## NTP TECHNICAL REPORT

ON THE


# TOXICOLOGY AND CARCINOGENESIS

STUDIES OF o -NITROTOLUENE

(CAS NO. 88-72-2)



# IN F344/N RATS AND B6C3F 1 

# MICE

(FEED STUDIES)

NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709

May 2002

NTP TR 504

NIH Publication No. 02-4438



## U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health

## FOREWORD

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention. In July 1981, the Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the relevant programs, staff, and resources from these Public Health Service agencies relating to basic and applied research and to biological assay development and validation.

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous chemicals. This knowledge is used for protecting the health of the American people and for the primary prevention of disease.

The studies described in this Technical Report were performed under the direction of the NIEHS and were conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use were in accordance with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic and chronic studies were conducted in compliance with Food and Drug Administration (FDA) Good Laboratory Practice Regulations, and all aspects of the chronic studies were subjected to retrospective quality assurance audits before being presented for public review.

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of human exposure, level of production, and chemical structure. The interpretive conclusions presented in this Technical Report are based only on the results of these NTP studies. Extrapolation of these results to other species and quantitative risk analyses for humans require wider analyses beyond the purview of these studies. Selection per se is not an indicator of a chemical’s carcinogenic potential.

Details about ongoing and completed NTP studies are available at the NTP’s World Wide Web site: http://ntp-server.niehs.nih.gov. Abstracts of all NTP Technical Reports and full versions of the most recent reports and other publications are available from the NIEHS’ Environmental Health Information Service (EHIS) http://ehis.niehs.nih.gov (800-315-3010 or 919-541-3841). In addition, printed copies of these reports are available from EHIS as supplies last. A listing of all the NTP Reports printed since 1982 appears on the inside back cover.

## NTP TECHNICAL REPORT

ON THE


# TOXICOLOGY AND CARCINOGENESIS

STUDIES OF o -NITROTOLUENE

(CAS NO. 88-72-2)



# IN F344/N RATS AND B6C3F 1 

# MICE

(FEED STUDIES)

NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709

May 2002

NTP TR 504

NIH Publication No. 02-4438



## U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health

### 2



# CONTRIBUTORS

NTP Pathology Working Group Evaluated slides and prepared pathology report on rats (August 31, 2000)


### P.K. Hildebrandt, D.V.M., Chairperson

ILS, Inc. 

### J. Everitt, D.V.M.

Chemical Industry Institute of Toxicology 

### G.P. Flake, M.D.

National Toxicology Program 

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

National Toxicology Program 

### J.E. Heath, D.V.M.

Southern Research Institute 

### R.A. Herbert, D.V.M., Ph.D.

National Toxicology Program 

### A. Nyska, D.V.M.

National Toxicology Program 

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

Experimental Pathology Laboratories, Inc. 

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

National Toxicology Program 

### T. Suwa, D.V.M., Ph.D., Observer

National Toxicology Program

Evaluated slides and prepared pathology report on mice (August 24, 2000)


### M.P. Jokinen, D.V.M., Chairperson

Pathology Associates International 

### D.R. Farnell, D.V.M., Ph.D.

Southern Research Institute 

### G.P. Flake, M.D.

National Toxicology Program 

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

National Toxicology Program 

### R.A. Herbert, D.V.M., Ph.D.

National Toxicology Program 

### A. Nyska, D.V.M.

National Toxicology Program 

### C. Okerberg, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc. 

### K. Ozaki, D.V.M., Ph.D., Observer

National Toxicology Program 

### J. Pletcher, D.V.M., M.P.H.

Pathology Associates International 

### C. Qualls, D.V.M., Ph.D.

Glaxo Wellcome, Inc. 

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

National Toxicology Program 

## National Toxicology Program Evaluated and interpreted results and reported findings



### J.K. Dunnick, Ph.D., Study Scientist 

### D.W. Bristol, Ph.D. J.R. Bucher, Ph.D. L.T. Burka, Ph.D. J.R. Hailey, D.V.M. J.K. Haseman, Ph.D. R.A. Herbert, D.V.M., Ph.D. R.R. Maronpot, D.V.M. D.P. Orzech, M.S. S.D. Peddada, Ph.D. G.N. Rao, D.V.M., Ph.D. J.H. Roycroft, Ph.D. R.C. Sills, D.V.M., Ph.D. C.S. Smith, Ph.D. G.S. Travlos, D.V.M. K.L. Witt, M.S., Integrated Laboratory Systems, Inc.



## Southern Research Institute Conducted studies and evaluated pathology findings


### C.D. Hébert, Ph.D., Study Director 

### J.D. Prejean, Ph.D., Principal Investigator 

### W.R. Richter, D.V.M., M.S., Principal Investigator 

### J.E. Heath, D.V.M. R.B. Thompson, D.V.M., Ph.D.


## Experimental Pathology Laboratories, Inc. Provided pathology quality assurance 

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

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


## Dynamac Corporation Prepared quality assurance audits


### S. Brecher, Ph.D., Principal Investigator

### o-Nitrotoluene, NTP TR 504 3



## Analytical Sciences, Inc. Provided statistical analyses


### R.W. Morris, M.S., Principal Investigator 

### L.J. Betz, M.S. K.P. McGowan, M.B.A. J.T. Scott, M.S. 

## Biotechnical Services, Inc. Prepared Technical Report


### S.R. Gunnels, M.A., Principal Investigator 

### M.P. Barker, B.A. L.M. Harper, B.S. E.S. Paal, M.S.J. D.C. Serbus, Ph.D. W.D. Sharp, B.A., B.S. R.A. Willis, B.A., B.S.

### 4



### ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . . . . . . . . . . . . . . . . 13

TECHNICAL REPORTS REVIEW SUBCOMMITTEE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . . . . . . . . . . . . 15

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Appendix A Summary of Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Appendix B Summary of Lesions in Female Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Appendix C Summary of Lesions in Male Mice in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

Appendix D Summary of Lesions in Female Mice in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

Appendix E Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

Appendix F o- Nitrobenzoic Acid, o-Nitrobenzylmercapturic Acid, and o-Aminobenzoic Acid — Biomarkers of Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

Appendix G Organ Weights and Organ-Weight-to-Body-Weight Ratios . . . . . . . . . . . . . . . . . . . . . . . 295

Appendix H Chemical Characterization and Dose Formulation Studies . . . . . . . . . . . . . . . . . . . . . . . . 297

Appendix I Feed and Compound Consumption in the 2-Year Feed Studies of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Appendix J Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

Appendix K Sentinel Animal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 

# CONTENTS

### o-Nitrotoluene, NTP TR 504 5



### Appendix L Comparative Metabolism Studies of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

Appendix M Genetic Alterations in ras , p53 , and $ -Catenin Genes in Hemangiosarcomas of B6C3F 1 

### Mice Following Exposure to o-Nitrotoluene for 2 Years . . . . . . . . . . . . . . . . . 347

### 6 o-Nitrotoluene, NTP TR 504



## SUMMARY

Background: Approximately 30 million pounds of ortho -nitrotoluene are used annually in the United States in the production of agricultural and rubber chemicals and dyes. We studied the effects of

o -nitrotoluene on male and female rats and mice to identify potential toxic or carcinogenic hazards to humans.

Methods: We gave feed containing o -nitrotoluene to 12 groups of 60 animals for 2 years. Male and female rats received 625, 1,250, or 2,000 parts per million (ppm) of o -nitrotoluene in their feed (equivalent to 0.0625%, 0.125%, or 0.2%); male and female mice received 1,250, 2,500, or 5,000 ppm (0.125%, 0.25%, or 0.5%). Groups of animals receiving untreated feed served as controls. Tissues from more than 40 sites were examined for every animal.

Results: All of the male rats and male mice receiving the highest concentrations died before the end of the study, mainly from chemically induced tumors. Survival rates were also lower in females receiving

o- nitrotoluene because of chemically induced tumors. Male rats had high incidences of malignant mesotheliomas and skin fibromas or fibrosarcomas; incidences of liver and lung tumors also increased. Most female rats receiving o -nitrotoluene developed mammary gland fibroadenomas; skin fibromas and liver adenomas were also seen. Virtually all male mice exposed to the two highest concentrations of

o- nitrotoluene developed hemangiosarcomas (circulatory system cancers) and died early. Carcinomas of the large intestine were seen in male mice receiving lower doses. Female mice receiving o -nitrotoluene also developed lethal hemangiosarcomas, liver adenomas and carcinomas, and large intestine carcinomas.

Conclusions: We conclude that o -nitrotoluene was clearly carcinogenic in male and female rats and mice, causing a variety of malignant tumors.

### 7



### o-Nitrotoluene is used to synthesize agricultural and rubber chemicals, azo and sulfur dyes, and dyes for cotton, wool, silk, leather, and paper. o-Nitrotoluene was nominated for study by NIOSH and the NTP based on its considerable human exposure as well as the absence of long-term studies of carcinogenicity in rodents. Male and female F344/N rats and B6C3F 1 

### mice were exposed
to o- nitrotoluene (greater than 99% pure) in feed for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium , cultured Chinese hamster ovary cells, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes.



# 2-Y EAR 

# S TUDY IN 

# R ATS In the core study, groups of 60 male and 60 female rats were fed diets containing 625, 1,250, or 2,000 ppm

o- nitrotoluene (equivalent to average daily doses of approximately 25, 50, or 90 mg o- nitrotoluene/kg body weight to males and 30, 60, or 100 mg/kg to females) for 105 weeks. In a 3-month stop-exposure study, groups of 70 male rats were fed diets containing 2,000 or 5,000 ppm o- nitrotoluene (equivalent to average daily doses of approximately 125 or 315 mg/kg) for 13 weeks followed by undosed feed for the remainder of the study. A group of 70 male rats receiving undosed feed served as a control group for both male rat studies; 60 female rats receiving undosed feed were the control group for the female core study. Ten control males and 10 males from each stop-exposure group were sacrificed at 3 months.


## Survival, Body Weights, and Feed Consumption All 2,000 ppm core study, all 5,000 ppm stop-exposure, and all but three core study 1,250 ppm male rats died before the end of the studies. Survival of 625 ppm core study and 2,000 ppm stop-exposure males and of 2,000 ppm females was significantly less than that of the controls. Mean body weights of all exposed groups of males except the 625 ppm group were generally less than those of the controls throughout the study. Mean body weights of 2,000 ppm females were less than thoseof the controls during year 2 of the study. Feed consumption by exposed groups of rats was similar to that by the controls. 

# ABSTRACT

o-NITROTOLUENE

CAS No. 88-72-2

Chemical Formula: C 7 

### H 7

### NO 2 

### Molecular Weight: 137.14

Synonyms: 2-Methylnitrobenzene; o -methylnitrobenzene; 2-nitrotoluene; 2-nitrotoluol 

## NO 2 

## CH 3

### 8 o-Nitrotoluene, NTP TR 504



## Biomarkers of Exposure Three urinary metabolites were followed during the study as biomarkers of exposure. The ratios of

o- nitrobenzoic acid to creatinine and of o- nitrobenzylmercapturic acid to creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure concentration in males and females. The ratio of o- aminobenzoic acid to creatinine was not related to exposure concentration.


## Pathology Findings The incidences of malignant mesothelioma in male ratsoccurred with positive trends in both the core and stopexposure studies and were significantly greater inexposed groups than in the controls. Incidences of subcutaneous skin neoplasms (fibroma, fibrosarcoma, and lipoma) were increased in exposed groups of males,while the incidences of fibroma or fibrosarcoma (combined) were increased in exposed females. In all exposed groups of males and females except 2,000 ppm core study males, the incidences of mammary glandfibroadenoma were significantly increased. The incidences of mammary gland hyperplasia were significantly increased in 625 and 1,250 ppm females.

Increased incidences of mesothelioma, skin neoplasms, and mammary gland fibroadenoma in the stop-exposure males indicated that 3 months of dosing were sufficient to produce a carcinogenic effect.

Liver weights of 5,000 ppm stop-exposure males were significantly greater than those of the controls at 3 months. The incidences of hepatocellular adenoma in2,000 ppm core study males and females and of hepatocellular adenoma or carcinoma (combined) in 2,000 ppmcore study and 5,000 ppm stop-exposure males were significantly increased. Cholangiocarcinoma occurred inthree 5,000 ppm stop-exposure males, and a single hepatocholangiocarcinoma occurred in a 625 ppm male and in a 2,000 ppm core study male. Nonneoplastic lesions of the liver included eosinophilic, mixed cell, and clear cell foci in exposed groups of males and females and mixed cell infiltrate in exposed males and basophilic focus in exposed females.

The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 5,000 ppm stop-exposure males, as were alveolar/bronchiolar hyperplasia in most exposed groups of males and females. The incidences of hematopoietic cell proliferation of the spleen and of hyperplasia of the mandibular lymph node (females) and bone marrow were increased in exposed groups of males at 3 months and/or 2 years and in exposed groups of females at 2 years.

The incidences of mononuclear cell leukemia were significantly decreased in all groups of males exposed to 1,250 ppm or greater and in all exposed groups offemales; the incidence of testicular interstitial cell adenoma was significantly decreased in 5,000 ppm stopexposure males.



# 2-Y EAR 

# S TUDY IN 

# M ICE Groups of 60 male and 60 female mice were fed diets containing 0, 1,250, 2,500, or 5,000 ppm o- nitrotoluene (equivalent to average daily doses of approximately 165, 360, or 700 mg/kg to males and 150, 320, or 710 mg/kg to females) for 105 weeks.


## Survival, Body Weights, and Feed Consumption All 2,500 and 5,000 ppm males died before the end of the study. Survival of 1,250 ppm males and 5,000 ppm females was significantly less than that of the controls. Mean body weights of exposed males and 5,000 ppm females were generally less than those of the controls throughout the study, and those of 2,500 ppm femaleswere less during the second year of the study. Feed consumption by 5,000 ppm males was less than that by the controls.


## Biomarkers of Exposure Three urinary metabolites were followed during the study as biomarkers of exposure. The ratios of

o- nitrobenzoic acid to creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related toexposure concentration in males and females. The concentrations of o- nitrobenzylmercapturic acid and

o- aminobenzoic acid were below the limit of quantitation at most time points.


## Pathology Findings The incidences of hemangiosarcoma in all exposedgroups of males and in 5,000 ppm females were significantly greater than those in the controls. Large intestine (cecum) carcinomas were observed in all exposed groups except 5,000 ppm males.

### o-Nitrotoluene, NTP TR 504 9



### The incidences of hepatocellular neoplasms were significantly increased in 2,500 and 5,000 ppm females. Nonneoplastic liver lesions including eosinophilic and basophilic foci and minimal to mild necrosis were enhanced in exposed males and females. Also present were focal hepatocyte syncytial alteration in exposed males and hepatocyte necrosis and focal hepatocyte cytoplasmic vacuolization in 5,000 ppm females.

Renal tubule pigmentation occurred more frequently in exposed groups of males and in 5,000 ppm females than in the controls. Olfactory epithelial degeneration occurred in every male and female mouse exposed to 2,500 or 5,000 ppm, and the severity of this lesion increased with increasing exposure concentration.



# G ENETIC 

# T OXICOLOGY

o-Nitrotoluene was not mutagenic in any of several strains of S. typhimurium , with or without metabolic activation enzymes (S9). Sister chromatid exchanges were significantly increased in cultured Chinese hamster ovary cells following exposure to o- nitrotoluene in the presence of S9; an equivocal response was seen without S9. o-Nitrotoluene did not induce chromosomal aberrations in cultured Chinese hamster ovary cells, with or without S9. o-Nitrotoluene did not induce a significant increase in the frequency of micronuclei in bone marrow polychromatic erythrocytes of male rats or male mice when administered by intraperitoneal injection. Results of a peripheral blood micronucleus test were equivocal for male mice and negative for female mice administered

o- nitrotoluene in feed for 13 weeks.



# C ONCLUSIONS Under the conditions of these studies, there was clear evidence of carcinogenic activity * of o- nitrotoluene in male rats based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma, and liver neoplasms. The increasedincidences of lung neoplasms in male rats were also considered to be exposure related. There was clear evidence of carcinogenic activity of o- nitrotoluene in female ratsbased on increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma. The increased incidence of hepatocellular adenoma in female rats was also considered to be exposure related. There

was clear evidence of carcinogenic activity of

o- nitrotoluene in male and female mice based on increased incidences of hemangiosarcoma, carcinoma ofthe large intestine (cecum), and hepatocellular neoplasms (females only). Exposure to o- nitrotoluene caused increased incidences of nonneoplastic lesions of the mammary gland (females only), liver, bone marrow, spleen, lung, and mandibular lymph node (females only) in male and female rats and of the liver, kidney, and nose in male and female mice.
Decreased incidences of mononuclear cell leukemiaoccurred in exposed groups of rats; the incidence of testicular interstitial cell adenoma was decreased in exposed male rats.

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 13. A summary of the Technical Reports Review Subcommittee comments and public discussion on this Technical Report appears on page 15.

### 10 o-Nitrotoluene, NTP TR 504



### Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of o- Nitrotoluene

Male Female Male Female F344/N Rats F344/N Rats B6C3F 1 

### Mice B6C3F 1 

### Mice

Concentrations in feed

Body weights

Survival rates

Nonneoplastic effects 0, 625, 1,250, or 2,000 ppm and 2,000 and 5,000 ppm stop-exposure

1,250, 2,000, and 2,000and 5,000 ppm stopexposure groups less than the control group

39/60, 18/60, 3/60, 0/60, 11/60, 0/60

Liver : eosinophilic focus (7/60, 18/60, 29/60, 24/60, 15/60, 13/60); mixed cell focus (5/60, 7/60, 12/60, 6/60, 12/60, 8/60); clear cell focus (29/60, 29/60, 34/60, 31/60, 30/60, 34/60); mixed cell cellular infiltration (1/60, 5/60, 11/60, 20/60, 15/60, 33/60) Bone marrow : hyperplasia (2/60, 25/60, 43/60, 45/60, 37/60, 33/60) Spleen : hematopoietic cell proliferation (7/60, 33/60, 38/60, 47/60, 36/60, 35/60) Lung : alveolar epithelial hyperplasia (2/60, 8/60, 3/60, 7/60, 15/60, 29/60) 0, 625, 1,250, or 2,000 ppm

2,000 ppm group less than the control group

47/60, 47/60, 39/60, 33/60

Mammary gland : hyperplasia (14/60, 36/60, 30/60, 19/60) Liver : eosinophilic focus (5/60, 12/59, 25/60, 32/60); mixed cell focus (6/60, 9/59, 11/60, 28/60); clear cell focus (16/60, 30/59, 28/60, 33/60); basophilic focus (51/60, 56/59, 60/60, 54/60) Bone marrow : hyperplasia (2/60, 7/60, 15/60, 24/60) Spleen : hematopoietic cell proliferation (22/60, 38/59, 48/60, 48/59) Lung : alveolar epithelial hyperplasia (6/60, 14/60, 16/60, 9/60) L

ymph node (mandibular) : lymphoid hyperplasia (3/60, 5/60, 6/59, 15/59) 0, 1,250, 2,500, or 5,000 ppm

Exposed groups less than the control group

52/60, 34/60, 0/60, 0/60

Liver : eosinophilic focus (3/60, 14/59, 1/57, 1/60); basophilic focus (0/60, 6/59, 4/57, 0/60); necrosis (1/60, 15/59, 27/57, 30/60); focal hepatocyte syncytial alteration (16/60, 26/59, 43/57, 39/60) Kidney : renal tubule pigmentation (1/58, 6/59, 32/58, 35/60) Nose : olfactory epithelial degeneration (0/60, 36/60, 60/60, 60/60) 0, 1,250, 2,500, or 5,000 ppm

2,500 and 5,000 ppm groups less than the control group

52/60, 46/60, 47/60, 5/60

Liver : eosinophilic focus (2/60, 3/59, 6/59, 28/60); basophilic focus (1/60, 6/59, 2/59, 6/60); necrosis (3/60, 0/59, 2/59, 13/60); focal hepatocyte necrosis (0/60, 0/59, 0/59, 6/60); focal hepatocyte cytoplasmic vacuolization (1/60, 2/59, 2/59, 9/60) Kidney : renal tubule pigmentation (0/59, 1/56, 3/58, 35/59) Nose : olfactory epithelial degeneration (0/60, 28/60, 59/59, 57/57)

### o-Nitrotoluene, NTP TR 504 11



### Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of o- Nitrotoluene

Male Female Male Female F344/N Rats F344/N Rats B6C3F 1 

### Mice B6C3F 1 

### Mice

Neoplastic effects

Decreased incidences

Level of evidence of carcinogenic activity Mesothelium : malignant mesothelioma (2/60, 20/60, 29/60, 44/60, 44/60, 54/60) Skin (subcutaneous) : lipoma (0/60, 4/60, 13/60, 13/60, 10/60, 12/60); fibroma (5/60, 46/60, 52/60, 59/60, 45/60, 52/60); fibrosarcoma (0/60, 7/60, 17/60, 20/60, 8/60, 12/60); fibroma or fibrosarcoma (5/60, 47/60, 55/60, 59/60, 47/60, 53/60) Mammary gland : fibroadenoma (0/60, 7/60, 10/60, 2/60, 13/60, 20/60) Liver : hepatocellular adenoma (2/60, 3/60, 3/60, 7/60, 3/60, 4/60); hepatocellular adenoma or carcinoma (3/60, 3/60, 3/60, 8/60, 3/60, 6/60); cholangiocarcinoma (0/60, 0/60, 0/60, 0/60,0/60, 3/60); hepatocholangiocarcinoma (0/60, 1/60, 0/60, 1/60, 0/60, 0/60) Lung : alveolar/ bronchiolar adenoma (1/60, 5/60, 1/60, 2/60, 3/60, 8/60); alveolar/ bronchiolar adenoma or carcinoma (2/60, 5/60, 1/60, 2/60, 3/60, 11/60)

Mononuclear cell leukemia : (30/60, 21/60, 3/60, 3/60, 13/60, 1/60) T

estis : Interstitial cell adenoma (55/60, 53/60, 51/60, 46/60, 50/60, 27/60)

Clear evidence Skin (subcutaneous) : fibroma (3/60, 3/60, 18/60, 20/60); fibroma or fibrosarcoma (3/60, 3/60, 21/60, 22/60) Mammary gland : fibroadenoma (23/60, 47/60, 52/60, 56/60) Liver : hepatocellular adenoma (1/60, 0/59, 1/60, 6/60)

Mononuclear cell leukemia : (21/60, 6/60, 4/60, 5/60)

Clear evidence Circulatory system : hemangiosarcoma (4/60, 17/60, 55/60, 60/60) Large intestine (cecum) : carcinoma (0/60, 12/60, 9/60, 0/60)

None

Clear evidence Circulatory system : hemangiosarcoma (0/60, 2/60, 3/60, 50/60) Large intestine (cecum) : carcinoma (0/60, 1/60, 4/60, 3/60) Liver : hepatocellular adenoma (7/60, 5/59, 19/59, 29/60); hepatocellular carcinoma (2/60, 4/59, 6/59, 16/60); hepatocellular adenoma or carcinoma (9/60, 9/59, 24/59, 39/60)

None

Clear evidence

### 12 o-Nitrotoluene, NTP TR 504

Genetic toxicology Salmonella typhimurium gene mutations: Sister chromatid exchanges

Cultured Chinese hamster ovary cells in vitro: Chromosomal aberrations

Cultured Chinese hamster ovary cells in vitro: Micronucleated erythrocytes

Rat bone marrow in vivo: Mouse bone marrow in vivo: Mouse peripheral blood in vivo: Negative in strains TA98, TA100, TA1535, and TA1537 with and without S9

Positive with S9; equivocal without S9

Negative with and without S9

Negative in two protocols Negative Equivocal in male mice; negative in female mice 

### Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of o- Nitrotoluene

### o-Nitrotoluene, NTP TR 504 13



## EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia th an control animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limitedset of conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency fo r Research on Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studi es such as those conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies.

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the e vidence observed in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised in March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic ac tivity. For each separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is selected to describe the findings. These categories refer to the strength of the experimental evidence and not to potency or mechanism.

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

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy.

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

incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for cl ear evidence.

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

neoplasms that may be chemical related.

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

malignant or benign neoplasms.

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

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

For studies showing multiple chemical-related neoplastic effects that if considered individually would be assigned to differentlevels of evidence categories, the following convention has been adopted to convey completely the study results. In a study with clear evidence of carcinogenic activity at some tissue sites, other responses that alone might be deemed some evidence are indicated as “were also related” to chemical exposure. In studies with clear or some evidence of carcinogenic activity, other responses that alone might be termed equivocalevidence are indicated as “may have been” related to chemical exposure.

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would exte nd the actual boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the bo rderline between two adjacent levels. These considerations should include:

• adequacy of the experimental design and conduct; • occurrence of common versus uncommon neoplasia; • progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; • some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is impo ssible to

identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to assume that benign neoplasms of those types have the potential to become malignant; • combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or tissue ; • latency in tumor induction; • multiplicity in site-specific neoplasia; • metastases; • supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same le sion in

another sex or species); • presence or absence of dose relationships; • statistical significance of the observed tumor increase; • concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm; • survival-adjusted analyses and false positive or false negative concerns; • structure-activity correlations; and • in some cases, genetic toxicology.

### 14 o-Nitrotoluene, NTP TR 504



## NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS TECHNICAL REPORTS REVIEW SUBCOMMITTEE

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on o- nitrotoluene on May 3, 2001 are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing the NTP studies:

• to ascertain that all relevant literature data have been adequately cited and interpreted, • to determine if the design and conditions of the NTP studies were appropriate, • to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, • to judge the significance of the experimental results by scientific criteria, and • to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses.



### Stephen S. Hecht, Ph.D., Chairperson

University of Minnesota Cancer Centers Minneapolis, MN


### Linda A. Chatman, D.V.M., Principal Reviewer

Pfizer, Inc. Groton, CT


### Harold Davis, D.V.M., Ph.D.

Preclinical Safety Assessment Amgen, Inc. Thousand Oaks, CA 

### Yvonne P. Dragan, Ph.D., Principal Reviewer

School of Public Health Ohio State University Columbus, OH


### Norman R. Drinkwater, Ph.D., Principal Reviewer

McArdle Laboratory for Cancer Research University of Wisconsin-Madison Madison, WI 

### James E. Klaunig, Ph.D.

Division of Toxicology Department of Pharmacology and Toxicology Indiana University/Purdue University at Indianapolis Indianapolis, IN

* Did not attend 

### David E. Malarkey, D.V.M., Ph.D. Department of Microbiology, Pathology, and Parasitology College of Veterinary Medicine North Carolina State University Raleigh, NC 

### Michele Medinsky, Ph.D.*

Durham, NC 

### Walter W. Piegorsch, Ph.D.

Department of Statistics University of South Carolina Columbia, SC 

### Mary Anna Thrall, D.V.M.

Department of Pathology College of Veterinary Medicine and Biomedical Sciences Colorado State University Fort Collins, CO

### o-Nitrotoluene, NTP TR 504 15



### On May 3, 2001, the draft Technical Report on the toxicology and carcinogenesis studies of o- nitrotoluene received public review by the National Toxicology Program’s Board of Scientific Counselors’ Technical Reports Review Subcommittee. The review meeting was held at the National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Dr. J.K. Dunnick, NIEHS, introduced the toxicology and carcinogenesis studies of o- nitrotoluene by discussing the uses of the chemical and the rationale for the study,describing the experimental design, reporting on survival and body weight effects, and commenting on chemical-related neoplastic and nonneoplastic lesions inmale and female rats and mice. The proposed conclusions for the 2-year studies of o- nitrotoluene were clear evidence of carcinogenic activity in male and female F344/N rats and B6C3F 1 

### mice.

Dr. R.C. Sills, NIEHS, presented results of analyses of mutations in ras oncogenes and p53 tumor suppressor genes extracted from hemangiosarcomas found in mice exposed to o- nitrotoluene and in control mice from previous NTP studies. While the mutation rate in ras oncogenes was similar in hemangiosarcomas from exposed and control mice, mutations occurred in almost all the p53 tumor suppressor genes from o- nitrotolueneinduced hemangiosarcomas, but only rarely in spontaneously occurring neoplasms. Dr. T.R. Devereux, NIEHS, showed results of analyses for mutations that disrupt the regulation of $ -catenin from the same set of hemangiosarcoma tissue samples. About half of the

o- nitrotoluene-induced hemangiosarcomas had suchmutations, while spontaneously occurring hemangiosarcomas had none.

Dr. Medinsky, a principal reviewer, was unable to attend the meeting and her comments were read into the record by Dr. M.S. Wolfe, NIEHS. Dr. Medinsky agreed with the proposed conclusions. She asked whether the test chemical was stable in the feed and if the feed was adversely affected by irradiation. Dr. Dunnick replied that the chemical was stored refrigerated and mixed withfeed every 2 or 3 days to minimize loss due to instability. Dr. G.N. Rao, NIEHS, explained that to prevent pathogen contamination, the NTP has begun irradiatingthe animal feed because the prepared diet is not formulated to be autoclaved. The nutrient composition of the diet was unaffected by irradiation. Dr. Medinsky also questioned the appropriateness ofanalyzing metabolite data normalized to creatinine concentrations rather than total metabolite excreted.Dr. L.T. Burka, NIEHS, explained that creatinine production, which is related to muscle mass, is a good surrogate measure of body weight, providing a convenient method for normalizing data from animals of different size due to age and sex differences. In addition, bulkmeasures of urine volume are less precise than metabolite concentrations and thus less useful for mathematical modeling.

Dr. Malarkey, the second principal reviewer, agreed withthe conclusions. He praised the inclusion of stop-exposure and molecular biology studies and asked for clarification of the specificity of the observed mutations and the sites of $ -catenin and p53 protein accumulation. Dr. Sills replied that the p53 protein was located primarily in the nucleus and $ -catenin predominantly in the cell membrane, although nuclear accumulation of $ -catenin was seen in some neoplasm types, such as hepatoblastomas. Dr. Malarkey suggested the lesions inthe reproductive tract and endocrine glands might indicate an endocrine-disrupting mechanism of toxicity by the chemical. As a general question for all NTP studies, he asked for details of monitoring for possible Helicobacter hepaticus contamination of the animal rooms. Dr. Rao described the monitoring procedures used to detect Helicobacter infection following an outbreak in the late 1980s and noted that NTP studies have been free of known viral and bacterial pathogens since 1991.

Dr. Klaunig, the third principal reviewer, also agreedwith the proposed conclusions. He asked for clarification of the cause of early death of the animals and for more information on the site of origin of the various mesotheliomas. Dr. Dunnick replied that mesotheliomas were a significant cause of mortality and that the majority of the mesotheliomas originated in the tunica vaginalis.

Dr. Chatman questioned the phrasing of the proposed conclusions and suggested that listing certain neoplasms as evidence of carcinogenic activity and others as “also exposure related” or “may have been related” was potentially confusing. Dr. Davis also questioned whether the additional statements implied that the lesions were treatment related. Dr. C.J. Portier, NIEHS, 

## SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS

### 16 o-Nitrotoluene, NTP TR 504



### explained that for each sex/species group, there is one overall conclusion category for the chemical, rather than different categories for different tissue sites. Thus, to convey fully the study results, the NTP mentions not only the neoplasms giving the strongest indication of carcinogenic activity but also other chemically related effects. Dr. Chatman proposed that the lung neoplasms in male rats and liver neoplasms in female rats be included in the clear evidence statements. Dr. J.R. Bucher, NIEHS, said the proposed phrasing was chosen to distinguish that the strength of response for the lung and liver neoplasms was less than that for the other neoplasm types. Dr. Dunnick noted that, were the other neoplasms not present, the lung neoplasms would be classified only as some evidence . Dr. Drinkwater agreed that the lung and liver effects were weaker than clear evidence and moved that the conclusions be accepted as written. Dr. Klaunig seconded the motion, which was approved by six yes votes to two no votes (Drs. Chatman and Davis).

### 17



# C HEMICAL AND 

# P HYSICAL 

# P ROPERTIES The nitrotoluenes are produced by the nitration of toluene with an aqueous acidic mixture of sulfuric acid and nitric acid at a temperature that starts at 25° C and is slowly raised to 37° C. The resulting product contains 55% to 60% o- nitrotoluene, 3% to 4% m- nitrotoluene, and 35% to 40% p- nitrotoluene. The isomers may be separated by a combination of fractional distillation and crystallization ( Kirk-Othmer , 1981). Isomers of nitrotoluene differ in the position of the nitro group in relation to the methyl group on the benzene ring. While thechemical formula is the same for all isomers, their chemical and physical properties vary (Table 1).



# P RODUCTION 

# , U SE 

# , AND 

# H UMAN 

# E XPOSURE

o-Nitrotoluene and p- nitrotoluene are important commercial chemicals used to synthesize agricultural andrubber chemicals, azo and sulfur dyes, and dyes for cotton, wool, silk, leather, and paper. An estimated 29 million pounds of the ortho isomer and 15 million pounds of the para isomer are used annually in the United States. The third isomer, m- nitrotoluene, is produced in negligible quantities (Kirk-Othmer , 1981; Abshire and Hughes, 1982). o-Nitrotoluene is on the U.S. Environmental Protection Agency’s (1999) list of high production volume chemicals with an estimated production in 1990 of 45 million to 62 million pounds
per year.

Environmental surveys have detected o- nitrotoluene inrivers and drinking water (USEPA, 1976); all three isomers of nitrotoluene have been found in wastewater effluent and atmospheric emissions from industrialplants (Forsten, 1973; USEPA, 1976). Microbial systems are capable of biodegrading nitroaromatic compounds (Spain, 1995). The Occupational Safety and Health Administration set an 8-hour, time-weighted average (TWA) permissible exposure limit of 5 ppm (30 mg/m 3 

### ) for nitrotoluenes (NIOSH, 1997), and the American Conference of Governmental Industrial Hygienists (2000) recommended a threshold limit value of 2 ppm (11 mg/m 3 

### ) for the 8-hour TWA. 

# INTRODUCTION

o-NITROTOLUENE

CAS No. 88-72-2

Chemical Formula: C 7 

### H 7

### NO 2 

### Molecular Weight: 137.14

Synonyms: 2-Methylnitrobenzene; o -methylnitrobenzene; 2-nitrotoluene; 2-nitrotoluol 

## NO 2 

## CH 3

### 18 o-Nitrotoluene, NTP TR 504



### The National Occupational Exposure Survey (1981- 1983) (NIOSH, 1990) found exposure to p- nitrotoluene among workers in five different occupational groups: biological technicians; painting and paint-sprayingmachine operators; machine operators; welders and cutters; and operators of separating, filtering, and clarifying machines. The latter group accounted for approximately60% of potential exposures. Data on potential workplace exposure for the ortho and meta isomers were not available. An estimated 4,350 people in the United States are potentially exposed to p- nitrotoluene in the workplace.



# A BSORPTION 

# , D ISTRIBUTION 

# , M ETABOLISM 

# , AND 

# E XCRETION 

## Experimental Animals The comparative metabolism of o- , m- , and
p- nitrotoluene administered orally was studied in F344 rats (Chism et al. , 1984; deBethizy and Rickert, 1984; Chism and Rickert, 1985; Rickert et al. , 1987).Following an oral dose of the three radiolabeled compounds as individual chemicals, 73% to 86% of the dosewas excreted in the urine within 72 hours. Fecal excretion accounted for 5% to 13% of the dose, and minimal amounts of radiolabel were captured in expired breath (Chism et al. , 1984).

Metabolism studies (Appendix L) were designed to compare the in vivo metabolism of o- nitrotoluene in F344/N rats and B6C3F 1 

### mice and to determine the effects of dose and of repeated dosing on rates and routes of excretion. The routes of excretion were similar in rats and mice, with the predominant route being via urine. The slightly higher amount of [ 14 

### C]- o- nitrotoluenederived radioactivity in feces of mice can be attributed tocontamination of the feces by urine. The rate of excretion was more rapid in rats, with about 90% of the administered radioactivity excreted in urine in the first24 hours. Less than 70% of the administered radioactivity was excreted in urine by mice in the same time period.

There is good agreement between the current comparative metabolism studies (Appendix L) and those reported by Chism et al. (1984) on the major urinary metabolites in male rats. o- Aminobenzyl alcohol, a major metabolite identified in the current comparative metabolism studies, was not identified by Chism et al. (1984) . o- Toluidine was also identified as a minor metabolite in some samples in the current studies. The urinary profile after 11 daily doses of 200 mg

o- nitrotoluene/kg body weight indicated a decreased excretion of o- nitrobenzylmercapturic acid, but no other obvious changes (Appendix L).

o-Nitrobenzoic acid and o- nitrobenzyl glucuronide are the only metabolites that could be identified in the urine of male mice (Table L3). The major urinary metabolitesof the three nitrotoluene isomers that have been identified in rats and mice are shown in Table 2. A detailed metabolic scheme for o- nitrotoluene is shown in Figure 1.

All three isomers apparently are converted to the corresponding benzyl alcohol and benzoic acid (Table 2); the
meta and para isomers undergo conjugation with T ABLE 

### 1 Chemical and Physical Properties of the Nitrotoluenes a



### o-Nitrotoluene m- Nitrotoluene p- Nitrotoluene

Boiling point 220.4° C 232.6° C 238.3° C Melting point –9.3° C 15° C 51.7° C Density (20o C) 1.163 1.157 1.286 Solubility (H 2 O, 30o C) 652 mg/L 498 mg/L 442 mg/L Volatility (20° C) 0.1 mm Hg 0.1 mm Hg 0.1 mm Hg Volatility (30° C) 0.25 mm Hg 0.25 mm Hg 0.25 mm Hg Log octanol/water

partition coefficient 2.30 2.40 2.37

a Verschueren, 1983; NTP, 1992

### o-Nitrotoluene, NTP TR 504 19



### T ABLE 

### 2 Urinary Metabolites of o- , m- , and p- Nitrotoluene in Male Rats and Mice Administered Gavage Doses of 200 mg/kg a



### o-Nitrotoluene m- Nitrotoluene p-Nitrotoluene

Rats b

o-Nitrobenzoic acid (29%) m-Nitrobenzoic acid (21%) p-Nitrobenzoic acid (28%)

o-Nitrobenzyl glucuronide (14%) m-Nitrohippuric acid (24%) p-Nitrohippuric acid (13%) S-( o- nitrobenzyl)-N-acetylcysteine (12%) m- Acetamidobenzoic acid (12%) p- Acetamidobenzoic acid (27%)



### Mice c

o-Nitrobenzoic acid (38%) m-Nitrohippuric acid (52%) p-Nitrohippuric acid (20%)

o-Nitrobenzyl glucuronide (24%) m-Nitrobenzoic acid (19%) 2-Methyl-5-nitrophenyl glucuronide (13%) 2-Methyl-5-nitrophenyl sulfate (19%)

a Percentage of administered dose b Chism et al. , 1984 c Appendix L; RTI, 1995, 1996a,b



### glycine to form the hippuric acid, or nitro reduction and acylation. For o- nitrotoluene, formation of

o- nitrobenzyl alcohol glucuronide is a major metabolic pathway. Conjugation with glucuronic acid is not a major metabolic route for the meta and para isomers.

o -Nitrobenzyl glucuronide is excreted via the bile intothe intestine, where bacterial enzymes hydrolyze the glucuronic acid and reduce the nitro group to form

o -aminobenzyl alcohol. o -Aminobenzyl alcohol is reabsorbed and further metabolized by hepatic enzymes to a species capable of covalent binding to hepatic DNA. Studies by Chism and Rickert (1985) suggested that

o- aminobenzyl sulfate is the metabolite of o- nitrotoluene responsible for binding covalently to DNA (Figure 2).

An analogous metabolic pathway is followed by 2,6-dinitrotoluene (2,6-DNT), which is oxidized in the liver to 2,6-dinitrobenzyl alcohol and then conjugated with glucuronic acid and excreted in the bile (Kedderis et al. , 1984). Intestinal microflora hydrolyze the glucuronide and reduce the nitro group to form 2-amino6-nitrobenzyl alcohol. A portion of this metabolite isreabsorbed from the intestine and oxidized to a hydroxylamine by hepatic enzymes. The hydroxylamine is then conjugated with sulfate by hepatic sulfotransferase. Theunstable N,O-sulfate decomposes to form an electrophilic nitrenium ion that can react with cellular nucleophiles such as DNA. This electrophilic ion is formed in the liver, hence the high carcinogenic activity of 2,6-DNT for rodent liver. 2,6-DNT is more active than 2,4-DNT in an in vivo/in vitro hepatocyte unscheduled DNA synthesis assay (Mirsalis and Butterworth, 1980).

The metabolic profiles for 2,6-DNT and o- nitrotoluene are similar (Rickert et al. , 1987). Both are excreted as glucuronides into the intestine where bacterial enzymes reduce the nitro groups; the aminobenzyl alcohols arereabsorbed and further metabolized in the liver to electrophilic compounds which presumably can interact with DNA. Binding of 2,6-DNT and o- nitrotoluene to rathepatic DNA is decreased by pretreatment with sulfotransferase inhibitors, suggesting that the final step in the activation of each chemical is formation of an unstable N,O-sulfate that decomposes to yield an electrophilic nitrenium ion.



## HumansNo information on the absorption, distribution, metabolism, or excretion of o- , m- , or p- nitrotoluene in humans was found in a review of the literature.



# T OXICITY 

## Experimental Animals Oral LD 50 

### values are 891 mg/kg (rats) and 2,463 mg/kg (mice) for o- nitrotoluene, 1,072 mg/kg (rats) and 330 mg/kg (mice) for m- nitrotoluene, and 2,144 mg/kg

### 20 o-Nitrotoluene, NTP TR 504



### F IGURE 

### 1 Composite Metabolic Scheme for o-Nitrotoluene in Rats and Mice (Chism et al. , 1984; Appendix L) Abbreviations: Major (R) or minor (r) urinary metabolite in rats; major (M) metabolite in mice.

### o-Nitrotoluene, NTP TR 504 21



### F IGURE 

### 2 Proposed Pathway for Bioactivation of o-Nitrotoluene (Chism and Rickert, 1985)

### 22 o-Nitrotoluene, NTP TR 504



### (rats) and 1,231 mg/kg (mice) for p- nitrotoluene. These acute toxicity studies did not include histopathologic examination of tissues (Ciss et al. , 1980a,b).

In 14-day studies (NTP, 1992), o- , m- , or p- nitrotoluene was administered in feed to male and female F344/N rats and B6C3F 1 

### mice at concentrations ranging from 388 to 20,000 ppm (equivalent to average daily doses of 55 to 900 mg nitrotoluene/kg body weight). There were no effects on survival or clinical findings of toxicity in thesestudies, although animals exposed to the higher concentrations showed decreases in body weight gains relative to the controls.

In 13-week studies (NTP, 1992), o- , m- , or p- nitrotoluene was given to male and female F344/N rats and B6C3F 1 

### mice in feed at concentrations from 625 to10,000 ppm. The estimated daily doses based on measures of feed consumption were similar for each of thethree isomers and ranged from 40 to 725 mg nitrotoluene/kg body weight per day for rats and 45 to 680 mg/kg per day for mice. There were no effects on survival, and clinical findings of toxicity were limited to decreases in feed consumption. Decreased body weight gains occurred in exposed rats and mice at the higher exposure concentrations and were most pronounced in rats receiving o- nitrotoluene.

In the 13-week NTP (1992) studies, toxicity to the kidney, spleen, and testis occurred in rats receiving any ofthe three isomers, and toxicity to the liver and mesothelium occurred in male rats given o- nitrotoluene(Table 3). Kidney toxicity in male rats was characterized by the presence of hyaline droplets in tubule epithelial cells, attributed to an increase in the level of

" 2u-globulin (as determined by ELISA). No granularcasts were seen, and this was considered to be only minimal toxicity to the kidney. In the p- nitrotoluene study, pigment, possibly lipofuscin, and karyomegaly were present in the renal tubule epithelium of exposed male and female rats. In the spleen of exposed male and female rats, there were mild increases in the incidencesof hematopoiesis, hemosiderin deposition, and/or congestion; this effect was most severe with the para isomer, followed by the ortho and then the meta isomer. Administration of o- , m- , or p- nitrotoluene impaired testicular function in the rat, shown by degeneration of the testis and reduction in sperm concentration, motility, and spermatid number. All three isomers increased the length of the estrous cycle in rats. Hepatic toxicity was characterized by cytoplasmic vacuolization, oval cell hyperplasia, an increase in the concentration of serum bile acids, and increased sorbitol dehydrogenase and alanine aminotransferase activities in male rats given

o- nitrotoluene. There was no histopathologic evidence of liver toxicity in male or female rats with the meta or para isomers or in female rats with the ortho isomer, but evidence of liver injury was observed in these groups, asindicated by increased relative liver weights and elevated bile acid concentrations and liver enzyme activities in serum. Mesotheliomas of the tunica vaginalis were observed in three of 10 male rats receiving 5,000 ppm o- nitrotoluene, and mesothelial cell hyperplasia was observed in two of 10 male rats receiving 10,000 ppm o- nitrotoluene (Table 4).

