - 3.00 

<0.10 - 0.60 

8.60 - 24.0 

0.90 - 5.40 

2.60 - 14.00 

3.60 - 19.40 

3.00 - 4:OO 

3.00 - 1,100 

3.00 - 23.0 

-<0.05 - .l.OO 

26 

26 

26 

26 

26 

25 

26 

26 

26 

26 

26 

26 

26 

26 

26 

26 

26 

26 

26 

26 

-26 

.26 

-26 

26 

26 

26 

:26 

-26 

26 

26 

26 

26 

-26 

26 

.26 

26 

26 

26 

26 

26 

26 

26 

26 

26 

26 

26 
Feed Analyses 263 

Contaminant Levels in NIH-07 Rat and Mouse Ration (continued) 

Table I4 

a For values less than the limit of detection, the detection limit is given as the mean. 

Sources of contamination: alfalfa, grains, and fish meal 

No aflatoxin measurement was recorded for the lot milled 2 October 1989. 

All values were less than the detection limit except the lot milled on 3 February 1989, which contained 0.11 ppm. 

e Sources of contamination: soy oil and fish meal 

k. All values were corrected for percent recovery. 

j All values were less than the detection limit except the lot milled on 4 April 1988, which contained 4.0 MPN/g. 

' Mean, standard deviation, and range include one large value of 1,100 MPN/g obtained in the lot milled 5 July 1988. 

h Mean, standard deviation, and range exclude one large value of 1,100 MPN/g obtained in the lot milled 5 July 1988. 

g MPN = most probable number 

CFU = colony forming units 

BHC = hexachlorocyclohexane or benzene hexachloride 
265 

APPENDIX J 

SENTINEL ANIMAL PROGRAM 

METHODS ..................................................................... 266 

RESULTS ..................................................................... 268 
266 Acetonitrile, NTP TR 447 

SENTINEL ANIMAL PROGRAM 

production source and weanling groups as the animals used for the studies of chemical compounds. 

are all subject to identical environmental conditions. The sentinel animals come from the same 

serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals 

evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via 

Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic 

optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel 

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in 

METHODS 

Associates, Inc. (Bethesda, MD), for viral titer screening. The following tests were performed: 

at terminal sacrifice. These samples were processed appropriately and were submitted to Microbiological 

For the 13-week study, samples were obtained from five male and five female controls during week 4 and 

Rats 

Complement Fixation 

Method of Analvsis 

LCM (lymphocytic choriomeningitis virus) Study termination 

Time of Analvsis 

ELISA 

Sendai 

RCV (rat coronavirus) 

PVM (pneumonia virus of mice) 

Mycoplasma pulntonis 

Mycoplasma arthritidis 

Polyoma virus 

MVM (minute virus of mice) 

KRV (Kilham rat virus) 

K (papovavirus) 

H-1 (Toolan’s H-1 virus) 

Hemagglutination Inhibition 

4 weeks and study termination 

4 weeks 

4 weeks and study termination 

4 weeks and study termination 

4 weeks and study termination 

Study termination 

Study termination 

4 weeks 

Study termination 

4 weeks and study termination 

Immunofluorescence Assay 

EDIM (epizootic diarrhea of infant mice) Study termination 
Sentinel Animal Program 267 

screened for the following: 

Blood from each collection was processed appropriately, shipped to Microbiological Associates, Inc., and 

Sera for the 24-month screening were obtained from five male and five female rats receiving 200 ppm. 

these animals at 6, 12, and 18 months. Following the 18-month bleeding, the animals were necropsied. 

allocation of the animals and were housed two per sex per exposure chamber. Sera were obtained from 

For the ?-year study, eight male and eight female rats were selected at the time of randomization and 

ELISA 

Method of Analvsis 

RCV/SDA (rat coronavirus/ 

PVM 

M. pulmonis 

M. arthritidis 

Sendai 

sialodacryoadenitis virus) 

Hemagglutination Inhibition 

KRV 

H-1 

Time of Analvsis 

6, 12, 18, and 24 months 

24 months 

24 months 

6, 12,18, and 24 months 

6,12,18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

Complement Fixation 

Method of Analvsis 

LCM Study termination 

Time of Analvsis 

ELISA 

Ectromelia virus Study termination 

GDVII (mouse encephalomyelitis virus) Study termination 

Mouse adenoma virus Study termination 

MHV (mouse hepatitis virus) Study termination 

Sendai 

Reovirus 3 

PVM 

M. pulmonis 

M. arthritidis 

Hemagglutination Inhibition 

Polyoma virus 

MVM 

K 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Immunofluorescence Assay 

EDIM Study termination 
268 Acetonitrile, NTP TR 447 

appropriately, shipped to Microbiological Associates, Inc., and screened for the following: 

obtained from five males and five females receiving 100 ppm. Blood from each collection was processed 

obtained from five males and five females at 6, 12, and 18 months. Sera for the 24-month screening were 

allocation of the animals to the various study groups and were housed in the control chamber. Sera were 

For the 2-year study, 15 male and 15 female mice were selected at the time of randomization and 

Method of Analysis Time of Analysis 

ELISA 

Sendai 

Reovirus 3 

PVM 

M.' pulmonis 

M. arthritidis 

MHV 

Mouse adenoma virus 

MVM 

LCM 

GDVII 

EDIM 

Ectromelia virus 

Hemagglutination Inhibition 

Polyoma virus 

K 

Immunofluorescence Assay 

Mouse adenoma virus 

MVM 

LCM 

EDIM 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

24 months 

24 months 

6, 12, 18, and 24 months 

6, 12, and 18 months 

6 and 12 months 

18 and 24 months 

6, 12, 18, and 24 months 

24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

18 and 24 months 

18 and 24 months 

6 and 12 months 

6, 12,18, and 24 months 

to be false positives. 

M. arthritidis infection in rats with positive titers. Accordingly, M. arthritidis-positive titers were considered 

sporadic samples were positive, and there were no clinical findings or histopathologic changes of 

have been due to a cross reaction with antibodies of nonpathogenic Mycoplasma or other agents. Only 

positive for M. arthritidis by immunoblot and Western blot procedures indicate that the positive titers may 

Three rats had positive titers to M. arthritidis at the end of the 2-year study. Further evaluation of samples 

RESULTS 
HEALTH & HUMAN SERVICES 

DEPARTMENT OF 

Central Data Management 

National Toxicology Program 

Public Health Service 

Research Triangle Park, NC 27709 

P.O. BOX 12233, MD El-02 

SPECIAL FOURTH-CLASS RATE 

Permit No. G-763 

DHHS/NIH 

POSTAGE AND FEES PAID 

April 1996 

NIH Publication No. 96-3363