The only histopathologic evidence of toxicity in mice in the 13-week studies (NTP, 1992) occurred in animals receiving o- nitrotoluene, which caused degeneration and metaplasia of the olfactory epithelium. No liver lesions were noted in mice, but the three isomers causedincreases in relative liver weights. There was no toxicity to the reproductive system in male or female mice treated with any of the nitrotoluene isomers. No immunotoxicity evaluation was conducted for

o- nitrotoluene.



## Humans No epidemiological studies or reports of adverse health effects related to exposure to o- , m- , or p- nitrotoluene were found in a review of the literature.



# C ARCINOGENICITY 

## Experimental Animals Interest in the carcinogenicity of mononitrotoluenes stems from the results of long-term rodent studies using technical-grade DNT, 2,4-DNT, or 2,6-DNT. Results ofthese studies suggest that 2,6-DNT is a potent carcinogen in rat liver (Rickert et al. , 1984). The results of Weisburger et al. (1978) suggested ortho -substituted aromatic compounds are more potent carcinogens than corresponding isomers with meta or para substitutions; this was seen with o- , m- , and p- toluidine in rats and mice, as well as with other compounds. The toluidine studies are of interest because reduction of the nitro group of the nitrotoluenes yields the corresponding toluidine. The National Cancer Institute (1979) reported that o- toluidine hydrochloride was carcinogenic in 2-year studies in male rats (mesotheliomas, splenic sarcomas, subcutaneous fibromas), in female rats

### o-Nitrotoluene, NTP TR 504 23



### T ABLE 

### 3 Summary of Selected Treatment-Related Effects in the 13-Week Nitrotoluene Feed Studies a



### o-Nitrotoluene m- Nitrotoluene p- Nitrotoluene Male Female Male Female Male Female

Rats

Final mean body weight (90% or less of controls) 2,500 2,500 10,000 10,000 5,000 10,000

Changes in hematology parameters 2,500 2,500 5,000 5,000 2,500 2,500

Liver Increased relative weight 625 625 10,000 10,000 5,000 10,000 Increased ALT 5,000 — b — 5,000 — 10,000 Increased SDH 2,500 — — — — — Increased bile acids 5,000 10,000 5,000 10,000 10,000 — Nonneoplastic lesions 2,500 — — — — —

Kidney Increased relative weight 2,500 1,250 10,000 5,000 5,000 10,000 Nonneoplastic lesions 1,250 2,500 625 — 625 625

Spleen Nonneoplastic lesions 1,250 2,500 2,500 2,500 625 625

Testis Decreased spermatid count 5,000 10,000 10,000 Nonneoplastic lesions 5,000 10,000 10,000

Epididymal mesothelium Neoplastic and preneoplastic

lesions 5,000 — —

Increased estrous cycle length 10,000 5,000 10,000



### Mice

Final mean body weight (90% or less of controls) 2,500 2,500 10,000 10,000 10,000 10,000

Nose Nonneoplastic lesions 1,250 1,250 — — — —

Liver Increased relative weight 2,500 1,250 625 625 625 625

a NTP, 1992; lowest exposure group (ppm) in which a chemical-related effect was seen; ALT=alanine aminotransferase; SDH=sorbitol dehydrogenase b Not observed in any exposure group

### 24 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 4 Treatment-Related Lesions in F344/N Rats and B6C3F 1 

### Mice in the 13-Week Feed Studies of o- Nitrotoluene a



### 0 ppm 625 ppm 1,250 ppm 2,500 ppm 5,000 ppm 10,000 ppm

Male Rats

Pancreatic Islets b 10 10 10 10 10 10 Cellular Hyperplasia c 0 0 0 1 (1.0) d 7** (1.0) 10** (1.6)

Liver 10 10 10 10 10 10 Inflammation 5 (1.8) 5 (1.0) 5 (1.6) 10* (1.5) 10* (1.8) 8 (1.8) Vacuolization 0 0 0 6** (1.3) 9** (1.8) 10** (3.0) Bile Duct Hyperplasia 0 0 0 2 (1.0) 10** (1.2) 10** (2.2)

Kidney 10 10 10 10 10 10 Nephropathy, Hyaline Droplet 0 0 6** (1.0) 10** (1.6) 10** (2.8) 9** (2.6) Regeneration 2 (1.0) 6 (1.0) 2 (1.0) 2 (1.0) 5 (1.0) 6 (1.1) Pigmentation 0 0 0 0 1 (1.0) 10** (1.0)

Spleen 10 10 10 10 10 10 Hematopoiesis 0 0 0 6** (1.3) 10** (2.0) 10** (2.0) Pigmentation 0 0 0 7** (1.3) 10** (2.0) 10** (2.0) Capsular Hyperplasia 0 0 1 (1.0) 1 (2.0) 1 (1.0) 9** (1.9)

Salivary Gland 10 10 10 10 10 10 Atrophy 0 0 10** (2.2) 10** (3.1) 10** (3.0) 10** (3.0)

Testis 10 10 10 10 10 10 Seminiferous Tubule Degeneration 0 0 0 0 10** (2.3) 10** (4.0)

Epididymis (Mesothelium) 10 10 10 10 10 10 Hyperplasia e 0 0 0 002 Mesothelioma f 0 0 0 030

Preputial Gland 10 10 10 10 10 10 Atrophy 0 0 4* (1.0) 6** (1.0) 10** (1.9) 10** (1.9)



### Female Rats

Pancreatic Islets 10 10 0 0 10 10 Cellular Hyperplasia 0 0 0 2 (1.0)

Kidney 10 10 10 10 10 10 Pigmentation 0 0 0 3 (1.0) 10** (1.1) 10** (1.8)

Salivary Gland 10 10 10 10 10 10 Atrophy 0 0 10** (1.5) 10** (3.0) 10** (3.0) 10** (2.0)

Spleen 10 10 10 10 10 10 Hematopoiesis 0 0 0 0 1 (1.0) 10** (1.0) Pigmentation 0 0 0 5* (1.0) 9** (2.0) 10** (2.0) Capsular Hyperplasia 0 0 0 0 1 (1.0) 2 (1.0)

Clitoral Gland 10 10 10 9 10 10 Atrophy 0 0 0 0 2 (1.0) 10** (1.5)

### o-Nitrotoluene, NTP TR 504 25



### T ABLE 

### 4 Treatment-Related Lesions in F344/N Rats and B6C3F 1 

### Mice in the 13-Week Feed Studies of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,500 ppm 5,000 ppm 10,000 ppm

Male Mice

Nose Olfactory Epithelium 10 10 10 10 10 10

Degeneration/Metaplasia 0 0 1 (1.0) 2 (1.0) 10** (2.0) 10** (3.0)



### Female Mice

Nose Olfactory Epithelium 10 10 10 10 10 10

Degeneration/Metaplasia 0 0 2 (1.5) 9** (1.0) 10** (1.9) 10** (2.9)

* Significantly different (P # 0.05) from the control group by the Fisher exact test ** P # 0.01 a Data presented by Dunnick et al. (1994) and PATHCO, Inc. (2000) b Number of animals with tissue examined microscopically c Number of animals with lesion d Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked e Preneoplastic lesion f Neoplasm



### (splenic sarcomas, urinary bladder transitional cell tumors, tumors of the mammary gland), in male mice (hemangioma, hemangiosarcoma), and in female mice (liver tumors).



## Humans No epidemiological studies of o- , m- , or p- nitrotoluene were found in a review of the literature.



# G ENETIC 

# T OXICITY Testing of the mononitrotoluenes in vitro for mutagenicity has generally yielded negative results, although occasional positive responses in various assays have been reported. A recent review of mutagenicity data for the mononitrotoluenes was provided by the International Agency for Research on Cancer (1996). The aromatic nitro group of the nitrotoluenes is considered a structural alert to potential DNA reactivity (Tennant and Ashby, 1991), but such activity would presumably be dependent upon the metabolic capability of the test system. Forexample, reduction of the nitro group to produce an aromatic amine would likely be necessary for a positive response in the Salmonella typhimurium assay. Although o- and m- nitrotoluene demonstrated no mutagenic activity in any of several strains of S. typhimurium
,with or without S9 metabolic activation, isolated positive responses were reported for p- nitrotoluene in strain TA100, with and without S9 (Chiu et al. , 1978; Miyata et al. , 1981; Spanggord et al. , 1982; Haworth et al.
, 1983; Suzuki et al. , 1983; Shimizu and Yano, 1986; Kawai et al. , 1987). p-Nitrotoluene also induced cell growth inhibition, a measure of DNA damage, in Bacillus subtilis M45/H17 in the absence of S9 (Shimizu and Yano, 1986); o- nitrotoluene was weakly positive in this assay, and results with m- nitrotoluene were negative.

All three mononitrotoluene isomers induced sister chromatid exchanges in cultured Chinese hamster ovary (CHO) cells; only m- nitrotoluene required S9 for a positive response (Galloway et al. , 1987). p-Nitrotoluene induced chromosomal aberrations in cultured CHO cells in the presence of S9 (Galloway et al. , 1987), but no increases in micronuclei or chromosomal aberrations were observed in bone marrow cells of male B6C3F 1 

### mice administered p- nitrotoluene as a single intraperitoneal injection (Furukawa et al. , 1989; Ohuchida et al. , 1989).

### 26 o-Nitrotoluene, NTP TR 504



### No induction of unscheduled DNA synthesis was observed in male F344 rat hepatocytes or spermatocytes treated with m- or p- nitrotoluene in the standard in vitro assay (Doolittle et al. , 1983; Working and Butterworth, 1984) or in vivo (Doolittle et al., 1983; Butterworth et al. , 1989; Mirsalis et al. , 1989). Positive results were obtained, however, in an in vitro unscheduled DNA synthesis assay employing serum-free medium (Parton et al. , 1995). o-Nitrotoluene was also negative in the in vitro unscheduled DNA synthesis assay, but in male F344 rats treated in vivo , a strongly positive response was observed (Doolittle et al. , 1983). No induction of unscheduled DNA synthesis by o- nitrotoluene was noted in germ free male rats, indicating that activation of

o- nitrotoluene or an intermediate metabolic conjugate byintestinal bacteria is necessary to the process. No induction of unscheduled DNA synthesis was observed in hepatocytes of female rats treated with o- nitrotoluene in vivo ; differences in in vivo results between males and females may be due to differences in hepatic metabolism or disposition of o- nitrotoluene. Different results between males and females have been attributed to males excreting more of the glucuronide conjugates of the nitrotoluenes into the bile and, subsequently, into the intestine where they are metabolized further by bacterial systems. Sex-related differences in metabolism have also been observed with the dinitrotoluenes in rats (Rickert and Long, 1981).

Covalent binding to DNA was measured in hepatocytes of male F344 rats after a single oral dose of o- , m- , or p- nitrotoluene (Rickert et al. , 1987). Only o- nitrotoluene bound DNA, whereas all three isomers bound protein. 

# S TUDY 

# R ATIONALE The National Institute for Occupational Safety and Health and the NTP nominated the nitrotoluenes for rodent toxicity and carcinogenicity studies based on the considerable human exposure to these chemicals as well as the lack of long-term studies of carcinogenicity in rodents. This Technical Report describes the results of the 2-year studies of o- nitrotoluene in F344/N rats and B6C3F 1 

### mice. The 2-year studies of p- nitrotoluene are reported in a companion Technical Report (NTP, 2002).

The exposure concentrations for the core 2-year

o- nitrotoluene studies were selected based on the findings from the 13-week studies (NTP, 1992). In thosestudies, body weight effects and/or liver, spleen, and kidney toxicities were seen primarily in rats at exposure concentrations of 2,500 ppm or greater. Male rats receiving 5,000 or 10,000 ppm in those studies had low incidences of mesothelioma or mesothelial hyperplasiaafter 13 weeks of exposure. The exposure concentrations selected for the 2-year rat studies were 625, 1,250, and 2,000 ppm. A 3-month stop-exposure study in male rats exposed to 2,000 or 5,000 ppm was included to determine the progression/regression of mesothelial lesions and to study the relationship between short-term and 2-year exposures in the development of neoplasms. In the 13-week study in mice, final mean body weights of 10,000 ppm mice were 22% to 30% less than those of the controls. The exposure concentrations selected for the 2-year study in mice were 1,250, 2,500, and 5,000 ppm, doses at which minimal or no toxicity was seen in the 13-week study.

### 27



# P ROCUREMENT
AND 

# C HARACTERIZATION
OF O


# -N ITROTOLUENE

o-Nitrotoluene was obtained from Aldrich Chemical Company (Milwaukee, WI) in one lot (8056-58-05RTI). Identity, purity, and stability analyses were conducted by the analytical chemistry laboratory, Research TriangleInstitute (Research Triangle Park, NC) and the study laboratory (Appendix H). Reports on analyses performed in support of the o- nitrotoluene studies are on file at the National Institute of Environmental Health Sciences.

The chemical, a yellow-green liquid, was identified as

o- nitrotoluene by infrared, ultraviolet/visible, protonnuclear magnetic resonance, and low- and high-resolution mass spectroscopy. The purity of lot 8056-58- 05RTI was determined by Karl Fischer water analysisand gas chromatography. Karl Fischer analysis indicated 0.29% water. Gas chromatography by one system indicated one major peak, one impurity with an area of 0.11%, and five minor impurities, each with an area less than 0.1% relative to the major peak; a second system indicated one major peak and six impurities, each with an area less than 0.1% relative to the major peak. A third gas chromatography system indicated no impurities. The overall purity was determined to be greater than 99%.

Stability studies of the bulk chemical were performed bythe analytical chemistry laboratory using gas chromatography. These studies indicated that o- nitrotoluene was stable as a bulk chemical for at least 2 weeks when stored protected from light at temperatures up to 25° C. To ensure stability, the bulk chemical was stored inamber glass bottles inside metal cans at room temperature. Stability was monitored during the studies usinggas chromatography. No degradation of the bulk chemical was detected. 

# P REPARATION AND 

# ANALYSIS
OF 

# D OSE 

# F ORMULATIONS The dose formulations were prepared every 2 weeks by mixing o- nitrotoluene with feed (Table H2). Homogeneity and stability studies of the 625 ppm dose formulation and homogeneity studies of the 5,000 ppm doseformulation in nonirradiated NTP-2000 feed were performed by the analytical chemistry laboratory using gas chromatography. Homogeneity studies of the 625, 2,000, and 5,000 ppm dose formulations in irradiated feed were performed by the study laboratory. Homogeneity was confirmed in each study. Stability of the dose formulations was confirmed for 36 days whenstored in sealed containers protected from light at temperatures up to 3° C; significant chemical losses due tovolatility were seen in the dose formulations under simulated animal room conditions.
Analyses of the dose formulations of o- nitrotoluene wereconducted at the study laboratory using gas chromatography every 8 to 12 weeks. All dose formulations used were 90% to 115% of the target concentrations. Because of the expected volatility losses during formulation, dose formulations were prepared at up to 115% of the target concentrations. Animal room samples for rats ranged from 75% to 94% and those for mice ranged from 53% to 81% of the target concentrations.



# 2-Y EAR 

# S TUDIES 

## Study Design In the core study, groups of 60 male and 60 female rats were fed diets containing 625, 1,250, or 2,000 ppm

o- nitrotoluene for 105 weeks. In a 3-month stop-exposure study, groups of 70 male rats were fed diets containing 2,000 or 5,000 ppm o- nitrotoluene for 13 weeks followed by undosed feed for the remainder of the study. A group of 70 male rats receiving undosed feed served as a control group for both male rat studies; 60 female 

# MATERIALS AND METHODS

### 28 o-Nitrotoluene, NTP TR 504



### rats receiving undosed feed were the control group for the female core study. Ten control males and 10 males from each stop-exposure group were sacrificed at 3 months. Groups of 60 male and 60 female mice were fed diets containing 0, 1,250, 2,500, or 5,000 ppm

o- nitrotoluene for 105 weeks. An interim evaluation planned for all groups at 15 months was not done due to high mortality in the exposed groups.



## Source and Specification of Animals Male and female F344/N rats and B6C3F 1 

### mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY) for use in the 2-year studies. Rats and mice were quarantined for 12 to 14 days before the beginning of the studies. Five male and five female rats and mice were randomly selected for parasite evaluation and gross observation of disease. Rats and mice were approximately 6 to 7 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program (Appendix K).



## Animal Maintenance Rats were housed up to three (males) or five (females) per cage; mice were housed individually (males) or five(females) per cage. Rats and mice were housed individually during urine collection periods. Feed and water were available ad libitum . Feed consumption was measured every 4 weeks. The estimate of dose delivered to the animals (mg/kg) was based on body weight and feed consumption data collected during the course of the 2-year studies and targeted chemical concentration in the feed. Animals were given nonirradiated feed from the beginning of the studies until 3 (rats) or 8 (mice) July1996 and irradiated feed thereafter. The feed was irradiated to reduce microbial contamination. Cages and racks were rotated every 2 weeks. Dose formulations were replaced in animal room feeders on a 2-day, 2-day, 3-day schedule, due to the formulations’ instability under animal room conditions. Further details of animal maintenance are given in Table 5. Information on feed composition and contaminants is provided in Appendix J.



## Clinical Examinations and Pathology All animals were observed twice daily. Clinical findings were recorded every 4 weeks. Animals were weighed initially, during week 4, and every 4 weeks thereafter. Ten male rats in the 0 ppm group and the 2,000 and 5,000 ppm stop-exposure groups were designated for interim evaluation at 3 months.

Complete necropsies and microscopic examinations were performed on all rats and mice. At the 3-month interim evaluation in male rats, the heart, right kidney, liver, lungs, right testis, and thymus were weighed. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples fromeach organ were examined. Tissues examined microscopically are listed in Table 5.

Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered into the Toxicology Data Management System. The slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/blockmatch, and wet tissue audit. The slides, individual animal data records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual animal records and tables were compared for accuracy, the slide and tissue counts were verified, and the histotechnique was evaluated. For the 2-year studies, a quality assessment pathologist evaluated slides from all tumors and all potential target organs, which included the bone marrow, kidney, liver, lung, mammary gland, pituitary gland, salivary gland, skin, and spleen of maleand female rats; the epididymis, pancreatic islets, peritoneum, preputial gland, prostate gland, and testis of male rats; the clitoral gland and mandibular lymph node of female rats; and the large intestine (cecum), kidney, liver, and stomach (forestomach and glandular) of male and female mice.

The quality assessment report and the reviewed slides were submitted to the NTP Pathology Working Group (PWG) chairperson, who reviewed the selected tissues and addressed any inconsistencies in the diagnoses made by the laboratory and quality assessment pathologists.Representative histopathology slides containing examples of lesions related to chemical administration, examples of disagreements in diagnoses between the laboratory and quality assessment pathologists, or lesions of general interest were presented by the chair person to the PWG for review. The PWG consisted of the quality assessment pathologist and other pathologists

### o-Nitrotoluene, NTP TR 504 29



### experienced in rodent toxicologic pathology. This group examined the tissues without any knowledge of dose groups or previously rendered diagnoses. When thePWG consensus differed from the opinion of the laboratory pathologist, the diagnosis was changed. Final diagnoses for reviewed lesions represent a consensusbetween the laboratory pathologist, reviewing pathologist(s), and the PWG. Details of these review procedures have been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses of the pathology data, the decision of whether to evaluate the diagnosed lesions for each tissue type separately or combined was generally based on the guidelines of McConnell et al. (1986).



## Urinary Metabolite Analyses Five male and five female rats and mice from each corestudy group were randomly selected for urinary metabolite analyses at 2 weeks and 3, 12, and 18 months (Appendix F). Animals were placed in metabolism cages for 24 hours. Urine samples were placed on ice,urine volume and creatinine concentration were measured, and then the samples were frozen pending metabolite analyses. To establish the correlation between exposure concentration and internal dose and to determine how metabolism of o- nitrotoluene may change with chronic exposure and age, three urinary metabolites, o -nitrobenzoic acid,

o -nitrobenzylmercapturic acid, and o -aminobenzoic acid were chosen as biomarkers based on the metabolism studies of Chism et al. (1984). Metabolite concentrations were determined by high-performance liquid chromatography (HPLC). An internal standard solution of anisic acid in water and sodium hydroxide was added tothe rat urine samples, which were then diluted with trifluoroacetic acid in methanol and water. Triethylaminewas used to adjust the pH to 2.3, and the resulting mixture was filtered and degassed by sonication. The samples were then analyzed by HPLC on a Zorbax 250 mm × 4.6 mm Rx-C 18 

### column (Varian, Palo Alto, CA) using ultraviolet detection at 254 nm (for

o -aminobenzoic acid concentration) or 266 nm. Mouseurine data were interpreted with a standard curve generated from the rat urine data. The ratios obtained by dividing the metabolite concentration by the creatinine concentration, rather than the mass of metabolite excreted per 24 hours, were analyzed to normalize the metabolite to body weight and determine the ratio more precisely.

### 30 o-Nitrotoluene, NTP TR 504

Study Laboratory Southern Research Institute (Birmingham, AL)


### Strain and Species F344/N rats B6C3F 1 mice



### Animal Source Taconic Laboratory Animals and Services (Germantown, NY)



### Time Held Before Studies Rats: 14 days (males) or 12 or 14 days (females) Mice: 12 days


### Average Age When Studies Began Rats: 6-7 weeks Mice: 6 weeks


### Date of First Exposure Rats: February 8, 1996 Mice: February 20, 1996


### Duration of Exposure 105 weeks, except 2,000 and 5,000 ppm stop-exposure groups which received undosed feed from week 14 until the end of the studies


### Date of Last Exposure Core study rats: February 5-11, 1998 Stop-exposure rats: May 8, 1996 Mice: February 17-23, 1998


### Average Age at Necropsy Rats: 110-112 weeks Mice: 110-111 weeks


### Size of Study Groups Rats: 60 males and 60 females [0 (females only), 625, 1,250, and 2,000 ppm groups]; 70 males (0 ppm and 2,000 and 5,000 ppm stop-exposure groups) Mice: 60 males and 60 females



### Method of Distribution Animals were distributed randomly into groups of approximately equal initial mean body weights.


### Animals per Cage Rats: up to 3 (males) or 5 (females); 1 rat per cage during urine collection periods Mice: 1 (males) or 5 (females); 1 mouse per cage during urine collection periods



### Method of Animal Identification Tail tattoo



### Diet NTP-2000 open formula meal/pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum. Animals received nonirradiated feed from the beginning of the studies until July 3 (rats) or 8 (mice), 1996 and irradiated feed thereafter. 

### T ABLE 

### 5 Experimental Design and Materials and Methods in the 2-Year Feed Studies of o -Nitrotoluene

### o-Nitrotoluene, NTP TR 504 31

Water Tap water (Birmingham, AL, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum


### Cages Polycarbonate (Lab Products, Inc., Maywood, NJ), changed twice weekly


### Bedding Heat-treated hardwood bedding chips (P.J. Murphy Forest Products, Inc., Montville, NJ), changed twice weekly


### Cage Filters Reemay ® (Andico; Birmingham, AL), changed every 2 weeks



### Racks Stainless steel drawer type (Lab Products, Inc., Maywood, NJ), rotated every 2 weeks



### Animal Room Environment Temperature: 72° ± 3° F Relative humidity: 50% ± 15% Room fluorescent light: 12 hours/day Room air changes: 10/hour


### Exposure Concentrations Core study rats: 0, 625, 1,250, or 2,000 ppm in feed Stop-exposure rats: 2,000 or 5,000 ppm in feed Mice: 0, 1,250, 2,500, or 5,000 ppm in feed


### Type and Frequency of Observation Observed twice daily; animals were weighed initially, during week 4, and every 4 weeks thereafter; clinical findings were recorded at 4-week intervals. Feed consumption was measured over a 1-week period every 4 weeks.



### Method of Sacrifice CO 2 asphyxiation


### Necropsy Necropsies were performed on all animals. Organs weighed at the 3-month interim evaluation were the heart, right kidney, liver, lungs, right testis, and thymus.


### Urinalysis Urine was collected during a 24-hour period from five male and five female rats and mice from each core study group at 2 weeks and 3, 12, and 18 months. Parameters evaluated included urine volume and creatinine, o -nitrobenzoic acid, o -nitrobenzylmercapturic acid, and

o -aminobenzoic acid concentrations.


### Histopathology Complete histopathology was performed on all rats and mice. In addition to gross lesions and tissue masses, the following tiss ues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), sm all intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (except male mice), nos e, ovary, pancreas, pancreatic islets, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. 

### T ABLE 

### 5 Experimental Design and Materials and Methods in the 2-Year Feed Studies of o -Nitrotoluene

### 32 o-Nitrotoluene, NTP TR 504



# S TATISTICAL 

# M ETHODS 

## Survival Analyses The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from naturalcauses were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.



## Calculation of Incidence The incidences of neoplasms or nonneoplastic lesions are presented in Tables A1, A5, B1, B5, C1, C5, D1, and D5 as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. For calculation of statistical significance, the incidences of most neoplasms(Tables A3a, A3b, B3, C3, and D3) and all nonneoplastic lesions are given as the numbers of animals affected at each site examined microscopically. However, whenmacroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian gland, intestine,mammary gland, and skin) before microscopic evaluation, or when neoplasms had multiple potential sites ofoccurrence (e.g., leukemia or lymphoma), the denominators consist of the number of animals on which a necropsy was performed. Tables A3a, A3b, B3, C3, and D3 also give the survival-adjusted neoplasm rate for each group and each site-specific neoplasm. This survival-adjusted rate (based on the Poly-3 method described below) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, to animals that do not reach terminal sacrifice. 

## Analysis of Neoplasm and Nonneoplastic Lesion Incidences The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence.This test is a survival-adjusted quantal-response procedure that modifies the Cochran-Armitage linear trend test to take survival differences into account. More specifically, this method modifies the denominator in the quantal estimate of lesion incidence to approximate more closely the total number of animal years at risk. For analysis of a given site, each animal is assigned a risk weight. This value is one if the animal had a lesion at that site or if it survived until terminal sacrifice; if the animal died prior to terminal sacrifice and did not have a lesion at that site, its risk weight is the fraction of the entire study time that it survived, raised to the kth power.

This method yields a lesion prevalence rate that depends only upon the choice of a shape parameter for a Weibull hazard function describing cumulative lesion incidence over time (Bailer and Portier, 1988). Unless otherwisespecified, a value of k=3 was used in the analysis of sitespecific lesions. This value was recommended by Bailer and Portier (1988) following an evaluation of neoplasmonset time distributions for a variety of site-specific neoplasms in control F344 rats and B6C3F 1 

### mice (Portier et al. , 1986). Bailer and Portier (1988) showed that the Poly-3 test gave valid results if the true value of k was anywhere in the range from 1 to 5. A further advantage of the Poly-3 method is that it does not require lesion lethality assumptions. Variation introduced by the use of risk weights, which reflect differential mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended by Bieler and Williams (1993).
Tests of significance included pairwise comparisons ofeach exposed group with controls and a test for an overall exposure-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance oflower incidences or decreasing trends in lesions are represented as 1-P with the letter N added (e.g., P=0.99 ispresented as P=0.01N). For neoplasms and nonneoplastic lesions detected at the interim evaluation, the Fisher exact test (Gart et al. , 1979), a procedure based on the overall proportion of affected animals, was used.



## Analysis of Continuous VariablesTwo approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). To makepairwise comparisons, urinary biomarker data were analyzed by Fisher’s least significant difference test (following initial ANOVA) (Miller, 1960). Average severityvalues were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973).

### o-Nitrotoluene, NTP TR 504 33



## Historical Control Data The concurrent control group represents the most valid comparison to the treated groups and is the only control group analyzed statistically in NTP bioassays. However, historical control data are often helpful in interpreting potential treatment-related effects, particularly for uncommon or rare neoplasm types. For meaningful comparisons, the conditions for studies in the historical database must be generally similar. Until recently, the NTP historical control database consisted of animals fed the NIH-07 diet. In 1995, the NTP changed the diet fed to animals used in toxicity and carcinogenesis studiesconducted by the NTP. This new diet (NTP-2000) contains less protein and more fiber and fat than the NIH-07 diet previously used (Rao, 1996, 1997). This dietary change was instituted primarily to increase longevity anddecrease the incidence and/or severity of some spontaneous neoplastic and nonneoplastic lesions in the rats and mice used in NTP studies. These studies of

o- nitrotoluene are among the first in which the animalson study were fed the NTP-2000 diet. Because the incidence of some neoplastic and nonneoplastic lesions maybe affected by the dietary change, use of the existing historical control database (NIH-07 diet) may not be appropriate for all neoplasm types.

Currently, the database includes 11 (10 for male rats) studies by various routes in which the NTP-2000 dietwas used. Based on the extensive NTP historical database using the NIH-07 diet, incidences of the vast majority of spontaneous neoplasms are not significantly different between control groups regardless of the route of administration. There is no reason to expect this to be different with the NTP-2000 diet. For example, control animals from dosed feed and dosed water studies are treated no differently and no differences in incidence ofneoplasms are expected. Exceptions exist for some neoplasms/routes, and if comparisons are necessary for these neoplasm types, only studies with similar routes of administration will be used.



## Irradiated Feed Ionizing energy (irradiation) is known to destroy most, if not all, bacterial and insect contamination without a significant loss of essential nutrients (Rao and Knapka, 1998). The NTP-2000 diet manufactured and used for the NTP studies was irradiated from May 1996 (fed to rats and mice after June 1996) at the FDA-approved level (25 to 50 kilogray) of ionizing radiation. Batches of diets were evaluated for nutrient concentrationsbefore and after irradiation. The concentrations of nutrients and their byproducts of irradiated diets were notsubstantially different from the same batches before irradiation and nutritionally adequate rodent diets.



# Q UALITY 

# A SSURANCE 

# M ETHODS The 2-year studies were conducted in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from the 2-year studies were submitted to theNTP Archives, these studies were audited retrospectively by an independent quality assurance contractor. Separate audits covered completeness and accuracy ofthe pathology data, pathology specimens, final pathology tables, and a draft of this NTP Technical Report. Audit procedures and findings are presented in the reports and are on file at NIEHS. The audit findingswere reviewed and assessed by NTP staff, and all comments were resolved or otherwise addressed during the preparation of this Technical Report.



# G ENETIC 

# T OXICOLOGY The genetic toxicity of o -nitrotoluene was assessed by testing the ability of the chemical to induce mutations in various strains of Salmonella typhimurium, sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells, micronucleated erythrocytes in rat and mouse bone marrow, andincreases in the frequency of micronucleated erythrocytes in mouse peripheral blood. The protocols for these studies and the results are given in Appendix E.

The genetic toxicity studies of o -nitrotoluene are part of a larger effort by the NTP to develop a comprehensive database that would permit a critical anticipation of a chemical’s carcinogenicity in experimental animalsbased on numerous considerations, including the molecular structure of the chemical and its observed effects in short-term in vitro and in vivo genetic toxicity tests (structure-activity relationships). These short-term genetic toxicity tests were originally developed to clarify mechanisms of chemical-induced DNA damage growingout of the earlier electrophilicity/mutagenicity relationship proposed by Miller and Miller (1977) and the somatic mutation theory of cancer (Straus, 1981;

### 34 o-Nitrotoluene, NTP TR 504



### Crawford, 1985). Therefore, the information obtained from these tests applies only to mutagenic carcinogens.

For mutagenic carcinogens, the combination of DNA reactivity and Salmonella mutagenicity is highly correlated with the induction of carcinogenicity in multiple species and genders of rodents and at multiple tissuesites (Ashby and Tennant, 1991). Data from NTP studies show that a positive response in Salmonella is the most predictive in vitro test for rodent carcinogenicity (89% of the Salmonella mutagens are rodent carcinogens) and that there is no complementarity among the in vitro genetic toxicity tests (Tennant et al., 1987; Zeiger et al., 1990). That is, no battery of tests that included the Salmonella test improved the predictivity of the Salmonella test alone. Although other in vitrogenetic toxicity tests correlate less well with rodent carcinogenicity compared with the Salmonella test, these other tests can provide useful information on the types of DNA and chromosomal effects induced by the chemical under investigation. The predictivity for carcinogenicity of a positive response in the acute in vivo bone marrow chromosome aberration test or micronucleus test appears to be less than that in the Salmonella test (Shelby et al., 1993; Shelby and Witt, 1995). However, clearly positive results in long-term peripheral blood micronucleus testsare associated with high predictivity for rodent carcinogenicity (Witt et al. , 2000); negative results in this assay do not correlate well with either negative or positive results in rodent carcinogenicity studies. Because of the theoretical and observed associations between induced genetic damage and adverse effects in somatic and germ cells, the determination of in vivo genetic effects isimportant to the overall understanding of the risks associated with exposure to a particular chemical. Most organic chemicals that are identified by the International Agency for Research on Cancer as human carcinogens, other than hormones, are genotoxic. The vast majority of these are detected by both the Salmonella assay and rodent bone marrow cytogenetics tests (Shelby, 1988; Shelby and Zeiger, 1990).

### 35



# R

ATS 

## Survival Estimates of 2-year survival probabilities for male andfemale rats are shown in Table 6 and in the KaplanMeier survival curves (Figure 3). All 2,000 ppm core study, all 5,000 ppm stop-exposure, and all but three core study 1,250 ppm male rats died before the end of the study. Survival of 625 ppm core study and 2,000 ppm stop-exposure males and of 2,000 ppm females was significantly less than that of the controls. Due to the poor survival of exposed rats, the planned 15-month interim evaluation was cancelled.



## Body Weights, Feed and Compound Consumption, and Clinical Findings Mean body weights of all exposed groups of males except the 625 ppm group were generally less than those of the controls throughout the study (Table 7 and Figure 4). Mean body weights of 2,000 ppm females were less than those of the controls during year 2 of the study (Table 8 and Figure 4). Feed consumption by exposed males and females was similar to that by the controls throughout the study (Tables I1 and I2). Dietary concentrations of 625, 1,250, or 2,000 ppm resulted in average daily doses of approximately 25, 50, or 90 mg

o -nitrotoluene/kg body weight to core study males and 30, 60, or 100 mg/kg to females. Dietary concentrations of 2,000 or 5,000 ppm to stop-exposure males resulted in average daily doses of approximately 125 or 315 mg/kg for the first 3 months of the study. Clinical findings included large subcutaneous masses in the torso, head,and appendages of exposed males and females; the numbers of these masses generally increased with exposure concentration. Males in both 2,000 ppm groups and the 5,000 ppm stop-exposure group had small ears and thin tails. Although the cause of the small ears and thin tails is unknown, the presence of this unusual clinical finding in both stop-exposure and core study groups suggests that the initial insult occurred during the first 3 months of o- nitrotoluene exposure. 

## Biomarkers of Exposure The results of urinary metabolite determinations in male and female rats are presented in Table F1. With the exception of the 2,000 ppm group at 18 months, the ratios of o- nitrobenzoic acid to creatinine excreted in the urine of male rats were significantly larger at 2 weeks than at the later time points. In females, by contrast, thedifferences between time points were generally not significant. Creatinine concentrations were generally lower at 2 weeks in males and females than at other time points, and the apparent time-related change in the ratios may not reflect a change in o- nitrotoluene metabolism. The o- nitrobenzoic acid/creatinine ratios were generally larger for females than for males and were linearly related to exposure concentration.

In contrast to the o- nitrobenzoic acid/creatinine ratios, larger ratios of o- nitrobenzylmercapturic acid to creatinine were seen in urine of all exposed groups at week 2 compared to later time points, with one exception. These ratios were significantly smaller for exposedfemales than for males. Because the first step in the formation of either o- nitrobenzoic acid or o- nitrobenzylmercapturic acid is oxidation of the methyl group to a benzyl alcohol, metabolic differences must be either in further oxidation to the carboxylic acid or in formation of conjugates of the alcohol and further reaction with reduced glutathione. The o- nitrobenzylmercapturic acid/creatinine ratios were linearly related to exposure concentration in males and females.

The ratios of o- aminobenzoic acid to creatinine for control and exposed male and female rats were generally similar. o- Aminobenzoic acid is a product of catabolism of tryptophan (White et al. , 1978) and is a relatively minor metabolite of o- nitrotoluene (Chism et al. , 1984). No time- or sex-related analyses of these data were performed. 

# RESULTS

### 36 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 6 Survival of Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Male

Animals initially in study 70 60 60 60 70 70

3-Month interim evaluation a 10 0 0 0 10 10 Accidental deaths a 0 030 00 Moribund 18 35 48 53 39 57 Natural deaths 3 7 6 7 10 3 Animals surviving to study termination 39 b 18 3 0 11 0 Percent probability of survival at end of study c 65 30 5 0 18 0 Mean survival (days) d 698 647 590 508 612 483

Survival analysis e P<0.001 P<0.001 P<0.001 P<0.001 Survival analysis f P<0.001 P<0.001 P<0.001



### Female

Animals initially in study 60 60 60 60

Moribund 10 11 19 22 Natural deaths 3 2 2 5 Animals surviving to study termination 47 g 47 b 39 33 Percent probability of survival at end of study 78 78 65 55 Mean survival (days) 712 706 696 697

Survival analysis e P=0.002 P=1.000 P=0.128 P=0.009

a Censored from survival analyses b Includes one animal that died during the last week of the study c Kaplan-Meier determinations d Mean of all deaths (uncensored, censored, and terminal sacrifice) e The result of the life table trend test (Tarone, 1975) for core study groups is in the control column, and the results of the l ife table pairwise comparisons (Cox, 1972) with the controls are in the exposed group columns. f Trend test and pairwise comparisons for stop-exposure groups g Includes two animals that died during the last week of the study

### o-Nitrotoluene, NTP TR 504 37



### F IGURE 

### 3 Kaplan-Meier Survival Curves for Male and Female Rats Exposed to o -Nitrotoluene in Feed for 2 Years

### 38 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 7 Mean Body Weights and Survival of Male Rats in the 2-Year Feed Study of o-Nitrotoluene

Weeks 0 ppm 625 ppm 1,250 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Study (g) Survivors (g) controls) Survivors (g) controls) Survivors

1 122 70 122 100 60 123 100 60 4 239 70 237 99 60 235 98 60 8 303 70 299 99 60 295 97 60 12 344 70 337 98 60 327 95 60 16 a 370 60 363 98 60 350 95 60 20 391 60 378 97 60 366 94 60 24 401 60 385 96 60 372 93 60 28 410 60 399 97 60 384 94 60 32 422 60 406 96 60 392 93 60 36 429 60 413 96 60 399 93 60 40 432 60 414 96 60 400 93 60 44 434 60 416 96 60 400 92 57 48 433 60 417 96 60 402 93 57 52 436 60 419 96 60 404 93 57 56 428 60 415 97 59 398 93 55 60 439 60 423 96 59 403 92 55 64 443 59 427 96 58 409 92 54 68 447 59 432 97 56 417 93 54 72 446 59 433 97 55 418 94 49 76 435 59 424 98 54 409 94 42 80 433 55 419 97 51 405 94 42 84 430 54 423 98 47 407 95 36 88 434 54 420 97 44 411 95 31 92 428 52 425 99 32 408 95 21 96 425 49 424 100 28 395 93 16 100 424 46 421 99 25 408 96 8 104 425 40 415 98 19 395 93 3

Mean for weeks 1-13 252 249 99 245 97 14-52 416 401 96 387 93 53-104 434 423 97 406 94

### o-Nitrotoluene, NTP TR 504 39



### T ABLE 

### 7 Mean Body Weights and Survival of Male Rats in the 2-Year Feed Study of o-Nitrotoluene

Weeks 2,000 ppm 2,000 ppm (Stop-Exposure) 5,000 ppm (Stop-Exposure)

on Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Study (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 122 100 60 123 101 70 121 99 70 4 228 96 60 230 97 70 211 88 70 8 282 93 60 285 94 70 247 81 70 12 315 92 60 316 92 70 263 77 70 16 a 329 89 60 341 92 60 279 76 60 20 350 90 60 359 92 60 297 76 60 24 357 89 60 371 92 60 305 76 60 28 363 88 60 381 93 60 312 76 60 32 371 88 60 390 93 60 321 76 60 36 377 88 60 398 93 60 328 76 60 40 378 88 60 400 93 58 329 76 60 44 378 87 60 403 93 58 331 76 58 48 378 87 60 402 93 58 334 77 57 52 383 88 59 408 94 58 336 77 52 56 377 88 57 402 94 57 329 77 45 60 380 86 50 411 94 56 342 78 40 64 388 88 46 417 94 55 346 78 38 68 394 88 40 422 94 54 356 80 33 72 396 89 34 421 94 52 364 82 25 76 393 90 23 415 96 49 367 84 22 80 386 89 15 408 94 46 360 83 14 84 374 87 8 403 94 38 368 86 8 88 368 85 4 413 95 32 349 80 4 92 423 99 2 408 95 28 321 75 1 96 412 97 22 349 82 1 100 412 97 18 104 404 95 11

Mean for weeks 1-13 237 94 239 95 211 84 14-52 366 88 385 93 317 76 53-104 388 89 411 95 350 81

a Interim evaluations occurred during week 14 (0 ppm group and 2,000 and 5,000 ppm stop-exposure groups).

### 40 o-Nitrotoluene, NTP TR 504



### F IGURE 

### 4 Growth Curves for Male and Female Rats Exposed to o -Nitrotoluene in Feed for 2 Years

### o-Nitrotoluene, NTP TR 504 41



### T ABLE 

### 8 Mean Body Weights and Survival of Female Rats in the 2-Year Feed Study of o- Nitrotoluene

Weeks 0 ppm 625 ppm 1,250 ppm 2,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 105 60 104 99 60 104 100 60 105 100 60 4 145 60 144 99 60 145 100 60 143 99 60 8 171 60 168 98 60 170 99 60 168 98 60 12 185 60 180 97 60 182 99 60 181 98 60 16 195 60 193 99 60 193 99 60 193 99 60 20 203 60 200 98 60 200 98 60 196 96 60 24 207 60 204 99 60 204 98 60 201 97 60 28 212 60 210 99 60 209 98 60 206 97 60 32 221 60 217 98 60 216 98 60 213 96 60 36 225 60 221 98 60 219 98 60 214 95 60 40 227 60 225 99 60 224 99 60 216 95 60 44 231 60 228 99 60 227 98 60 220 95 60 48 236 60 232 98 60 231 98 60 222 94 60 52 239 60 235 98 60 235 98 60 224 93 60 56 244 60 241 99 60 241 99 59 227 93 60 60 254 59 247 97 60 247 97 59 230 90 60 64 258 59 256 99 59 251 97 58 234 91 60 68 266 58 263 99 59 259 97 58 241 91 59 72 271 58 267 99 58 267 98 58 243 89 58 76 276 58 271 98 57 268 97 56 243 88 58 80 281 58 274 97 56 272 97 56 247 88 58 84 284 58 275 97 56 272 96 55 247 87 57 88 284 58 278 98 55 274 96 53 250 88 55 92 290 56 285 98 55 282 97 52 257 89 52 96 291 56 287 99 55 287 99 51 264 91 48 100 294 54 291 99 52 293 100 49 273 93 43 104 299 48 302 101 47 298 100 39 275 92 33

Mean for weeks 1-13 152 149 98 150 99 149 98 14-52 220 217 99 216 98 211 96 53-104 276 272 99 270 98 249 90

### 42 o-Nitrotoluene, NTP TR 504



## Pathology and Statistical AnalysesThis section describes the statistically significant or biologically noteworthy changes in the incidences ofmononuclear cell leukemia and neoplasms and/or nonneoplastic lesions of the mesothelium, skin, mammary gland, liver, lung, circulatory system, testis, and other organs. Summaries of the incidences of neoplasms andnonneoplastic lesions, individual animal tumor diagnoses, statistical analyses of primary neoplasms that occurred with an incidence of at least 5% in at least oneanimal group, and historical incidences for the neoplasms mentioned in this section are presented in Appendix A for male rats and Appendix B for female
rats.

Mesothelium: The incidences of malignant mesothelioma occurred with positive trends in males, and the incidences in exposed groups were significantly greater than that in the control group at 2 years (Tables 9, A3a, and A3b). The incidences in the exposed groups exceeded the 2-year historical ranges in control (all routes) male rats given NTP-2000 diet and in feed study controls given NIH-07 diet (Tables 9 and A4a). The mesotheliomas were associated with the tunica vaginalis of the testis or epididymis (Plate 1). In some males, mesotheliomas were observed on the abdominal wall and on the surface of abdominal organs. The majority of mesotheliomas were large and consisted of papillary or solid areas of pleomorphic mesothelial cells (Plate 2). In some areas, beginning formation of tubules was seen.Extension or implantation of neoplastic cells and invasion of adjacent organs were limited to the serosa or occasionally the capsule of the involved organ. In male F344/N rats, almost all spontaneous mesotheliomas arisefrom the tunica vaginalis and, consequently, early neoplasms are found adherent to the epididymis or tunica albuginea of the testis, consistent with the predominant location of mesotheliomas in this study. In NTP studies, chemically induced mesotheliomas have been seen almost exclusively in male F344/N rats. Mesotheliomas were not observed in female rats in this study (Table B1).

T ABLE 

### 9 Incidences of Malignant Mesothelioma in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Malignant Mesothelioma a
Overall rate b 2/60 (3%) 20/60 (33%) 29/60 (48%) 44/60 (73%) 44/60 (73%) 54/60 (90%) Adjusted rate c 3.7% 40.6% 62.4% 87.1% 80.3% 95.1% Terminal rate d 2/39 (5%) 5/18 (28%) 1/3 (33%) 0/0 10/11 (91%) 0/0 First incidence (days) 729 (T) 428 432 359 275 299 Poly-3 test e P<0.001 P<0.001 P<0.001 P<0.001 Poly-3 test f P<0.001 P<0.001 P<0.001

(T)Terminal sacrifice a Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 23/609 (3.7% ± 2.9%), ran ge 0%-10%; with feed study controls given NIH-07 diet: 25/1,004 (2.5% ± 2.0%), range 0%-8% b Number of animals with neoplasm per number of animals necropsied c Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d Observed incidence at terminal kill e Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. f Trend test and pairwise comparisons for stop-exposure groups

### o-Nitrotoluene, NTP TR 504 43



### Skin (Subcutaneous): The incidences of fibroma, fibrosarcoma, and fibroma or fibrosarcoma (combined) of the subcutaneous skin in males and of fibroma and fibroma or fibrosarcoma (combined) in females occurred with positive trends, and the incidences in the exposed groups were generally significantly greater than those in the controls at 2 years (Tables 10, A3a, A3b, and B3).Incidences of multiple fibroma and of lipoma were significantly increased in exposed groups of males. The incidences of these neoplasms in the exposed groupsgenerally exceeded the 2-year historical ranges in control (all routes) rats given NTP-2000 diet and in feed study controls given NIH-07 diet (Tables 10, A4b, and B4a).

Fibromas occurred in the subcutis of the skin of thetorso, head, and appendages. Fibromas were noninvasive, expansile neoplasms consisting of proliferations of fibrocytes with an abundant collagenous stroma (Plate 3). Neoplastic cells and collagen formed interlacing bundles or whorling patterns. Mitoses were rare, and cellular atypia was not a feature of fibroma. Fibrosarcomas were generally more cellular and the large, well-formed bundles of mature collagen were less prominent than in fibromas (Plate 4). Fusiform cells were densely packed in interlacing bundles; pleomorphic cells with large nuclei were present in some instances. Mitotic figures were common and often atypical. Lipomas were characterized by focal nodules of variably sized adipocytes often infiltrated with streaks of fibrous tissue; occasionally, a few chronic inflammatory cells were present (Plate 5). Fibromas and fibrosarcomas are the most common neoplasms found in the skin of F344/N rats. Fibromas occur more frequently than fibrosarcomas and are more common in male than female control F344/N rats. Lipomas occur more often in males than in females and are most commonly found in the subcutis (Elwell et al. , 1990).

### 44 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 10 Incidences of Subcutaneous Skin Neoplasms in Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Male

Number Necropsied 60 60 60 60 60 60 Lipoma, Multiple a 0 0 1 00 3 Lipoma (includes multiple) b 0 4* 13** 13** 10** 12** Fibroma, Multiple 1 25** 39** 35** 22** 28** Fibroma (includes multiple) c 5 46** 52** 59** 45** 52** Fibrosarcoma, Multiple 0 0 4* 5* 0 4* Fibrosarcoma (includes multiple) d 0 7* 17** 20** 8** 12**

Fibroma or Fibrosarcoma e

Overall rate f 5/60 (8%) 47/60 (78%) 55/60 (92%) 59/60 (98%) 47/60 (78%) 53/60 (88%) k Adjusted rate g 9.3% 86.3% 98.7% 99.8% 89.0% 97.8% Terminal rate h 3/39 (8%) 15/18 (83%) 3/3 (100%) 0/0 10/11 (91%) 0/0 First incidence (days) 705 465 384 391 275 299 Poly-3 test i P<0.001 P<0.001 P<0.001 P<0.001 Poly-3 test j P<0.001 P<0.001 P<0.001



### Female

Number Necropsied 60 60 60 60 Fibroma, Multiple 0 0 0 1 Fibroma (includes multiple) l 3 3 18** 20** Fibrosarcoma, Multiple 0 0 0 1 Fibrosarcoma (includes multiple) 0 0 4 3

Fibroma or Fibrosarcomam

Overall rate 3/60 (5%) 3/60 (5%) 21/60 (35%) 22/60 (37%) Adjusted rate 5.3% 5.4% 37.6% 40.6% Terminal rate 3/47 (6%) 3/47 (6%) 16/39 (41%) 16/33 First incidence (days) 729 (T) 729 (T) 432 663 Poly-3 test i P<0.001 P=0.652 P<0.001 P<0.001

* Significantly different (P # 0.05) from the control group by the Poly-3 test ** P # 0.01 (T)Terminal sacrifice a Number of animals with lesion b Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 8/609 (1.5% ± 1.3%), ran ge 0%-4%; with feed study controls given NIH-07 diet: 2/1,004 (0.2% ± 0.6%), range 0%-2% c Historical incidence for NTP-2000 diet: 33/609 (5.1% ± 4.0%), range 0%-12%; for NIH-07 diet: 56/1,004 (5.6% ± 3.2%), range 0%-1 0% d Historical incidence for NTP-2000 diet: 8/609 (1.3% ± 1.4%), range 0%-4%; for NIH-07 diet: 9/1,004 (0.9% ± 1.4%), range 0%-4% e Historical incidence for NTP-2000 diet: 41/609 (6.4% ± 4.3%), range 2%-14%; for NIH-07 diet: 65/1,004 (6.5% ± 3.1%), range 2% -10% f Number of animals with neoplasm per number of animals necropsied g Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality h Observed incidence at terminal kill i Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. j Trend test and pairwise comparisons for stop-exposure groups k A single sarcoma occurred in an animal that also had a fibroma. l Historical incidence for NTP-2000 diet: 14/659 (2.0% ± 2.1%), range 0%-6%; for NIH-07 diet: 15/1,001 (1.5% ± 1.6%), range 0%- 4% m Historical incidence for NTP-2000 diet: 18/659 (2.6% ± 2.1%), range 0%-6%; for NIH-07 diet: 19/1,001 (1.9% ± 2.0%), range 0%- 6%

### o-Nitrotoluene, NTP TR 504 45



### Mammary Gland: In all exposed groups of males andfemales except 2,000 ppm core study males, the incidences of fibroadenoma of the mammary gland were significantly greater than those in the controls at 2 years (Tables 11, A3a, A3b, and B3). The incidences occurredwith positive trends in males and females. The incidences of multiple fibroadenoma were significantly increased in exposed groups of females. The incidences in these groups generally exceeded the 2-year historical ranges in control (all routes) rats given NTP-2000 diet and in feed study controls given NIH-07 diet (Tables 11, A4c, and B4b). Fibroadenomas were characterized by collections of glandular epithelium arranged in acini and ducts and were surrounded by fibrous connective tissue (Plate 6). The relative amounts of glandular and fibrouselements varied greatly among the neoplasms. Epithelial cells were usually uniform and arranged in a single layer (Plate 7); a few fibroadenomas had small areasof cellular atypia. The incidences of hyperplasia, a precursor of fibroadenoma, were significantly increased in 625 and 1,250 ppm females (Tables 11 and B5). Hyperplasia was characterized by enlarged lobules withan increase in the number of alveoli separated by collagenous stroma.

### 46 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 11 Incidences of Mammary Gland Neoplasms in Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Male

Number Necropsied 60 60 60 60 60 60 Hyperplasia a 6 (2.2) b 2 (1.5) 2 (2.5) 2 (2.0) 1 (1.0) 4 (1.5)

Fibroadenoma, Multiple 0 0 1 0 1 0

Fibroadenoma (includes multiple) c

Overall rate d 0/60 (0%) 7/60 (12%) 10/60 (17%) 2/60 (3%) 13/60 (22%) 20/60 (33%) Adjusted rate e 0.0% 15.6% 26.2% 9.0% 31.2% 61.1% Terminal rate f 0/39 (0%) 4/18 (22%) 1/3 (33%) 0/0 5/11 (46%) 0/0 First incidence (days) —i 621 576 608 523 299 Poly-3 test g P<0.001 P=0.004 P<0.001 P=0.110 Poly-3 testh P<0.001 P<0.001 P<0.001



### Female

Number Necropsied 60 60 60 60 Hyperplasia 14 (1.7) 36** (2.3) 30** (2.3) 19 (2.1)

Fibroadenoma, Multiple 6 35** 39** 42**

Fibroadenoma (includes multiple) j Overall rate 23/60 (38%) 47/60 (78%) 52/60 (87%) 56/60 k (93%) Adjusted rate 4 0.0% 82.0% 91.7% 96.2% Terminal rate 18/47 (38%) 39/47 (83%) 36/39 (92%) 33/33 (100%) First incidence (days) 620 505 525 464 Poly-3 test g P<0.001 P<0.001 P<0.001 P<0.001

Carcinoma l 001 1

** Significantly different (P # 0.01) from the control group by the Poly-3 test a Number of animals with lesion b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked c Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 26/609 (3.8% ± 2.9%), ran ge 0%-8%; with feed study controls given NIH-07 diet: 42/1,004 (4.2% ± 3.5%), range 0%-12% d Number of animals with neoplasm per number of animals necropsied e Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality f Observed incidence at terminal kill g Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. h Trend test and pairwise comparisons for stop-exposure groups i Not applicable; no neoplasms in animal group j Historical incidence for NTP-2000 diet: 284/659 (41.1% ± 10.1%), range 28%-56%; for NIH-07 diet: 431/1,001 (43.1% ± 10.7%), range 24%-60% k A single adenoma occurred in an animal that also had a fibroadenoma. l Historical incidence for NTP-2000 diet: 15/659 (2.6% ± 2.2%), range 0%-6%; for NIH-07 diet: 32/1,001 (3.2% ± 2.6%), range 0%-8%

### o-Nitrotoluene, NTP TR 504 47



### Liver: At 3 months, liver weights of 5,000 ppm stopexposure males were significantly greater than those ofthe controls (Table G1). The incidences of hepatocellular adenoma in 2,000 ppm core study males and females and of hepatocellular adenoma or carcinoma (combined) in 2,000 ppm core study and 5,000 ppm stop-exposuremales were significantly greater than those in the controls at 2 years (Tables 12, A3a, A3b, and B3). The incidences in these groups generally exceeded the 2-year historical ranges in control (all routes) rats given NTP-2000 diet and in feed study controls given NIH-07diet (Tables 12, A4d, and B4c). Hepatocellular adenomas consisted of nodules of hepatocytes that compressedadjacent hepatic parenchyma and lacked the normal lobular and sinusoidal pattern (Plate 8). Hepatocellular carcinomas consisted of solid sheets of hepatocytes ortrabeculae three or more cells thick. Neoplastic hepatocytes were anaplastic, with prominent nuclei containing one or more nucleoli and variable amounts of cytoplasm. A single hepatocholangiocarcinoma occurred in a 625 ppm male and in a 2,000 ppm core study male, andcholangiocarcinomas occurred in three 5,000 ppm stopexposure males. Although the incidences of these neoplasms were low, they were considered exposure related. Hepatocholangiocarcinomas were malignant neoplasms containing cells resembling both hepatocyte and biliaryepithelium (Plate 9), while cholangiocarcinomas consisted primarily of neoplastic biliary epithelium. In cholangiocarcinomas, the bile duct epithelium was atypical, infiltrative, and often surrounded by collagenous connective tissue (Plate 10). No hepatocholangiocarcinomas or cholangiocarcinomas have been observed in male controls given NTP-2000 or NIH-07 diet for 2 years. However, 2 of 20 male F344/N rats given 5,000 ppm o- nitrotoluene for 13 weeks followed by a 13-week recovery period and one male F344/N rat given 5,000 ppm o- nitrotoluene for 26 weeks had cholangiocarcinomas (NTP, 1996).

At the 3-month interim evaluation, the incidence ofhepatocellular cytoplasmic vacuolization was significantly increased in 5,000 ppm stop-exposure males. The vacuolated hepatocytes were observed uniformly throughout the liver lobules. Hepatocellular vacuoles were suggestive of hydropic degeneration and were of moderate severity, and the incidences correlated with the increased liver weights. At 2 years, the incidences of several nonneoplastic lesions of the liver were increased in exposed groups of males and females (Tables 12, A5, and B5). Hepatocellular foci are considered potentialpreneoplastic lesions and are relatively discrete aggregates of hepatocytes, often enlarged; the lobular pattern of the liver is generally retained. Mixed cell infiltration in males was focal and characterized by minimal chronic active inflammation. Centrilobular necrosis was seen ina small number of exposed rats at all exposure concentrations (except 625 ppm females) and was characterized by focal mild coagulative necrosis of hepatocytes.

### 48 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 12 Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Male

3-Month Interim Evaluation Number Examined Microscopically 10 10 10 Hepatocellular Cytoplasmic

Vacuolization a 0 0 10** (2.8) b

2-Year Study Number Examined Microscopically 60 60 60 60 60 60 Clear Cell Focus 29 29 34 31* 30 34** Eosinophilic Focus 7 18** 29** 24** 15** 13** Mixed Cell Focus 5 7 12** 6 12* 8* Hematopoietic Cell Proliferation 0 6* (1.8) 2 (2.0) 2 (1.5) 11** (1.5) 6** (1.5) Infiltration Cellular, Mixed Cell 1 (1.0) 5 (1.2) 11** (1.5) 20** (1.7) 15** (1.7) 33** (1.7) Centrilobular, Necrosis 1 (2.0) 3 (2.3) 8** (1.9) 5* (1.8) 9** (2.6) 3 (2.7)

Hepatocholangiocarcinoma c 0 1 0 10 0 Cholangiocarcinoma c 0 0 0 0 0 3* Hepatocellular Adenoma, Multiple 0 1 0 0 1 1

Hepatocellular Adenoma (includes multiple) d

Overall rate e 2/60 (3%) 3/60 (5%) 3/60 (5%) 7/60 (12%) 3/60 (5%) 4/60 (7%) Adjusted rate f 3.7% 6.8% 8.4% 27.1% 7.6% 18.4% Terminal rate g 1/39 (3%) 1/18 (6%) 0/3 (0%) 0/0 1/11 (9%) 0/0 First incidence (days) 707 621 615 391 579 499 Poly-3 testh P=0.007 P=0.413 P=0.325 P=0.006 Poly-3 test i P=0.062 P=0.362 P=0.079

Hepatocellular Carcinoma, Multiple 0 0 0 1 0 0 Hepatocellular Carcinoma

(includes multiple) 1 0 0 1 0 2

Hepatocellular Adenoma or Carcinoma j Overall rate 3/60 (5%) 3/60 (5%) 3/60 (5%) 8/60 (13%) 3/60 (5%) 6/60 (13%) Adjusted rate 5.6% 6.8% 8.4% 30.2% 7.6% 25.9% Terminal rate 2/39 (5%) 1/18 (6%) 0/3 (0%) 0/0 1/11 (9%) 0/0 First incidence (days) 707 621 615 391 579 391 Poly-3 testh P=0.009 P=0.570 P=0.466 P=0.007 Poly-3 test i P=0.030 P=0.511 P=0.029

### o-Nitrotoluene, NTP TR 504 49

Female

Number Examined Microscopically 60 59 60 60 Basophilic Focus 51 56* 60** 54 Clear Cell Focus 16 30** 28* 33** Eosinophilic Focus 5 12 25** 32** Mixed Cell Focus 6 9 11 28** Centrilobular, Necrosis 3 (1.7) 0 2 (2.5) 2 (3.0)

Hepatocellular Adenoma, Multiple 0 0 0 1

Hepatocellular Adenoma (includes multiple) k

Overall rate 1/60 (2%) 0/59 (0%) 1/60 (2%) 6/60 (10%) Adjusted rate 1.8% 0.0% 1.9% 11.2% Terminal rate 1/47 (2%) 0/47 (0%) 1/39 (3%) 4/33 (12%) First incidence (days) 729 (T) —l 729 (T) 687 Poly-3 testh P=0.005 P=0.507N P=0.748 P=0.048

* Significantly different (P # 0.05) from the control group by the Fisher exact test (interim evaluation) or the Poly-3 test (2-year study) ** P # 0.01 (T)Terminal sacrifice a Number of animals with lesion b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked c Historical incidence for 2-year studies with control groups given NTP-2000 diet: 0/609; with feed study controls given NIH-07 diet: 0/1,002 d Historical incidence for NTP-2000 diet (mean ± standard deviation): 5/609 (0.8% ± 1.2%), range 0%-3%; for NIH-07 diet: 23/1,0 02 (2.3% ± 3.0%), range 0%-10% e Number of animals with neoplasm per number of animals with liver examined microscopically f Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality g Observed incidence at terminal kill h Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. A lower incidence in an exposure group is indicated by N

. i Trend test and pairwise comparisons for stop-exposure groups j Historical incidence for NTP-2000 diet: 10/609 (1.8% ± 1.9%), range 0%-5%; for NIH-07 diet: 28/1,002 (2.8% ± 3.3%), range 0%- 10% k Historical incidence for NTP-2000 diet: 4/659 (0.7% ± 1.0%), range 0%-2%; for NIH-07 diet: 4/1,000 (0.4% ± 1.1%), range 0%-4% l Not applicable; no neoplasms in animal group 

### T ABLE 

### 12 Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

### T ABLE 

### 13 Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Male

Number Examined Microscopically 60 60 60 60 60 60 Alveolar Epithelium, Hyperplasia a 2 (3.0) b 8* (2.4) 3 (1.7) 7** (2.3) 15** (1.5) 29** (1.6)

Alveolar/bronchiolar Adenoma,

Multiple 0 0 0 0 0 1

Alveolar/bronchiolar Adenoma (includes multiple) c

Overall rate d 1/60 (2%) 5/60 (8%) 1/60 (2%) 2/60 (3%) 3/60 (5%) 8/60 (13%) Adjusted rate e 1.9% 11.2% 2.9% 8.7% 7.6% 33.0% Terminal rate f 1/39 (3%) 2/18 (11%) 1/3 (33%) 0/0 1/11 (9%) 0/0 First incidence (days) 729 (T) 639 729 (T) 491 615 419 Poly-3 test g P=0.237 P=0.066 P=0.654 P=0.254 Poly-3 testh P<0.001 P=0.205 P<0.001

Alveolar/bronchiolar Carcinoma

(includes multiple) 1 0 0 0 0 3

Alveolar/bronchiolar Adenoma or Carcinoma i

Overall rate 2/60 (3%) 5/60 (8%) 1/60 (2%) 2/60 (3%) 3/60 (5%) 11/60 (18%) Adjusted rate 3.7% 11/2% 2.9% 8.7% 7.6% 42.0% Terminal rate 2/39 (5%) 2/18 (11%) 1/3 (33%) 0/0 1/11 (9%) 0/0 First incidence (days) 729 (T) 639 729 (T) 419 615 419 Poly-3 test g P=0.390 P=0.149 P=0.644N P=0.386 Poly-3 testh P<0.001 P=0.362 P<0.001 

### 50 o-Nitrotoluene, NTP TR 504



### Lung: The incidences of alveolar/bronchiolar adenoma and adenoma or carcinoma (combined) in 5,000 ppm stop-exposure males were significantly greater than those in the controls (Tables 13 and A3b). The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in the 5,000 ppm stop-exposure group exceeded the 2-year historical ranges in control (all routes) male rats given NTP-2000 diet and in feed study controls given NIH-07 diet (Tables 13 and A4e). The incidences of alveolar epithelial hyperplasia in all exposed groups except 1,250 ppm males and 2,000 ppm females were increased (Tables 13, A5 and B5). Alveolar/bronchiolar adenomas were usually papillary and distorted the alveolar architecture (Plate 11).Neoplastic cells were cuboidal to columnar and well differentiated (Plate 12). Alveolar/bronchiolar carcinomaswere solid or papillary, obliterated the normal pulmonary architecture, and sometimes invaded adjacentstructures. Alveolar epithelial hyperplasia was characterized by increased numbers of type II pneumocytes lining the alveoli; however, the alveolar architecture was maintained.

### o-Nitrotoluene, NTP TR 504 51



### T ABLE 

### 13 Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Female

Number Examined Microscopically 60 60 60 60 Alveolar Epithelium, Hyperplasia 6 (1.7) 14* (2.1) 16* (1.8) 9 (1.8)

Alveolar/bronchiolar Adenoma,

Multiple 0 1 0 0 Alveolar/bronchiolar Adenoma

(includes multiple) 1 2 0 3 Alveolar/bronchiolar Carcinoma

(includes multiple) 0 0 0 1 Alveolar/bronchiolar Adenoma

or Carcinoma 1 2 0 4

* Significantly different (P # 0.05) from the control group by the Poly-3 test ** P # 0.01 (T)Terminal sacrifice a Number of animals with lesion b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked c Historical incidence for 2-year studies with control groups given NTP-2000 diet: 24/609 (4.2% ± 3.5%), range 0%-12%; with feed study controls given NIH-07 diet: 24/1,002 (2.4% ± 3.2%), range 0%-14% d Number of animals with neoplasm per number of animals with liver examined microscopically e Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality f Observed incidence at terminal kill g Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. A lower incidence in an exposure group is indicated by N

. h Trend test and pairwise comparisons for stop-exposure groups i Historical incidence for NTP-2000 diet: 26/609 (4.5% ± 3.9%), range 0%-14%; for NIH-07 diet: 31/1,002 (3.1% ± 3.5%), range 0% -16%

### 52 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 14 Incidences of Circulatory System Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Hemangioma, All Organs a

Overall rate b 0/60 (0%) 3/60 (5%) 0/60 (0%) 2/60 (3%) 0/60 (0%) 3/60 (5%) Adjusted rate c 0.0% 6.7% 0.0% 8.7% 0.0% 14.0% Terminal rate d 0/39 (0%) 1/18 (6%) 0/3 (0%) 0/0 0/11 (9%) 0/0 First incidence (days) —g 632 — 439 — 417 Poly-3 test e P=0.158 P=0.090 — h P=0.112 Poly-3 test f P=0.025 — P=0.034

Hemangioma or Hemangiosarcoma, All Organs i

Overall rate 1/60 (2%) 3/60 (5%) 1/60 (2%) 2/60 (3%) 0/60 (0%) 4/60 (7%) Adjusted rate 1.9% 6.7% 2.9% 8.7% 0.0% 18.0% Terminal rate 1/39 (3%) 1/18 (6%) 0/3 (0%) 0/0 0/11 (9%) 0/0 First incidence (days) 729 (T) 632 714 439 — 391 Poly-3 test e P=0.232 P=0.243 P=0.654 P=0.254 Poly-3 test f P=0.041 P=0.565N P=0.037

(T)Terminal sacrifice a Historical incidence for 2-year studies with control groups given NTP-2000 diet: 3/609 (0.5% ± 1.3%), range 0%-4%; with feed s tudy controls given NIH-07 diet: 3/1,004 (0.3% ± 0.7%), range 0%-2% b Number of animals with neoplasm per number of animals necropsied c Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d Observed incidence at terminal kill e Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. A lower incidence in an exposure group is indicated by N

. f Trend test and pairwise comparisons for stop-exposure groups g Not applicable; no neoplasms in animal group h Value of statistic cannot be computed. i Historical incidence for NTP-2000 diet: 6/609 (1.1% ± 1.4%), range 0%-4%; for NIH-07 diet: 10/1,004 (1.0% ± 1.4%), range 0%-4 % 

### Circulatory System : The incidences of hemangioma and hemangioma or hemangiosarcoma (combined) were significantly greater in 5,000 ppm stop-exposure males than in the controls (Tables 14, A3a, and A3b). In the 5,000 ppm stop-exposure males, a hemangioma occurred in the spleen of one animal, a second had a hemangioma of the skin and of the skeletal muscle, and a third had a hemangioma in an unspecified tissue (Table A2). These incidences exceeded the 2-year historical ranges in control (all routes) male rats given NTP-2000 diet and in feed study controls given NIH-07 diet (Tables 14 and A4f). The hemangiosarcoma in the 5,000 ppm stop-exposure male occurred in the skeletal muscle. The presence of benign hemangiomas and a single hemangiosarcoma in a variety of organs in the 5,000 ppm stop-exposure group was not considered to be related to exposure to o- nitrotoluene.

### o-Nitrotoluene, NTP TR 504 53



### Mononuclear Cell Leukemia: The incidences of mononuclear cell leukemia were significantly decreased in all groups of males exposed to 1,250 ppm or greaterand in all exposed groups of females, and these incidences were less than the 2-year historical ranges in control (all routes) rats given NTP-2000 diet and in feed study controls given NIH-07 diet (Tables 15, A3a, A3b, A4g, B3, and B4d). Mononuclear cell leukemia is one of the most common neoplasms in the F344/N rat. A number of factors may contribute to decreases inleukemia incidences in F344/N rats, including splenectomy, X-irradiation, and corn oil gavage. Chemicals that cause toxicity of the spleen may also be associated with decreased incidences of leukemia (Elwell et al. , 1990). The decreased incidences of leukemia in both males and females was most likely related to the splenic toxicity previously discussed.

T ABLE 

### 15 Incidences of Mononuclear Cell Leukemia in Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Male

Mononuclear Cell Leukemia a
Overall rate b 30/60 (50%) 21/60 (35%) 3/60 (5%) 3/60 (5%) 13/60 (22%) 1/60 (2%) Adjusted rate c 52.2% 42.8% 8.5% 12.7% 31.1% 5.0% Terminal rate d 16/39 (41%) 7/18 (39%) 0/3 (0%) 0/0 4/11 (36%) 0/0 First incidence (days) 533 465 676 419 531 569 Poly-3 test e P<0.001N P=0.214N P<0.001N P=0.003N Poly-3 test f P<0.001N P=0.027N P<0.001N



### Female

Mononuclear Cell Leukemia g Overall rate 21/60 (35%) 6/60 (10%) 4/60 (7%) 5/60 (8%) Adjusted rate 35.6% 10.6% 7.3% 9.1% Terminal rate 11/47 (23%) 3/47 (6%) 1/39 (3%) 1/33 (3%) First incidence (days) 453 429 523 488 Poly-3 test e P=0.001N P=0.001N P=0.001N P=0.001N

a Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 300/609 (47.3% ± 10.5%), range 32%-68%; with feed study controls given NIH-07 diet: 547/1,004 (54.5% ± 10.7%), range 32%-74% b Number of animals with neoplasm per number of animals necropsied c Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d Observed incidence at terminal kill e Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. f Trend test and pairwise comparisons for stop-exposure groups g Historical incidence for NTP-2000 diet: 185/659 (29.1% ± 8.4%), range 16%-42%; for NIH-07 diet: 293/1,001 (29.3% ± 7.6%), range 16%-42%

### 54 o-Nitrotoluene, NTP TR 504



### Testis : The combined incidence of interstitial cell adenoma (bilateral or unilateral) of the testis was significantly less in the 5,000 ppm stop-exposure group than in the controls (Tables 16 and A3b). The decrease resultedprimarily from the lower incidence of bilateral interstitial cell adenoma in the 5,000 ppm stop-exposure group. The incidence of bilateral interstitial adenoma in the 2,000 ppm core study males was also slightly less than that in the controls; however, the combined incidence of unilateral and bilateral interstitial cell adenoma in thisgroup did not differ significantly from that in the controls. The decreased incidence in the 5,000 ppm stopexposure group could not be explained solely by reduced survival, as the survival rates in that group and the 2,000 ppm core study males were similar. Thedecreased incidence was likely related to testicular toxicity observed early in the study in the 5,000 ppm stopexposure group. Interstitial cell adenoma is the most common testicular neoplasm in F344/N rats, occurringwith an incidence of 88% in 2-year carcinogenicity studies (Table A4h) and 96% in lifetime studies. Chemicals that cause testicular toxicity characterized by atrophy and degeneration have been associated with decreases in the incidence of interstitial cell adenoma in 2-year studies (Boorman et al. , 1985).

At 3 months, the incidence of testicular atrophy was significantly increased in the 5,000 ppm stop-exposure group (Tables 16 and A5); this lesion was characterizedby a reduction in the numbers of normal-appearing spermatozoa and of seminiferous tubules, which varied from a few seminiferous tubules to a reduction of all tubules in some testes. The majority of tubules had undergone degeneration and were lined only by Sertoli cells, withfew to no spermatogenic cells remaining. Mineralization was associated with tubular degeneration. In some tubules, cellular debris and multinucleated giantcells were observed, but these were not prominent findings in the atrophic testes. As a result of degeneration and atrophy, the luminal contents of the epididymis wereatypical; cellular atypia was characterized by the presence of rounded and enlarged or multinucleated degenerated spermatozoa. In addition to increased incidences of testicular atrophy in most exposed groups at 2 years, the incidence of interstitial cell hyperplasia was increased in the 2,000 ppm core study group.

### o-Nitrotoluene, NTP TR 504 55



### T ABLE 

### 16 Incidences of Neoplasms and Nonneoplastic Lesions of the Testis and Epididymis in Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

3-Month Interim Evaluation Epididymis a 10 10 10 Atrophyb 0 0 5* (2.4) c
Testis 10 10 10 Mineralization 0 0 5* (2.0) Germinal Epithelium, Atrophy 0 1 (1.0) 9** (2.4)

2-Year Study Testis 60 60 60 60 60 60 Germinal Epithelium, Atrophy 13 (2.8) 21* (3.2) 12 (3.2) 19** (3.0) 18* (3.6) 53** (3.9) Interstitial Cell, Hyperplasia 10 (1.4) 14 (1.6) 13 (1.5) 31** (1.4) 15 (1.5) 4 (1.0)

Interstitial Cell Adenoma,

Bilateral 43 34 32 17 31 4** Interstitial Cell Adenoma,

Unilateral 12 19 19 29 19 23

Interstitial Cell Adenoma (bilateral or unilateral) d

Overall rate e 55/60 (92%) 53/60 (88%) 51/60 (85%) 46/60 (77%) 50/60 (83%) 27/60 (45%) Adjusted rate f 94.3% 93.7% 96.5% 92.2% 92.6% 74.7% Terminal rate g 38/39 (97%) 18/18 (100%) 3/3 (100%) 0/0 10/11 (91%) 0/0 First incidence (days) 533 465 483 404 370 384 Poly-3 testh P=0.453N P=0.610N P=0.457 P=0.475N Poly-3 test i P<0.001N P=0.506N P<0.001N

* Significantly different (P # 0.05) from the control group by the Fisher exact test (interim evaluation) or the Poly-3 test (2-year study) ** P # 0.01 a Number of animals with tissue examined microscopically b Number of animals with lesion c Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked d Historical incidence for 2-year studies with control groups given NTP-2000 diet: 535/609 (86.4% ± 9.1%), range 72%-98%; with f eed study controls given NIH-07 diet: 889/1,003 (88.6% ± 6.0%), range 74%-96% e Number of animals with neoplasm per number of animals with testis examined microscopically f Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality g Observed incidence at terminal kill h Beneath the control incidence is the P value associated with the trend test for the core study groups. Beneath the exposed gro up incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts f or differential mortality in animals that do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. i Trend test and pairwise comparisons for stop-exposure groups

### 56 o-Nitrotoluene, NTP TR 504



### Other Organs: The incidences of nonneoplastic lesions of the bone marrow, kidney, mediastinal and mandibular lymph nodes, nose, pancreatic islets, pituitary gland, preputial and clitoral glands, salivary gland, and spleen were increased in exposed groups of males and/or females at 3 months and/or 2 years (Tables 17, A5, and B5).

Bone marrow hyperplasia in exposed groups of males and females was characterized by hematopoietic cell proliferation. Hematopoiesis of the spleen in exposed groups of males and females involved primarily the erythropoietic cell series. Although congestion of thespleen occurred in both stop-exposure groups, the pathogenesis of this lesion was not determined. Hematopoietic cell proliferation in the bone marrow and spleen may have been a secondary response because many rats had subcutaneous ulcerated neoplasms with varying degrees of inflammation.

The incidences and severity of lymphoid hyperplasia of the mandibular lymph node were significantly increased in 2,000 ppm females.

At 3 months, hyaline degeneration of the kidney in 5,000 ppm stop-exposure males was characterized by a slight increase in the presence of hyaline droplets within renal tubule epithelial cells. The hyaline droplets were homogeneous, eosinophilic, and rounded (3 to 5 μm in diameter). Larger droplets appeared to be extruded into the tubular lumina. Associated with the hyaline dropletdegeneration was minimal nephropathy, which was considered part of the degeneration and, therefore, was not given a separate diagnosis. At 2 years, pigmentation of the kidney of exposed groups of males consisted of fine, dark brown granules within the cytoplasm of cortical renal tubule epithelial cells.

Olfactory epithelial degeneration of the nose occurred in levels II and III of the nasal cavity of stop-exposure males at 3 months and was characterized by disruption, vacuolization, or atrophy of the olfactory epithelium. In some areas, epithelial cells were somewhat flattened, suggesting very early development of squamous cell metaplasia. The degenerative changes were mostly unilateral and primarily involved the most dorsal aspect of the ethmoid turbinates, nasal septum, and dorsal meatus.

Hyperplasia of the pancreatic islets occurred in 5,000 ppm stop-exposure males at 3 months and 2 years and was characterized by a greater number and size of islets (up to 500 μm in diameter). Some hyperplastic islets were twice the size of those in the controls. Larger islets had slightly enlarged islet cells arranged in a less orderly fashion than the normal glandular pattern seen in the controls. Pancreatic islet pigmentation was observed at 2 years in 5,000 ppm stop-exposure males.

The incidences of atrophy of the preputial gland were significantly increased in most groups of exposed males at 3 months and at 2 years. Atrophy was characterizedby glandular epithelial cells with fewer and smaller cytoplasmic granules than normal. The interstitial tissue was more prominent than normal and was likely due to the decreased size of the glandular epithelial tissue. Incidences of atrophy of the clitoral gland, analogous to the preputial gland, were significantly increased in 1,250 and 2,000 ppm females.

Incidences of cytoplasmic alteration of the pituitary gland (pars distalis) were significantly increased in the 2,000 ppm core study and 5,000 ppm stop-exposure male groups at 2 years. The pituitary gland cytoplasmic alteration in exposed male rats consisted of hypertrophy, increased eosinophilia, and cytoplasmic vacuolization of cells in the pars distalis, and may have been related to the atrophy of the testis.

Atrophy of the salivary gland was observed in exposed groups of males and females and was characterized by areduction in the size and number of eosinophilic cytoplasmic granules in the epithelial cells of secretory ducts in the submandibular salivary gland. In some salivary glands, few or no eosinophilic granules were found and the epithelial cells were reduced in size.

The incidences of pigmentation of the spleen were increased in 2,000 ppm core-study males and females.

### o-Nitrotoluene, NTP TR 504 57



### T ABLE 

### 17 Incidences of Selected Nonneoplastic Lesions in Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Male

3-Month Interim Evaluation Kidney a 10 10 10 Renal Tubule, Degeneration, Hyaline b 1 (1.0) c 0 10** (2.0) Nose 10 10 10 Olfactory Epithelium, Degeneration 0 4* (1.5) 10** (3.0) Pancreatic Islets 10 10 10 Hyperplasia 0 0 8** (1.6) Preputial Gland 10 10 10 Atrophy 0 1 (2.0) 8** (2.0) Salivary Gland 10 10 10 Atrophy 0 10** (3.0) 10** (3.8) Spleen 10 10 10 Congestion 0 3 (1.7) 10** (1.9) Hematopoietic Cell Proliferation 0 2 (1.0) 10** (1.8)

2-Year Study Bone Marrow 60 60 60 60 60 60 Hyperplasia 2 (3.5) 25** (3.0) 43** (2.9) 45** (2.9) 37** (3.0) 33** (2.9) Kidney 60 60 60 60 60 60 Renal Tubule, Pigmentation 5 (2.4) 12* (1.8) 14** (1.6) 9* (1.9) 18** (2.2) 6 (2.7) Lymph Node 60 60 60 60 60 60 Mediastinal, Pigmentation 6 (2.7) 9 (3.1) 3 (3.0) 8* (3.4) 13* (3.6) 13** (3.2) Pancreatic Islets 60 60 60 60 60 60 Hyperplasia 2 (1.5) 0 0 2 (1.5) 2 (1.5) 10** (1.9) Pigmentation 1 (1.0) 0 0 2 (2.0) 0 11** (2.0) Pituitary Gland 59 60 58 59 57 59 Pars Distalis, Cytoplasmic Alteration 1 (3.0) 0 1 (1.0) 4* (1.3) 1 (1.0) 42** (2.1) Preputial Gland 60 59 58 56 60 59 Atrophy 7 (2.0) 9 (2.3) 35** (2.3) 41** (2.6) 38** (2.3) 54** (2.9) Salivary Gland 60 60 59 60 58 59 Atrophy 0 2 (2.0) 18** (2.1) 43** (2.2) 16** (2.1) 49** (2.4) Spleen 60 60 60 60 60 60 Hematopoietic Cell Proliferation 7 (2.9) 33** (2.6) 38** (2.4) 47** (2.3) 36** (2.6) 35** (2.2) Pigmentation 9 (2.6) 8 (2.5) 13 (2.6) 16** (2.3) 4 (2.8) 7 (2.7)

### 58 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 17 Incidences of Selected Nonneoplastic Lesions in Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Female

Bone Marrow 60 60 60 60 Hyperplasia 2 (3.0) 7 (2.4) 15** (2.7) 24** (2.7) Clitoral Gland 59 57 54 53 Atrophy 1 (2.0) 3 (2.0) 6* (2.3) 25** (2.4) Lymph Node 60 60 59 59 Mandibular, Lymphoid Hyperplasia 3 (2.0) 5 (2.2) 6 (2.2) 15** (2.7) Salivary Gland 60 60 60 60 Atrophy 2 (3.5) 3 (2.0) 9* (2.1) 48** (2.4) Spleen 60 59 60 59 Hematopoietic Cell Proliferation 22 (1.6) 38** (1.4) 48** (1.9) 48** (2.1) Pigmentation 36 (2.5) 44 (2.2) 43 (2.4) 46* (2.3)

* Significantly different (P # 0.05) from the control group by the Fisher exact test (interim evaluation) or the Poly-3 test (2-year study) ** P # 0.01 a Number of animals with tissue examined microscopically b Number of animals with lesion c Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

### o-Nitrotoluene, NTP TR 504 59



# M ICE 

## Survival Estimates of 2-year survival probabilities for male andfemale mice are shown in Table 18 and in the KaplanMeier survival curves (Figure 5). All 2,500 ppm males died by week 101 and all 5,000 ppm males died by week 66. Survival of 1,250 ppm males and 5,000 ppm females was significantly less than that of the controls. Due to the poor survival of exposed mice, the planned 15-month interim evaluation was cancelled.



## Body Weights, Feed and Compound Consumption, and Clinical Findings Mean body weights of exposed males and 5,000 ppm females were generally less than those of the controls throughout the study; mean body weights of 2,500 ppm females were less than those of the controls during the second year of the study (Figure 6; Tables 19 and 20). Feed consumption by 5,000 ppm males was less than that by the controls; feed consumption by other exposed groups of males and females was similar to that by the controls (Tables I3 and I4). Dietary concentrations of 1,250, 2,500, or 5,000 ppm resulted in average daily doses of approximately 165, 360, or 700 mg

o- nitrotoluene/kg body weight to males and 150, 320, or 710 mg/kg to females. Clinical findings included large subcutaneous masses in the torso, head, and appendages of males and females; incidences of clinical findings were generally exposure-concentration related.



## Biomarkers of Exposure The results of urinary metabolite determinations in male and female mice are presented in Table F2. At time points with sufficient data to permit determinations, the ratios of o- nitrobenzoic acid to creatinine excreted inurine appeared to be linearly related to exposure concentration. The concentrations of o- nitrobenzylmercapturic acid and o- aminobenzoic acid were generally below the limit of quantitation in male and female mice.

T ABLE 

### 18 Survival of Mice in the 2-Year Feed Study of o -Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Male

Animals initially in study 60 60 60 60

Moribund 3 5 21 31 Natural deaths 5 21 39 29 Animals surviving to study termination 52 34 0 0 Percent probability of survival at end of study a 87 57 0 0 Mean survival (days) b 712 674 523 386

Survival analysis c P<0.001 P<0.001 P<0.001 P<0.001



### Female

Animals initially in study 60 60 60 60

Moribund 4 6 7 22 Natural deaths 4 8 6 33 Animals surviving to study termination 52 46 47 5 Percent probability of survival at end of study 87 77 78 8 Mean survival (days) 706 692 716 626

Survival analysis P<0.001 P=0.236 P=0.355 P<0.001

a Kaplan-Meier determinations b Mean of all deaths (uncensored, censored, and terminal sacrifice) c The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons (Cox, 1972) with the controls are in the exposed group columns.

### 60 o-Nitrotoluene, NTP TR 504



### F IGURE 

### 5 Kaplan-Meier Survival Curves for Male and Female Mice Exposed to o-Nitrotoluene in Feed for 2 Years

### o-Nitrotoluene, NTP TR 504 61



### F IGURE 

### 6 Growth Curves for Male and Female Mice Exposed to o-Nitrotoluene in Feed for 2 Years

### 62 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 19 Mean Body Weights and Survival of Male Mice in the 2-Year Feed Study of o-Nitrotoluene

Weeks 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 20.9 60 20.8 100 60 20.9 100 60 20.8 100 60 4 24.0 60 23.4 98 60 22.6 94 60 21.4 89 60 8 27.7 60 27.1 98 60 26.2 95 60 25.1 91 60 12 30.6 60 29.0 95 60 28.3 93 60 26.7 87 60 16 34.2 60 32.3 94 60 30.9 90 60 28.3 83 60 20 37.3 60 35.9 96 60 33.8 91 60 29.7 80 60 24 38.7 60 36.4 94 60 34.3 89 60 30.0 78 60 28 40.5 60 38.4 95 60 35.6 88 60 30.8 76 60 32 41.9 60 39.9 95 60 36.8 88 60 31.1 74 60 36 43.6 60 41.0 94 60 37.7 87 60 31.7 73 60 40 44.7 59 42.1 94 60 38.3 86 59 31.9 71 60 44 44.8 59 42.7 95 60 39.0 87 59 32.2 72 57 48 45.8 59 43.2 94 60 39.4 86 58 32.8 72 54 52 46.6 59 43.8 94 60 39.4 85 58 32.7 70 49 56 47.1 59 44.4 94 60 40.2 85 57 32.4 69 35 60 47.0 59 44.0 94 60 40.1 85 56 33.0 70 8 64 46.7 59 43.4 93 60 40.4 87 53 33.3 71 3 68 47.7 59 45.0 94 56 40.7 85 48 72 47.3 59 44.6 94 55 41.0 87 41 76 47.6 59 44.2 93 54 39.5 83 34 80 48.0 59 44.8 93 52 38.9 81 24 84 46.3 59 43.4 94 49 37.3 81 9 88 46.5 57 43.6 94 47 37.1 80 4 92 46.5 55 42.1 91 45 31.4 68 2 96 46.6 54 43.1 93 42 31.2 67 1 100 46.8 54 43.7 93 38 29.4 63 1 104 46.5 52 42.6 92 36

Mean for weeks 1-13 25.8 25.1 97 24.5 95 23.5 91 14-52 41.8 39.6 95 36.5 87 31.1 74 53-104 47.0 43.8 93 37.3 79 32.9 70

### o-Nitrotoluene, NTP TR 504 63



### T ABLE 

### 20 Mean Body Weights and Survival of Female Mice in the 2-Year Feed Study of o- Nitrotoluene

Weeks 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 16.9 60 16.7 99 60 16.8 99 60 16.7 99 60 4 20.6 60 21.1 102 60 20.4 99 60 19.4 94 60 8 22.4 60 23.2 104 60 23.3 104 60 22.1 99 60 12 25.7 60 25.5 99 60 25.1 98 60 23.2 90 60 16 27.8 60 27.4 99 60 26.8 96 60 24.3 87 60 20 29.4 60 29.3 100 60 29.0 99 60 25.4 86 60 24 31.8 60 32.2 101 60 30.1 95 60 26.4 83 60 28 33.2 60 33.6 101 60 32.0 96 60 27.1 82 60 32 35.4 60 35.8 101 60 33.6 95 60 28.0 79 60 36 37.3 60 36.7 98 60 35.0 94 60 29.0 78 60 40 38.1 60 37.7 99 59 35.9 94 60 29.5 77 60 44 38.1 59 38.6 101 59 37.0 97 60 30.5 80 60 48 39.3 59 38.8 99 58 37.5 95 60 30.4 77 60 52 40.4 59 40.7 101 57 38.1 94 60 30.6 76 60 56 41.8 58 42.6 102 57 38.8 93 60 31.5 75 60 60 42.4 58 42.5 100 57 39.3 93 60 31.6 75 58 64 43.7 58 43.5 100 57 39.9 91 60 32.2 74 57 68 45.2 58 44.4 98 57 41.3 91 60 33.4 74 56 72 44.5 57 44.4 100 56 41.2 93 60 33.3 75 55 76 44.8 57 43.9 98 56 40.8 91 60 33.8 75 52 80 44.8 57 44.2 99 55 40.9 91 59 33.6 75 51 84 43.8 56 42.2 96 55 38.7 88 59 32.8 75 43 88 44.7 55 43.1 96 54 39.3 88 59 33.4 75 35 92 45.1 55 43.9 97 53 39.9 89 57 33.0 73 30 96 45.0 55 45.3 101 50 39.7 88 56 32.7 73 23 100 46.2 55 47.4 103 49 41.3 89 52 32.3 70 10 104 46.6 53 46.9 101 46 41.2 88 47 32.3 69 6

Mean for weeks 1-13 21.4 21.6 101 21.4 100 20.4 95 14-52 35.1 35.1 100 33.5 95 28.1 80 53-104 44.5 44.2 99 40.2 90 32.8 74

### 64 o-Nitrotoluene, NTP TR 504



## Pathology and Statistical AnalysesThis section describes the statistically significant or biologically noteworthy changes in the incidences of neoplasms and/or nonneoplastic lesions of the circulatory system, large intestine (cecum), liver, kidney, nose,spleen, skin, and prostate gland. Summaries of the incidences of neoplasms and nonneoplastic lesions, individual animal tumor diagnoses, statistical analyses of primary neoplasms that occurred with an incidence of atleast 5% in at least one animal group, and historical incidences for the neoplasms mentioned in this section are presented in Appendix C for male mice and Appendix D for female mice.

Circulatory System: The incidences of hemangiosarcoma (all organs) in all exposed groups of males and in 5,000 ppm females were significantly greater than those in the controls (Tables 21, C3, and D3). The incidences exceeded the 2-year historical ranges in control (allroutes) mice given NTP-2000 diet and in feed study controls given NIH-07 diet (Tables 21, C4a, and D4a). Hemangiosarcomas in exposed males and females occurred primarily in the mesentery (Plates 13 and 14), skeletal muscle (Plates 15 and 16), and subcutis of the skin (Tables 21, C3, and D3). In some cases, mice with multiple and/or large hemangiosarcomas had small hemangiosarcomas in other tissues, primarily the lung (Tables C1 and D1). Many of these neoplasms were considered to be metastases. Hemangiosarcomas ranged from small neoplasms, approximately 1 to 2 mm in diameter, to large masses with diameters greater than 1 cm. Microscopically, hemangiosarcomas consisted of numerous, irregular, variably sized, blood-filled vascular channels lined by large, pleomorphic endothelial cells and separated by variable amounts of fibrous stroma. The central portions of most hemangiosarcomas had undergone necrosis, leaving a large, blood-filled cavity surrounded by a rim of neoplastic vascular tissue. Generally, in the B6C3F 1 

### mouse, hemangiosarcomas can be multicentric, and often arise primarily in the spleen and liver (Ward et al. , 1999).

### o-Nitrotoluene, NTP TR 504 65



### T ABLE 

### 21 Incidences of Hemangiosarcoma in Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Male

Number Necropsied 60 60 60 60

Hemangiosarcoma, Mesentery, Multiple a 0 0 8** 14** Hemangiosarcoma, Mesentery

(includes multiple) 0 8** 38** 38** Hemangiosarcoma, Skeletal Muscle, Multiple 0 0 8** 26** Hemangiosarcoma, Skeletal Muscle

(includes multiple) 0 6* 33** 45** Hemangiosarcoma, Subcutaneous Skin,

Multiple 0 0 0 8** Hemangiosarcoma, Subcutaneous Skin,

(includes multiple) 0 4* 8** 20**

Hemangiosarcoma (includes multiple), All Organs b

Overall rate c 4/60 (7%) 17/60 (28%) 55/60 (92%) 60/60 (100%) Adjusted rate d 7.0% 32.7% 97.9% 100.0% Terminal rate e 3/52 (6%) 9/34 (27%) 0/0 0/0 First incidence (days) 710 542 365 277 Poly-3 test f P<0.001 P<0.001 P<0.001 P<0.001



### Female

Number Necropsied 60 60 60 60

Hemangiosarcoma, Mesentery, Multiple 0 0 0 7* Hemangiosarcoma, Mesentery

(includes multiple) 0 0 0 32** Hemangiosarcoma, Skeletal Muscle, Multiple 0 0 0 2 Hemangiosarcoma, Skeletal Muscle

(includes multiple) 0 0 0 16** Hemangiosarcoma, Subcutaneous Skin,

Multiple 0 0 0 3 Hemangiosarcoma, Subcutaneous Skin

(includes multiple) 0 0 2 19**

Hemangiosarcoma (includes multiple), All Organs g

Overall rate 0/60 (0%) 2/60 (3%) 3/60 (5%) 50/60 (83%) Adjusted rate 0.0% 3.6% 5.2% 90.2% Terminal rate 0/52 (0%) 1/46 (2%) 2/47 (4%) 4/5 (80%) First incidence (days) — h 343 624 409 Poly-3 test P<0.001 P=0.232 P=0.124 P<0.001

* Significantly different (P # 0.05) from the control group by the Poly-3 test ** P # 0.01 a Number of animals with lesion b Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 37/659 (5.8% ± 3.2%), range 2%-14%; with feed study controls given NIH-07 diet: 53/952 (5.6% ± 3.5%), range 2%-14% c Number of animals with neoplasm per number of animals necropsied d Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality e Observed incidence at terminal kill f Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for differential m ortality in animals that do not reach terminal sacrifice. g Historical incidence for NTP-2000 diet: 15/659 (2.6% ± 2.7%), range 0%-8%; for NIH-07 diet: 31/953 (3.3% ± 2.4%), range 0%-8% h Not applicable; no neoplasms in animal group

### 66 o-Nitrotoluene, NTP TR 504



### Large Intestine (Cecum): The incidences of carcinomaof the cecum in 1,250 and 2,500 ppm males were significantly increased and exceeded the 2-year historical range in control (all routes) male mice given NTP-2000 diet; this neoplasm has not been observed in feed study controls given NIH-07 diet (Tables 22, C3, and C4b). Although the increased incidences of carcinoma in exposed groups of females were not significant, theywere considered to be exposure related because this neoplasm is very rare and has not been observed in female controls given NTP-2000 diet (0/659; all routes) orNIH-07 diet (0/953; feed studies) for 2 years. In the current study, no cecal carcinomas were observed in either control group or in 5,000 ppm males. All 5,000 ppm males died by week 66; hemangiosarcomas may have caused death before cecal carcinomas developed. Microscopically, the carcinomas were characterized by proliferation of glandular structures composed of moderately pleomorphic mucosal epithelial cells that invaded the cecal wall. In some smaller carcinomas,minimal invasion was observed, while in large carcinomas, extensive invasion was observed and was characterized by neoplastic cells extending through the cecal wall into the attached mesentery (Plates 17 and 18) orinvaded vascular structure (Plate 19). In some carcinomas, the submucosa was diffusely thickened by solid masses of large cells resembling “signet ring cells” with abundant clear cytoplasm and a peripherally located nucleus and mixed with smaller numbers of epithelial cells with large nuclei and small amounts of cytoplasm. Invasion of the cecal wall by these neoplasms was notapparent, although similar cells were seen in other carcinomas with extensive invasion. All carcinomas of the cecum were confirmed to be of epithelial cell origin (cytokeratin-20 positive) and were negative for lysozyme (macrophage marker).

T ABLE 

### 22 Incidences of Carcinoma of the Large Intestine (Cecum) in Mice in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Male

Carcinoma a
Overall rate b 0/60 (0%) 12/60 (20%) 9/60 (15%) 0/60 (0%) Adjusted rate c 0.0% 22.7% 31.6% 0.0% Terminal rate d 0/52 (0%) 4/34 (12%) 0/0 0/0 First incidence (days) —e 485 270 — Poly-3 test f P<0.001 P<0.001 P<0.001 —g



### Female

Carcinomah Overall rate 0/60 (0%) 1/60 (2%) 4/60 (7%) 3/60 (5%) Adjusted rate 0.0% 1.9% 7.0% 7.4% Terminal rate 0/52 (0%) 1/46 (2%) 3/47 (6%) 1/5 (20%) First incidence (days) — 729 (T) 715 619 Poly-3 test P=0.024 P=0.492 P=0.064 P=0.072

(T) Terminal sacrifice a Historical incidence for 2-year studies with controls given NTP-2000 diet (mean ± standard deviation): 1/659 (0.2% ± 0.6%), range 0%-2%; with feed study controls given NIH-07 diet: 0/952 b Number of animals with neoplasm per number of animals necropsied c Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d Observed incidence at terminal kill e Not applicable; no neoplasms in animal group f Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for differential m ortality in animals that do not reach terminal sacrifice. g Value of statistic cannot be computed. h Historical incidence for NTP-2000 diet: 0/659; for NIH-07 diet: 0/953

### o-Nitrotoluene, NTP TR 504 67



### Liver: The incidences of hepatocellular adenoma in 2,500 and 5,000 ppm females, hepatocellular carcinoma in 5,000 ppm females, and hepatocellular adenoma or carcinoma (combined) in 2,500 and 5,000 ppm females were significantly greater than those in the controls and exceeded the 2-year historical ranges for these neoplasms in control (all routes) female mice given NTP-2000 diet (Tables 23, D3, and D4b). In addition, the incidences of hepatocellular carcinoma and hepatocellular adenoma or carcinoma (combined) in 5,000 ppm females exceeded the historical ranges for feed study controls given NIH-07 diet. Due to low survival, these neoplasms occurred lessfrequently in 2,500 and 5,000 ppm males than in the concurrent or historical controls (Tables 18, 23, C3, and C4c). The incidences of eosinophilic foci in 1,250 ppm males and 5,000 ppm females and of basophilic foci in 1,250 and 2,500 ppm males and 1,250 and 5,000 ppm females were significantly increased (Tables 23, C5, and D5). Microscopically, foci, hepatocellular adenoma, and hepatocellular carcinoma represent a continuum. These lesions had an appearance typical of that seen in B6C3F 1 

### mice. Eosinophilic and basophilic foci were small to moderately large lesions composed of hepatocytes with eosinophilic and basophilic cytoplasm; generally, these hepatocytes were somewhat enlarged. The hepatocytes were arranged in normal hepatic cords that merged with the surrounding normal hepatocytes. Most foci had littleor no compression of the surrounding normal hepatocytes, although some degree of compression was present in some larger foci. Adenomas were discrete masses withdistinct borders that caused compression of the surrounding normal hepatic parenchyma. Adenomas usually were composed of hepatocytes that appeared similar to those seen in eosinophilic foci, except that in adenomas the normal lobular architecture was not apparent, and plates of neoplastic hepatocytes intersected the surrounding normal hepatocytes at sharp angles rather than merging with them as in foci. Carcinomas were discrete masses that generally had irregular borders due to localized areas of growth of neoplastic hepatocytes into the surrounding normal parenchyma. The neoplastic hepatocytes often were somewhat atypical, but the major distinguishingfeature of carcinomas was the presence of abnormal patterns of growth. The most common abnormal growthpattern was formation of trabeculae of neoplastic hepatocytes that were three or more cell layers thick (Plate 20),while less commonly the neoplastic cells formed glandular structures or solid masses.

Incidences of focal syncytial alteration of hepatocytes were significantly increased in all groups of exposed males (Tables 23 and C5). Microscopically, syncytial alteration was generally a minimal change consisting of one to several scattered hepatocytes containing multiple (usually four to six) small nuclei. The significance of this lesion is uncertain; the detection of this minimal changewas most likely due to the lower number of hepatocellular neoplasms in males that died early, making it possible to detect more syncytial cells microscopically. The incidences of necrosis in all exposed male groups and the incidences of necrosis, focal hepatocyte necrosis, and focal cytoplasmic vacuolization in 5,000 ppm females were significantly increased (Tables 23, C5, andD5). Necrosis was characterized by one or more scattered discrete foci of coagulative necrosis consisting of hepatocytes with uniformly eosinophilic cytoplasm and pyknotic or karyorrhectic nuclei. Focal hepatocyte necrosis was characterized by small numbers of necrotichepatocytes that were associated with mixed inflammatory cells.

### 68 o-Nitrotoluene, NTP TR 504



### T ABLE 

### 23 Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Male

Number Examined Microscopically 60 59 57 60

Basophilic Focus a 0 6* 4* 0 Eosinophilic Focus 3 14** 1 1 Necrosis 1 (1.0) b 15** (2.0) 27** (1.9) 30** (1.5) Hepatocyte, Syncytial Alteration, Focal 16 (1.0) 26* (1.1) 43** (1.2) 39** (1.4)

Hepatocellular Adenoma, Multiple 3 6 2 0 Hepatocellular Adenoma (includes multiple) 18 18 3 0 Hepatocellular Carcinoma, Multiple 3 7 1 0 Hepatocellular Carcinoma (includes multiple) 12 16 5 2

Hepatocellular Adenoma or Carcinoma c 27 28 7 2



### Female

Number Examined Microscopically 60 59 59 60

Basophilic Focus 1 6* 2 6* Eosinophilic Focus 2 3 6 28** Necrosis 3 (1.7) 0 2 (1.5) 13** (2.2) Hepatocyte, Necrosis, Focal 0 0 0 6** (1.5) Hepatocyte, Cytoplasmic Vacuolization,

Focal 1 (2.0) 2 (2.5) 2 (1.5) 9** (1.4)

Hepatocellular Adenoma, Multiple 0 0 3 20** Hepatocellular Adenoma (includes multiple) d 7 5 19** 29** Hepatocellular Carcinoma, Multiple 0 1 2 7 Hepatocellular Carcinoma (includes multiple) e 2 4 6 16**

Hepatocellular Adenoma or Carcinoma f

Overall rate g 9/60 (15%) 9/59 (15%) 24/59 (41%) 39/60 (65%) Adjusted rate h 15.7% 16.9% 42.1% 79.1% Terminal rate i 8/52 (15%) 9/46 (20%) 20/47 (43%) 5/5 (100%) First incidence (days) 362 729 (T) 661 567 Poly-3 test j P<0.001 P=0.538 P<0.001 P<0.001

* Significantly different (P # 0.05) from the control group by the Poly-3 test ** P # 0.01 (T)Terminal sacrifice a Number of animals with lesion b Average severity grade of lesions in affected animals: 1=minimal, 2=mild; 3=moderate, 4=marked c Historical incidence for 2-year studies with control groups given NTP-2000 diet (mean ± standard deviation): 304/659 (47.8% ± 12 .9%), range 28%-72%; with feed study controls given NIH-07 diet: 493/950 (51.9% ± 7.9%), range 40%-68% d Historical incidence for NTP-2000 diet: 101/655 (16.0% ± 6.3%), range 8%-28%; for NIH-07 diet: 218/951 (22.9% ± 9.9%), range 12%-50% e Historical incidence for NTP-2000 diet: 49/655 (8.0% ± 4.7%), range 3%-16%; for NIH-07 diet: 104/951 (10.9% ± 4.6%), range 4% -20% f Historical incidence for NTP-2000 diet: 143/655 (22.8% ± 9.6%), range 12%-40%; for NIH-07 diet: 292/951 (30.7% ± 10.2%), range 12%-56% g Number of animals with neoplasm per number of animals with liver examined microscopically h Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality i Observed incidence at terminal kill j Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for differential m ortality in animals that do not reach terminal sacrifice.

### o-Nitrotoluene, NTP TR 504 69



### Kidney: The incidences of renal tubule pigmentation in all exposed groups of males and 5,000 ppm females were significantly greater than those in the controls (Tables 24, C5, and D5). Microscopically, pigmentation was a minimal to moderate change consisting of fine, dark brown granules within the cytoplasm of cortical renal tubular epithelial cells. The incidence of hyalinedroplet accumulation in renal tubule cells was significantly increased in 2,500 ppm males and in 5,000 ppm females. Microscopically, hyaline droplet accumulation consisted of small, homogeneous, brightly eosinophilic droplets within the epithelium of renal cortical tubules.

T ABLE 

### 24 Incidences of Selected Nonneoplastic Lesions of the Kidney in Mice in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Male

Number Examined Microscopically 58 59 58 60

Renal Tubule Pigmentation a 1 (1.0) b 6* (2.8) 32** (2.3) 35** (2.2) Renal Tubule, Hyaline Droplet Accumulation 1 (4.0) 2 (3.0) 5* (2.0) 3 (1.3)



### Female

Number Examined Microscopically 59 56 58 59

Renal Tubule Pigmentation 0 1 (2.0) 3 (2.0) 35** (2.9) Renal Tubule, Hyaline Droplet Accumulation 1 (3.0) 3 (3.0) 2 (2.0) 10** (2.3)

* Significantly different (P # 0.05) from the control group by the Poly-3 test ** P # 0.01 a Number of animals with lesion b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked



### Nose: Olfactory epithelial degeneration occurred in all mice that received 2,500 or 5,000 ppm (males: 0/60, 36/60, 60/60, 60/60; females: 0/60, 28/60, 59/59, 57/57; Tables C5 and D5). Severities of this lesion increased with increasing exposure concentration (males: 1.1, 1.8, 3.0; females: 1.0, 2.0, 3.0). Olfactory degeneration was a complex lesion and consisted of necrosis, atrophy, regeneration, hyperplasia, hypertrophy, and metaplasia.The most prominent changes were atrophy of the olfactory epithelium, Bowman’s glands, and olfactory nerve bundles accompanied by replacement of the olfactory epithelium with a respiratory-type epithelium composed of ciliated columnar cells that covered the dorsal meatus surface and extended into the mucosa to form dilated pseudoglands. Olfactory degeneration was similar to that seen in mice exposed to o- nitrotoluene for 13 weeks (NTP, 1992). Spleen: The incidences of hematopoietic cell proliferation were significantly increased in all exposed groups of males (13/60, 24/60, 49/58, 60/60; Table C5) and the 2,500 and 5,000 ppm females (11/59, 19/57, 21/58,54/57; Table D5). These increases were considered secondary to the increased incidences of hemangiosarcoma in these groups.

Skin: The incidences of edema of the subcutaneous tissue in 2,500 and 5,000 ppm males (0/60, 3/60, 14/60, 22/60; Table C5) and in the 5,000 ppm females (0/60, 1/60, 2/60, 4/60; Table D5) were significantly greater than those in the control groups. Microscopically, thethickened subcutaneous tissue was characterized by separation of the connective tissue by abundant clear spaces caused by the accumulation of fluid. The cause of the edema was unclear. However, the presence of dilated

### 70 o-Nitrotoluene, NTP TR 504



### lymphatics in the areas of edema suggested that the edema may have resulted from lymphatic obstructioncaused by the presence of subcutaneous hemangiosarcomas.

Prostate Gland : The incidences of chronic inflammation in 2,500 and 5,000 ppm males were significantly increased (0 ppm, 0/60; 1,250 ppm, 1/59; 2,500 ppm, 7/57; 5,000 ppm, 4/58; Table C5). These increases were minimal and were not considered to be exposure related.



# G ENETIC 

# T OXICOLOGY

o-Nitrotoluene (3.0 to 1,000 μg/plate) was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535, or TA1537, with or without Aroclor-induced rat or hamster liver S9 (Table E1; Haworth et al. , 1983). Significantly increased sister chromatid exchange frequencies were observed in cultured Chinese hamster ovary cells treated with o- nitrotoluene with S9; an equivocal response was seen without S9 (Table E2; Galloway et al. , 1987). Due to o- nitrotoluene-inducedcell cycle delay in the trial without S9, an extended culture time was employed to permit accumulation of sufficient second-division metaphase cells for analysis.

o-Nitrotoluene did not induce chromosomal aberrations in cultured Chinese hamster ovary cells with or without S9 (Table E3; Galloway et al. , 1987). No increases in the frequencies of micronucleated polychromatic erythrocytes were observed in vivo in the bone marrow of male rats or male mice treated with o- nitrotoluene. Inmale F344/N rats, an acute micronucleus test was performed using two protocols (Table E4). The initial test used a single intraperitoneal injection of o- nitrotoluene followed by bone marrow analysis 24 hours later; in the second test, bone marrow was harvested for analysis 48 hours after a single intraperitoneal injection of

o- nitrotoluene. In neither test was a positive response observed. In male mice, three intraperitoneal injections also yielded negative results, although a small increasein the frequency of micronucleated polychromatic erythrocytes was observed at all doses tested (Table E5).

o-Nitrotoluene, administered in feed for 13 weeks, didnot increase the frequency of micronucleated normochromatic (mature) erythrocytes in peripheral bloodof female mice. However, a small increase in the frequency of micronucleated normochromatic erythrocyteswas noted in male mice at the highest exposure concentration tested, 10,000 ppm (Table E6). The increase in male mice was sufficient to generate a significant trend test (P=0.003), but because none of the frequencies in individual exposed groups were significantly increased over the corresponding control group and because the increase in the frequency of micronuclei in the 10,000 ppm group was small, the test in male mice was judged to be equivocal.

### P LATE 

### 1Malignant mesothelioma in a male F344/N rat exposed to 5,000 ppm (stopexposure) o -nitrotoluene in the 2-year feed study. Note the mesothelioma extending from the tunica vaginalis (arrow). H&E; 16 x 

### P LATE 

### 2 Higher magnification of papillary portion of the mesothelioma in Plate 1. Note the malignant mesothelial cells covering the stroma. H&E; 80 x



### P LATE 

### 3 Subcutaneous skin fibroma in a male F344/N rat exposed to 2,000 ppm

o -nitrotoluene in feed for 2 years. Note the proliferation of fibrocytes, lack of cellular atypia, and the abundant collagenous stroma. H&E; 80 x 

### P LATE 

### 4 Subcutaneous skin fibrosarcoma in a male F344/N rat exposed to 1,250 ppm

o -nitrotoluene in feed for 2 years. Compared to the fibroma in Plate 3, the fibrosarcoma is more cellular, neoplastic cells are more pleomorphic, and the collageneous stroma is less prominent. H&E; 80 x 

### o-Nitrotoluene, NTP TR 504

### P LATE 

### 8 Hepatocellular adenoma in a male F344/N rat exposed to 2,000 ppm

o -nitrotoluene in feed for 2 years. Note the compression of adjacent hepatocytes and loss of normal lobular parenchyma. H&E; 5 x 

### P LATE 

### 6 Mammary gland fibroadenoma in a male F344/N rat exposed to 1,250 ppm

o -nitrotoluene in feed for 2 years. Note the collection of glandular epithelium surrounded by fibrous connective tissue. H&E; 10 x



### P LATE 

### 7 Higher magnification of the fibroadenoma in Plate 6. Uniform epithelial cells are arranged in a single layer forming glands and ducts. H&E; 40 x 

### P LATE 

### 5 Subcutaneous lipoma in a male F344/N rat exposed to 2,000 ppm

o -nitrotoluene in feed for 2 years. Note the sheet of variably sized adipocytes within the subcutis. H&E; 80 x 

### o-Nitrotoluene, NTP TR 504

### P LATE 

### 12 Higher magnification of the alveolar/bronchiolar adenoma in Plate 11. Note the papillary arrangement of well-differentiated neoplastic cells. H&E; 80 x 

### P LATE 

### 9 Hepatocholangiocarcinoma in a male F344/N rat exposed to 2,000 ppm

o -nitrotoluene in feed for 2 years. Note the presence of cells resembling hepatocytes and bile ductules. H&E; 80 x 

### P LATE 

### 10 Cholangiocarcinoma in the liver of a male F344/N rat exposed to 5,000 ppm (stop-exposure) o -nitrotoluene in the 2-year feed study. Note the atypical bile duct cells surrounded by scirrhous-type response. H&E; 80 x



### P LATE 

### 11Alveolar/bronchiolar adenoma in a male F344/N rat exposed to 5,000 ppm (stopexposure) o -nitrotoluene in the 2-year feed study. Note that the normal alveolar achitecture is distorted. H&E; 8 x



### o-Nitrotoluene, NTP TR 504

### P LATE 

### 15 Hemangiosarcoma in the skeletal muscle (thorax) of a male B6C3F 1 mouse exposed to 5,000 ppm o -nitrotoluene in feed for 2 years. Note that the centralportion of the hemangiosarcoma has undergone necrosis, leaving a large bloodfilled cavity surrounded by a rim of neoplastic endothelial cells. H&E; 8 x 

### P LATE 

### 13 Hemangiosarcoma in the mesentery from a male B6C3F 1 mouse exposed to 5,000 ppm o -nitrotoluene in feed for 2 years. Note the variably sized and large blood-filled cavernous spaces lined by malignant endothelial cells. H&E; 10 x



### P LATE 

### 16 Higher magnification of Plate 15. A sheet of malignant endothelial cells is associated with the skeletal muscle (right bottom part of figure). H&E; 80 x 

### P LATE 

### 14 Higher magnification of Plate 13. Malignant endothelial cells line the cavernous spaces. Note the nuclear pleomorphism and hyperchromatic nuclei. H&E; 80 x 

### o-Nitrotoluene, NTP TR 504

### P LATE 

### 19 Carcinoma in the cecum of a female B6C3F 1 mouse exposed to 2,500 ppm

o -nitrotoluene in feed for 2 years. Note the neoplastic epithelial cells within the lymphatic vasculature (arrows). H&E; 40 x 

### P LATE 

### 17 Carcinoma in the cecum of a male B6C3F 1 mouse exposed to 2,500 ppm

o -nitrotoluene in feed for 2 years. Note the neoplastic epithelial cells extending through muscle layers of the cecum and within the mesentery. H&E; 10 x



### P LATE 

### 20 Hepatocellular carcinoma in a female B6C3F 1 mouse exposed to 5,000 ppm

o -nitrotoluene in feed for 2 years. Note the trabeculae of neoplastic hepatocytes that are three or more cell layers thick. H&E; 66 x 

### P LATE 

### 18 Higher magnification of Plate 17. Note the infiltrative nature of the anaplastic epithelial cells within the cecal wall. H&E; 16 x 

### o-Nitrotoluene, NTP TR 504

### 71



### This NTP Technical Report continues the reporting ofthe comparative biologic effects of the nitrotoluene isomers. The results for the 2-year carcinogenicity studies of o- nitrotoluene in F344/N rats and B6C3F 1 

### mice are described here. The results of the 2-year studies of
p- nitrotoluene in rats and mice are reported in NTP Technical Report 498 (NTP, 2002).

Comparative 13-week toxicity studies of o- , m- , and
p- nitrotoluene (NTP, 1992) showed that all three isomers caused mild toxicity in the kidney, spleen, liver, and/orreproductive system, particularly at the highest concentration of 10,000 ppm. In general, toxicity was most severe with the ortho isomer. o-Nitrotoluene was carcinogenic in male rats, as indicated by the occurrence of mesotheliomas in the 5,000 ppm group and mesothelial cell hyperplasia in the 10,000 ppm group. The 2-yearstudies were conducted to further evaluate the carcinogenic potential of o- nitrotoluene.

In the 2-year studies, o- nitrotoluene caused multiple carcinogenic effects in rats and mice at all exposure concentrations. The average daily dose for core study malerats receiving 625, 1,250, or 2,000 ppm was approximately 25, 50, or 90 mg o- nitrotoluene/kg body weight,respectively; male rats in the 2,000 or 5,000 ppm stopexposure groups received approximately 125 or 315 mg/kg for the first 3 months of the study. Female rats exposed to 625, 1,250, or 2,000 ppm received an average daily dose of approximately 30, 60, or100 mg/kg. The average daily doses were approximately 165, 360, or 700 mg/kg for male mice and 150, 320, or 710 mg/kg for female mice receiving 1,250, 2,500, or 5,000 ppm.

Survival was reduced in all exposed groups of male ratsand male mice because of the development of neoplasms. The development of neoplasms also reduced survival of 2,000 ppm female rats and 5,000 ppm female mice. Reduced survival of exposed rats and mice led to cancellation of the planned 15-month interim evaluations. Increased incidences of neoplasms in stop-exposure males, including mesothelioma, skin neoplasms, and mammary gland fibroadenoma, indicated that 3 months of o -nitrotoluene exposure were sufficient to produce a carcinogenic effect.

Mean body weights of exposed male rats except the 625 ppm group were generally less than those of the controls throughout the study, and those of 2,000 ppm females were less during the second year of the study. Mean body weights of exposed groups of male mice and 5,000 ppm females were less than those of controls throughout most of the study; those of 2,500 ppm females were less during the second year of the study.

Malignant mesothelioma is a neoplasm arising from thesurface serosal cells of the pleural, peritoneal, and pericardial cavities and from the tunica vaginalis (Attanoosand Gibbs, 1997). Mesotheliomas or mesothelial hyperplasia occurred in male rats after 13 or 26 weeks of exposure to 5,000 or 10,000 ppm o- nitrotoluene (NTP, 1992, 1996). In these studies, mesotheliomas did notoccur in control rats and occurred in less than 4% of historical control male rats in 2-year studies. At the 3-month interim sacrifice in the current 2-year study, no mesotheliomas or mesothelial hyperplasia wereobserved in exposed male rats. However, as time progressed, chemical-induced mesotheliomas occurred in all exposed male rat groups, including the stop-exposure groups, and the incidences were significantly increased by both the pairwise and trend statistics. The incidences in the exposed male rats exceeded the historical ranges for control male rats given NTP-2000 or NIH-07 diet.The occurrence of mesothelioma in male rats was considered to be clear evidence of carcinogenic activity.

Malignant mesotheliomas occurred with incidences of 33%, 48%, and 73% in the 625, 1,250, and 2,000 ppm core study male rat groups, respectively. The incidences of malignant mesothelioma were 73% and 90% in the 2,000 and 5,000 ppm stop-exposure male rat groups. The incidence of mesothelioma was higher in the 

# DISCUSSION AND CONCLUSIONS

### 72 o-Nitrotoluene, NTP TR 504



### 2,000 ppm stop-exposure group than in the 625 ppmgroup even though the latter group received approximately 50% more total exposure to o- nitrotoluene. The incidences of mesothelioma were similar in the 2,000 ppm core study and stop-exposure groups of male rats. Thus, critical events leading to mesothelioma occurred early in the study, and this damage was irreversible.

The mesotheliomas varied from small lesions localized to the serosa of the epididymis or testis to massive mesothelial cell proliferation overlying the serosa of multiple organs. These malignant and invasive forms of mesothelioma are typically found in the surface of theepididymis and testis, although they may occur less frequently in the abdominal cavity and metastasize to the lymph nodes in the thorax. Malignant mesotheliomas of the tunica vaginalis also occur in humans (Jones et al.
, 1995).
In this study, as well as in other NTP studies, chemical induction of mesothelioma is found primarily in male rats. Mesotheliomas have been reported in male ratsafter exposure to 10 other chemicals in 2-year NTP studies: acronycine (NCI, 1978a); cytembena (NTP, 1981); 1,2-dibromoethane (NTP, 1982); 3,3 N -dimethoxybenzidine dihydrochloride (NTP, 1990a), 2,3-dibromo1-propanol (NTP, 1993); 3,3 N -dimethylbenzidine dihydrochloride (NTP, 1991); glycidol (NTP, 1990b); pentachlorophenol (NTP, 1999); phenoxybenzamine hydrochloride (NCI, 1978b); and o- toluidine hydrochloride (NCI, 1979).
Studies of other aromatic amines show that the position of the substitution on the aromatic ring is important for predicting the carcinogenic effect. o- Toluidine hydrochloride induced a high incidence of mesothelioma in male rats, while no mesotheliomas were observed after treatment with m- or p- toluidine (Weisburger et al.
, 1978). In studies of o- and p- anisidine hydrochloride, only the ortho isomer induced a carcinogenic response (urinary bladder neoplasms in rats and mice; kidney and thyroid gland neoplasms in male rats) (NCI, 1978c,d). The structural similarity of o- toluidine hydrochloride,

o- anisidine hydrochloride, and o- nitrotoluene (all contain a benzene ring with a methyl or methoxy and nitrogen-containing group on adjacent carbons) suggests asimilar intermediate may be responsible for the carcinogenic effects. Chemicals with an ortho substitution on the aromatic ring are more likely to be carcinogenic than chemicals with meta or para substitution on the aromatic ring (Weisburger et al. , 1978).

The molecular pathogenesis of mesotheliomas is notwell understood. Active-oxygen free radicals are considered to be important mediators in asbestos-induced mesotheliomas (Attanoos and Gibbs, 1997). Human mesotheliomas have cyclooxygenase-2 and nitric oxide synthase gene alterations (Marrogi et al. , 2000) and p16 gene deletions (Xiao et al. , 1995). Studies of humanmesotheliomas have also found deletions on chromosomes 1p, 3p, and 22q, which may be the loci of yetunidentified tumor suppressor genes important in the neoplastic transformation process (Attanoos and Gibbs, 1997). Trisomy of chromosome 7 is another frequent change in mesothelioma (Walker et al. , 1992). Mutations in the ras gene are not commonly seen in mesothelioma cells in humans (Ramael et al. , 1993; Cristaudo et al. , 1995) or in rat mesotheliomas induced by ferric nitrilotriacetate (Nishiyama et al. , 1995). Mutations of the p53 gene also were not seen in a sample of rat mesothelioma (Nishiyama et al. , 1995) or in human malignant mesotheliomas (Ramael et al. , 1992; Mor et al. , 1997).

At 2 years, significant increases were seen in the incidences of subcutaneous skin fibroma and fibrosarcoma in all exposed groups of male rats, fibroma in 1,250 and2,000 ppm female rats, and mammary gland fibroadenoma in most exposed groups of male and female rats. The subcutaneous skin and mammary gland neoplasms were also increased in multiplicity. The incidencesexceeded the historical control ranges for these neoplasms in control rats given NTP-2000 or NIH-07 diet. Subcutaneous skin fibrosarcomas and mammary glandfibroadenomas in male and female rats were clear evidence of a carcinogenic response because the effectswere seen in both sexes at 1,250 and 2,000 ppm and incidences for these neoplasms exceeded historical rates.

There were increases in the incidences of hepatocellular and/or bile duct neoplasms in 2,000 ppm core study and 5,000 ppm stop-exposure male rats. The incidence of hepatocellular adenoma or carcinoma (combined) in the2,000 ppm core study group exceeded the historical control ranges in control rats given NTP-2000 or NIH-07diet. In the 5,000 ppm stop-exposure group, three animals had cholangiocarcinoma, and six had hepatocellular neoplasms.

### o-Nitrotoluene, NTP TR 504 73



### In a previous study in which male rats received 5,000 ppm o- nitrotoluene in feed for 13 weeks and undosed feed for 13 weeks, cholangiocarcinomas occurred in two of 20 male rats (NTP, 1996). Cholangiocarcinomas have also been seen in a 2-year study of dinitrotoluene (Rickert, 1984a). Liver and bile duct neoplasms in male rats were considered to be clear evidence for a carcinogenic effect because the increased incidences were significant, even though many of the male rats died early due to other neoplasms, such asmesotheliomas. The incidence of hepatocellular adenoma was also increased in 2,000 ppm female rats and was considered related to exposure to o -nitrotoluene. No cholangiocarcinomas were seen in female rats.

The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was increased in 5,000 ppm stopexposure male rats, and this incidence exceeded the historical ranges in control rats given either NTP-2000 or NIH-07 diet. In addition, the incidences of alveolar epithelial hyperplasia were increased in the 625 and 2,000 ppm core study male rats and in the 2,000 and5,000 ppm stop-exposure groups. The increased incidences of the lung neoplasms were considered related to chemical exposure. While there were increases in the incidences of alveolar epithelial hyperplasia in 625 and 1,250 ppm female rats, there was no evidence for an increased incidence of alveolar/bronchiolar neoplasms in female rats.

The incidences of hemangioma and hemangioma orhemangiosarcoma (combined) were significantly increased in 5,000 ppm stop-exposure male rats. Thepresence of benign hemangiomas and a single hemangiosarcoma in a variety of organs in this group was not considered to be related to exposure to o- nitrotoluene.

Nitroaromatic chemicals that cause hematopoiesis and hemosiderin pigment accumulation in the spleen also may cause a decreased incidence of mononuclear cell leukemia (Elwell et al. , 1996). The spleen plays a critical role in the pathogenesis of mononuclear cell leukemia. Although the stem cell for mononuclear cellleukemia in Fischer rats is considered to be a lymphocyte of bone marrow origin, the initial histologic evidence for proliferation and expansion of these neoplastic cells occurs in the spleen, where the leukemia cells fillthe sinusoids. Alteration of the spleen microenvironment can effect the development of mononuclear cell leukemia (Elwell et al. , 1996). The findings of increased spleen toxicity and decreased incidences of mononuclearcell leukemia are consistent with similar response patterns seen with other nitroaromatic compounds, including p- nitrotoluene. Decreases in the incidence of interstitial cell adenoma of the testis were also seen in exposed male rats.

At 3 months, testicular and epididymal atrophy and salivary gland atrophy probably were related to the lower body weights of male rats exposed to 2,000 or 5,000 ppm. The pituitary gland cytoplasmic alteration in exposed male rats consisted of hypertrophy, increased eosinophilia, and cytoplasmic vacuolization of cells in the pars distalis and may have been related to the atrophy of the testis. The clitoral gland atrophy observed in exposed female rats may also have been related to reduced body weights. The salivary, preputial, and clitoral gland atrophy and pituitary gland alterations inmale and female rats may have been related to the compromised state of the animals with the development of neoplasms.

There were exposure-related increased incidences ofhemangiosarcoma in male and female mice. Hemangiosarcomas are malignant vascular neoplasms of the blood vessels (Booth and Sundberg, 1995) and may occur in many tissues. In this study, the hemangiosarcomasoccurred primarily in the skeletal muscle, skin (subcutaneous tissue), and/or mesentery. This was considered to be clear evidence of a carcinogenic effect because theneoplasms were malignant, the incidences were significant by both the pairwise and trend statistics and exceeded historical control ranges, and the effect was seen in males and females.

The NTP has identified other chemicals that caused hemangiosarcomas in B6C3F 1 

### mice: 1,3-butadiene (NTP, 1984), pentachlorophenol Dowicide EC-7 (NTP, 1989), 1,2-propylene oxide (NTP, 1985), chloroprene (NTP, 1998), and tetrafluoroethylene (NTP, 1997). The neoplasm site varied with the study. For example, in the 1,3-butadiene study, hemangiosarcomas occurred in the heart; in the chloroprene studies hemangiosarcomas were observed most frequently in the liver, mesentery,and skin; and in the tetrafluoroethylene study, hemangiosarcomas were observed primarily in the liver.

The carcinogenic effects of 1,3-butadiene and chloroprene have been attributed to a mutagenic epoxide intermediate (Melnick and Kohn, 1995). Vinyl chloride,

### 74 o-Nitrotoluene, NTP TR 504



### another epoxide-forming chemical, also causes hemangiosarcomas in mice (Guengerich, 1992; Singer, 1996). Recent studies demonstrated that both ras and p53 mutations may play a role in the pathogenesis of hemangiosarcomas following exposure to 1,3-butadiene or vinyl chloride in rodents (Hollstein et al. , 1994; Hong et al. , 2000), and to vinyl chloride in humans (Barbin et al. , 1997; Marion et al. , 1991).

In a study to determine if the same mechanism might be applicable to o- nitrotoluene, 15 hemangiosarcomas from

o- nitrotoluene-exposed mice and 13 hemangiosarcomasfrom control mice from other NTP studies were examined for genetic alterations (Appendix M). The purpose of these genetic toxicity studies was to compare thespectrum of genetic changes in spontaneous hemangiosarcomas with that in o- nitrotoluene-induced hemangiosarcomas. Alterations in p53 and $ -catenin protein distribution were screened using immunohistochemical analysis of formalin-fixed sections.

No indication of p53 or $ -catenin protein accumulation was observed in the hemangiosarcomas from the control mice, but p53 protein had accumulated in all 15 hemangiosarcomas from o- nitrotoluene-exposed mice. $ -Catenin had accumulated in seven of the

o- nitrotoluene-induced hemangiosarcomas.

Wild-type p53 protein has a short half-life, and protein accumulation in the cytoplasm or nucleus does not occur in a normal cell. The p53 protein accumulation in the

o- nitrotoluene-induced hemangiosarcomas suggests that a mutation has occurred in the p53 gene, leading to the formation of an altered protein that accumulates in the nucleus. Similarly, $ -catenin is usually rapidly degraded in normal cells, and the accumulation of this protein in the o- nitrotoluene-induced hemangiosarcomas suggests that a mutation may have occurred. Further analysis ofthe DNA from these hemangiosarcomas indicated specific mutations in the p53 and $ -catenin genes. The occurrence of p53 and $ -catenin mutations in the

o- nitrotoluene-induced hemangiosarcomas, but not in the spontaneous hemangiosarcomas, suggests that the pathways leading to o- nitrotoluene-induced cancer differ from the pathways in spontaneous hemangiosarcomas.

Exposure to o- nitrotoluene caused carcinomas of thececum in male and female mice. None of the approximately 500 other chemicals evaluated by the NTP havecaused carcinomas of the cecum in mice. The occurrence of cecum carcinomas in the current study was considered clear evidence of a carcinogenic effectbecause these are malignant neoplasms and the incidences were significantly increased in 1,250 and 2,500 ppm male mice. Carcinomas of the cecum were not observed in 5,000 ppm male mice, probably due tothe early hemangiosarcoma-related deaths of those animals; mean survival was 712 days in control male mice and 386 days in 5,000 ppm males.

In the 2-year study, the incidences of hepatocellular adenoma, carcinoma, and/or the combined incidence of hepatocellular neoplasms were increased in 2,500 and5,000 ppm female mice. The incidence of hepatocellular carcinoma in 5,000 ppm female mice exceeded the historical range for control mice given NTP-2000 or NIH-07 diet. The increases in the incidences of benignand malignant liver neoplasms in female mice were considered to be clear evidence for a carcinogenic effect.Exposed male mice died early because of the development of hemangiosarcomas, which may explain why the later-developing hepatocellular neoplasms were not seen in male mice.

The predominant metabolic pathway for o- nitrotoluene is oxidation of the methyl group, first to an alcohol and subsequently to a carboxylic acid (Figure 1).

o-Nitrobenzylmercapturic acid is likely formed fromglutathione transferase-catalyzed conjugation of glutathione with o -nitrobenzyl sulfate and subsequent catabolism via the mercapturic acid pathway. Conjugation of p -nitrobenzyl sulfate with glutathione has been demonstrated in vitro (deBethizy and Rickert, 1983). Nitro-group reduction appears to be a relatively minor pathway, at least as determined from the urinary metabolite profile. o- Aminobenzyl alcohol and

o- aminobenzoic acid are representatives of this pathway. In the current studies, o- nitrobenzoic acid,

o- nitrobenzylmercapturic acid, and o- aminobenzoic acid were measured in the urine of rats and mice at 2 weeksand at 3, 12, and 18 months (Appendix F). The contribution of o- nitrotoluene metabolism to the

o- aminobenzoic acid in the urine was overshadowed by endogenous o -aminobenzoic acid produced via catabolism of tryptophan. For the other two metabolites in rats, and in mice where there were sufficient data, the metabolite to creatinine ratio generally increased with increasing exposure concentration. In rats, the metabolite/creatinine ratio was generally larger at the 2-week time point than at later times. At this age, theanimals are at about half their final weight, but food consumption is high. Therefore, exposure is highest on a

### o-Nitrotoluene, NTP TR 504 75



### weight basis for the younger animals. This fact is not as obvious from the daily metabolite data. There appear to be differences in metabolism between male and female rats, with males excreting more o -nitrobenzylmercapturic acid and females excreting more o- nitrobenzoic acid.

o-Nitrotoluene generally is not genotoxic in Salmonella or other in vitro short-term tests. Metabolic activation of the chemical requires reduction of the nitro group to anaromatic amine before activation to the ultimate carcinogen. The reduction of this nitro group is thought to be catalyzed by bacterial nitroreductases in the intestine (Doolittle et al. , 1983).

Measurement of chemical-induced DNA repair as unscheduled DNA synthesis is an indicator of genotoxic and carcinogenic potential (Mirsalis and Butterworth, 1980; Mirsalis et al. , 1982a,b). o-Nitrotoluene’s induction of unscheduled DNA synthesis in rat hepatocytes after an oral dose of 200 mg/kg to male rats, but not in cultured hepatocytes exposed in vitro or in germ free rats, suggests intestinal bacteria are essential for the metabolic activation of this chemical to a genotoxicant (Doolittle et al. , 1983).

In the 2-year studies of o- and p- nitrotoluene, the ortho isomer caused a spectrum of carcinogenic responses in rats and mice that differed from that seen with the para isomer (Table 25). Earlier studies showed that covalent binding of radioactivity from [ 14 

### C]- o- nitrotoluene to total rat hepatic macromolecules is 3.5 times greater than that of m- or p- nitrotoluene. o-Nitrotoluene also binds to male F344 rat hepatic DNA, but this binding could not be detected for m- or p- nitrotoluene (Rickert et al.
, 1984b). o-Nitrotoluene, but not m- or p- nitrotoluene, induced DNA repair in the in vivo/in vitro hepatocyte unscheduled DNA synthesis assay in male, but not female, F344 rats (Doolittle et al. , 1983).

Quantitative differences in metabolism of the mononitrotoluenes are a result of differences in the hepatic conjugation or further oxidation of the first metabolic intermediates, the mononitrobenzyl alcohols. The

o- nitrotoluene metabolite o- nitrobenzyl alcohol is a better substrate for microsomal glucuronyl transferase than are the m- or p- nitrobenzyl alcohols (Rickert et al.
, 1985). Once formed, part of the o- nitrobenzyl glucuronide is excreted via the bile into the intestine, where intestinal microflora hydrolyze the glucuronide and reduce the nitro group, resulting in the formation of

o- aminobenzyl alcohol, which may be reabsorbed (Chism and Rickert, 1985).

o- Aminobenzyl alcohol has received considerable attention as a key intermediate in the bioactivation of

o- nitrotoluene. It has been suggested that sulfation of

o- aminobenzyl alcohol may lead to covalent binding to DNA (Chism and Rickert, 1989). The reactivity of

o- aminobenzyl sulfate could be related to the ease of formation of a reactive benzyl cation due to the electrondonating ability of the amino group. Alternatively, sulfation could lead to formation of a quinone-imine via elimination of sulfate. o- Aminobenzyl alcohol also provides a link between o- toluidine and o- nitrotoluene, as it is likely a metabolite of both chemicals. Although

o- aminobenzyl alcohol has not been identified as a metabolite of o- toluidine, the presence of N-acetylo- aminobenzyl alcohol as a urinary metabolite in rats is evidence that it may be present in vivo (Son et al. , 1980). Chism and Rickert (1989) also provided evidence thatoxidation of the amino group to a hydroxylamine followed by sulfation, a metabolic activation process for some aryl amines, was not responsible for DNA covalent binding by o- aminobenzyl alcohol.

The difference in genotoxic responses of ortho- versus meta- or para- substituted arylamines has been studied by Marques et al. (1997). Their data indicated that the conformation of the DNA adduct is a determinant factor in the genotoxic response. Adducts of ortho- analogues tend to adopt a syn conformation while meta- and paraanalogues tend to adopt an anti conformation. While the work of Marques et al. (1997) focused on reaction at the nitrogen of the aryl amine, the same principle may apply for reaction at the benzylic carbon of an o- nitrotoluene metabolite.

No o- nitrotoluene epidemiology studies have been reported in the literature. However, excess cancers have been found in workers exposed to a related chemical,

o- toluidine (Ward et al. , 1991).

### 76 o-Nitrotoluene, NTP TR 504TABLE25Comparison of Selected Neoplasms in the 2-Year Feed Studies ofp-Nitrotoluene ando-Nitrotoluenep-Nitrotolueneao-Nitrotoluene0 ppm 1,250 ppm 2,500 ppm 5,000 ppm 0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (Stop-Exposure) Exposure)Male RatsAverage Daily Dose (mg/kg) 0 55 110 240 0 25 50 90 125 315Survival 31/50 38/50 38/50 40/50 39/60 18/60 3/60 0/60 11/60 0/60MesotheliumMalignantMesotheliomab5/50 2/50 1/50 4/50 2/60 20/60 29/60 44/60 44/60 54/60Skin (Subcutaneous)Lipoma 0/50 0/50 0/50 0/50 0/60 4/60 13/60 13/60 10/60 12/60Fibroma 1/50 2/50 7/50 1/50 5/60 46/60 52/60 59/60 45/60 52/60Fibrosarcoma 0/50 2/50 7/50 1/50 0/60 7/60 17/60 20/60 8/60 12/60Fibroma or Fibrosarcoma 1/50 2/50 9/50 1/50 5/60 47/60 55/60 59/60 47/60 53/60Mammary GlandFibroadenoma 0/50 0/50 0/50 0/50 0/60 7/60 10/60 2/60 13/60 20/60LiverHepatocellular Adenoma 0/50 0/50 0/50 0/50 2/60 3/60 3/60 7/60 3/60 4/60Hepatocellular Adenoma orCarcinoma 1/50 0/50 0/50 0/50 3/60 3/60 3/60 8/60 3/60 6/60Cholangiocarcinoma 0/50 0/50 0/50 0/50 0/60 0/60 0/60 0/60 0/60 3/60Hepatocholangiocarcinoma 0/50 0/50 0/50 0/50 0/60 1/60 0/60 1/60 0/60 0/60LungAlveolar/bronchiolar Adenoma 1/50 0/50 2/50 2/50 1/60 5/60 1/60 2/60 3/60 8/60Alveolar/bronchiolar Adenomaor Carcinoma 1/50 1/50 2/50 3/50 2/60 5/60 1/60 2/60 3/60 11/60aNTP, 2002bNumber of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver and lung; for other tissues, denominator isnumber of animals necropsied.

### o-Nitrotoluene, NTP TR 504 77TABLE25Comparison of Selected Neoplasms in the 2-Year Feed Studies ofp-Nitrotoluene ando-Nitrotoluenep-Nitrotolueneo-Nitrotoluene0 ppm 1,250 ppm 2,500 ppm 5,000 ppm 0 ppm 625 ppm 1,250 ppm 2,000 ppmFemale RatsAverage Daily Dose (mg/kg) 0 60 125 265 0 30 60 100Survival 39/50 37/50 39/50 41/50 47/60 47/60 39/60 33/60Skin (Subcutaneous)Fibroma 0/50 0/50 0/50 1/49 3/59 3/60 18/60 19/60Fibroma or Fibrosarcoma 1/50 1/50 0/50 2/50 3/60 3/60 21/60 22/60Mammary GlandFibroadenoma 14/50 17/50 20/50 5/50 23/60 47/60 52/60 56/60LiverHepatocellular Adenoma 0/50 0/50 1/50 0/50 1/60 0/59 1/60 6/60Clitoral GlandAdenoma or Carcinoma 8/50 12/50 20/50 8/49 14/59 13/57 6/54 3/530 ppm 1,250 ppm 2,500 ppm 5,000 ppm 0 ppm 1,250 ppm 2,500 ppm 5,000 ppmMale MiceAverage Daily Dose (mg/kg) 0 170 345 690 0 165 360 700Survival 46/50 46/50 45/50 42/50 52/60 46/60 47/60 5/60Circulatory SystemHemangiosarcoma 1/50 1/50 3/50 2/50 4/60 17/60 55/60 60/60Large Intestine (Cecum)Carcinoma 1/48 0/48 0/48 0/49 0/56 12/49 9/36 0/44LungAlveolar/bronchiolar Adenomaor Carcinoma 8/50 14/50 12/50 19/50 14/60 7/60 6/60 0/60

### 78 o-Nitrotoluene, NTP TR 504TABLE25Comparison of Selected Neoplasms in the 2-Year Feed Studies ofp-Nitrotoluene ando-Nitrotoluenep-Nitrotolueneo-Nitrotoluene0 ppm 1,250 ppm 2,500 ppm 5,000 ppm 0 ppm 1,250 ppm 2,500 ppm 5,000 ppmFemale MiceAverage Daily Dose (mg/kg) 0 155 315 660 0 150 320 710Survival 46/50 47/50 43/50 49/50 52/60 46/60 47/60 5/60Circulatory SystemHemangiosarcoma 1/50 1/50 0/50 1/50 0/60 2/60 3/60 50/60Large Intestine (Cecum)Carcinoma 0/49 0/48 0/48 0/50 0/60 1/60 4/60 3/60LiverHepatocellular Adenoma 6/49 3/50 2/50 5/50 7/60 5/59 19/59 29/60Hepatocellular Carcinoma 3/49 4/50 0/50 1/50 2/60 4/59 6/59 16/60Hepatocellular Adenoma orCarcinoma 8/49 6/50 2/50 6/50 9/60 9/59 24/59 39/60

### o-Nitrotoluene, NTP TR 504 79



# C ONCLUSIONS Under the conditions of these studies, there was clear evidence of carcinogenic activity * of o- nitrotoluene in male rats based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma, and liver neoplasms. The increasedincidences of lung neoplasms in male rats were also considered to be exposure related. There was clear evidence of carcinogenic activity of o- nitrotoluene in female ratsbased on increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma. The increased incidence of hepatocellular adenoma in female rats was also considered to be exposure related. There

was clear evidence of carcinogenic activity of o- nitrotoluene in male and female mice based on increased incidences of hemangiosarcoma, carcinoma ofthe large intestine (cecum), and hepatocellular neoplasms (females only).
Exposure to o- nitrotoluene caused increased incidences of nonneoplastic lesions of the mammary gland (females only), liver, bone marrow, spleen, lung, and mandibular lymph node (females only) in male and female rats and of the liver, kidney, and nose in male and female mice.

Decreased incidences of mononuclear cell leukemiaoccurred in exposed groups of rats; the incidence of testicular interstitial cell adenoma was decreased in exposed male rats.

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 13. A summary of the Technical Reports Review Subcommittee comments and public discussion on this Technical Report appears on page 15.

### 80 o-Nitrotoluene, NTP TR 504

### 81



### Abshire, A.D., and Hughes, C.S. (1982). Toluene. In Chemical Economics Handbook, pp. 300.7202z300.7203a. SRI International, Palo Alto, CA.

The Aldrich Library of Infrared Spectra (1981). 3rd ed. (C.J. Pouchert, Ed.), Spectrum No. 803h. Aldrich Chemical Company, Inc., Milwaukee.

American Conference of Governmental Industrial Hygienists (2000). 2000 TLVs ® 

### and BEIs ® 

### . Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices, p. 52. Cincinnati, OH.

Ashby, J., and Tennant, R.W. (1991). Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutat. Res. 257 , 229-306.

Attanoos, R.L., and Gibbs, A.R. (1997). Pathology of malignant mesothelioma. Histopathology 30 , 403-418.
Bailer, A.J., and Portier, C.J. (1988). Effects of treatment-induced mortality and tumor-induced mortality on tests for carcinogenicity in small samples. Biometrics 44 , 417-431.
Barbin, A., Froment, O., Boivin, S., Marion, M., Belpoggi, F., Maltoni, F., and Montesano, R. (1997). p53 gene mutation pattern in rat liver tumors induced by vinyl chloride. Cancer Res. 57 , 1695-1698.
Bieler, G.S., and Williams, R.L. (1993). Ratio of estimates, the delta method, and quantal response tests for increased carcinogenicity. Biometrics 49 , 793-801.
Boorman, G.A., Montgomery, C.A., Jr., Eustis, S.L., Wolfe, M.J., McConnell, E.E., and Hardisty, J.F. (1985).Quality assurance in pathology for rodent carcinogenicity studies. In Handbook of Carcinogen Testing (H.A. Milman and E.K. Weisburger, Eds.), pp. 345-357. Noyes Publications, Park Ridge, NJ. Booth, C.J., and Sundberg, J.P. (1995). Hemangiomas and hemangiosarcomas in inbred laboratory mice. Lab. Anim. Sci. 45 , 497-502.

Butterworth, B.E., Smith-Oliver, T., Earle, L., Loury, D.J., White, R.D., Doolittle, D.J., Working, P.K., Cattley, R.C., Jirtle, R., Michalopoulos, G., and Strom, S. (1989). Use of primary cultures of human hepatocytes in toxicology studies. Cancer Res

. 49
, 1075-1084.

Chism, J.P., and Rickert, D.E. (1985). Isomer- and sexspecific bioactivation of mononitrotoluenes: Role of enterohepatic circulation. Drug Metab. Dispos. 13
, 651-657.

Chism, J.P., and Rickert, D.E. (1989). In vitro activation of 2-aminobenzyl alcohol and 2-amino-6-nitrobenzylalcohol, metabolites of 2-nitrotoluene and 2,6-dinitrotoluene. Chem. Res. Toxicol. 2 , 150-156.

Chism, J.P., Turner, M.J., Jr., and Rickert, D.E. (1984). The metabolism and excretion of mononitrotoluenes by Fisher 344 rats. Drug Metab. Dispos. 12 , 596-602.

Chiu, C.W., Lee, L.H., Wang, C.Y., and Bryan, G.T. (1978). Mutagenicity of some commercially available nitro compounds for Salmonella typhimurium. Mutat. Res. 58 , 11-22.

Ciss, M., Dutertre, H., Huyen, N., Phu-Lich, N., andTruhaut, R. (1980a). Etude toxicologique des nitrotoluènes: Toxicité aiguë et toxicité subaiguë. Dakar Méd. 25 , 303-311.

Ciss, M., Huyen, N., Dutertre, H., Phu-Lich, N., andTruhaut, R. (1980b). Etude toxicologique des nitrotoluènes: Toxicité à long terme. Dakar Méd. 25
, 293-302.

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

# REFERENCES

### 82 o-Nitrotoluene, NTP TR 504



### Cox, D.R. (1972). Regression models and life-tables. J. R. Stat. Soc. B34 , 187-220.

Crawford, B.D. (1985). Perspectives on the somatic mutation model of carcinogenesis. In Advances in Modern Environmental Toxicology. Mechanisms and Toxicity of Chemical Carcinogens and Mutagens (M.A. Mehlman, W.G. Flamm, and R.J. Lorentzen, Eds.), pp. 13-59. Princeton Scientific Publishing Co., Inc., Princeton, NJ.

Cristaudo, A., Vivaldi, A., Sensales, G., Guglielmi, G., Ciancia, E., Elisei, R., and Ottenga, F. (1995).Molecular biology studies on mesothelioma tumor samples: Preliminary data on H-Ras, p21, and SV40. J. Environ. Pathol. Toxicol. Oncol. 14 , 29-34.

deBethizy, J.D., and Rickert, D.E. (1983). Sequential enzyme-catalyzed metabolism of 4-nitrotoluene to S-(4-nitrobenzyl)glutathione. Biochem. Biophys. Res. Commun. 114 , 500-504.

deBethizy, J.D., and Rickert, D.E. (1984). Metabolismof nitrotoluenes by freshly isolated Fisher 344 rat hepatocytes. Drug Metab. Dispos. 12 , 45-50.

Doolittle, D.J., Sherrill, J.M., and Butterworth, B.E.(1983). Influence of intestinal bacteria, sex of the animal, and position of the nitro group on the hepatic genotoxicity of nitrotoluene isomers in vivo

. Cancer Res

. 43
, 2836-2842.

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50 , 1096-1121.

Dunnick, J.K., Elwell, M.R., and Bucher, J.R. (1994). Comparative toxicities of o- , m- , and p- nitrotoluene in 13-week feed studies in F344 rats and B6C3F 1 

### mice. Fundam. Appl. Toxicol. 22 , 411-421.

Elwell, M.R., Stedham, M.A., and Kovatch, R.M. (1990). Skin and subcutis. In Pathology of the Fischer Rat. Reference and Atlas (G.A. Boorman, S.L. Eustis, M.R. Elwell, C.A. Montgomery, Jr., and W.F. MacKenzie, Eds.), pp. 261-277. Academic Press, Inc., San Diego. Elwell, M.R., Dunnick, J.K., Hailey, J.R., and Haseman, J.K. (1996). Chemicals associated with decreases in the incidence of mononuclear cell leukemia in the Fischer rat. Toxicol. Pathol. 24 , 238-245.

Forsten, I. (1973). Pollution abatement in a munitions plant. Environ. Sci. Technol. 7 , 806-810.

Furukawa, A., Ohuchida, A., and Wierzba, K. (1989). In vivo mutagenicity tests on polyploid inducers. Environ. Mol. Mutagen. 14 , 63-64.

Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A.D., Nakamura, F., Ahmed, M., Duk, S., Rimpo, J., Margolin, B.H., Resnick, M.A., Anderson, B., and Zeiger, E. (1987). Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ. Mol. Mutagen. 10 (Suppl. 10), 1-175.

Gart, J.J., Chu, K.C., and Tarone, R.E. (1979). Statistical issues in interpretation of chronic bioassay tests for carcinogenicity. JNCI 62 , 957-974.

Guengerich, F.P. (1992). Roles of the vinyl chlorideoxidation products 2-chlorooxirane and 2-chloroacetaldehyde in the in vitro formation of etheno adducts of nucleic acid bases. Chem. Res. Toxicol. 5 , 2-5.

Handbook of Proton NMR Spectra and Data (1985). Vol. 3 (Asahi Research Center Co., Ed.). Academic Press, New York.

Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. (1983). Salmonella mutagenicity test results for 250 chemicals. Environ. Mutagen. 5 , 3-142.

Hollander, M., and Wolfe, D.A. (1973). Nonparametric Statistical Methods, pp. 120-123. John Wiley and Sons, New York.

Hollstein, M., Marion, M.J., Lehman, T., Welsh, J., Harris, C.C., Martel-Planch, G., Kusters, I., and Montesano, R. (1994). p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride-exposed factory workers. Carcinogenesis 15 , 1-3.

### o-Nitrotoluene, NTP TR 504 83



### Hong, H.-H.L., Devereux, T.R., Melnick, R.L., Moomaw, C.R., Boorman, G.A., and Sills, R.C. (2000). Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years. Toxicol. Pathol. 28 , 529-534.
Integrated Laboratory Systems (ILS) (1990). Micronucleus Data Management and Statistical Analysis Software, Version 1.4. ILS, P.O. Box 13501, Research Triangle Park, NC 27707.
International Agency for Research on Cancer (IARC) (1996). IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man: Printing Processes and Printing Inks, Carbon Black and Some Nitro Compounds, Vol. 65. IARC, Lyon, France.
Jones, M.A., Young, R.H., and Scully, R.E. (1995).Malignant mesothelioma of the tunica vaginalis: A clinicopathologic analysis of 11 cases with review of the literature. Am. J. Surg. Pathol. 19 , 815-825.
Kaplan, E.L., and Meier, P. (1958). Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 53 , 457-481.
Kawai, A., Goto, S., Matsumoto, Y., and Matsushita, H. (1987). Mutagenicity of aliphatic and aromatic nitrocompounds: Industrial materials and related compounds. Jpn. J. Ind. Health 29 , 34-54.
Kedderis, G.L., Dyroff, M.C., and Rickert, D.E. (1984).Hepatic macromolecular covalent binding of the hepatocarcinogen 2,6-dinitrotoluene and its 2,4-isomer in vivo:Modulation by the sulfotransferase inhibitors pentachlorophenol and 2,6-dichloro-4-nitrophenol. Carcinogenesis 5 , 1199-1204.
Kirk-Othmer Encyclopedia of Chemical Technology (1981). 3rd ed. (M. Grayson and D. Eckroth, Eds.), Vol. 15, pp. 925-933. John Wiley and Sons, New York.
McConnell, E.E., Solleveld, H.A., Swenberg, J.A., andBoorman, G.A. (1986). Guidelines for combining neoplasms for evaluation of rodent carcinogenesis studies. JNCI 76 , 283-289.
MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. (1990). The in vivo erythrocyte micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundam. Appl. Toxicol. 14 , 513-522. Marion, M.J., Froment, O., and Trepo, C. (1991). Activation of Ki-ras gene by point mutation in humanliver angiosarcoma associated with vinyl chloride exposure. Mol. Carcinog. 4 , 450-454.

Maronpot, R.R., and Boorman, G.A. (1982).Interpretation of rodent hepatocellular proliferative alterations and hepatocellular tumors in chemical safety
assessment. Toxicol. Pathol. 10 , 71-80.

Marques, M.M., Mourato, L.L.G., Amorim, M.T., Santos, M.A., Melchior, W.B., Jr., and Beland, F.A. (1997). Effect of substitution site upon the oxidation potentials of alkylanilines, the mutagenicities of

n -hydroxyalkylanilines, and the conformations of alkylaniline-DNA adducts. Chem. Res. Toxicol. 10
, 1266-1274.

Marrogi, A., Pass, H.I., Khan, M., MethenyBarlow, L.J., Harris, C.C., and Gerwin, B.I. (2000). Human mesothelioma samples overexpress both cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (NOS2): In vitro antiproliferative effects of a COX-2 inhibitor. Cancer Res. 60 , 3696-3700.

Melnick, R.L., and Kohn, M.C. (1995). Mechanistic data indicate that 1,3-butadiene is a human carcinogen. Carcinogenesis 16 , 157-163.

The Merck Index (1996). 12th ed., p. 1141. Merck and Company, Inc., Whitehouse Station, NJ.

Miller, J.A., and Miller, E.C. (1977). Ultimate chemical carcinogens as reactive mutagenic electrophiles. In Origins of Human Cancer (H.H. Hiatt, J.D. Watson, and J.A. Winsten, Eds.), pp. 605-627. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Miller, R.G., Jr. (1960). Simultaneous Statistical Inference. McGraw Hill, New York.

Mirsalis, J.C., and Butterworth, B.E. (1980). Detection of unscheduled DNA synthesis in hepatocytes isolated from rats treated with genotoxic agents: An in vivoin vitro assay for potential carcinogens and mutagens. Carcinogenesis 1 , 621-625.

Mirsalis, J.C., Tyson, C.K., and Butterworth, B.E. (1982a). Detection of genotoxic carcinogens in the in vivo-in vitro hepatocyte DNA repair assay. Environ. Mutagen. 4 , 553-562.

### 84 o-Nitrotoluene, NTP TR 504



### Mirsalis, J.C., Hamm, T.E., Jr., Sherrill, J.M., andButterworth, B.E. (1982b). Role of gut flora in the genotoxicity of dinitrotoluene. Nature 295 , 322-323.

Mirsalis, J.C., Tyson, C.K., Steinmetz, K.L., Loh, E.N., Hamilton, C.M., Bakke, J.P., and Spaldin, J.W. (1989). Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds. Environ. Mol. Mutagen. 14 , 155-164.

Miyata, R., Nohmi, T., Yoshikawa, K., and Ishidate, M., Jr. (1981). Metabolic activation of nitrotoluene andtrichlorethylene by rat-liver S9 or mouse-liver S9 fractions in Salmonella typhimurium strains. Eisei Shikenji Jokoku 99 , 60-65.

Mor, O., Yaron, P., Huszar, M., Yellin, A., Jakobovitz, O., Brok-Simoni, F., Rechavi, G., andReichert, N. (1997). Absence of p53 mutations in malignant mesotheliomas. Am. J. Respir. Cell Mol. Biol. 16
, 9-13.

National Cancer Institute (NCI) (1978a). Bioassay of Acronycine for Possible Carcinogenicity (CAS No. 7008-42-6). Technical Report Series No. 49. NIH Publication No. 78-849. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD.
National Cancer Institute (NCI) (1978b). Bioassay of Phenoxybenzamine Hydrochloride for Possible Carcinogenicity (CAS No. 63-92-3). Technical Report Series No. 72. NIH Publication No. 78-1322. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD.
National Cancer Institute (NCI) (1978c). Bioassay of

o -Anisidine Hydrochloride for Possible Carcinogenicity (CAS No. 134-29-0). Technical Report Series No. 89. NIH Publication No. 78-1339. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD.
National Cancer Institute (NCI) (1978d). Bioassay of
p -Anisidine Hydrochloride for Possible Carcinogenicity (CAS No. 20265-97-8). Technical Report Series No. 116. NIH Publication No. 78-1371. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD. National Cancer Institute (NCI) (1979). Bioassay of

o -Toluidine Hydrochloride for Possible Carcinogenicity (CAS No. 636-21-5). Technical Report Series No. 153. NIH Publication No. 79-1709. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD.

National Institute for Occupational Safety and Health (NIOSH) (1990). National Occupational Exposure Survey (1981-1983), unpublished provisional data as of July 1, 1990. NIOSH, Cincinnati, OH.

National Institute for Occupational Safety and Health (NIOSH) (1997). NIOSH Pocket Guide to Chemical Hazards. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control, Washington, D.C.
National Toxicology Program (NTP) (1981). Carcinogenesis Bioassay of Cytembena (CAS No. 21739-91-3). Technical Report Series No. 207. NIH Publication No. 81-1763. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC, and Bethesda, MD.
National Toxicology Program (NTP) (1982). Carcinogenesis Bioassay of 1,2-Dibromoethane (CAS No. 106-93-4) in F344 Rats and B6C3F 1 

### Mice (Inhalation Study). Technical Report Series No. 210. NIH Publication No. 82-1766. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC, and Bethesda, MD.
National Toxicology Program (NTP) (1984). Toxicology and Carcinogenesis Studies of 1,3-Butadiene (CAS No. 106-99-0) in B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 288. NIH Publication No. 84-2544. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.
National Toxicology Program (NTP) (1985). Toxicology and Carcinogenesis Studies of Propylene Oxide in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). (CAS No. 75-56-9). Technical Report Series No. 267. NIH Publication No. 85-2527. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

### o-Nitrotoluene, NTP TR 504 85



### National Toxicology Program (NTP) (1989). Toxicology and Carcinogenesis Studies of Two Pentachlorophenol Technical-Grade Mixtures (CAS No. 87-86-5) in B6C3F 1 

### Mice (Feed Studies). Technical Report Series No. 349. NIH Publication No. 89-2804. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1990a). Toxicology and Carcinogenesis Studies of 3,3 N -Dimethoxybenzidine Dihydrochloride (CAS No. 20325-40-0) in F344/N Rats (Drinking Water Studies). Technical Report Series No. 372. NIH Publication No. 90-2827. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1990b). Toxicology and Carcinogenesis Studies of Glycidol (CAS No. 556-52-5) in F344/N Rats and B6C3F 1 

### Mice (Gavage Studies). Technical Report Series No. 374. NIH Publication No. 90-2829. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1991). Toxicology and Carcinogenesis Studies of 3,3 N- Dimethylbenzidine Dihydrochloride (CAS No. 612-82-8) in F344/N Rats (Drinking Water Studies). Technical Report Series No. 390. NIH Publication No. 91-2845. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1992). NTP Technical Report on Toxicity Studies of o-, m- , and
p-Nitrotoluenes (CAS Nos. 88-72-2, 99-08-1, 99-99-0) Administered in Dosed Feed to F344/N Rats and B6C3F 1 

### Mice. Toxicity Report Series No. 23. NIH Publication No. 93-3346. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. National Toxicology Program (NTP) (1993).Toxicology and Carcinogenesis Studies of 2,3-Dibromo1-propanol (CAS No. 96-13-9) in F344/N Rats and B6C3F1 Mice (Dermal Studies). Technical Report Series No. 400. NIH Publication No. 94-2855. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1996). NTP Technical Report on Comparative Toxicity and Carcinogenicity Studies of o -Nitrotoluene and

o -Toluidine Hydrochloride (CAS Nos. 88-72-2 and 636- 21-5) Administered in Feed to Male F344/N Rats. Toxicity Report Series No. 44. NIH Publication No. 96-3936. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1997). Toxicology and Carcinogenesis Studies of Tetrafluoroethylene (CAS No. 116-14-3) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 450. NIH Publication No. 97-3366. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1998). Toxicology and Carcinogenesis Studies of Chloroprene (CAS No. 126-99-8) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 467. NIH Publication No. 98-3957. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1999). Toxicology and Carcinogenesis Studies of Pentachlorophenol (CAS No. 87-86-5) in F344/N Rats (Feed Studies). Technical Report Series No. 483. NIH Publication No. 99-3973. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

### 86 o-Nitrotoluene, NTP TR 504



### National Toxicology Program (NTP) (2002). Toxicology and Carcinogenesis Studies of
p-Nitrotoluene (CAS No. 99-99-0) in F344/N Rats and B6C3F 1 

### Mice (Feed Studies). Technical Report Series No. 498. NIH Publication No. 02-4432. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.
Nishiyama, Y., Suwa, H., Okamoto, K., Fukumoto, M., Hiai, H., and Toyokuni, S. (1995). Low incidence of point mutations in H-, K- and N- ras oncogenes and p53tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate. Jpn. J. Cancer Res. 86 , 1150-1158.
NIST Standard Reference Database. NBS/EPA/MSDC Mass Spectral Database, PC Version (Database 1-A). National Institute of Standards and Technology, Gaithersburg, MD.
Ohuchida, A., Furukawa, A., and Yoshida, R. (1989). Micronucleus test of polyploidy inducers. Mutat. Res. 216 , 371-372.
Parton, J.W., Yount, D.J., and Garriott, M.L. (1995). Improved sensitivity of the unscheduled DNA synthesis assay in primary rat hepatocytes following culture in serum-free defined media. Environ. Mol. Mutagen. 26
, 147-154.
PATHCO, Inc. (2000). Pathology report on selected organs in the rat 90-day subchronic o-nitrotoluene study. PATHCO, Inc., Frederick, MD.
Piegorsch, W.W., and Bailer, A.J. (1997). Statistics for Environmental Biology and Toxicology , Section 6.3.2. Chapman and Hall, London.
Portier, C.J., and Bailer, A.J. (1989). Testing for increased carcinogenicity using a survival-adjusted quantal response test. Fundam. Appl. Toxicol. 12
, 731-737.
Portier, C.J., Hedges, J.C., and Hoel, D.G. (1986). Agespecific models of mortality and tumor onset for historical control animals in the National Toxicology Program’s carcinogenicity experiments. Cancer Res. 46
, 4372-4378. Ramael, M., Lemmens, G., Eerdekens, C., Buysse, C., Deblier, I., Werner, J., and Van Marck, E. (1992).Immunoreactivity for p53 protein in malignant mesothelioma and non-neoplastic mesothelium. J. Pathol. 168
, 371-375.

Ramael, M., Deblier, I., Eerdekens, C., Lemmens, G., Jacobs, W., and Van Marck, E. (1993). Immunohistochemical staining of ras oncogene product in neoplastic and non-neoplastic mesothelial tissues: Immunoreactivity for N- ras and lack of immunohistochemical staining for Ha- ras and K- ras

. J. Pathol. 169
, 421-424.

Rao, G.N. (1996). New diet (NTP-2000) for rats in theNational Toxicology Program toxicity and carcinogenicity studies. Fundam. Appl. Toxicol. 32 , 102-108.

Rao, G.N. (1997). New nonpurified diet (NTP-2000) for rodents in the National Toxicology Program’s toxicology and carcinogenesis studies. J. Nutr. 127 , 842s-846s.

Rao, G.N., and Knapka, J.J. (1988). Animal diets in safety evaluation studies. In Nutrition and Chemical Toxicity (C. Ioannides, Ed.), pp. 345-374. Wiley, New York.

Research Triangle Institute (RTI) (1995). Comparative Metabolism and Excretion of [ 14 

### C]ortho-Nitrotoluene (ONT) and [ 14 

### C]ortho-Toluidine (OTL) in Rats and Mice. Project Report No. 06/13, NIEHS Contract No. N01-ES-15329. Research Triangle Park, NC.

Research Triangle Institute (RTI) (1996a). Comparative Metabolism and Excretion of [ 14 

### C]p-Nitrotoluene Following Oral Administration to Fischer 344 Rats and B6C3F 1 

### Mice. Project Report No. 16P, NIEHS Contract No. N01-ES-15329.

Research Triangle Institute (RTI) (1996b). Comparative Metabolism and Excretion of [ 14 

### C]m-Nitrotoluene (MNT) in Rats and Mice. Project Report No. 17, NIEHS Contract No. N01-ES-15329. Research Triangle Park, NC.

Rickert, D.E. (1987). Metabolism of nitroaromatic compounds. Drug. Metab. Rev

. 18 , 23-53.

### o-Nitrotoluene, NTP TR 504 87



### Rickert, D.E., and Long, R.M. (1981). Metabolism and excretion of 2,4-dinitrotoluene in male and female Fischer 344 rats after different doses. Drug Metab. Dispos. 9 , 226-232.

Rickert, D.E., Butterworth, B.E., and Popp, J.A. (1984a).Dinitrotoluene: Acute toxicity, oncogenicity, genotoxicity, and metabolism. CRC Crit. Rev. Toxicol. 13
, 217-234.

Rickert, D.E., Long, R.M., Dyroff, M.C., andKedderis, G.L. (1984b). Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer 344 rats. Chem. Biol. Interact. 52 , 131-139.

Rickert, D.E., DeBethizy, J.D., Glover, M.R., andKedderis, G.L. (1985). Kinetics of conjugation and oxidation of nitrobenzyl alcohols by rat hepatic enzymes. Biochem. Pharmacol. 34 , 4163-4168.

The Sadtler Handbook of Ultraviolet Spectra (1979). Spectrum No. 1042 (W. Simons, Ed.). Sadtler Research Laboratories, Philadelphia.

Shelby, M.D. (1988). The genetic toxicity of human carcinogens and its implications. Mutat. Res

. 204 , 3-15.

Shelby, M.D., and Witt, K.L. (1995). Comparison ofresults from mouse bone marrow chromosome aberration and micronucleus tests. Environ. Mol. Mutagen. 25
, 302-313.

Shelby, M.D., and Zeiger, E. (1990). Activity of human carcinogens in the Salmonella and rodent bone marrow cytogenetics tests. Mutat. Res. 234 , 257-261.

Shelby, M.D., Erexson, G.L., Hook, G.J., and Tice, R.R. (1993). Evaluation of a three-exposure mouse bonemarrow micronucleus protocol: Results with 49 chemicals. Environ. Mol. Mutagen. 21 , 160-179.

Shimizu, M., and Yano, E. (1986). Mutagenicity of mono-nitrobenzene derivatives in the Ames test and rec
assay. Mutat. Res. 170 , 11-22.

Singer, B. (1996). DNA damage: Chemistry, repair, and mutagenic potential. Regul. Toxicol. Pharmacol. 23
, 2-13. Son, O.S., Everett, D.W., and Fiala, E.S. (1980). Metabolism of o -[ methyl

- 14 

### C]toluidine in the F344 rat. Xenobiotica 10 , 457-468.

Spain, J.C. (1995). Biodegradation of nitroaromatic compounds. Annu. Rev. Microbiol. 49 , 523-555.

Spanggord, R.J., Mortelmans, K.E., Griffin, A.F., and Simmon, V.F. (1982). Mutagenicity in Salmonella typhimurium and structure-activity relationships ofwastewater components emanating from the manufacture of trinitrotoluene. Environ. Mutagen. 4 , 163-179.

Straus, D.S. (1981). Somatic mutation, cellular differentiation, and cancer causation. JNCI 67 , 233-241.

Suzuki, J., Koyama, T., and Suzuki, S. (1983). Mutagenicities of mono-nitrobenzene derivatives in the presence of norharman. Mutat. Res. 120 , 105-110.

Tarone, R.E. (1975). Tests for trend in life table analysis. Biometrika 62 , 679-682.

Tennant, R.W., and Ashby, J. (1991). Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program. Mutat. Res. 257
, 209-227.
Tennant, R.W., Margolin, B.H., Shelby, M.D., Zeiger, E., Haseman, J.K., Spalding, J., Caspary, W., Resnick, M., Stasiewicz, S., Anderson, B., and Minor, R. (1987). Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science 236 , 933-941.
U.S. Environmental Protection Agency (USEPA) (1976). Frequency of organic compounds identified in water. EPA-600/4-76-062. USEPA, Athens, GA.
U.S. Environmental Protection Agency (USEPA) (1999). Office of Pollution Prevention and Toxics High Production Volume Chemicals. Retrieved 30 March 1999 from the World Wide Web: <http://www.epa.gov/opptintr/chemtrk/cbiguid2.htm>
Verschueren, K. (1983). Handbook of Environmental Data on Organic Chemicals, 2nd ed. Van Nostrand Reinhold Company, New York.

### 88 o-Nitrotoluene, NTP TR 504



### Walker, C., Everitt, J., and Barrett, J.C. (1992). Possiblecellular and molecular mechanisms for asbestos carcinogenicity. Am. J. Ind. Med. 21 , 253-273. Ward, E., Carpenter, A., Markowitz, S., Roberts, D., and Halperin, W. (1991). Excess number of bladder cancers in workers exposed to ortho-toluidine and aniline. J. Natl. Cancer Inst. 83 , 501-506.
Ward, J.M., Mann, P.C., Morishima, H., and Frith, C.H. (1999). Thymus, spleen, and lymph nodes. In Pathology of the Mouse (R.R. Maronpot, Ed.), pp. 333-360. Cache River Press, Vienna, IL. Weisburger, E.K., Russfield, A.B., Homburger F., Weisburger, J.H., Boger, E., Van Dongen, C.G., and Chu, K.C. (1978). Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity. J. Environ. Pathol. Toxicol. 2 , 325-356.
White, A., Handler, P., Smith, E.L., Hill, R.L., and Lehman, I.R. (1978). Principles of Biochemistry , 6th ed., p. 751. McGraw-Hill Book Company, New York. Williams, D.A. (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27 , 103-117. Williams, D.A. (1972). The comparison of several dose levels with a zero dose control. Biometrics 28 , 519-531.

Witt, K.L., Knapton, A., Wehr, C.M., Hook, G.J., Mirsalis, J., Shelby, M.D., and MacGregor, J.T. (2000). Micronucleated erythrocyte frequency in peripheral blood of B6C3F 1 

### mice from short-term, prechronic, and chronic studies of the NTP Carcinogenesis Bioassay Program. Environ. Mol. Mutagen. 36 , 163-194.

Working, P.K., and Butterworth, B.E. (1984). An assayto detect chemically induced DNA repair in rat spermatocytes. Environ. Mutagen. 6 , 273-286.

Xiao, S., Li, D., Vijg, J., Sugarbaker, D.J., Corson, J.M., and Fletcher, J.A. (1995). Codeletion of p15 and p16 in primary malignant mesothelioma. Oncogene 11
, 511-515.

Zeiger, E., Haseman, J.K., Shelby, M.D., Margolin, B.H., and Tennant, R.W. (1990). Evaluation of four in vitrogenetic toxicity tests for predicting rodent carcinogenicity: Confirmation of earlier results with 41 additional chemicals. Environ. Mol. Mutagen. 16 (Suppl. 18), 1-14.

### 89



# APPENDIX A SUMMARY OF LESIONS IN MALE RATS IN THE 2-YEAR FEED STUDY OF o-NITROTOLUENE

T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 T ABLE 

### A3a Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 T ABLE 

### A3b Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Stop-Exposure Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . 136 T ABLE 

### A4a Historical Incidence of Malignant Mesothelioma in Control Male F344/N Rats . . . . . . . . . 141 T ABLE 

### A4b Historical Incidence of Subcutaneous Skin Neoplasms in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 T ABLE 

### A4c Historical Incidence of Mammary Gland Fibroadenoma in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 T ABLE 

### A4d Historical Incidence of Liver Neoplasms in Control Male F344/N Rats . . . . . . . . . . . . . . . . 144 T ABLE 

### A4e Historical Incidence of Alveolar/bronchiolar Neoplasms in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 T ABLE 

### A4f Historical Incidence of Circulatory System Neoplasms in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 T ABLE 

### A4g Historical Incidence of Mononuclear Cell Leukemia in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 T ABLE 

### A4h Historical Incidence of Interstitial Cell Adenoma of the Testis in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

### o-Nitrotoluene, NTP TR 504 90



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene a



### 0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Disposition Summary Animals initially in study 70 60 60 60 70 70 3-Month interim evaluation 10 10 10 Early deaths

Accidental deaths 3 Moribund 18 35 48 53 39 57 Natural deaths 3 7 6 7 10 3 Survivors

Died last week of study 1 Terminal sacrifice 38 18 3 11

Animals examined microscopically 70 60 60 60 70 70



## Systems Examined at 3 Months with No Neoplasms Observed Alimentary System Cardiovascular System Endocrine System General Body System Genital System Hematopoietic System Integumentary System Musculoskeletal System Nervous System Respiratory System Special Senses System Urinary System



## 2-Year Study Alimentary System Intestine large, colon (60) (59) (60) (60) (59) (60)

Polyp adenomatous 1 (2%) 1 (2%) 2 (3%) Intestine large, rectum (59) (60) (60) (59) (60) (58)

Leiomyosarcoma 1 (2%) Intestine large, cecum (60) (60) (60) (58) (59) (60)

Leiomyoma 1 (2%) 1 (2%) Leiomyosarcoma 1 (2%) Intestine small, jejunum (59) (60) (58) (53) (57) (60)

Carcinoma 1 (2%) Intestine small, ileum (58) (58) (58) (58) (56) (58) Liver (60) (60) (60) (60) (60) (60)

Cholangiocarcinoma 3 (5%) Hemangiosarcoma 1 (2%) Hepatocellular carcinoma 1 (2%) 2 (3%) Hepatocellular carcinoma, multiple 1 (2%) Hepatocellular adenoma 2 (3%) 2 (3%) 3 (5%) 7 (12%) 2 (3%) 3 (5%) Hepatocellular adenoma, multiple 1 (2%) 1 (2%) 1 (2%) Hepatocholangiocarcinoma 1 (2%) 1 (2%) Histiocytic sarcoma 1 (2%) 1 (2%)

### 91 o- Nitrotoluene, NTP TR 504



## 2-Year Study (continued) 

### Alimentary System (continued) Mesentery (16) (6) (5) (4) (1) (3)

Fibroma 1 (20%) Fibrosarcoma 1 (17%) 2 (67%) Lipoma 1 (17%) Oral mucosa (1) (1)

Hemangioma 1 (100%) Pancreas (60) (60) (60) (60) (60) (60)

Histiocytic sarcoma 1 (2%) Mixed tumor benign 2 (3%) Acinus, adenoma 1 (2%) 3 (5%) Islets, pancreatic, adenoma 1 (2%) Salivary glands (60) (60) (59) (60) (58) (59)

Fibrosarcoma 1 (2%) Fibrosarcoma, metastatic, skin 1 (2%) Rhabdomyosarcoma, metastatic,

skeletal muscle 1 (2%) Sarcoma 1 (2%) Schwannoma malignant, metastatic, skin 1 (2%) Stomach, forestomach (59) (60) (60) (60) (60) (60)

Squamous cell papilloma 1 (2%) Stomach, glandular (59) (60) (60) (60) (60) (60) Tongue (1) (1)

Squamous cell papilloma 1 (100%) Tooth (1) (1)

Fibroma 1 (100%)



### Cardiovascular System Heart (60) (60) (60) (60) (60) (60)

Schwannoma malignant 1 (2%)



### Endocrine System Adrenal cortex (60) (60) (60) (60) (60) (60)

Adenoma 1 (2%) Adrenal medulla (60) (60) (60) (59) (60) (60)

Neuroblastoma 1 (2%) Pheochromocytoma malignant 2 (3%) 1 (2%) Pheochromocytoma benign 1 (2%) 4 (7%) 6 (10%) 2 (3%) 4 (7%) 2 (3%) Bilateral, pheochromocytoma benign 1 (2%) Islets, pancreatic (60) (60) (60) (60) (60) (60)

Adenoma 2 (3%) 1 (2%) Carcinoma 2 (3%) Parathyroid gland (58) (55) (59) (58) (58) (57)

Adenoma 1 (2%) 1 (2%) Pituitary gland (59) (60) (58) (59) (57) (59)

Craniopharyngioma 1 (2%) Pars distalis, adenoma 11 (19%) 9 (15%) 3 (5%) 9 (15%) 10 (18%) 5 (8%) Pars intermedia, adenoma 1 (2%) 

### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### o-Nitrotoluene, NTP TR 504 92



## 2-Year Study (continued) 

### Endocrine System (continued) Thyroid gland (59) (60) (60) (60) (60) (60)

Rhabdomyosarcoma, metastatic,

skeletal muscle 1 (2%) Sarcoma, metastatic, salivary glands 1 (2%) Bilateral, C-cell, adenoma 1 (2%) C-cell, adenoma 9 (15%) 6 (10%) 1 (2%) 3 (5%) 4 (7%) C-cell, carcinoma 4 (7%) 1 (2%) 1 (2%) Follicular cell, adenoma 1 (2%) Follicular cell, carcinoma 1 (2%) 1 (2%) 1 (2%)



### General Body System Peritoneum (3) (21) (33) (46) (47) (54) Tissue NOS (1) (1)

Hemangioma 1 (100%)



### Genital System Preputial gland (60) (59) (58) (56) (60) (59)

Adenoma 2 (3%) 3 (5%) 2 (3%) 1 (2%) 1 (2%) 1 (2%) Carcinoma 2 (3%) 2 (3%) 1 (2%) 1 (2%) 2 (3%) Prostate (60) (59) (60) (60) (60) (60)

Ventral, adenoma 2 (3%) 1 (2%) Seminal vesicle (60) (59) (60) (60) (60) (59) Testes (60) (60) (60) (60) (60) (60)

Bilateral, interstitial cell, adenoma 43 (72%) 34 (57%) 32 (53%) 17 (28%) 31 (52%) 4 (7%) Interstitial cell, adenoma 12 (20%) 19 (32%) 19 (32%) 29 (48%) 19 (32%) 23 (38%)



### Hematopoietic System Bone marrow (60) (60) (60) (60) (60) (60)

Histiocytic sarcoma 1 (2%) Lymph node (16) (14) (4) (9) (18) (16)

Squamous cell carcinoma, metastatic, skin 1 (6%) Mediastinal, histiocytic sarcoma 1 (6%) Lymph node, mandibular (58) (59) (58) (59) (58) (59)

Histiocytic sarcoma 1 (2%) Rhabdomyosarcoma, metastatic,

skeletal muscle 1 (2%) Lymph node, mesenteric (60) (60) (59) (58) (60) (59) Spleen (60) (60) (60) (60) (60) (60)

Fibroma 1 (2%) Fibrosarcoma 1 (2%) 1 (2%) Hemangioma 1 (2%) Hemangiosarcoma 1 (2%) Histiocytic sarcoma 1 (2%) 1 (2%) Thymus (56) (54) (55) (55) (56) (56)

Alveolar/bronchiolar carcinoma,

metastatic, lung 1 (2%) Fibrosarcoma, metastatic, spleen 1 (2%) Thymoma benign 1 (2%) 

### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### 93 o- Nitrotoluene, NTP TR 504



## 2-Year Study (continued) 

### Integumentary System Mammary gland (57) (47) (46) (43) (51) (50)

Fibroadenoma 7 (15%) 9 (20%) 2 (5%) 12 (24%) 20 (40%) Fibroadenoma, multiple 1 (2%) 1 (2%) Hemangioma 1 (2%) Skin (60) (60) (60) (60) (60) (60)

Basal cell adenoma 2 (3%) 1 (2%) Hemangioma 1 (2%) Histiocytic sarcoma 1 (2%) 1 (2%) Keratoacanthoma 5 (8%) 3 (5%) 1 (2%) 1 (2%) Schwannoma malignant 1 (2%) Squamous cell carcinoma 1 (2%) Squamous cell papilloma 2 (3%) Subcutaneous tissue, fibroma 4 (7%) 21 (35%) 13 (22%) 24 (40%) 45 (75%) 24 (40%) Subcutaneous tissue, fibroma, multiple 1 (2%) 25 (42%) 39 (65%) 35 (58%) 22 (37%) 28 (47%) Subcutaneous tissue, fibrosarcoma 7 (12%) 13 (22%) 15 (25%) 8 (13%) 8 (13%) Subcutaneous tissue, fibrosarcoma, multiple 4 (7%) 5 (8%) 4 (7%) Subcutaneous tissue, hemangioma 2 (3%) Subcutaneous tissue, hemangiopericytoma 1 (2%) Subcutaneous tissue, hemangiosarcoma 1 (2%) Subcutaneous tissue, lipoma 4 (7%) 12 (20%) 13 (22%) 10 (17%) 9 (15%) Subcutaneous tissue, lipoma, multiple 1 (2%) 3 (5%) Subcutaneous tissue, osteosarcoma 1 (2%) Subcutaneous tissue, sarcoma 1 (2%) Subcutaneous tissue, schwannoma malignant 1 (2%) 1 (2%) 1 (2%)



### Musculoskeletal System Bone (60) (60) (60) (60) (60) (60)

Osteosarcoma 1 (2%) 1 (2%) 1 (2%) 2 (3%) 3 (5%) Skeletal muscle (1) (5) (3) (6) (5)

Alveolar/bronchiolar carcinoma,

metastatic, lung 1 (20%) Hemangioma 1 (20%) Hemangiosarcoma 1 (20%) Rhabdomyosarcoma 1 (20%) 1 (17%) 1 (20%)



### Nervous System Brain (60) (60) (60) (60) (60) (60)

Carcinoma, metastatic, pituitary gland 1 (2%) Granular cell tumor benign 1 (2%) Histiocytic sarcoma 1 (2%) Oligodendroglioma malignant 1 (2%) Rhabdomyosarcoma, metastatic,

skeletal muscle 1 (2%) Cranial nerve, schwannoma malignant 1 (2%) 

### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### o-Nitrotoluene, NTP TR 504 94



## 2-Year Study (continued) 

### Respiratory System Lung (60) (60) (60) (60) (60) (60)

Alveolar/bronchiolar adenoma 1 (2%) 5 (8%) 1 (2%) 2 (3%) 3 (5%) 7 (12%) Alveolar/bronchiolar adenoma, multiple 1 (2%) Alveolar/bronchiolar carcinoma 1 (2%) 1 (2%) Alveolar/bronchiolar carcinoma, multiple 2 (3%) Carcinoma, metastatic, Zymbal’s gland 1 (2%) Histiocytic sarcoma 1 (2%) 1 (2%) Squamous cell carcinoma 1 (2%) Nose (60) (60) (60) (60) (60) (60)



### Special Senses System Eye (2) (1) (2) (1)

Schwannoma malignant, metastatic, brain 1 (50%) Harderian gland (1) (1)

Schwannoma malignant, metastatic, brain 1 (100%) Zymbal’s gland (2) (2) (2) (1)

Carcinoma 2 (100%) 2 (100%) 2 (100%) 1 (100%)



### Urinary System Kidney (60) (60) (60) (60) (60) (60) Urinary bladder (60) (60) (60) (59) (60) (59)

Hemangioma 1 (2%) Leiomyoma 1 (2%) Papilloma 1 (2%) 1 (2%) Squamous cell papilloma 1 (2%) Transitional epithelium, papilloma 2 (3%)



### Systemic Lesions Multiple organs b (60) (60) (60) (60) (60) (60)

Histiocytic sarcoma 1 (2%) 2 (3%) 2 (3%) Leukemia mononuclear 30 (50%) 21 (35%) 3 (5%) 3 (5%) 13 (22%) 1 (2%) Lymphoma malignant 1 (2%) Mesothelioma malignant 2 (3%) 20 (33%) 29 (48%) 44 (73%) 44 (73%) 54 (90%) 

### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### 95 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Neoplasm Summary Total animals with primary neoplasms c

2-Year study 60 60 57 60 60 60 Total primary neoplasms

2-Year study 144 221 202 224 235 239 Total animals with benign neoplasms

2-Year study 60 58 55 59 57 56 Total benign neoplasms

2-Year study 102 154 147 148 152 149 Total animals with malignant neoplasms

2-Year study 37 47 42 51 54 58 Total malignant neoplasms

2-Year study 42 67 55 76 83 90 Total animals with metastatic neoplasms

2-Year study 4 3 1 6 5 Total metastatic neoplasms

2-Year study 7 4 1 8 11

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms

### o-Nitrotoluene, NTP TR 504 96

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon ++++++++++++++++++++++++++++++

Polyp adenomatous Intestine large, rectum ++++++++++++++++++++++++++++++ Intestine large, cecum ++++++++++++++++++++++++++++++ Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum ++++++++++++++++++++++++++++++ Intestine small, ileum + + + + + + + +A+ + + + + + +M+ + + + + + + + + + + + + Liver ++++++++++++++++++++++++++++++

Hepatocellular carcinoma Hepatocellular adenoma X Histiocytic sarcoma X Mesentery + + + + + + + + Pancreas ++++++++++++++++++++++++++++++

Islets, pancreatic, adenoma X Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular ++++++++++++++++++++++++++++++ Tongue


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++

Pheochromocytoma malignant Pheochromocytoma benign Bilateral, pheochromocytoma benign Islets, pancreatic ++++++++++++++++++++++++++++++

Adenoma X Parathyroid gland ++++++++++++++++++++++++++++++ Pituitary gland + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X Thyroid gland ++++++++++++++++++++++++++++++

Bilateral, C-cell, adenoma X C-cell, adenoma X X X Follicular cell, carcinoma X


### General Body System Peritoneum

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

455555666666667777777777777777 

### Number of Days on Study 433358344467790001222222222222 133756104933415574027999999999

000000000000000000000000000000 

### Carcass ID Number 225554310635502354434000001112 508544375151714905973467893452

### 97 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Polyp adenomatous X 1 Intestine large, rectum + + + + + + + + + + + + + + + + I + + + + + + + + + + + + + 59 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 59 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + A + + + 59 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular carcinoma X 1 Hepatocellular adenoma X 2 Histiocytic sarcoma 1 Mesentery + + + + + + + + 16 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Islets, pancreatic, adenoma 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 59 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 59 Tongue + 1


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pheochromocytoma malignant X X 2 Pheochromocytoma benign X 1 Bilateral, pheochromocytoma benign X 1 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 2 Parathyroid gland + + + + + + + + + + + + + + + + + M + + + + + + + + + M + + 58 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Pars distalis, adenoma X X X X X 11 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + M + + 59

Bilateral, C-cell, adenoma 1 C-cell, adenoma X X X X X X 9 Follicular cell, carcinoma 1


### General Body System Peritoneum + + + 3 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 222222222333333333333333333333 999999999000000000000000000000

000000000000000000000000000000 T otal 

### Carcass ID Number 233355556001112222233344466666 Tissues/ 346823690236891678901267823789 T umors

### o-Nitrotoluene, NTP TR 504 98

Genital System Epididymis ++++++++++++++++++++++++++++++ Preputial gland ++++++++++++++++++++++++++++++

Adenoma X Carcinoma X Prostate ++++++++++++++++++++++++++++++

Ventral, adenoma X Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++

Bilateral, interstitial cell, adenoma X X X X X XXXX XX XXXXXX Interstitial cell, adenoma X XXXX XX X X


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++

Histiocytic sarcoma X Lymph node + + + ++++++ +++ Lymph node, mandibular ++++++++++++++++I+++++++++++++

Histiocytic sarcoma X Lymph node, mesenteric ++++++++++++++++++++++++++++++ Spleen ++++++++++++++++++++++++++++++ Thymus + + M + + + +M+ + + + + + + + + + + +M+ + + + + + + + +


### Integumentary System Mammary gland + M + + + M + + + + + + + + + + + + +M+ + + + + + + + + + Skin ++++++++++++++++++++++++++++++

Basal cell adenoma Keratoacanthoma X X Subcutaneous tissue, fibroma X Subcutaneous tissue, fibroma, multiple X Subcutaneous tissue, hemangiosarcoma


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++

Osteosarcoma X Skeletal muscle +


### Nervous System Brain ++++++++++++++++++++++++++++++

Histiocytic sarcoma X Peripheral nerve + + Spinal cord + + 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

455555666666667777777777777777 

### Number of Days on Study 433358344467790001222222222222 133756104933415574027999999999

000000000000000000000000000000 

### Carcass ID Number 225554310635502354434000001112 508544375151714905973467893452

### 99 o- Nitrotoluene, NTP TR 504

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 2 Carcinoma X 2 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Ventral, adenoma X 2 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X X X X 43 Interstitial cell, adenoma X X X 12


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma 1 Lymph node + + + + 16 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 58

Histiocytic sarcoma 1 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Thymus + + + + + + + + + + + + + + + + + + + + + + + + + + + M + + 56


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Basal cell adenoma X X 2 Keratoacanthoma X X X 5 Subcutaneous tissue, fibroma X XX 4 Subcutaneous tissue, fibroma, multiple 1 Subcutaneous tissue, hemangiosarcoma X 1


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Osteosarcoma 1 Skeletal muscle 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma 1 Peripheral nerve 2 Spinal cord 2 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 222222222333333333333333333333 999999999000000000000000000000

000000000000000000000000000000 T otal 

### Carcass ID Number 233355556001112222233344466666 Tissues/ 346823690236891678901267823789 T umors

### o-Nitrotoluene, NTP TR 504 100

Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Histiocytic sarcoma X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++

Papilloma


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Histiocytic sarcoma X Leukemia mononuclear X X X X XXXXXX XXXX Mesothelioma malignant 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

455555666666667777777777777777 

### Number of Days on Study 433358344467790001222222222222 133756104933415574027999999999

000000000000000000000000000000 

### Carcass ID Number 225554310635502354434000001112 508544375151714905973467893452

### 101 o- Nitrotoluene, NTP TR 504

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X 1 Alveolar/bronchiolar carcinoma X 1 Histiocytic sarcoma 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye + + 2


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Papilloma X 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma 1 Leukemia mononuclear X X X X X X X X X X X X X X X X 30 Mesothelioma malignant X X 2 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 222222222333333333333333333333 999999999000000000000000000000

000000000000000000000000000000 T otal 

### Carcass ID Number 233355556001112222233344466666 Tissues/ 346823690236891678901267823789 T umors

### o-Nitrotoluene, NTP TR 504 102

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon ++++++++++++++++++++++++++++A+ Intestine large, rectum ++++++++++++++++++++++++++++++ Intestine large, cecum ++++++++++++++++++++++++++++++ Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum ++++++++++++++++++++++++++++++ Intestine small, ileum +++++++++++++++++++++++++++A++ Liver ++++++++++++++++++++++++++++++

Hemangiosarcoma X Hepatocellular adenoma X Hepatocellular adenoma, multiple Hepatocholangiocarcinoma X Mesothelioma malignant, metastatic,

peritoneum X Mesentery + + + +

Fibrosarcoma X Lipoma Pancreas ++++++++++++++++++++++++++++++ Salivary glands ++++++++++++++++++++++++++++++

Rhabdomyosarcoma, metastatic,

skeletal muscle X Schwannoma malignant, metastatic, skin X Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular ++++++++++++++++++++++++++++++


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++

Pheochromocytoma benign X X Islets, pancreatic ++++++++++++++++++++++++++++++

Carcinoma Parathyroid gland + + + + + + + + + + + +M+ + + + + + + + + + + + I + + + +

Adenoma Pituitary gland ++++++++++++++++++++++++++++++

Pars distalis, adenoma X X X X X Pars intermedia, adenoma Thyroid gland ++++++++++++++++++++++++++++++

Rhabdomyosarcoma, metastatic,

skeletal muscle X C-cell, adenoma C-cell, carcinoma Follicular cell, carcinoma 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

344445555555556666666666666666 

### Number of Days on Study 726792355666680011111223333455 885199459779990355666112669106

100011011000110100001010010110 

### Carcass ID Number 289920902899028079791807819318 585270681907293611233599474096

### 103 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 59 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + M + + + + + + + + + 58 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma 1 Hepatocellular adenoma X 2 Hepatocellular adenoma, multiple X 1 Hepatocholangiocarcinoma 1 Mesothelioma malignant, metastatic,

peritoneum 1 Mesentery + + 6

Fibrosarcoma 1 Lipoma X 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Rhabdomyosarcoma, metastatic,

skeletal muscle 1 Schwannoma malignant, metastatic, skin 1 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pheochromocytoma benign X X 4 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Carcinoma XX 2 Parathyroid gland + + + + + + + + M + + + + + + M + + + + + + + + + + + + + M 55

Adenoma X 1 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pars distalis, adenoma X X X X 9 Pars intermedia, adenoma X 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Rhabdomyosarcoma, metastatic,

skeletal muscle 1 C-cell, adenoma X X X X X X 6 C-cell, carcinoma X X X X 4 Follicular cell, carcinoma X 1 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

666667777777777777777777777777 

### Number of Days on Study 558890011222222222223333333333 684417734667999999990000000000

110111111110000000110000111111 T otal 

### Carcass ID Number 028212211028778899127777000111 Tissues/ 527440881330781289663456147025 T umors

### o-Nitrotoluene, NTP TR 504 104

General Body System Peritoneum + + + + + + + + + Tissue NOS


### Genital System Coagulating gland Epididymis ++++++++++++++++++++++++++++++ Preputial gland ++++++++++++++++++++++++++++++

Adenoma X X Carcinoma X Prostate + + + + + + + + + + + + + + + + + + + + + +M+ + + + + + + Seminal vesicle + + + + + + + + + + + + + + + + + + + + + +M+ + + + + + + Testes ++++++++++++++++++++++++++++++

Bilateral, interstitial cell, adenoma X X X X X X X X X X Interstitial cell, adenoma X X X X X X X X X X X X X X


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + + + + + + Lymph node, mandibular ++++++++++++++++++++++++++++++

Rhabdomyosarcoma, metastatic,

skeletal muscle X Lymph node, mesenteric ++++++++++++++++++++++++++++++ Spleen ++++++++++++++++++++++++++++++

Histiocytic sarcoma X Thymus + + + + + + + + + + + + + + + +M+ + + + + + + + +M+ + +

Thymoma benign X


### Integumentary System Mammary gland + + M + + + +M+ + + + +M+ + +M+M+M+ +M+ + + + +

Fibroadenoma X X Hemangioma X Skin ++++++++++++++++++++++++++++++

Histiocytic sarcoma Keratoacanthoma X Subcutaneous tissue, fibroma X X X X X X X X X X X X X X Subcutaneous tissue, fibroma, multiple X X X X X Subcutaneous tissue, fibrosarcoma X X X Subcutaneous tissue, hemangioma X Subcutaneous tissue, lipoma X X Subcutaneous tissue,

schwannoma malignant X 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

344445555555556666666666666666 

### Number of Days on Study 726792355666680011111223333455 885199459779990355666112669106

100011011000110100001010010110 

### Carcass ID Number 289920902899028079791807819318 585270681907293611233599474096

### 105 o- Nitrotoluene, NTP TR 504

General Body System Peritoneum + + + + + + + + + + + + 21 Tissue NOS + 1


### Genital System Coagulating gland + 1 Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + M + + + + + + + + + + + + + + + + 59

Adenoma X 3 Carcinoma X 2 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X X 34 Interstitial cell, adenoma X X X X X 19


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + + + + + 14 Lymph node, mandibular + + + + + + + + + + + + + + + + + M + + + + + + + + + + + + 59

Rhabdomyosarcoma, metastatic,

skeletal muscle 1 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma 1 Thymus + M + + + M + + + + + + + + + + + + + M + + + + + + + M + + 54

Thymoma benign 1


### Integumentary System Mammary gland M + M + + M + + + M + + + + + + + + + + M M + + + + + + + + 47

Fibroadenoma X X X X X 7 Hemangioma 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma X 1 Keratoacanthoma X X 3 Subcutaneous tissue, fibroma X X X X X X X 21 Subcutaneous tissue, fibroma, multiple X X X X X X X X X X X X X X X X X X X X 25 Subcutaneous tissue, fibrosarcoma X X X X 7 Subcutaneous tissue, hemangioma X 2 Subcutaneous tissue, lipoma X X 4 Subcutaneous tissue,

schwannoma malignant 1 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

666667777777777777777777777777 

### Number of Days on Study 558890011222222222223333333333 684417734667999999990000000000

110111111110000000110000111111 T otal 

### Carcass ID Number 028212211028778899127777000111 Tissues/ 527440881330781289663456147025 T umors

### o-Nitrotoluene, NTP TR 504 106

Musculoskeletal System Bone ++++++++++++++++++++++++++++++

Osteosarcoma Skeletal muscle + + + +

Rhabdomyosarcoma X


### Nervous System Brain ++++++++++++++++++++++++++++++

Granular cell tumor benign Rhabdomyosarcoma, metastatic,

skeletal muscle X Peripheral nerve + + + + Spinal cord + + + +


### Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X Carcinoma, metastatic,

Zymbal’s gland X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Ear + Eye + Harderian gland + Lacrimal gland + Zymbal’s gland +

Carcinoma X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++

Squamous cell papilloma X Transitional epithelium, papilloma


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Histiocytic sarcoma X Leukemia mononuclear X X X X X X X X X Mesothelioma malignant X X X X X X X X X 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

344445555555556666666666666666 

### Number of Days on Study 726792355666680011111223333455 885199459779990355666112669106

100011011000110100001010010110 

### Carcass ID Number 289920902899028079791807819318 585270681907293611233599474096

### 107 o- Nitrotoluene, NTP TR 504

Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Osteosarcoma X 1 Skeletal muscle + 5

Rhabdomyosarcoma 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Granular cell tumor benign X 1 Rhabdomyosarcoma, metastatic,

skeletal muscle 1 Peripheral nerve 4 Spinal cord 4


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X X X X 5 Carcinoma, metastatic,

Zymbal’s gland 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Ear 1 Eye 1 Harderian gland 1 Lacrimal gland 1 Zymbal’s gland + 2

Carcinoma X 2


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Squamous cell papilloma 1 Transitional epithelium, papilloma X X 2


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma X 2 Leukemia mononuclear X X X X X X X X X X X X 21 Mesothelioma malignant X X X X X X X X X X X 20 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

666667777777777777777777777777 

### Number of Days on Study 558890011222222222223333333333 684417734667999999990000000000

110111111110000000110000111111 T otal 

### Carcass ID Number 028212211028778899127777000111 Tissues/ 527440881330781289663456147025 T umors

### o-Nitrotoluene, NTP TR 504 108

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon ++++++++++++++++++++++++++++++ Intestine large, rectum ++++++++++++++++++++++++++++++ Intestine large, cecum ++++++++++++++++++++++++++++++

Leiomyosarcoma Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum +++++++++++++++++++A++++++++++ Intestine small, ileum + + + + + + + + + + + + + + +M+ + + + + + + + + + + + + + Liver ++++++++++++++++++++++++++++++

Hepatocellular adenoma X Mesentery +

Fibroma Oral mucosa + Pancreas ++++++++++++++++++++++++++++++

Acinus, adenoma Salivary glands ++++++++++++++++++++++++++++++

Sarcoma X Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular ++++++++++++++++++++++++++++++ Tongue

Squamous cell papilloma


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++

Pheochromocytoma benign X Islets, pancreatic ++++++++++++++++++++++++++++++ Parathyroid gland M +++++++++++++++++++++++++++++ Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + +M+ + + + +

Pars distalis, adenoma Thyroid gland ++++++++++++++++++++++++++++++

Sarcoma, metastatic, salivary glands X Follicular cell, adenoma


### General Body System Peritoneum + + + +++++ + +++++ ++ 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

222334444555555555555555555566 

### Number of Days on Study 999893888001111223666788899901 899412333222599371479611977735

111111111111111111111111111111 

### Carcass ID Number 888636677668578937556637838854 567786415184380024895993811912

### 109 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leiomyosarcoma X 1 Intestine small, duodenum + + + + + + + M + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, jejunum + + + + + + + + + A + + + + + + + + + + + + + + + + + + + + 58 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + M + + + + 58 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular adenoma X X 3 Mesentery + + + + 5

Fibroma X 1 Oral mucosa 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Acinus, adenoma X 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + M + + + + + + + + 59

Sarcoma 1 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Tongue + 1

Squamous cell papilloma X 1


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pheochromocytoma benign X X X X X 6 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + M + + + + + + + 58

Pars distalis, adenoma X XX 3 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Sarcoma, metastatic, salivary glands 1 Follicular cell, adenoma X 1


### General Body System Peritoneum + + + + + + + + + + + + + + + + + 33 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

666666666666666666666677777777 

### Number of Days on Study 112233333555557788899901111223 661826677266883655511164444990

111111111111111111111111111111 T otal 

### Carcass ID Number 578563536544473734446853447574 Tissues/ 062605562458194774603323392707 T umors

### o-Nitrotoluene, NTP TR 504 110

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + +M+ + Preputial gland ++++++++++++++++++++++++++++++

Adenoma X Prostate ++++++++++++++++++++++++++++++ Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++

Bilateral, interstitial cell, adenoma X X X X X X X X X X X Interstitial cell, adenoma X X X X X X X X X X


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + +M+ + + + + Lymph node, mesenteric +++++++++++++++++++++++I++++++ Spleen ++++++++++++++++++++++++++++++

Fibrosarcoma Hemangiosarcoma Thymus + + + M + + + + + + + + + + + + + + + + + + + + + + +M+ +


### Integumentary System Mammary gland + + + M + + + + +M+M+ + + + + + +M+ + + + +M+ + + +

Fibroadenoma X X Fibroadenoma, multiple X Skin ++++++++++++++++++++++++++++++

Keratoacanthoma X Subcutaneous tissue, fibroma X X X X X X X Subcutaneous tissue, fibroma, multiple X X X X X X X X X XXXXXXX Subcutaneous tissue, fibrosarcoma X X X X X Subcutaneous tissue, fibrosarcoma, multiple X Subcutaneous tissue, lipoma X X X X Subcutaneous tissue, lipoma, multiple X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle

Mesothelioma malignant, metastatic,

peritoneum


### Nervous System Brain ++++++++++++++++++++++++++++++

Cranial nerve, schwannoma malignant X Peripheral nerve + Spinal cord + 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

222334444555555555555555555566 

### Number of Days on Study 999893888001111223666788899901 899412333222599371479611977735

111111111111111111111111111111 

### Carcass ID Number 888636677668578937556637838854 567786415184380024895993811912

### 111 o- Nitrotoluene, NTP TR 504

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Preputial gland + + + + + + + + + + + + + + + + + M + + + + M + + + + + + + 58

Adenoma X 2 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X 32 Interstitial cell, adenoma X X X X X X X X X 19


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + 4 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + M + + + + + + + + 58 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Fibrosarcoma X 1 Hemangiosarcoma X 1 Thymus + + + + + + + + + + M + + + + + I + + + M + + + + + + + + + 55


### Integumentary System Mammary gland + + + + + M + + + + + M M M + + + + + M + M + + + M M + + M 46

Fibroadenoma X X X X X X X 9 Fibroadenoma, multiple 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Keratoacanthoma 1 Subcutaneous tissue, fibroma X X X X X X 13 Subcutaneous tissue, fibroma, multiple X X X X X X X X X X X X X X X X X X X X X X X 39 Subcutaneous tissue, fibrosarcoma X X X X X X X X 13 Subcutaneous tissue, fibrosarcoma, multiple X XX 4 Subcutaneous tissue, lipoma X X X X X X X X 12 Subcutaneous tissue, lipoma, multiple 1


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle + + + 3

Mesothelioma malignant, metastatic, 1

peritoneum X


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Cranial nerve, schwannoma malignant 1 Peripheral nerve 1 Spinal cord 1 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

666666666666666666666677777777 

### Number of Days on Study 112233333555557788899901111223 661826677266883655511164444990

111111111111111111111111111111 T otal 

### Carcass ID Number 578563536544473734446853447574 Tissues/ 062605562458194774603323392707 T umors

### o-Nitrotoluene, NTP TR 504 112

Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye + +

Schwannoma malignant, metastatic, brain X Harderian gland +

Schwannoma malignant, metastatic, brain X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++

Papilloma


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear Lymphoma malignant X Mesothelioma malignant X X X XXXX X XXXX XX 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

222334444555555555555555555566 

### Number of Days on Study 999893888001111223666788899901 899412333222599371479611977735

111111111111111111111111111111 

### Carcass ID Number 888636677668578937556637838854 567786415184380024895993811912

### 113 o- Nitrotoluene, NTP TR 504

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye 2

Schwannoma malignant, metastatic, brain 1 Harderian gland 1

Schwannoma malignant, metastatic, brain 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Papilloma X 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X X X 3 Lymphoma malignant 1 Mesothelioma malignant X X X X X X X X X X X X X X X 29 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

666666666666666666666677777777 

### Number of Days on Study 112233333555557788899901111223 661826677266883655511164444990

111111111111111111111111111111 T otal 

### Carcass ID Number 578563536544473734446853447574 Tissues/ 062605562458194774603323392707 T umors

### o-Nitrotoluene, NTP TR 504 114

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon ++++++++++++++++++++++++++++++

Polyp adenomatous X Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + +M+ + + + + Intestine large, cecum ++++++++++++++++++++++++++++++ Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum + + + A + + A + + + + + + + + + + + +AA+ + + + + + + + + Intestine small, ileum +++++++++++++++++++A++++++++++ Liver ++++++++++++++++++++++++++++++

Hepatocellular carcinoma, multiple Hepatocellular adenoma X X X X Hepatocholangiocarcinoma X Mesentery + + + + Oral mucosa

Hemangioma Pancreas ++++++++++++++++++++++++++++++ Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular ++++++++++++++++++++++++++++++ Tooth


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++

Adenoma X Adrenal medulla + + + + + + + + + + + + + + + + + + +M+ + + + + + + + + +

Neuroblastoma Pheochromocytoma benign X Islets, pancreatic ++++++++++++++++++++++++++++++ Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +M+ + + + Pituitary gland ++++++++++++++++++++++++++++++

Pars distalis, adenoma X X X Thyroid gland ++++++++++++++++++++++++++++++

C-cell, adenoma X C-cell, carcinoma Follicular cell, carcinoma


### General Body System Peritoneum + + + ++++ ++++++++++++++ + +++ 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

333344444444444444444444455555 

### Number of Days on Study 599900111123336677777889900001 911644499912993411149335529992

222212222222122222222222212222 

### Carcass ID Number 313091511121942012440240293332 162754013789147953294016463455

### 115 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 59 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Polyp adenomatous 1 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine large, cecum + + + + + + + + + + + + + + A + + + + + + + + + + + + + A + 58 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, jejunum + + + + + + + + + A + + + + A + + + + + + + + + + + + + A + 53 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + A + 58 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular carcinoma, multiple X 1 Hepatocellular adenoma X X X 7 Hepatocholangiocarcinoma 1 Mesentery 4 Oral mucosa + 1

Hemangioma X 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Tooth + 1


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma 1 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Neuroblastoma X 1 Pheochromocytoma benign X 2 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + M + + + 58 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + M + + + + + + + 59

Pars distalis, adenoma X X X X X X 9 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

C-cell, adenoma 1 C-cell, carcinoma X 1 Follicular cell, carcinoma X 1


### General Body System Peritoneum + + + + + + + + + + + + + + + + + + + + + 46 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

555555555555555555555555666666 

### Number of Days on Study 111122333344444666678888003355 222937144804444277961699382756

222122212211222222122212222222 T otal 

### Carcass ID Number 333914292499012203944094010340 Tissues/ 679426976339101280258287580803 T umors

### o-Nitrotoluene, NTP TR 504 116

Genital System Epididymis ++++++++++++++++++++++++++++++ Preputial gland + + + + + + + + + + + + + + + + + + + + + + +M+ + + I + +

Adenoma X Carcinoma Prostate ++++++++++++++++++++++++++++++ Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++

Bilateral, interstitial cell, adenoma X X X X Interstitial cell, adenoma X X X X X X X X X X X X X X X


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + Lymph node, mandibular + + + + + + + + + + + + + + + + + + +M+ + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + +M+ + + +M+ + + + + Spleen ++++++++++++++++++++++++++++++ Thymus + + + + + + + + + +M+ + + + + +M+ + + + + + +M+M+ +


### Integumentary System Mammary gland + MMMM + + M + + + + +M+ + + + + + + + + + + + MMMM

Fibroadenoma Skin ++++++++++++++++++++++++++++++

Basal cell adenoma Subcutaneous tissue, fibroma X X X X X X X X X X X X X X X Subcutaneous tissue, fibroma, multiple X X X X X X X X X X X X X X Subcutaneous tissue, fibrosarcoma X X X X X X X Subcutaneous tissue, fibrosarcoma, multiple Subcutaneous tissue, hemangiopericytoma X Subcutaneous tissue, lipoma X X X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++

Osteosarcoma X


### Nervous System Brain ++++++++++++++++++++++++++++++

Carcinoma, metastatic, pituitary gland Peripheral nerve Spinal cord 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

333344444444444444444444455555 

### Number of Days on Study 599900111123336677777889900001 911644499912993411149335529992

222212222222122222222222212222 

### Carcass ID Number 313091511121942012440240293332 162754013789147953294016463455

### 117 o- Nitrotoluene, NTP TR 504

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + + + M + M 56

Adenoma 1 Carcinoma X 1 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X 17 Interstitial cell, adenoma X X X X X X X X X X X X X X 29


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + ++++ + 9 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Thymus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + M 55


### Integumentary System Mammary gland M M + M + + + + + + + M + + + + + + + M M + + M + + + + + + 43

Fibroadenoma X X2 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Basal cell adenoma X 1 Subcutaneous tissue, fibroma X X X X X X X X X 24 Subcutaneous tissue, fibroma, multiple X X X X X X X X X X X X X X X X X X X X X 35 Subcutaneous tissue, fibrosarcoma X X X X X X X X 15 Subcutaneous tissue, fibrosarcoma, multiple X X X X X 5 Subcutaneous tissue, hemangiopericytoma 1 Subcutaneous tissue, lipoma X X X X X X X X X X 13


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Osteosarcoma 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Carcinoma, metastatic, pituitary gland X 1 Peripheral nerve + 1 Spinal cord + 1 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

555555555555555555555555666666 

### Number of Days on Study 111122333344444666678888003355 222937144804444277961699382756

222122212211222222122212222222 T otal 

### Carcass ID Number 333914292499012203944094010340 Tissues/ 679426976339101280258287580803 T umors

### o-Nitrotoluene, NTP TR 504 118

Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye + Zymbal’s gland + +

Carcinoma X X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++

Hemangioma X


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear X X Mesothelioma malignant X X X X X X XXXXXXXXXXXXXX X XX 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

333344444444444444444444455555 

### Number of Days on Study 599900111123336677777889900001 911644499912993411149335529992

222212222222122222222222212222 

### Carcass ID Number 313091511121942012440240293332 162754013789147953294016463455

### 119 o- Nitrotoluene, NTP TR 504

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma 2 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye 1 Zymbal’s gland 2

Carcinoma 2


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + A + 59

Hemangioma 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X 3 Mesothelioma malignant X X X X X X X X X X X X X X X X X X X X X 44 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

555555555555555555555555666666 

### Number of Days on Study 111122333344444666678888003355 222937144804444277961699382756

222122212211222222122212222222 T otal 

### Carcass ID Number 333914292499012203944094010340 Tissues/ 679426976339101280258287580803 T umors

### o-Nitrotoluene, NTP TR 504 120

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon +++++A++++++++++++++++++++++++ Intestine large, rectum ++++++++++++++++++++++++++++++

Leiomyosarcoma Intestine large, cecum ++++++++++++++++++++++++++++++

Leiomyoma Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum +++++++++++++++++++++++++A++++

Carcinoma Intestine small, ileum +++++A++++++A++++++A++++++++++ Liver ++++++++++++++++++++++++++++++

Hepatocellular adenoma X Hepatocellular adenoma, multiple Histiocytic sarcoma Mesothelioma malignant, metastatic,

peritoneum Mesentery Pancreas ++++++++++++++++++++++++++++++

Histiocytic sarcoma Mesothelioma malignant, metastatic,

peritoneum Mixed tumor benign Salivary glands + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + I

Fibrosarcoma X Fibrosarcoma, metastatic, skin Stomach, forestomach ++++++++++++++++++++++++++++++

Squamous cell papilloma Stomach, glandular ++++++++++++++++++++++++++++++


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++

Pheochromocytoma malignant Pheochromocytoma benign X Islets, pancreatic ++++++++++++++++++++++++++++++ Parathyroid gland + M + + + + + + + + + +M+ + + + + + + + + + + + + + + + +

Adenoma X Pituitary gland + + + + MM + + + + + + + + + + + + + + + + + + + + + + + +

Craniopharyngioma Pars distalis, adenoma X X Thyroid gland ++++++++++++++++++++++++++++++

C-cell, adenoma X C-cell, carcinoma 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm (Stop-Exposure)

223444455555555555555555555666 

### Number of Days on Study 677127902233356666777788999012 050900923314757999699999255351

555555555555555555555555555545 

### Carcass ID Number 434201051342112114000102245390 762961105067841674308072852174

### 121 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leiomyosarcoma X 1 Intestine large, cecum + + + + + + + + + + A + + + + + + + + + + + + + + + + + + + 59

Leiomyoma X 1 Intestine small, duodenum + + + + + + + + + + + + + M + + + + + + + + + + + + + + + + 59 Intestine small, jejunum + + A + + + + + + + A + + + + + + + + + + + + + + + + + + + 57

Carcinoma X 1 Intestine small, ileum + + A + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular adenoma X 2 Hepatocellular adenoma, multiple X 1 Histiocytic sarcoma X 1 Mesothelioma malignant, metastatic,

peritoneum X 1 Mesentery + 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma X 1 Mesothelioma malignant, metastatic,

peritoneum X 1 Mixed tumor benign X X 2 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58

Fibrosarcoma 1 Fibrosarcoma, metastatic, skin X 1 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Squamous cell papilloma X 1 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pheochromocytoma malignant X 1 Pheochromocytoma benign X X X 4 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58

Adenoma 1 Pituitary gland + + + + + + + + + M + + + + + + + + + + + + + + + + + + + + 57

Craniopharyngioma X 1 Pars distalis, adenoma X X X X X X X X 10 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

C-cell, adenoma XX 3 C-cell, carcinoma X 1 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm (Stop-Exposure)

666666666666777777777777777777 

### Number of Days on Study 234555577788000011122222223333 179688803745238806699999990000

555555554455455555545555554555 T otal 

### Carcass ID Number 045103439923951534590244559155 Tissues/ 538297021938269599482018793313 T umors

### o-Nitrotoluene, NTP TR 504 122

General Body System Peritoneum ++++ ++++++ + ++++++++++ +++


### Genital System Epididymis ++++++++++++++++++++++++++++++ Preputial gland ++++++++++++++++++++++++++++++

Adenoma Carcinoma Prostate ++++++++++++++++++++++++++++++

Ventral, adenoma Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++

Bilateral, interstitial cell, adenoma X X X X X X X X X X Interstitial cell, adenoma X X X X X X X X X X X X


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + + + + + + + + +

Squamous cell carcinoma, metastatic,

skin X Mediastinal, histiocytic sarcoma Lymph node, mandibular + + + + + +M+ + + + + + + +M+ + + + + + + + + + + + + + Lymph node, mesenteric ++++++++++++++++++++++++++++++ Spleen ++++++++++++++++++++++++++++++

Fibroma Fibrosarcoma X Histiocytic sarcoma Thymus + + + + + + + + + + + I + + + + + + + + + +M+ + + + + + +

Fibrosarcoma, metastatic, spleen X


### Integumentary System Mammary gland + + + + +M+ + + + + + + +M+ + + + + + + + + + + + +M+

Fibroadenoma X X X X Fibroadenoma, multiple Skin ++++++++++++++++++++++++++++++

Histiocytic sarcoma Keratoacanthoma X Squamous cell carcinoma X Subcutaneous tissue, fibroma X X X X X X X X X X X XXXX XX Subcutaneous tissue, fibrosarcoma X X Subcutaneous tissue, lipoma X X X Subcutaneous tissue, osteosarcoma X Subcutaneous tissue,

schwannoma malignant X 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm (Stop-Exposure)

223444455555555555555555555666 

### Number of Days on Study 677127902233356666777788999012 050900923314757999699999255351

555555555555555555555555555545 

### Carcass ID Number 434201051342112114000102245390 762961105067841674308072852174

### 123 o- Nitrotoluene, NTP TR 504

General Body System Peritoneum + + + + + + + + + + + + + + + + + + + + + + + 47


### Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 1 Carcinoma X 1 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Ventral, adenoma X 1 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X 31 Interstitial cell, adenoma X X X X X X X 19


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + + + + + + 18

Squamous cell carcinoma, metastatic,

skin 1 Mediastinal, histiocytic sarcoma X 1 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Fibroma X 1 Fibrosarcoma 1 Histiocytic sarcoma X 1 Thymus + + + + + + M + + + + + + + + + + + + + + + + + + + + + M + 56

Fibrosarcoma, metastatic, spleen 1


### Integumentary System Mammary gland + + + + + + + M M + M + + + M M + M + + + + + + + + + + + + 51

Fibroadenoma X X X X X X X X 12 Fibroadenoma, multiple X 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma X 1 Keratoacanthoma 1 Squamous cell carcinoma 1 Subcutaneous tissue, fibroma X X X X X X X X X X X X X X X X X X X X X X X X X X X X 45 Subcutaneous tissue, fibrosarcoma X X X X X X 8 Subcutaneous tissue, lipoma X X X X X X X 10 Subcutaneous tissue, osteosarcoma 1 Subcutaneous tissue,

schwannoma malignant 1 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm (Stop-Exposure)

666666666666777777777777777777 

### Number of Days on Study 234555577788000011122222223333 179688803745238806699999990000

555555554455455555545555554555 T otal 

### Carcass ID Number 045103439923951534590244559155 Tissues/ 538297021938269599482018793313 T umors

### o-Nitrotoluene, NTP TR 504 124

Musculoskeletal System Bone ++++++++++++++++++++++++++++++

Osteosarcoma X X Skeletal muscle + + +

Mesothelioma malignant, metastatic,

peritoneum X Rhabdomyosarcoma X


### Nervous System Brain ++++++++++++++++++++++++++++++ Peripheral nerve + Spinal cord +


### Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X Histiocytic sarcoma Squamous cell carcinoma X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Lacrimal gland Zymbal’s gland + +

Carcinoma X X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++

Leiomyoma


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Histiocytic sarcoma Leukemia mononuclear X X X X Mesothelioma malignant XXXX XXXXXX X XXXXXXXXXX XXX 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm (Stop-Exposure)

223444455555555555555555555666 

### Number of Days on Study 677127902233356666777788999012 050900923314757999699999255351

555555555555555555555555555545 

### Carcass ID Number 434201051342112114000102245390 762961105067841674308072852174

### 125 o- Nitrotoluene, NTP TR 504

Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Osteosarcoma 2 Skeletal muscle + ++ 6

Mesothelioma malignant, metastatic,

peritoneum XX 3 Rhabdomyosarcoma 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Peripheral nerve + + 3 Spinal cord + + 3


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X X 3 Histiocytic sarcoma X 1 Squamous cell carcinoma 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Lacrimal gland + 1 Zymbal’s gland 2

Carcinoma 2


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leiomyoma X 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma X X 2 Leukemia mononuclear X X X X X X X X X 13 Mesothelioma malignant X X X X X X X X X X X X X X X X X X X X 44 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm (Stop-Exposure)

666666666666777777777777777777 

### Number of Days on Study 234555577788000011122222223333 179688803745238806699999990000

555555554455455555545555554555 T otal 

### Carcass ID Number 045103439923951534590244559155 Tissues/ 538297021938269599482018793313 T umors

### o-Nitrotoluene, NTP TR 504 126

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon ++++++++++++++++++++++++++++++

Mesothelioma malignant, metastatic,

peritoneum Polyp adenomatous Intestine large, rectum +++++++++I+++++++I++++++++++++ Intestine large, cecum ++++++++++++++++++++++++++++++

Leiomyoma Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum ++++++++++++++++++++++++++++++ Intestine small, ileum ++++++++++++++++++++++++++++++ Liver ++++++++++++++++++++++++++++++

Cholangiocarcinoma X X Hepatocellular carcinoma X Hepatocellular adenoma Hepatocellular adenoma, multiple X Mesothelioma malignant, metastatic,

peritoneum X Mesentery

Fibrosarcoma Pancreas ++++++++++++++++++++++++++++++

Mesothelioma malignant, metastatic,

peritoneum X Acinus, adenoma X Salivary glands + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular ++++++++++++++++++++++++++++++ Tooth

Fibroma


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++

Mesothelioma malignant, metastatic,

peritoneum Schwannoma malignant X


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++

Pheochromocytoma benign Islets, pancreatic ++++++++++++++++++++++++++++++

Adenoma Parathyroid gland + + + + + + + +M+ + + + +M+ + + + + + + + + + + + + + + Pituitary gland ++++++++++++++++++++++++++++++

Pars distalis, adenoma Thyroid gland ++++++++++++++++++++++++++++++

C-cell, adenoma X X 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm (Stop-Exposure)

223333333333333444444444444444 

### Number of Days on Study 991555568888999011113356667889 996999924444111127992934441559

666566655566555665555565565556 

### Carcass ID Number 001722289911889007696906717990 181724562609298948631939152047

### 127 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + M + + + + + + + + + + + + + + + + + + + M + + + M + + + 57 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Mesothelioma malignant, metastatic,

peritoneum X 1 Polyp adenomatous X X 2 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leiomyoma X 1 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, ileum M + + + + + + + + + M + + + + + + + + + + + + + + + + + + + 58 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Cholangiocarcinoma X 3 Hepatocellular carcinoma X 2 Hepatocellular adenoma X X X 3 Hepatocellular adenoma, multiple 1 Mesothelioma malignant, metastatic,

peritoneum X X 3 Mesentery ++ + 3

Fibrosarcoma X X2 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Mesothelioma malignant, metastatic,

peritoneum 1 Acinus, adenoma X X 3 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Tooth + 1

Fibroma X 1


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Mesothelioma malignant, metastatic,

peritoneum X 1 Schwannoma malignant 1


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pheochromocytoma benign X X 2 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 1 Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + M 57 Pituitary gland M + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Pars distalis, adenoma X XX X X 5 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

C-cell, adenoma X X4 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm (Stop-Exposure)

555555555555555555555555566666 

### Number of Days on Study 000000114444455566667899902339 122225220004755577799157731261

555555565565556656665656656666 T otal 

### Carcass ID Number 678897826626892281106290260012 Tissues/ 367850404592311356767772880586 T umors

### o-Nitrotoluene, NTP TR 504 128

General Body System Peritoneum +++++++++++++++ +++++++++ ++++ Tissue NOS

Hemangioma


### Genital System Epididymis ++++++++++++++++++++++++++++++ Preputial gland ++++++++++++++++++++++++++++++

Adenoma X Carcinoma X X Prostate ++++++++++++++++++++++++++++++ Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++

Bilateral, interstitial cell, adenoma X Interstitial cell, adenoma X X XXXXXXX


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + + + + + + + Lymph node, mandibular + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric ++++++++++++++++++++++++++++++ Spleen ++++++++++++++++++++++++++++++

Hemangioma X Thymus + + + + + + + + + + + + + + +M+ + + + + + + + +M+ + + +

Alveolar/bronchiolar carcinoma,

metastatic, lung X


### Integumentary System Mammary gland + + + + + M + MM + + + M + + + + + + + + + + + + + + + + +

Fibroadenoma X X X X X X X X X X X Skin ++++++++++++++++++++++++++++++

Hemangioma Schwannoma malignant Squamous cell papilloma Subcutaneous tissue, fibroma X X XXXXXX XXXX X XXX X Subcutaneous tissue, fibroma, multiple X X X X X X Subcutaneous tissue, fibrosarcoma X X X X X Subcutaneous tissue, fibrosarcoma, multiple X Subcutaneous tissue, lipoma X X X Subcutaneous tissue, lipoma, multiple X Subcutaneous tissue, sarcoma Subcutaneous tissue,

schwannoma malignant X 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm (Stop-Exposure)

223333333333333444444444444444 

### Number of Days on Study 991555568888999011113356667889 996999924444111127992934441559

666566655566555665555565565556 

### Carcass ID Number 001722289911889007696906717990 181724562609298948631939152047

### 129 o- Nitrotoluene, NTP TR 504

General Body System Peritoneum + + + + + + + + + + + + + + + + + + + + + + + + + + 54 Tissue NOS + 1

Hemangioma X 1


### Genital System Epididymis + M + + + + + + + + + + + + + + + + + + + + + + + + I + + + 58 Preputial gland + + + + + + + M + + + + + + + + + + + + + + + + + + + + + + 59

Adenoma 1 Carcinoma 2 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Seminal vesicle + + + + M + + + + + + + + + + + + + + + + + + + + + + + + + 59 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, interstitial cell, adenoma X X X 4 Interstitial cell, adenoma X X X X X X X X X X X X X X 23


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + + + + + + 16 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Lymph node, mesenteric M + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangioma 1 Thymus + + + + + + + + + + M + + + + + + + + + + + + + + + + M + + 56

Alveolar/bronchiolar carcinoma,

metastatic, lung 1


### Integumentary System Mammary gland + + M M + + + + M + + + + + + + + + M + + + + + M + + + M + 50

Fibroadenoma X X X X X X X X X 20 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangioma X 1 Schwannoma malignant X 1 Squamous cell papilloma X X 2 Subcutaneous tissue, fibroma X X X X X X X 24 Subcutaneous tissue, fibroma, multiple X X X X X X X X X X X X X X X X X X X X X X 28 Subcutaneous tissue, fibrosarcoma X XX 8 Subcutaneous tissue, fibrosarcoma, multiple X X X 4 Subcutaneous tissue, lipoma X X X X X X 9 Subcutaneous tissue, lipoma, multiple X X 3 Subcutaneous tissue, sarcoma X 1 Subcutaneous tissue,

schwannoma malignant 1 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm (Stop-Exposure)

555555555555555555555555566666 

### Number of Days on Study 000000114444455566667899902339 122225220004755577799157731261

555555565565556656665656656666 T otal 

### Carcass ID Number 678897826626892281106290260012 Tissues/ 367850404592311356767772880586 T umors

### o-Nitrotoluene, NTP TR 504 130

Musculoskeletal System Bone ++++++++++++++++++++++++++++++

Osteosarcoma X X Skeletal muscle + + +

Alveolar/bronchiolar carcinoma,

metastatic, lung Hemangioma Hemangiosarcoma X Mesothelioma malignant, metastatic,

peritoneum X Rhabdomyosarcoma X


### Nervous System Brain ++++++++++++++++++++++++++++++

Oligodendroglioma malignant


### Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X Alveolar/bronchiolar adenoma,

multiple Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma,

multiple X Mesothelioma malignant, metastatic,

peritoneum X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Zymbal’s gland +

Carcinoma X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear Mesothelioma malignant XXXXXXXXXXXXXXX XXXXXXXXX XXXX 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm (Stop-Exposure)

223333333333333444444444444444 

### Number of Days on Study 991555568888999011113356667889 996999924444111127992934441559

666566655566555665555565565556 

### Carcass ID Number 001722289911889007696906717990 181724562609298948631939152047

### 131 o- Nitrotoluene, NTP TR 504

Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Osteosarcoma X 3 Skeletal muscle + + 5

Alveolar/bronchiolar carcinoma,

metastatic, lung X 1 Hemangioma X 1 Hemangiosarcoma 1 Mesothelioma malignant, metastatic,

peritoneum 1 Rhabdomyosarcoma 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Oligodendroglioma malignant X 1


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X X X X X X 7 Alveolar/bronchiolar adenoma,

multiple X 1 Alveolar/bronchiolar carcinoma X 1 Alveolar/bronchiolar carcinoma,

multiple X 2 Mesothelioma malignant, metastatic,

peritoneum X 2 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Zymbal’s gland 1

Carcinoma 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + M + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X 1 Mesothelioma malignant X X X X X X X X X X X X X X X X X X X X X X X X X X 54 

### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm (Stop-Exposure)

555555555555555555555555566666 

### Number of Days on Study 000000114444455566667899902339 122225220004755577799157731261

555555565565556656665656656666 T otal 

### Carcass ID Number 678897826626892281106290260012 Tissues/ 367850404592311356767772880586 T umors

### o-Nitrotoluene, NTP TR 504 132



### T ABLE 

### A3a Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Adrenal Medulla: Benign Pheochromocytoma Overall rate a 2/60 (3%) 4/60 (7%) 6/60 (10%) 2/59 (3%) Adjusted rate b 3.7% 8.9% 16.3% 8.9% Terminal rate c 2/39 (5%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) 729 (T) 567 531 471 Poly-3 test d P=0.064 P=0.261 P=0.048 P=0.380


### Adrenal Medulla: Benign or Malignant Pheochromocytoma Overall rate 4/60 (7%) 4/60 (7%) 6/60 (10%) 2/59 (3%) Adjusted rate 7.4% 8.9% 16.3% 8.9% Terminal rate 4/39 (10%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) 729 (T) 567 531 471 Poly-3 test P=0.220 P=0.543 P=0.170 P=0.584


### Liver: Hepatocellular Adenoma Overall rate 2/60 (3%) 3/60 (5%) 3/60 (5%) 7/60 (12%) Adjusted rate 3.7% 6.8% 8.4% 27.1% Terminal rate 1/39 (3%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) 707 621 615 391 Poly-3 test P=0.007 P=0.413 P=0.325 P=0.006


### Liver: Hepatocellular Adenoma or Carcinoma Overall rate 3/60 (5%) 3/60 (5%) 3/60 (5%) 8/60 (13%) Adjusted rate 5.6% 6.8% 8.4% 30.2% Terminal rate 2/39 (5%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) 707 621 615 391 Poly-3 test P=0.009 P=0.570 P=0.466 P=0.007


### Lung: Alveolar/bronchiolar Adenoma Overall rate 1/60 (2%) 5/60 (8%) 1/60 (2%) 2/60 (3%) Adjusted rate 1.9% 11.2% 2.9% 8.7% Terminal rate 1/39 (3%) 2/18 (11%) 1/3 (33%) 0/0 First incidence (days) 729 (T) 639 729 (T) 419 Poly-3 test P=0.237 P=0.066 P=0.654 P=0.254


### Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 2/60 (3%) 5/60 (8%) 1/60 (2%) 2/60 (3%) Adjusted rate 3.7% 11.2% 2.9% 8.7% Terminal rate 2/39 (5%) 2/18 (11%) 1/3 (33%) 0/0 First incidence (days) 729 (T) 639 729 (T) 419 Poly-3 test P=0.390 P=0.149 P=0.644N P=0.386


### Mammary Gland: Fibroadenoma Overall rate 0/60 (0%) 7/60 (12%) 10/60 (17%) 2/60 (3%) Adjusted rate 0.0% 15.6% 26.2% 9.0% Terminal rate 0/39 (0%) 4/18 (22%) 1/3 (33%) 0/0 First incidence (days) —e 621 576 608 Poly-3 test P<0.001 P=0.004 P<0.001 P=0.110


### Pituitary Gland (Pars Distalis): Adenoma Overall rate 11/59 (19%) 9/60 (15%) 3/58 (5%) 9/59 (15%) Adjusted rate 19.9% 19.6% 8.9% 34.2% Terminal rate 6/39 (15%) 3/18 (17%) 1/3 (33%) 0/0 First incidence (days) 441 569 621 391 Poly-3 test P=0.390 P=0.581N P=0.149N P=0.155

### 133 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A3a Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Preputial Gland: Adenoma Overall rate 2/60 (3%) 3/59 (5%) 2/58 (3%) 1/56 (2%) Adjusted rate 3.7% 6.8% 5.9% 5.0% Terminal rate 2/39 (5%) 1/17 (6%) 0/3 (0%) 0/0 First incidence (days) 729 (T) 465 519 471 Poly-3 test P=0.420 P=0.413 P=0.519 P=0.634


### Preputial Gland: Adenoma or Carcinoma Overall rate 4/60 (7%) 5/59 (8%) 2/58 (3%) 2/56 (4%) Adjusted rate 7.4% 11.2% 5.9% 9.7% Terminal rate 3/39 (8%) 2/17 (12%) 0/3 (0%) 0/0 First incidence (days) 720 465 519 471 Poly-3 test P=0.542 P=0.387 P=0.559N P=0.551



### Skin: Keratoacanthoma Overall rate 5/60 (8%) 3/60 (5%) 1/60 (2%) 0/60 (0%) Adjusted rate 9.2% 6.7% 2.8% 0.0% Terminal rate 3/39 (8%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) 537 616 564 — Poly-3 test P=0.079N P=0.470N P=0.238N P=0.218N



### Skin: Keratoacanthoma or Basal Cell Adenoma Overall rate 7/60 (12%) 3/60 (5%) 1/60 (2%) 1/60 (2%) Adjusted rate 12.9% 6.7% 2.8% 4.6% Terminal rate 5/39 (13%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) 537 616 564 608 Poly-3 test P=0.053N P=0.252N P=0.114N P=0.291N


### Skin (Subcutaneous Tissue): Lipoma Overall rate 0/60 (0%) 4/60 (7%) 13/60 (22%) 13/60 (22%) Adjusted rate 0.0% 8.9% 33.0% 44.8% Terminal rate 0/39 (0%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) — 569 502 414 Poly-3 test P<0.001 P=0.041 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibroma Overall rate 5/60 (8%) 46/60 (77%) 52/60 (87%) 59/60 (98%) Adjusted rate 9.3% 85.0% 96.2% 99.8% Terminal rate 3/39 (8%) 15/18 (83%) 3/3 (100%) 0/0 First incidence (days) 705 465 432 391 Poly-3 test P<0.001 P<0.001 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibrosarcoma Overall rate 0/60 (0%) 7/60 (12%) 17/60 (28%) 20/60 (33%) Adjusted rate 0.0% 15.3% 42.3% 59.4% Terminal rate 0/39 (0%) 1/18 (6%) 1/3 (33%) 0/0 First incidence (days) — 569 384 439 Poly-3 test P<0.001 P=0.004 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibroma or Fibrosarcoma Overall rate 5/60 (8%) 47/60 (78%) 55/60 (92%) 59/60 (98%) Adjusted rate 9.3% 86.3% 98.7% 99.8% Terminal rate 3/39 (8%) 15/18 (83%) 3/3 (100%) 0/0 First incidence (days) 705 465 384 391 Poly-3 test P<0.001 P<0.001 P<0.001 P<0.001

### o-Nitrotoluene, NTP TR 504 134



### T ABLE 

### A3a Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Testes: Adenoma Overall rate 55/60 (92%) 53/60 (88%) 51/60 (85%) 46/60 (77%) Adjusted rate 94.3% 93.7% 96.5% 92.2% Terminal rate 38/39 (97%) 18/18 (100%) 3/3 (100%) 0/0 First incidence (days) 533 465 483 404 Poly-3 test P=0.453N P=0.610N P=0.457 P=0.475N


### Thyroid Gland (C-Cell): Adenoma Overall rate 10/59 (17%) 6/60 (10%) 0/60 (0%) 1/60 (2%) Adjusted rate 18.6% 13.6% 0.0% 4.5% Terminal rate 6/38 (16%) 6/18 (33%) 0/3 (0%) 0/0 First incidence (days) 533 729 (T) — 404 Poly-3 test P=0.006N P=0.352N P=0.010N P=0.147N


### Thyroid Gland (C-Cell): Carcinoma Overall rate 0/59 (0%) 4/60 (7%) 0/60 (0%) 1/60 (2%) Adjusted rate 0.0% 9.1% 0.0% 4.6% Terminal rate 0/38 (0%) 3/18 (17%) 0/3 (0%) 0/0 First incidence (days) — 691 — 632 Poly-3 test P=0.323 P=0.042 — f P=0.350


### Thyroid Gland (C-Cell): Adenoma or Carcinoma Overall rate 10/59 (17%) 10/60 (17%) 0/60 (0%) 2/60 (3%) Adjusted rate 18.6% 22.6% 0.0% 8.8% Terminal rate 6/38 (16%) 9/18 (50%) 0/3 (0%) 0/0 First incidence (days) 533 691 — 404 Poly-3 test P=0.029N P=0.403 P=0.010N P=0.269N


### All Organs: Hemangioma Overall rate 0/60 (0%) 3/60 (5%) 0/60 (0%) 2/60 (3%) Adjusted rate 0.0% 6.7% 0.0% 8.7% Terminal rate 0/39 (0%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) — 632 — 439 Poly-3 test P=0.158 P=0.090 — P=0.112


### All Organs: Hemangioma or Hemangiosarcoma Overall rate 1/60 (2%) 3/60 (5%) 1/60 (2/%) 2/60 (3%) Adjusted rate 1.9% 6.7% 2.9% 8.7% Terminal rate 1/39 (3%) 1/18 (6%) 0/3 (0%) 0/0 First incidence (days) 729 (T) 632 714 439 Poly-3 test P=0.232 P=0.243 P=0.654 P=0.254


### All Organs: Mononuclear Cell Leukemia Overall rate 30/60 (50%) 21/60 (35%) 3/60 (5%) 3/60 (5%) Adjusted rate 52.2% 42.8% 8.5% 12.7% Terminal rate 16/39 (41%) 7/18 (39%) 0/3 (0%) 0/0 First incidence (days) 533 465 676 419 Poly-3 test P<0.001N P=0.214N P<0.001N P=0.003N


### All Organs: Malignant Mesothelioma Overall rate 2/60 (3%) 20/60 (33%) 29/60 (48%) 44/60 (73%) Adjusted rate 3.7% 40.6% 62.4% 87.1% Terminal rate 2/39 (5%) 5/18 (28%) 1/3 (33%) 0/0 First incidence (days) 729 (T) 428 432 359 Poly-3 test P<0.001 P<0.001 P<0.001 P<0.001

### 135 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A3a Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

All Organs: Benign Neoplasms Overall rate 60/60 (100%) 58/60 (97%) 55/60 (92%) 59/60 (98%) Adjusted rate 100.0% 99.4% 99.1% 99.8% Terminal rate 39/39 (100%) 18/18 (100%) 3/3 (100%) 0/0 First incidence (days) 441 465 432 391 Poly-3 test P=0.799N P=0.996N P=0.974N P=1.000N


### All Organs: Malignant Neoplasms Overall rate 37/60 (62%) 47/60 (78%) 42/60 (70%) 51/60 (85%) Adjusted rate 63.7% 83.6% 81.8% 94.0% Terminal rate 21/39 (54%) 14/18 (78%) 2/3 (67%) 0/0 First incidence (days) 533 378 384 359 Poly-3 test P<0.001 P=0.010 P=0.023 P<0.001


### All Organs: Benign or Malignant Neoplasms Overall rate 60/60 (100%) 60/60 (100%) 57/60 (95%) 60/60 (100%) Adjusted rate 100.0% 100.0% 99.6% 100.0% Terminal rate 39/39 (100%) 18/18 (100%) 3/3 (100%) 0/0 First incidence (days) 441 378 384 359 Poly-3 test P=1.000N — P=1.000N —

(T)Terminal sacrifice a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for a drenal gland, liver, lung, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals necropsi ed. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals th at do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal group f Value of statistic cannot be computed.

### o-Nitrotoluene, NTP TR 504 136



### T ABLE 

### A3b Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Stop-Exposure Feed Study of o- Nitrotoluene

0 ppm 2,000 ppm 5,000 ppm

Adrenal Medulla: Benign Pheochromocytoma Overall rate a 2/60 (3%) 4/60 (7%) 2/60 (3%) Adjusted rate b 3.7% 10.1% 9.9% Terminal rate c 2/39 (5%) 2/11 (18%) 0/0 First incidence (days) 729 (T) 569 567 Poly-3 test d P=0.187 P=0.210 P=0.352


### Adrenal Medulla: Benign or Malignant Pheochromocytoma Overall rate 4/60 (7%) 5/60 (8%) 2/60 (3%) Adjusted rate 7.4% 12.7% 9.9% Terminal rate 4/39 (10%) 3/11 (27%) 0/0 First incidence (days) 729 (T) 569 567 Poly-3 test P=0.359 P=0.315 P=0.544



### Bone: Osteosarcoma Overall rate 1/60 (2%) 2/60 (3%) 3/60 (5%) Adjusted rate 1.9% 5.0% 14.0% Terminal rate 0/39 (0%) 0/11 (0%) 0/0 First incidence (days) 727 470 359 Poly-3 test P=0.074 P=0.399 P=0.097


### Liver: Cholangiocarcinoma Overall rate 0/60 (0%) 0/60 (0%) 3/60 (5%) Adjusted rate 0.0% 0.0% 13.9% Terminal rate 0/39 (0%) 0/11 (0%) 0/0 First incidence (days) —e — 384 Poly-3 test P=0.025 — f P=0.034


### Liver: Hepatocellular Adenoma Overall rate 2/60 (3%) 3/60 (5%) 4/60 (7%) Adjusted rate 3.7% 7.6% 18.4% Terminal rate 1/39 (3%) 1/11 (9%) 0/0 First incidence (days) 707 579 499 Poly-3 test P=0.062 P=0.362 P=0.079


### Liver: Hepatocellular Adenoma or Carcinoma Overall rate 3/60 (5%) 3/60 (5%) 6/60 (10%) Adjusted rate 5.6% 7.6% 25.9% Terminal rate 2/39 (5%) 1/11 (9%) 0/0 First incidence (days) 707 579 391 Poly-3 test P=0.030 P=0.511 P=0.029


### Lung: Alveolar/bronchiolar Adenoma Overall rate 1/60 (2%) 3/60 (5%) 8/60 (13%) Adjusted rate 1.9% 7.6% 33.0% Terminal rate 1/39 (3%) 1/11 (9%) 0/0 First incidence (days) 729 (T) 615 419 Poly-3 test P<0.001 P=0.205 P<0.001


### Lung: Alveolar/bronchiolar Carcinoma Overall rate 1/60 (2%) 0/60 (0%) 3/60 (5%) Adjusted rate 1.9% 0.0% 14.1% Terminal rate 1/39 (3%) 0/11 (0%) 0/0 First incidence (days) 729 (T) — 499 Poly-3 test P=0.115 P=0.565N P=0.096

### 137 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A3b Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Stop-Exposure Feed Study of o- Nitrotoluene

0 ppm 2,000 ppm 5,000 ppm

Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 2/60 (3%) 3/60 (5%) 11/60 (18%) Adjusted rate 3.7% 7.6% 42.0% Terminal rate 2/39 (5%) 1/11 (9%) 0/0 First incidence (days) 729 (T) 615 419 Poly-3 test P<0.001 P=0.362 P<0.001


### Mammary Gland: Fibroadenoma Overall rate 0/60 (3%) 13/60 (22%) 20/60 (33%) Adjusted rate 0.0% 31.2% 61.1% Terminal rate 0/39 (0%) 5/11 (46%) 0/0 First incidence (days) — 523 299 Poly-3 test P<0.001 P<0.001 P<0.001



### Pancreas: Adenoma Overall rate 1/60 (2%) 0/60 (0%) 3/60 (5%) Adjusted rate 1.9% 0.0% 14.3% Terminal rate 0/39 (0%) 0/11 (0%) 0/0 First incidence (days) 705 — 471 Poly-3 test P=0.117 P=0.565N P=0.094


### Pituitary Gland (Pars Distalis): Adenoma Overall rate 11/59 (19%) 10/57 (18%) 5/59 (8%) Adjusted rate 19.9% 25.5% 23.6% Terminal rate 6/39 (15%) 4/11 (36%) 0/0 First incidence (days) 441 579 567 Poly-3 test P=0.360 P=0.351 P=0.486


### Preputial Gland: Adenoma or Carcinoma Overall rate 4/60 (7%) 2/60 (3%) 3/59 (5%) Adjusted rate 7.4% 5.1% 14.1% Terminal rate 3/39 (8%) 0/11 (0%) 0/0 First incidence (days) 720 649 384 Poly-3 test P=0.411 P=0.491N P=0.352



### Skin: Keratoacanthoma Overall rate 5/60 (8%) 1/60 (2%) 0/60 (0%) Adjusted rate 9.2% 2.6% 0.0% Terminal rate 3/39 (8%) 0/11 (0%) 0/0 First incidence (days) 537 579 — Poly-3 test P=0.087N P=0.200N P=0.248N


### Skin: Squamous Cell Papilloma or Keratoacanthoma Overall rate 5/60 (8%) 1/60 (2%) 2/60 (3%) Adjusted rate 9.2% 2.6% 9.8% Terminal rate 3/39 (8%) 0/11 (0%) 0/0 First incidence (days) 537 579 540 Poly-3 test P=0.406N P=0.200N P=0.615


### Skin: Squamous Cell Papilloma, Keratoacanthoma, or Squamous Cell Carcinoma Overall rate 5/60 (8%) 2/60 (3%) 2/60 (3%) Adjusted rate 9.2% 5.0% 9.8% Terminal rate 3/39 (8%) 0/11 (0%) 0/0 First incidence (days) 537 569 540 Poly-3 test P=0.492N P=0.364N P=0.615

### o-Nitrotoluene, NTP TR 504 138



### T ABLE 

### A3b Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Stop-Exposure Feed Study of o- Nitrotoluene

0 ppm 2,000 ppm 5,000 ppm

Skin: Squamous Cell Papilloma, Keratoacanthoma, Basal Cell Adenoma, or Squamous Cell Carcinoma Overall rate 7/60 (12%) 2/60 (3%) 2/60 (3%) Adjusted rate 12.9% 5.0% 9.8% Terminal rate 5/39 (13%) 0/11 (0%) 0/0 First incidence (days) 537 569 540 Poly-3 test P=0.268N P=0.184N P=0.513N


### Skin (Subcutaneous Tissue): Lipoma Overall rate 0/60 (0%) 10/60 (17%) 12/60 (20%) Adjusted rate 0.0% 24.6% 44.7% Terminal rate 0/39 (0%) 3/11 (27%) 0/0 First incidence (days) — 523 362 Poly-3 test P<0.001 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibroma Overall rate 5/60 (8%) 45/60 (75%) 52/60 (87%) Adjusted rate 9.3% 87.6% 97.2% Terminal rate 3/39 (8%) 10/11 (91%) 0/0 First incidence (days) 705 370 299 Poly-3 test P<0.001 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibrosarcoma Overall rate 0/60 (0%) 8/60 (13%) 12/60 (20%) Adjusted rate 0.0% 19.6% 44.7% Terminal rate 0/39 (0%) 2/11 (18%) 0/0 First incidence (days) — 275 359 Poly-3 test P<0.001 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibrosarcoma or Sarcoma Overall rate 0/60 (0%) 8/60 (13%) 13/60 (22%) Adjusted rate 0.0% 19.6% 47.5% Terminal rate 0/39 (0%) 2/11 (18%) 0/0 First incidence (days) — 275 359 Poly-3 test P<0.001 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibroma, Fibrosarcoma, or Sarcoma Overall rate 5/60 (8%) 47/60 (78%) 53/60 (88%) Adjusted rate 9.3% 89.0% 97.8% Terminal rate 3/39 (8%) 10/11 (91%) 0/0 First incidence (days) 705 275 299 Poly-3 test P<0.001 P<0.001 P<0.001



### Testes: Adenoma Overall rate 55/60 (92%) 50/60 (83%) 27/60 (45%) Adjusted rate 94.3% 92.6% 74.7% Terminal rate 38/39 (97%) 10/11 (91%) 0/0 First incidence (days) 533 370 384 Poly-3 test P<0.001N P=0.506N P<0.001N


### Thyroid Gland (C-Cell): Adenoma Overall rate 10/59 (17%) 3/60 (5%) 4/60 (7%) Adjusted rate 18.6% 7.7% 18.4% Terminal rate 6/38 (16%) 2/11 (18%) 0/0 First incidence (days) 533 531 417 Poly-3 test P=0.326N P=0.119N P=0.603N

### 139 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A3b Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Stop-Exposure Feed Study of o- Nitrotoluene

0 ppm 2,000 ppm 5,000 ppm

Thyroid Gland (C-Cell): Adenoma or Carcinoma Overall rate 10/59 (17%) 4/60 (7%) 4/60 (7%) Adjusted rate 18.6% 10.2% 18.4% Terminal rate 6/38 (16%) 2/11 (18%) 0/0 First incidence (days) 533 531 417 Poly-3 test P=0.384N P=0.209N P=0.603N


### All Organs: Hemangioma Overall rate 0/60 (0%) 0/60 (0%) 3/60 (5%) Adjusted rate 0.0% 0.0% 14.0% Terminal rate 0/39 (0%) 0/11 (0%) 0/0 First incidence (days) — — 417 Poly-3 test P=0.025 — P=0.034


### All Organs: Hemangioma or Hemangiosarcoma Overall rate 1/60 (2%) 0/60 (0%) 4/60 (7%) Adjusted rate 1.9% 0.0% 18.0% Terminal rate 1/39 (3%) 0/11 (0%) 0/0 First incidence (days) 729 (T) — 391 Poly-3 test P=0.041 P=0.565N P=0.037


### All Organs: Osteosarcoma Overall rate 1/60 (2%) 3/60 (5%) 3/60 (5%) Adjusted rate 1.9% 7.3% 14.0% Terminal rate 0/39 (0%) 0/11 (0%) 0/0 First incidence (days) 727 260 359 Poly-3 test P=0.060 P=0.217 P=0.097


### All Organs: Mononuclear Cell Leukemia Overall rate 30/60 (50%) 13/60 (22%) 1/60 (2%) Adjusted rate 52.2% 31.1% 5.0% Terminal rate 16/39 (41%) 4/11 (36%) 0/0 First incidence (days) 533 531 569 Poly-3 test P<0.001N P=0.027N P<0.001N


### All Organs: Malignant Mesothelioma Overall rate 2/60 (3%) 44/60 (73%) 54/60 (90%) Adjusted rate 3.7% 80.3% 95.1% Terminal rate 2/39 (5%) 10/11 (91%) 0/0 First incidence (days) 729 (T) 275 299 Poly-3 test P<0.001 P<0.001 P<0.001


### All Organs: Benign Neoplasms Overall rate 60/60 (100%) 57/60 (95%) 56/60 (93%) Adjusted rate 100.0% 99.5% 98.7% Terminal rate 39/39 (100%) 11/11 (100%) 0/0 First incidence (days) 441 370 299 Poly-3 test P=0.337N P=1.000N P=0.746N


### All Organs: Malignant Neoplasms Overall rate 37/60 (62%) 54/60 (90%) 58/60 (97%) Adjusted rate 63.7% 92.1% 97.9% Terminal rate 21/39 (54%) 11/11 (100%) 0/0 First incidence (days) 533 260 299 Poly-3 test P<0.001 P<0.001 P<0.001

### o-Nitrotoluene, NTP TR 504 140



### T ABLE 

### A3b Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Stop-Exposure Feed Study of o- Nitrotoluene

0 ppm 2,000 ppm 5,000 ppm

All Organs: Benign or Malignant Neoplasms Overall rate 60/60 (100%) 60/60 (100%) 60/60 (100%) Adjusted rate 100.0% 100.0% 100.0% Terminal rate 39/39 (100%) 11/11 (100%) 0/0 First incidence (days) 441 260 299 Poly-3 test — — —

(T)Terminal sacrifice a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for a drenal gland, liver, lung, pancreas, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animal s necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals th at do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal group f Value of statistic cannot be computed.

### 141 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A4a Historical Incidence of Malignant Mesothelioma in Control Male F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 4/100
p,p N -Dichlorodiphenyl sulfone (feed) 2/50 Indium phosphide (inhalation) 2/50 60-Hz Magnetic fields (whole body exposure) 4/100 Methacrylonitrile (gavage) 0/50 Naphthalene (inhalation) 0/49

o -Nitrotoluene (feed) 2/60
p-Nitrotoluene (feed) 5/50 Sodium nitrite (drinking water) 3/50 Vanadium pentoxide (inhalation) 1/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 23/609 (3.8%) Mean ± standard deviation 3.7% ± 2.9% Range 0%-10%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 0/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/51 Butyl benzyl phthalate 1/50 D&C Yellow No. 11 2/50 Emodin 2/50

o-Nitroanisole 1/50
p-Nitrobenzoic acid 0/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 25/1,004 (2.5%) Mean ± standard deviation 2.5% ± 2.0% Range 0%-8%

a Data as of January 18, 2001 b Data as of December 21, 1999

### o-Nitrotoluene, NTP TR 504 142



### T ABLE 

### A4b Historical Incidence of Subcutaneous Skin Neoplasms in Control Male F344/N Rats

Incidence in Controls Study Lipoma Fibroma Fibrosarcoma Fibroma or Fibrosarcoma

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 0/100 2/100 2/100 4/100
p,p N -Dichlorodiphenyl sulfone (feed)) 1/50 2/50 0/50 2/50 Indium phosphide (inhalation) 1/50 1/50 1/50 2/50 60-Hz Magnetic fields (whole body exposure) 1/100 12/100 1/100 13/100 Methacrylonitrile (gavage) 1/50 3/50 0/50 3/50 Naphthalene (inhalation) 1/49 5/49 2/49 7/49

o -Nitrotoluene (feed) 0/60 5/60 0/60 5/60
p-Nitrotoluene (feed) 0/50 1/50 0/50 1/50 Sodium nitrite (drinking water) 1/50 0/50 1/50 1/50 Vanadium pentoxide (inhalation) 2/50 2/50 1/50 3/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 8/609 (1.3%) 33/609 (5.4%) 8/609 (1.3%) 41/609 (6.7%) Mean ± standard deviation 1.5% ± 1.3% 5.1% ± 4.0% 1.3% ± 1.4% 6.4% ± 4.3% Range 0%-4% 0%-12% 0%-4% 2%-14%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 0/50 4/50 0/50 4/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/51 2/51 0/51 2/51 Butyl benzyl phthalate 0/50 5/50 0/50 5/50 D&C Yellow No. 11 0/50 3/50 0/50 3/50 Emodin 1/50 1/50 0/50 1/50

o-Nitroanisole 1/50 1/50 0/50 1/50
p-Nitrobenzoic acid 0/50 4/50 1/50 5/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 2/1,004 (0.2%) 56/1,004 (5.6%) 9/1,004 (0.9%) 65/1,004 (6.5%) Mean ± standard deviation 0.2% ± 0.6% 5.6% ± 3.2% 0.9% ± 1.4% 6.5% ± 3.1% Range 0%-2% 0%-10% 0%-4% 2%-10%

a Data as of January 18, 2001 b Data as of December 21, 1999

### 143 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A4c Historical Incidence of Mammary Gland Fibroadenoma in Control Male F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 8/100
p,p N -Dichlorodiphenyl sulfone (feed) 3/50 Indium phosphide (inhalation) 2/50 60-Hz Magnetic fields (whole body exposure) 6/100 Methacrylonitrile (gavage) 2/50 Naphthalene (inhalation) 0/49

o -Nitrotoluene (feed) 0/60
p-Nitrotoluene (feed) 0/50 Sodium nitrite (drinking water) 3/50 Vanadium pentoxide (inhalation) 2/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 26/609 (4.3%) Mean ± standard deviation 3.8% ± 2.9% Range 0%-8%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 4/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/51 Butyl benzyl phthalate 2/50 D&C Yellow No. 11 3/50 Emodin 1/50

o-Nitroanisole 3/50
p-Nitrobenzoic acid 2/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 42/1,004 (4.2%) Mean ± standard deviation 4.2% ± 3.5% Range 0%-12%

a Data as of January 18, 2001 b Data as of December 21, 1999

### o-Nitrotoluene, NTP TR 504 144



### T ABLE 

### A4d Historical Incidence of Liver Neoplasms in Control Male F344/N Rats

Incidence in Controls Study Hepatocholangio- Cholangio- Hepatocellular Hepatocellular Hepatocellular carcinoma carcinoma Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 0/100 0/100 0/100 0/100 0/100
p,p N -Dichlorodiphenyl sulfone (feed) 0/50 0/50 0/50 2/50 2/50 Indium phosphide (inhalation) 0/50 0/50 0/50 0/50 0/50 60-Hz Magnetic fields

(whole body exposure) 0/100 0/100 1/100 0/100 1/100 Methacrylonitrile (gavage) 0/50 0/50 1/50 0/50 1/50 Naphthalene (inhalation) 0/49 0/49 1/49 1/49 2/49

o -Nitrotoluene (feed) 0/60 0/60 2/60 1/60 3/60
p-Nitrotoluene (feed) 0/50 0/50 0/50 1/50 1/50 Sodium nitrite (drinking water) 0/50 0/50 0/50 0/50 0/50 Vanadium pentoxide (inhalation) 0/50 0/50 0/50 0/50 0/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 0/609 0/609 5/609 (0.8%) 5/609 (0.8%) 10/609 (1.6%) Mean ± standard deviation 0.8% ± 1.2% 1.0% ± 1.4% 1.8% ± 1.9% Range 0%-3% 0%-4% 0%-5%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 0/50 0/50 5/50 1/50 5/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/51 0/51 0/51 0/51 0/51 Butyl benzyl phthalate 0/50 0/50 2/50 0/50 2/50 D&C Yellow No. 11 0/50 0/50 1/50 0/50 1/50 Emodin 0/50 0/50 1/50 0/50 1/50

o-Nitroanisole 0/50 0/50 0/50 0/50 0/50
p-Nitrobenzoic acid 0/49 0/49 2/49 3/49 4/49



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 0/1,002 0/1,002 23/1,002 (2.3%) 7/1,002 (0.7%) 28/1,002 (2.8%) Mean ± standard deviation 2.3% ± 3.0% 0.7% ± 1.5% 2.8% ± 3.3% Range 0%-10% 0%-6% 0%-10%

a Data as of January 18, 2001 b Data as of December 21, 1999

### T ABLE 

### A4e Historical Incidence of Alveolar/bronchiolar Neoplasms in Control Male F344/N Rats

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 3/100 0/100 3/100
p,p N

- Dichlorodiphenyl sulfone (feed) 2/50 0/50 2/50 Indium phosphide (inhalation) 6/50 1/50 7/50 60-Hz Magnetic fields (whole body exposure) 3/100 0/100 3/100 Methacrylonitrile (gavage) 0/50 0/50 0/50 Naphthalene (inhalation) 2/49 0/49 2/49

o -Nitrotoluene (feed) 1/60 1/60 2/60
p-Nitrotoluene (feed) 1/50 0/50 1/50 Sodium nitrite (drinking water) 2/50 0/50 2/50 Vanadium pentoxide (inhalation) 4/50 0/50 4/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 24/609 (3.9%) 2/609 (0.3%) 26/609 (4.3%) Mean ± standard deviation 4.2% ± 3.5% 0.4% ± 0.8% 4.5% ± 3.9% Range 0%-12% 0%-2% 0%-14%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 1/50 0/50 1/50 2,2-Bis(bromomethyl)-1,3-propanediol 1/51 0/51 1/51 Butyl benzyl phthalate 0/50 0/50 0/50 D&C Yellow No. 11 7/50 1/50 8/50 Emodin 0/50 0/50 0/50

o-Nitroanisole 2/50 1/50 3/50
p-Nitrobenzoic acid 0/50 0/50 0/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 24/1,002 (2.4%) 7/1,002 (0.7%) 31/1,002 (3.1%) Mean ± standard deviation 2.4% ± 3.2% 0.7% ± 1.2% 3.1% ± 3.5% Range 0%-14% 0%-4% 0%-16%

a Data as of January 18, 2001 b Data as of December 21, 1999 

### 145 o- Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 146



### T ABLE 

### A4f Historical Incidence of Circulatory System Neoplasms in Control Male F344/N Rats

Incidence in Controls Study Hemangioma Hemangiosarcoma Hemangioma or Hemangiosarcoma

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 1/100 0/100 1/100
p,p N

- Dichlorodiphenyl sulfone (feed) 0/50 1/50 1/50 Indium phosphide (inhalation) 0/50 1/50 1/50 60-Hz Magnetic fields (whole body exposure) 0/100 0/100 0/100 Methacrylonitrile (gavage) 0/50 0/50 0/50 Naphthalene (inhalation) 0/49 0/49 0/49

o -Nitrotoluene (feed) 0/60 1/60 1/60
p-Nitrotoluene (feed) 2/50 0/50 2/50 Sodium nitrite (drinking water) 0/50 0/50 0/50 Vanadium pentoxide (inhalation) 0/50 0/50 0/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 3/609 (0.5%) 3/609 (0.5%) 6/609 (1.0%) Mean ± standard deviation 0.5% ± 1.3% 0.6% ± 0.9% 1.1% ± 1.4% Range 0%-4% 0%-2% 0%-4%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 0/50 0/50 0/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/51 0/51 0/51 Butyl benzyl phthalate 0/50 1/50 1/50 D&C Yellow No. 11 1/50 0/50 1/50 Emodin 0/50 1/50 1/50

o-Nitroanisole 1/50 1/50 2/50
p-Nitrobenzoic acid 0/50 1/50 1/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 3/1,004 (0.3%) 7/1,004 (0.7%) 10/1,004 (1.0%) Mean ± standard deviation 0.3% ± 0.7% 0.7% ± 1.2% 1.0% ± 1.4% Range 0%-2% 0%-4% 0%-4%

a Data as of January 18, 2001 b Data as of December 21, 1999

### 147 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A4g Historical Incidence of Mononuclear Cell Leukemia in Control Male F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 68/100
p,p N -Dichlorodiphenyl sulfone (feed) 27/50 Indium phosphide (inhalation) 16/50 60-Hz Magnetic fields (whole body exposure) 50/100 Methacrylonitrile (gavage) 20/50 Naphthalene (inhalation) 26/49

o -Nitrotoluene (feed) 30/60
p-Nitrotoluene (feed) 24/50 Sodium nitrite (drinking water) 17/50 Vanadium pentoxide (inhalation) 22/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 300/609 (49.3%) Mean ± standard deviation 47.3% ± 10.5% Range 32%-68%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 16/50 2,2-Bis(bromomethyl)-1,3-propanediol 27/51 Butyl benzyl phthalate 31/50 D&C Yellow No. 11 37/50 Emodin 28/50

o-Nitroanisole 26/50
p-Nitrobenzoic acid 29/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 547/1,004 (54.5%) Mean ± standard deviation 54.5% ± 10.7% Range 32%-74%

a Data as of January 18, 2001; includes data for lymphocytic, monocytic, and undifferentiated leukemia b Data as of December 21, 1999; includes data for lymphocytic, monocytic, and undifferentiated leukemia

### o-Nitrotoluene, NTP TR 504 148



### T ABLE 

### A4h Historical Incidence of Interstitial Cell Adenoma of the Testis in Control Male F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 96/100
p,p N

- Dichlorodiphenyl sulfone (feed) 41/50 Indium phosphide (inhalation) 40/50 60-Hz Magnetic fields (whole body exposure) 93/100 Methacrylonitrile (gavage) 40/50 Naphthalene (inhalation) 38/49

o -Nitrotoluene (feed) 55/60
p-Nitrotoluene (feed) 49/50 Sodium nitrite (drinking water) 47/50 Vanadium pentoxide (inhalation) 36/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 535/609 (87.9%) Mean ± standard deviation 86.4% ± 9.1% Range 72%-98%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 47/50 2,2-Bis(bromomethyl)-1,3-propanediol 49/51 Butyl benzyl phthalate 44/50 D&C Yellow No. 11 39/49 Emodin 41/50

o-Nitroanisole 48/50
p-Nitrobenzoic acid 44/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 889/1,003 (88.6%) Mean ± standard deviation 88.6% ± 6.0% Range 74%-96%

a Data as of January 18, 2001 b Data as of December 21, 1999

### 149 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene a



### 0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

Disposition Summary Animals initially in study 70 60 60 60 70 70 3-Month interim evaluation 10 10 10 Early deaths

Accidental deaths 3 Moribund 18 35 48 53 39 57 Natural deaths 3 7 6 7 10 3 Survivors

Died last week of study 1 Terminal sacrifice 38 18 3 11

Animals examined microscopically 70 60 60 60 70 70



## 3-Month Interim Evaluation Alimentary System Liver (10) (10) (10)

Hepatodiaphragmatic nodule 1 (10%) 2 (20%) Hepatocyte, vacuolization cytoplasmic 10 (100%) Mesentery (1)

Accessory spleen 1 (100%) Pancreas (10) (10) (10)

Atrophy 1 (10%) Salivary glands (10) (10) (10)

Atrophy 10 (100%) 10 (100%)



### Cardiovascular System Heart (10) (10) (10)

Cardiomyopathy 5 (50%) 3 (30%) 6 (60%)



### Endocrine System Adrenal cortex (10) (10) (10)

Accessory adrenal cortical nodule 1 (10%) 2 (20%) 2 (20%) Islets, pancreatic (10) (10) (10)

Hyperplasia 8 (80%) Pituitary gland (10) (10) (10)

Pars distalis, cyst 1 (10%) Thyroid gland (10) (10) (10)

Ultimobranchial cyst 1 (10%) 2 (20%)



### Genital System Epididymis (10) (10) (10)

Atrophy 5 (50%) Atypia cellular 2 (20%) 10 (100%) Preputial gland (10) (10) (10)

Atrophy 1 (10%) 8 (80%)

a Number of animals examined microscopically at the site and the number of animals with lesion

### o-Nitrotoluene, NTP TR 504 150



## 2-Year Study Alimentary System Intestine large, cecum (60) (60) (60) (58) (59) (60)

Edema 1 (2%) 1 (2%) Liver (60) (60) (60) (60) (60) (60)

Angiectasis 1 (2%) 2 (3%) 2 (3%) 3 (5%) 2 (3%) Atrophy 1 (2%) 1 (2%) 1 (2%) 4 (7%) 3 (5%) Basophilic focus 38 (63%) 22 (37%) 21 (35%) 25 (42%) 31 (52%) 17 (28%) 

## 3-Month Interim Evaluation (continued) 

### Genital System (continued) Prostate (10) (10) (10)

Inflammation, chronic 1 (10%) Testes (10) (10) (10)

Mineralization 5 (50%) Germinal epithelium, atrophy 1 (10%) 9 (90%)



### Hematopoietic System Lymph node (1) (1)

Mediastinal, hemorrhage 1 (100%) Mediastinal, pigmentation 1 (100%) Renal, hemorrhage 1 (100%) Renal, pigmentation 1 (100%) Spleen (10) (10) (10)

Congestion 3 (30%) 10 (100%) Hematopoietic cell proliferation 2 (20%) 10 (100%) Pigmentation 1 (10%) Thymus (10) (10) (10)

Hemorrhage 1 (10%)



### Respiratory System Nose (10) (10) (10)

Olfactory epithelium, degeneration 4 (40%) 10 (100%)



### Urinary System Kidney (10) (10) (10)

Nephropathy 8 (80%) Renal tubule, degeneration, hyaline 1 (10%) 10 (100%)



## Systems Examined with No Lesions Observed General Body System Integumentary System Musculoskeletal System Nervous System Special Senses System 

### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### 151 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)



## 2-Year Study (continued) 

### Alimentary System (continued) Liver (continued) (60) (60) (60) (60) (60) (60)

Clear cell focus 29 (48%) 29 (48%) 34 (57%) 31 (52%) 30 (50%) 34 (57%) Cyst 1 (2%) Degeneration, cystic 3 (5%) 3 (5%) 1 (2%) 2 (3%) 6 (10%) 3 (5%) Eosinophilic focus 7 (12%) 18 (30%) 29 (48%) 24 (40%) 15 (25%) 13 (22%) Fibrosis 1 (2%) 2 (3%) Hematopoietic cell proliferation 6 (10%) 2 (3%) 2 (3%) 11 (18%) 6 (10%) Hemorrhage 2 (3%) 2 (3%) 2 (3%) 2 (3%) Hepatodiaphragmatic nodule 5 (8%) 8 (13%) 8 (13%) 5 (8%) 10 (17%) 4 (7%) Infiltration cellular, mixed cell 1 (2%) 5 (8%) 11 (18%) 20 (33%) 15 (25%) 33 (55%) Mixed cell focus 5 (8%) 7 (12%) 12 (20%) 6 (10%) 12 (20%) 8 (13%) Necrosis, focal 3 (5%) 4 (7%) 6 (10%) 6 (10%) 2 (3%) 3 (5%) Bile duct, hyperplasia 53 (88%) 37 (62%) 7 (12%) 6 (10%) 1 (2%) Centrilobular, necrosis 1 (2%) 3 (5%) 8 (13%) 5 (8%) 9 (15%) 3 (5%) Hepatocyte, vacuolization cytoplasmic 4 (7%) 2 (3%) 1 (2%) 1 (2%) 1 (2%) 6 (10%) Kupffer cell, pigmentation 4 (7%) 1 (2%) 3 (5%) 2 (3%) Mesentery (16) (6) (5) (4) (1) (3)

Accessory spleen 3 (19%) 3 (75%) Congestion 3 (19%) Hemorrhage 1 (17%) Fat, necrosis 13 (81%) 3 (50%) 5 (100%) 1 (25%) 1 (100%) Oral mucosa (1) (1)

Hyperplasia 1 (100%) Pancreas (60) (60) (60) (60) (60) (60)

Atrophy 11 (18%) 14 (23%) 7 (12%) 4 (7%) 11 (18%) 9 (15%) Acinus, hyperplasia, focal 1 (2%) 2 (3%) 3 (5%) 4 (7%) 6 (10%) 3 (5%) Salivary glands (60) (60) (59) (60) (58) (59)

Atrophy 2 (3%) 18 (31%) 43 (72%) 16 (28%) 49 (83%) Stomach, forestomach (59) (60) (60) (60) (60) (60)

Edema 4 (7%) 2 (3%) 1 (2%) 2 (3%) Ulcer 2 (3%) 2 (3%) 1 (2%) 1 (2%) 1 (2%) Epithelium, hyperplasia 6 (10%) 2 (3%) 1 (2%) 2 (3%) 4 (7%) 1 (2%) Stomach, glandular (59) (60) (60) (60) (60) (60)

Edema 1 (2%) 1 (2%) Erosion 4 (7%) 2 (3%) 7 (12%) 7 (12%) 7 (12%) 3 (5%) Infiltration cellular, lymphoid 1 (2%) Perforation 1 (2%) Ulcer 1 (2%) 2 (3%) 3 (5%) 1 (2%) 1 (2%) Tongue (1) (1)

Epithelium, hyperplasia 1 (100%) Tooth (1) (1)

Malformation 1 (100%)



### Cardiovascular System Heart (60) (60) (60) (60) (60) (60)

Cardiomyopathy 40 (67%) 43 (72%) 43 (72%) 37 (62%) 39 (65%) 46 (77%) Thrombosis 1 (2%) 1 (2%) 1 (2%) 4 (7%) 1 (2%)

### o-Nitrotoluene, NTP TR 504 152



## 2-Year Study (continued) 

### Endocrine System Adrenal cortex (60) (60) (60) (60) (60) (60)

Accessory adrenal cortical nodule 13 (22%) 12 (20%) 13 (22%) 10 (17%) 14 (23%) 9 (15%) Degeneration, fatty 16 (27%) 10 (17%) 11 (18%) 4 (7%) 14 (23%) 12 (20%) Hyperplasia, diffuse 1 (2%) 3 (5%) 2 (3%) Hyperplasia, focal 6 (10%) 2 (3%) 6 (10%) 3 (5%) 4 (7%) 6 (10%) Hypertrophy, focal 3 (5%) 2 (3%) 2 (3%) 3 (5%) 2 (3%) Necrosis 1 (2%) Adrenal medulla (60) (60) (60) (59) (60) (60)

Hyperplasia 7 (12%) 4 (7%) 2 (3%) 3 (5%) 1 (2%) Islets, pancreatic (60) (60) (60) (60) (60) (60)

Hyperplasia 2 (3%) 2 (3%) 2 (3%) 10 (17%) Pigmentation 1 (2%) 2 (3%) 11 (18%) Parathyroid gland (58) (55) (59) (58) (58) (57)

Hyperplasia 1 (2%) Pituitary gland (59) (60) (58) (59) (57) (59)

Pars distalis, angiectasis 3 (5%) 7 (12%) 2 (3%) 4 (7%) 4 (7%) Pars distalis, cyst 2 (3%) 6 (10%) 3 (5%) 4 (7%) 3 (5%) 6 (10%) Pars distalis, cytoplasmic alteration 1 (2%) 1 (2%) 4 (7%) 1 (2%) 42 (71%) Pars distalis, hyperplasia, focal 10 (17%) 13 (22%) 13 (22%) 10 (17%) 9 (16%) 15 (25%) Pars intermedia, cyst 1 (2%) 1 (2%) 1 (2%) 2 (3%) Thyroid gland (59) (60) (60) (60) (60) (60)

Ultimobranchial cyst 1 (2%) 2 (3%) 3 (5%) 4 (7%) 3 (5%) C-cell, hyperplasia 10 (17%) 11 (18%) 9 (15%) 7 (12%) 9 (15%) 6 (10%) Follicle, cyst 3 (5%) 2 (3%) Follicular cell, hyperplasia 1 (2%)



### General Body System Peritoneum (3) (21) (33) (46) (47) (54)

Mesothelium, hyperplasia 1 (33%) 1 (5%) 4 (12%) 2 (4%) 3 (6%)



### Genital System Epididymis (60) (60) (59) (60) (60) (58)

Atypia cellular 39 (65%) 32 (53%) 27 (46%) 21 (35%) 24 (40%) 28 (48%) Fibrosis 2 (3%) Inflammation, chronic 1 (2%) 2 (3%) Preputial gland (60) (59) (58) (56) (60) (59)

Atrophy 7 (12%) 9 (15%) 35 (60%) 41 (73%) 38 (63%) 54 (92%) Cyst 1 (2%) 4 (7%) 1 (2%) 1 (2%) 1 (2%) Hyperplasia 1 (2%) Inflammation, chronic 40 (67%) 43 (73%) 36 (62%) 27 (48%) 33 (55%) 14 (24%) Prostate (60) (59) (60) (60) (60) (60)

Inflammation, chronic 24 (40%) 14 (24%) 18 (30%) 9 (15%) 17 (28%) 8 (13%) Epithelium, hyperplasia 1 (2%) Ventral, hyperplasia 18 (30%) 3 (5%) 9 (15%) 1 (2%) 4 (7%) 1 (2%) Testes (60) (60) (60) (60) (60) (60)

Angiectasis 2 (3%) 1 (2%) Hemorrhage 2 (3%) Germinal epithelium, atrophy 13 (22%) 21 (35%) 12 (20%) 19 (32%) 18 (30%) 53 (88%) Interstitial cell, hyperplasia 10 (17%) 14 (23%) 13 (22%) 31 (52%) 15 (25%) 4 (7%) 

### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### 153 o- Nitrotoluene, NTP TR 504



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)



## 2-Year Study (continued) 

### Hematopoietic System Bone marrow (60) (60) (60) (60) (60) (60)

Hyperplasia 2 (3%) 25 (42%) 43 (72%) 45 (75%) 37 (62%) 33 (55%) Lymph node (16) (14) (4) (9) (18) (16)

Iliac, hyperplasia, lymphoid 1 (11%) Mediastinal, atrophy 1 (11%) Mediastinal, ectasia 1 (6%) Mediastinal, hemorrhage 2 (13%) 3 (21%) 1 (25%) 2 (22%) 4 (22%) 9 (56%) Mediastinal, hyperplasia, lymphoid 1 (6%) Mediastinal, pigmentation 6 (38%) 9 (64%) 3 (75%) 8 (89%) 13 (72%) 13 (81%) Pancreatic, hyperplasia, histiocytic 2 (13%) Pancreatic, hyperplasia, lymphoid 1 (6%) Pancreatic, pigmentation 1 (6%) 1 (7%) Renal, ectasia 1 (25%) Renal, pigmentation 1 (6%) 1 (25%) Lymph node, mandibular (58) (59) (58) (59) (58) (59)

Atrophy 1 (2%) 1 (2%) Ectasia 2 (3%) 1 (2%) Hemorrhage 2 (3%) 2 (3%) 1 (2%) 1 (2%) 4 (7%) 3 (5%) Hyperplasia, lymphoid 3 (5%) 4 (7%) 3 (5%) 2 (3%) 5 (9%) 3 (5%) Pigmentation 9 (16%) 2 (3%) 6 (10%) 12 (20%) 5 (9%) 5 (8%) Lymph node, mesenteric (60) (60) (59) (58) (60) (59)

Atrophy 1 (2%) 2 (3%) 1 (2%) Ectasia 2 (3%) Hemorrhage 2 (3%) 6 (10%) 3 (5%) 2 (3%) 4 (7%) 4 (7%) Hyperplasia, lymphoid 2 (3%) Pigmentation 3 (5%) 3 (5%) Spleen (60) (60) (60) (60) (60) (60)

Accessory spleen 1 (2%) Fibrosis 6 (10%) 6 (10%) 9 (15%) 7 (12%) 14 (23%) 27 (45%) Hematopoietic cell proliferation 7 (12%) 33 (55%) 38 (63%) 47 (78%) 36 (60%) 35 (58%) Hemorrhage 2 (3%) Necrosis 1 (2%) Pigmentation 9 (15%) 8 (13%) 13 (22%) 16 (27%) 4 (7%) 7 (12%) Capsule, fibrosis 1 (2%) Lymphoid follicle, atrophy 1 (2%) 1 (2%) Thymus (56) (54) (55) (55) (56) (56)

Cyst 2 (4%) 1 (2%)



### Integumentary System Mammary gland (57) (47) (46) (43) (51) (50)

Dilatation 3 (5%) 10 (21%) 1 (2%) 1 (2%) 3 (6%) 1 (2%) Galactocele 2 (4%) Hyperplasia 6 (11%) 2 (4%) 2 (4%) 2 (5%) 1 (2%) 4 (8%) Skin (60) (60) (60) (60) (60) (60)

Cyst 1 (2%) Cyst epithelial inclusion 1 (2%) Inflammation, chronic 1 (2%) 1 (2%) Subcutaneous tissue, edema 1 (2%) Subcutaneous tissue, inflammation, chronic 1 (2%)

### o-Nitrotoluene, NTP TR 504 154



## 2-Year Study (continued) 

### Musculoskeletal System Bone (60) (60) (60) (60) (60) (60)

Cranium, osteopetrosis 1 (2%) 2 (3%) 1 (2%) Femur, osteopetrosis 1 (2%) Skeletal muscle (1) (5) (3) (6) (5)

Atrophy 1 (100%) Hemorrhage 1 (20%)



### Nervous System Brain (60) (60) (60) (60) (60) (60)

Compression 6 (10%) 3 (5%) 2 (3%) 1 (2%) 2 (3%) 1 (2%) Developmental malformation 1 (2%) Gliosis 1 (2%) Hemorrhage 1 (2%) 1 (2%) Hydrocephalus 3 (5%) 1 (2%) 1 (2%) 2 (3%) 2 (3%) 1 (2%) Necrosis 1 (2%) Spinal cord (2) (4) (1) (1) (3)

Cyst epithelial inclusion 1 (33%)



### Respiratory System Lung (60) (60) (60) (60) (60) (60)

Congestion 1 (2%) 2 (3%) 1 (2%) Hemorrhage 3 (5%) 1 (2%) 3 (5%) 4 (7%) Infiltration cellular, histiocyte 25 (42%) 42 (70%) 40 (67%) 22 (37%) 23 (38%) 15 (25%) Metaplasia, osseous 1 (2%) 1 (2%) 1 (2%) Alveolar epithelium, hyperplasia 2 (3%) 8 (13%) 3 (5%) 7 (12%) 15 (25%) 29 (48%) Nose (60) (60) (60) (60) (60) (60)

Foreign body 9 (15%) 13 (22%) 10 (17%) 11 (18%) 14 (23%) 5 (8%) Inflammation, chronic 9 (15%) 9 (15%) 8 (13%) 9 (15%) 10 (17%) 2 (3%) Inflammation, suppurative 2 (3%) Respiratory epithelium, hyperplasia 6 (10%) 5 (8%) 7 (12%) 8 (13%) 7 (12%) 3 (5%) Respiratory epithelium, metaplasia,

squamous 2 (3%) 1 (2%) 1 (2%) 1 (2%)



### Special Senses System Eye (2) (1) (2) (1)

Atrophy 1 (50%) Cataract 1 (100%) 1 (50%) 1 (100%) Inflammation, chronic 1 (50%) Retina, degeneration 1 (50%) 1 (100%) 1 (50%) 1 (100%) Lacrimal gland (1) (1)

Cyst 1 (100%) 

### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### 155 o- Nitrotoluene, NTP TR 504



## 2-Year Study (continued) 

### Urinary System Kidney (60) (60) (60) (60) (60) (60)

Cyst 1 (2%) 3 (5%) 2 (3%) 1 (2%) 2 (3%) Hydronephrosis 3 (5%) Infarct 1 (2%) 1 (2%) 1 (2%) Inflammation, chronic 1 (2%) 2 (3%) 4 (7%) Mineralization 1 (2%) 1 (2%) Nephropathy 56 (93%) 59 (98%) 56 (93%) 56 (93%) 60 (100%) 60 (100%) Renal tubule, degeneration, hyaline 3 (5%) 3 (5%) 1 (2%) 2 (3%) 1 (2%) Renal tubule, dilatation 1 (2%) 3 (5%) 2 (3%) 2 (3%) 1 (2%) Renal tubule, necrosis 1 (2%) 1 (2%) 2 (3%) Renal tubule, pigmentation 5 (8%) 12 (20%) 14 (23%) 9 (15%) 18 (30%) 6 (10%) Transitional epithelium, hyperplasia 1 (2%) 1 (2%) 2 (3%) 1 (2%) Urinary bladder (60) (60) (60) (59) (60) (59)

Fibrosis 1 (2%) 2 (3%) Inflammation, chronic 2 (3%) Transitional epithelium, hyperplasia 2 (3%) 2 (3%) 3 (5%) 1 (2%) Transitional epithelium, inflammation,

chronic 1 (2%) 

### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm 2,000 ppm 5,000 ppm(Stop- (StopExposure) Exposure)

### o-Nitrotoluene, NTP TR 504 156

### 157



# APPENDIX B SUMMARY OF LESIONS IN FEMALE RATS IN THE 2-YEAR FEED STUDY OF o-NITROTOLUENE

T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 T ABLE 

### B4a Historical Incidence of Subcutaneous Skin Neoplasms in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 T ABLE 

### B4b Historical Incidence of Mammary Gland Neoplasms in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 T ABLE 

### B4c Historical Incidence of Hepatocellular Adenoma in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 T ABLE 

### B4d Historical Incidence of Mononuclear Cell Leukemia in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

### o-Nitrotoluene, NTP TR 504 158



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene a



### 0 ppm 625 ppm 1,250 ppm 2,000 ppm

Disposition Summary Animals initially in study 60 60 60 60 Early deaths

Moribund 10 11 19 22 Natural deaths 3 2 2 5 Survivors

Died last week of study 2 1 Terminal sacrifice 45 46 39 33

Animals examined microscopically 60 60 60 60



### Alimentary System Intestine large, colon (58) (59) (60) (59)

Polyp adenomatous 1 (2%) 1 (2%) Intestine large, rectum (60) (60) (60) (58)

Polyp adenomatous 1 (2%) Intestine small, ileum (57) (59) (59) (58) Liver (60) (59) (60) (60)

Carcinoma, metastatic, adrenal cortex 1 (2%) Hepatocellular adenoma 1 (2%) 1 (2%) 5 (8%) Hepatocellular adenoma, multiple 1 (2%) Sarcoma 1 (2%) Mesentery (9) (1) (3) (3)

Lipoma 2 (67%) Pancreas (60) (59) (60) (60)

Acinus, adenoma 1 (2%) 1 (2%) Salivary glands (60) (60) (60) (60) Stomach, forestomach (59) (59) (60) (60)

Squamous cell papilloma 1 (2%) Tongue (1) (1)

Squamous cell carcinoma 1 (100%) Squamous cell papilloma 1 (100%)



### Cardiovascular System Heart (60) (60) (60) (60)

Schwannoma malignant 1 (2%)



### Endocrine System Adrenal cortex (60) (60) (60) (59)

Adenoma 2 (3%) 1 (2%) Carcinoma 1 (2%) Adrenal medulla (60) (60) (60) (58)

Pheochromocytoma benign 2 (3%) 1 (2%) Islets, pancreatic (60) (59) (60) (60)

Adenoma 1 (2%) 1 (2%) 1 (2%) Carcinoma 1 (2%) Pituitary gland (59) (60) (60) (60)

Pars distalis, adenoma 19 (32%) 30 (50%) 26 (43%) 20 (33%) Pars distalis, adenoma, multiple 2 (3%) Pars intermedia, adenoma 1 (2%) 1 (2%)

### 159 o- Nitrotoluene, NTP TR 504

Endocrine System (continued) Thyroid gland (60) (60) (60) (60)

Bilateral, C-cell, adenoma 1 (2%) 1 (2%) C-cell, adenoma 9 (15%) 5 (8%) 2 (3%) 5 (8%) C-cell, carcinoma 1 (2%) 3 (5%) 3 (5%) 1 (2%) Follicular cell, adenoma 1 (2%) Follicular cell, carcinoma 1 (2%) 1 (2%)



### General Body System None



### Genital System Clitoral gland (59) (57) (54) (53)

Adenoma 12 (20%) 8 (14%) 3 (6%) 3 (6%) Carcinoma 2 (3%) 4 (7%) 3 (6%) Bilateral, adenoma 1 (2%) Ovary (60) (59) (60) (60)

Granulosa cell tumor malignant 1 (2%) Granulosa-theca tumor malignant 1 (2%) Uterus (60) (59) (60) (60)

Hemangioma 1 (2%) Polyp stromal 11 (18%) 7 (12%) 15 (25%) 16 (27%) Sarcoma stromal 1 (2%) 1 (2%) Vagina (2) (1) (3) (6)

Fibroma 1 (33%) Granular cell tumor benign 1 (17%) Hemangioma 1 (17%) Leiomyoma 1 (50%) Leiomyosarcoma 1 (17%) Squamous cell papilloma 1 (33%) 1 (17%)



### Hematopoietic System Bone marrow (60) (60) (60) (60) Lymph node (8) (4) (4) (4)

Deep cervical, carcinoma, metastatic, thyroid gland 1 (25%) Lymph node, mandibular (60) (60) (59) (59) Lymph node, mesenteric (59) (58) (59) (59) Spleen (60) (59) (60) (59)

Carcinoma, metastatic, adrenal cortex 1 (2%) Thymus (57) (55) (58) (58)



### Integumentary System Mammary gland (60) (60) (59) (60)

Adenoma 1 (2%) Carcinoma 1 (2%) 1 (2%) Fibroadenoma 17 (28%) 12 (20%) 13 (22%) 14 (23%) Fibroadenoma, multiple 6 (10%) 35 (58%) 39 (66%) 42 (70%) 

### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

### o-Nitrotoluene, NTP TR 504 160

Integumentary System (continued) Skin (59) (60) (60) (60)

Basal cell adenoma 1 (2%) Pinna, melanoma malignant 1 (2%) Pinna, neural crest tumor 1 (2%) Sebaceous gland, adenoma 1 (2%) Subcutaneous tissue, fibroma 3 (5%) 3 (5%) 18 (30%) 19 (32%) Subcutaneous tissue, fibroma, multiple 1 (2%) Subcutaneous tissue, fibrosarcoma 4 (7%) 2 (3%) Subcutaneous tissue, fibrosarcoma, multiple 1 (2%) Subcutaneous tissue, hemangiosarcoma 1 (2%) Subcutaneous tissue, neural crest tumor 1 (2%)



### Musculoskeletal System Skeletal muscle (1) (1) (1) (1)

Lipoma 1 (100%) Rhabdomyosarcoma 1 (100%)



### Nervous System Brain (60) (60) (60) (60)

Astrocytoma malignant 1 (2%)



### Respiratory System Lung (60) (60) (60) (60)

Alveolar/bronchiolar adenoma 1 (2%) 1 (2%) 3 (5%) Alveolar/bronchiolar adenoma, multiple 1 (2%) Alveolar/bronchiolar carcinoma 1 (2%) Sarcoma, metastatic, liver 1 (2%)



### Special Senses System Zymbal’s gland (2) (1) (3)

Adenoma 1 (50%) 1 (33%) Carcinoma 1 (50%) 1 (100%) 2 (67%)



### Urinary System Kidney (60) (59) (60) (60) Urinary bladder (60) (59) (60) (60)

Papilloma 1 (2%) 1 (2%) Squamous cell papilloma, multiple 1 (2%)



### Systemic Lesions Multiple organs b (60) (60) (60) (60)

Leukemia mononuclear 21 (35%) 6 (10%) 4 (7%) 5 (8%) Lymphoma malignant 1 (2%) 1 (2%) 

### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

### 161 o- Nitrotoluene, NTP TR 504

Neoplasm Summary Total animals with primary neoplasms c 54 58 60 60

Total primary neoplasms 113 131 147 165 Total animals with benign neoplasms 47 57 59 59

Total benign neoplasms 88 112 126 143 Total animals with malignant neoplasms 24 16 18 20

Total malignant neoplasms 25 19 20 21 Total animals with metastatic neoplasms 2 1

Total metastatic neoplasms 3 1Total animals with uncertain neoplasmsbenign or malignant 1 1 Total uncertain neoplasms 1 1

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms 

### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

### o-Nitrotoluene, NTP TR 504 162

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon ++++++A++++++++++++++++++++A++ Intestine large, rectum ++++++++++++++++++++++++++++++ Intestine large, cecum ++++++A++++++++++++++++++++A++ Intestine small, duodenum +++++++++++++++++++++++++++A++ Intestine small, jejunum ++++++A+++++++++++++++++++++++ Intestine small, ileum + + + + + +A+ + + +M+ + + + + + + + + + + + + + +A+ + Liver ++++++++++++++++++++++++++++++

Hepatocellular adenoma Mesentery + + + Pancreas ++++++++++++++++++++++++++++++ Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach + + + +M+ + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular ++++++++++++++++++++++++++++++ Tooth + +


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++

Adenoma X Adrenal medulla ++++++++++++++++++++++++++++++

Pheochromocytoma benign X Islets, pancreatic ++++++++++++++++++++++++++++++

Adenoma Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +M+ + + + Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + +M+ + +

Pars distalis, adenoma X X X XXXXXX X Pars intermedia, adenoma X Thyroid gland ++++++++++++++++++++++++++++++

C-cell, adenoma X X C-cell, carcinoma


### General Body System None


### Genital System Clitoral gland ++++++++++++++++++++++++++++++

Adenoma X X X X X Carcinoma X Ovary ++++++++++++++++++++++++++++++ Uterus ++++++++++++++++++++++++++++++

Polyp stromal X X X Vagina +

Leiomyoma

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

446666777777777777777777777777 

### Number of Days on Study 052279000112233333333333333333 130041067992601111111111111233

222222222232322222222223333222 

### Carcass ID Number 956958767609196667777780000955 960647066508077891234596789512

### 163 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + I + + + + + + + + 59 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular adenoma X 1 Mesentery + + + + + + 9 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Tooth 2


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 2 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pheochromocytoma benign X 2 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 1 Parathyroid gland + + + + M + + + + + + + + + + + + + + + + + M + + + + + + + 57 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Pars distalis, adenoma X X X X X X X X X 19 Pars intermedia, adenoma 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

C-cell, adenoma X X XXXXX 9 C-cell, carcinoma X 1


### General Body System None


### Genital System Clitoral gland + + + + + + M + + + + + + + + + + + + + + + + + + + + + + + 59

Adenoma X X X X X X X 12 Carcinoma X 2 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Polyp stromal X X X X X X X X 11 Vagina + 2

Leiomyoma X 1 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333333333444455555555555555

222222233333222222222222222222 T otal 

### Carcass ID Number 558888800000777855566668899999 Tissues/ 351234512345789078912346801234 T umors

### o-Nitrotoluene, NTP TR 504 164

Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + + + + + Lymph node, mandibular ++++++++++++++++++++++++++++++ Lymph node, mesenteric ++++++A+++++++++++++++++++++++ Spleen ++++++++++++++++++++++++++++++ Thymus + + + + + + + + +M+ + + + + + +M+ + + +M+ + + + + + +


### Integumentary System Mammary gland ++++++++++++++++++++++++++++++

Fibroadenoma X X X X X X X X X Fibroadenoma, multiple X X Skin ++++++++++++++++++++++++++++++

Subcutaneous tissue, fibroma X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle +

Lipoma X


### Nervous System Brain ++++++++++++++++++++++++++++++ Peripheral nerve + Spinal cord +


### Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye + Zymbal’s gland + +

Adenoma X Carcinoma X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear XXXXXXX XXX X X X X 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

446666777777777777777777777777 

### Number of Days on Study 052279000112233333333333333333 130041067992601111111111111233

222222222232322222222223333222 

### Carcass ID Number 956958767609196667777780000955 960647066508077891234596789512

### 165 o- Nitrotoluene, NTP TR 504

Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + 8 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Thymus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Fibroadenoma X X X X X X X X 17 Fibroadenoma, multiple X X X X 6 Skin + + + + + + + + + + + + + + + M + + + + + + + + + + + + + + 59

Subcutaneous tissue, fibroma X X 3


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle 1

Lipoma 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Peripheral nerve 1 Spinal cord 1


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye + + 3 Zymbal’s gland 2

Adenoma 1 Carcinoma 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X X X X X X X 21 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333333333444455555555555555

222222233333222222222222222222 T otal 

### Carcass ID Number 558888800000777855566668899999 Tissues/ 351234512345789078912346801234 T umors

### o-Nitrotoluene, NTP TR 504 166

Alimentary System Esophagus + + + + + + + +M+ + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +

Polyp adenomatous X Intestine large, rectum ++++++++++++++++++++++++++++++

Polyp adenomatous X Intestine large, cecum + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Intestine small, ileum + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Liver + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +

Carcinoma, metastatic, adrenal cortex Sarcoma Mesentery Pancreas + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +

Acinus, adenoma X Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++

Carcinoma Adrenal medulla ++++++++++++++++++++++++++++++ Islets, pancreatic + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +

Adenoma Parathyroid gland + + + + + + + + + + + + + + + + + + + +M+ + + + + + + +M Pituitary gland ++++++++++++++++++++++++++++++

Pars distalis, adenoma X X X X X X X X XXXX XXXXX X Pars distalis, adenoma, multiple Pars intermedia, adenoma Thyroid gland ++++++++++++++++++++++++++++++

Bilateral, C-cell, adenoma X C-cell, adenoma X X C-cell, carcinoma


### General Body System None 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

445556666777777777777777777777 

### Number of Days on Study 290386889000133333333333333333 955129245258611111111113333333

333333333333333333333333333333 

### Carcass ID Number 436412153634355555666661112222 948189377734112345123456790123

### 167 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + I + + 58 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Polyp adenomatous 1 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Polyp adenomatous 1 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Carcinoma, metastatic, adrenal cortex X 1 Sarcoma X 1 Mesentery + 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Acinus, adenoma 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Carcinoma X 1 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Adenoma X 1 Parathyroid gland + + + + + M + + + + + + + + + + + M + + + + + + + + + + + + 56 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pars distalis, adenoma X X X X X X X X X X X X 30 Pars distalis, adenoma, multiple X X 2 Pars intermedia, adenoma X 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, C-cell, adenoma 1 C-cell, adenoma X X X 5 C-cell, carcinoma X XX 3


### General Body System None 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333444444445555555555555555

333333333333333333333333333333 T otal 

### Carcass ID Number 226667111133342223334444445555 Tissues/ 450690124568906780252356780689 T umors

### o-Nitrotoluene, NTP TR 504 168

Genital System Clitoral gland + + + + + + +M+ + + + + + + + + + + + + + + + + + +M+ +

Adenoma X X Carcinoma X X Bilateral, adenoma Ovary + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +

Granulosa-theca tumor malignant Uterus + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X X Sarcoma stromal Vagina


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + M Lymph node, mandibular ++++++++++++++++++++++++++++++ Lymph node, mesenteric + + + + + +M+ + + + + + + + + + + + + + + + + + + + +M+ Spleen + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +

Carcinoma, metastatic, adrenal cortex Thymus + M M +++++++++++++++++++++++++++


### Integumentary System Mammary gland ++++++++++++++++++++++++++++++

Fibroadenoma X X X X X Fibroadenoma, multiple X X X X X X X XXXXX XX XX X Skin ++++++++++++++++++++++++++++++

Basal cell adenoma X Pinna, melanoma malignant Subcutaneous tissue, fibroma


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle +


### Nervous System Brain ++++++++++++++++++++++++++++++ Peripheral nerve + + Spinal cord + +


### Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X Alveolar/bronchiolar adenoma,

multiple Sarcoma, metastatic, liver Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++ 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

445556666777777777777777777777 

### Number of Days on Study 290386889000133333333333333333 955129245258611111111113333333

333333333333333333333333333333 

### Carcass ID Number 436412153634355555666661112222 948189377734112345123456790123

### 169 o- Nitrotoluene, NTP TR 504

Genital System Clitoral gland + + + + + + + + + + + + + + + + M + + + + + + + + + + + + + 57

Adenoma X X X X X X 8 Carcinoma X X 4 Bilateral, adenoma X 1 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Granulosa-theca tumor malignant X 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Polyp stromal X X X X X 7 Sarcoma stromal X 1 Vagina + 1


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node ++ 4 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Carcinoma, metastatic, adrenal cortex X 1 Thymus I + + + + + + + I + + + + + + + + + + + + + + + + + + M + + 55


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Fibroadenoma X X X X X X X 12 Fibroadenoma, multiple X X X X X X X X X X X X X X X X X X 35 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Basal cell adenoma 1 Pinna, melanoma malignant X 1 Subcutaneous tissue, fibroma X X X 3


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Peripheral nerve 2 Spinal cord 2


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma 1 Alveolar/bronchiolar adenoma,

multiple X 1 Sarcoma, metastatic, liver X 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333444444445555555555555555

333333333333333333333333333333 T otal 

### Carcass ID Number 226667111133342223334444445555 Tissues/ 450690124568906780252356780689 T umors

### o-Nitrotoluene, NTP TR 504 170

Special Senses System Eye + + Zymbal’s gland +

Carcinoma X


### Urinary System Kidney + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + +


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear X X X 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

445556666777777777777777777777 

### Number of Days on Study 290386889000133333333333333333 955129245258611111111113333333

333333333333333333333333333333 

### Carcass ID Number 436412153634355555666661112222 948189377734112345123456790123

### 171 o- Nitrotoluene, NTP TR 504

Special Senses System Eye + 3 Zymbal’s gland 1

Carcinoma 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X X X 6 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 625 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333444444445555555555555555

333333333333333333333333333333 T otal 

### Carcass ID Number 226667111133342223334444445555 Tissues/ 450690124568906780252356780689 T umors

### o-Nitrotoluene, NTP TR 504 172



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

345555566666777777777777777777 

### Number of Days on Study 732268935789000000000333333333 023596766258667788999000111111

334343333443443344444444444444 

### Carcass ID Number 990718989128117702223222001111 366192192721042391670345781235



### Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon ++++++++++++++++++++++++++++++ Intestine large, rectum ++++++++++++++++++++++++++++++ Intestine large, cecum ++++++++++++++++++++++++++++++ Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + +M+ + Intestine small, ileum ++++++++++++++++++++++++++++++ Liver ++++++++++++++++++++++++++++++

Hepatocellular adenoma X Mesentery + Pancreas ++++++++++++++++++++++++++++++

Acinus, adenoma Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++

Squamous cell papilloma Stomach, glandular ++++++++++++++++++++++++++++++ Tongue +

Squamous cell papilloma X


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++ Islets, pancreatic ++++++++++++++++++++++++++++++

Adenoma Carcinoma X Parathyroid gland + + + + + + + + + + + + +M+M+ + + + + + + + + + + + + + Pituitary gland ++++++++++++++++++++++++++++++

Pars distalis, adenoma X X X X X X X X X X X Thyroid gland ++++++++++++++++++++++++++++++

Bilateral, C-cell, adenoma X C-cell, adenoma X X C-cell, carcinoma X Follicular cell, adenoma X Follicular cell, carcinoma


### General Body System None

### 173 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + I + + + + + + + + + + + + + + + + 59 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, ileum + + + + + + + + + M + + + + + + + + + + + + + + + + + + + + 59 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular adenoma 1 Mesentery + + 3 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Acinus, adenoma X 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Squamous cell papilloma X 1 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Tongue 1

Squamous cell papilloma 1


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 1 Carcinoma 1 Parathyroid gland + + + + + + + + + M + + + + + + + M + + + + M + + + + + + + 55 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pars distalis, adenoma X X X X X X X X X X X X X X X 26 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Bilateral, C-cell, adenoma 1 C-cell, adenoma 2 C-cell, carcinoma XX 3 Follicular cell, adenoma 1 Follicular cell, carcinoma X 1


### General Body System None 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 111333333333344444444444455555

444333333344433333333333344444 T otal 

### Carcass ID Number 112888999902277777788889900000 Tissues/ 680678078908945678903454512345 T umors

### o-Nitrotoluene, NTP TR 504 174

Genital System Clitoral gland + + + + + + + + M + + + + + + + + MM + + + + + + + M + + +

Adenoma X Carcinoma X Ovary ++++++++++++++++++++++++++++++ Uterus ++++++++++++++++++++++++++++++

Polyp stromal X X X Vagina +

Fibroma Squamous cell papilloma


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + +

Deep cervical, carcinoma, metastatic,

thyroid gland Lymph node, mandibular ++++++++++++++++++++++++++++++ Lymph node, mesenteric ++++++++++++++++++++++++++++++ Spleen ++++++++++++++++++++++++++++++ Thymus ++++++++++++++++++++++++++++++


### Integumentary System Mammary gland + M ++++++++++++++++++++++++++++

Carcinoma X Fibroadenoma X X X X X X Fibroadenoma, multiple X XXXX XXXXXX XXXXXXXX Skin ++++++++++++++++++++++++++++++

Subcutaneous tissue, fibroma X X X XXXX X Subcutaneous tissue, fibrosarcoma X X Subcutaneous tissue, hemangiosarcoma Subcutaneous tissue, neural crest tumor X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle

Rhabdomyosarcoma


### Nervous System Brain ++++++++++++++++++++++++++++++


### Respiratory System Lung ++++++++++++++++++++++++++++++ Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye + + 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

345555566666777777777777777777 

### Number of Days on Study 732268935789000000000333333333 023596766258667788999000111111

334343333443443344444444444444 

### Carcass ID Number 990718989128117702223222001111 366192192721042391670345781235

### 175 o- Nitrotoluene, NTP TR 504

Genital System Clitoral gland + + + + + + M + + + + + + + + + + + + + + + + + M + + + + + 54

Adenoma X X 3 Carcinoma X X 3 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Polyp stromal X X X X X X X X X X X X 15 Vagina + + 3

Fibroma X 1 Squamous cell papilloma X 1


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + 4

Deep cervical, carcinoma, metastatic,

thyroid gland X 1 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + M + 59 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + M + + + 59 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Thymus + + + + + + + + I + M + + + + + + + + + + + + + + + + + + + 58


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Carcinoma 1 Fibroadenoma X X X X X X X 13 Fibroadenoma, multiple XXXX XX XXXX X X X X X X X X X X 39 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Subcutaneous tissue, fibroma X X X X X X X X X X 18 Subcutaneous tissue, fibrosarcoma X X 4 Subcutaneous tissue, hemangiosarcoma X 1 Subcutaneous tissue, neural crest tumor 1


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle + 1

Rhabdomyosarcoma X 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye 2 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 111333333333344444444444455555

444333333344433333333333344444 T otal 

### Carcass ID Number 112888999902277777788889900000 Tissues/ 680678078908945678903454512345 T umors

### o-Nitrotoluene, NTP TR 504 176

Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++

Papilloma


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear X X X Lymphoma malignant 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

345555566666777777777777777777 

### Number of Days on Study 732268935789000000000333333333 023596766258667788999000111111

334343333443443344444444444444 

### Carcass ID Number 990718989128117702223222001111 366192192721042391670345781235

### 177 o- Nitrotoluene, NTP TR 504

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Papilloma X 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X 4 Lymphoma malignant X 1 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 111333333333344444444444455555

444333333344433333333333344444 T otal 

### Carcass ID Number 112888999902277777788889900000 Tissues/ 680678078908945678903454512345 T umors

### o-Nitrotoluene, NTP TR 504 178

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Intestine large, colon + + + + + + + + + + + +M+ + + + + + + + + + + + + + + + +

Polyp adenomatous Intestine large, rectum + + + + + + + +M+ + + + + + + + + + + + + + + + + + + + + Intestine large, cecum ++++++++++++A+++++++++++++++++ Intestine small, duodenum ++++++++++++++++++++++++++++++ Intestine small, jejunum ++++++++++A+A+++++++++++++A+++ Intestine small, ileum + + + + + + + + + +A+M+ + + + + + + + + + + + + + + + + Liver ++++++++++++++++++++++++++++++

Hepatocellular adenoma X X Hepatocellular adenoma, multiple Mesentery

Lipoma Pancreas ++++++++++++++++++++++++++++++ Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular ++++++++++++++++++++++++++++++ Tongue

Squamous cell carcinoma


### Cardiovascular System Blood vessel ++++++++++++++++++++++++++++++ Heart ++++++++++++++++++++++++++++++

Schwannoma malignant


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++

Adenoma X Adrenal medulla + M ++++++++++++++++++++++++++++

Pheochromocytoma benign X Islets, pancreatic ++++++++++++++++++++++++++++++ Parathyroid gland + + + + + + + + + + +M+ + + + + + + + +M+ + + + + + + + Pituitary gland ++++++++++++++++++++++++++++++

Pars distalis, adenoma X X X X X X X X X X X X Thyroid gland ++++++++++++++++++++++++++++++

C-cell, adenoma X X C-cell, carcinoma X Follicular cell, carcinoma X


### General Body System None 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

445566666666666666777777777777 

### Number of Days on Study 686803333666788899000000002222 489632669335655718268888892999

444444444444444444444444444444 

### Carcass ID Number 765387677464433446363355558888 525322836274714536795607941678

### 179 o- Nitrotoluene, NTP TR 504



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

777777777777777777777777777777 

### Number of Days on Study 223333333333333333333333333333 991113333344444444444444445555

444444444444444444444444444444 T otal 

### Carcass ID Number 898883347734444555556666677778 Tissues/ 903458901421689123680134507890 T umors



### Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Polyp adenomatous X 1 Intestine large, rectum I + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular adenoma X X X 5 Hepatocellular adenoma, multiple X 1 Mesentery + + + 3

Lipoma X X2 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Tongue + 1

Squamous cell carcinoma X 1


### Cardiovascular System Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Schwannoma malignant X 1


### Endocrine System Adrenal cortex + + + M + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Adenoma 1 Adrenal medulla + + + M + + + + + + + + + + + + + + + + + + + + + + + + + + 58

Pheochromocytoma benign 1 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Parathyroid gland + + + + + + + + + + + + M + M + + + + + + + + + + + + + + + 56 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pars distalis, adenoma X X X X X X X X 20 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

C-cell, adenoma X X X 5 C-cell, carcinoma 1 Follicular cell, carcinoma 1


### General Body System None

### o-Nitrotoluene, NTP TR 504 180

Genital System Clitoral gland + + + + + + MM + + M + + + + + + + + + + MM + M + + + + +

Adenoma Ovary ++++++++++++++++++++++++++++++

Granulosa cell tumor malignant X Uterus ++++++++++++++++++++++++++++++

Hemangioma X Polyp stromal X X X X X X X Sarcoma stromal Vagina + + +

Granular cell tumor benign Hemangioma X Leiomyosarcoma Squamous cell papilloma


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + M Lymph node, mandibular ++++++++++++++++++++++++++++++ Lymph node, mesenteric ++++++++++++I+++++++++++++++++ Spleen ++++++++++++A+++++++++++++++++ Thymus ++++++++++++++++++++++++++++++


### Integumentary System Mammary gland ++++++++++++++++++++++++++++++

Adenoma X Carcinoma X Fibroadenoma X X X X X X X X Fibroadenoma, multiple X X X X X X XXXXXXXXXXXX Skin ++++++++++++++++++++++++++++++

Pinna, neural crest tumor X Sebaceous gland, adenoma X Subcutaneous tissue, fibroma X X X X X X Subcutaneous tissue, fibroma, multiple X Subcutaneous tissue, fibrosarcoma Subcutaneous tissue, fibrosarcoma, multiple


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle +


### Nervous System Brain ++++++++++++++++++++++++++++++

Astrocytoma malignant X 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

445566666666666666777777777777 

### Number of Days on Study 686803333666788899000000002222 489632669335655718268888892999

444444444444444444444444444444 

### Carcass ID Number 765387677464433446363355558888 525322836274714536795607941678

### 181 o- Nitrotoluene, NTP TR 504



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

777777777777777777777777777777 

### Number of Days on Study 223333333333333333333333333333 991113333344444444444444445555

444444444444444444444444444444 T otal 

### Carcass ID Number 898883347734444555556666677778 Tissues/ 903458901421689123680134507890 T umors



### Genital System Clitoral gland + + + + + + M + + + + + + + + + + + + + + + + + + + + + + + 53

Adenoma X X X 3 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Granulosa cell tumor malignant 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangioma 1 Polyp stromal X X X X X X X X X 16 Sarcoma stromal X 1 Vagina + + + 6

Granular cell tumor benign X 1 Hemangioma 1 Leiomyosarcoma X 1 Squamous cell papilloma X 1


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + 4 Lymph node, mandibular + + + + + + + + + + + + + M + + + + + + + + + + + + + + + + 59 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Thymus + + + + + + + + + + + + + + + + + + + + M + M + + + + + + + 58


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma 1 Carcinoma 1 Fibroadenoma X X X X X X 14 Fibroadenoma, multiple X X X X X X X X X X X X X X X X X X X X X X X X 42 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pinna, neural crest tumor 1 Sebaceous gland, adenoma 1 Subcutaneous tissue, fibroma X X X X X X X X X X X X X 19 Subcutaneous tissue, fibroma, multiple 1 Subcutaneous tissue, fibrosarcoma X X 2 Subcutaneous tissue, fibrosarcoma, multiple X 1


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Astrocytoma malignant 1

### o-Nitrotoluene, NTP TR 504 182

Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X Alveolar/bronchiolar carcinoma Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye + Zymbal’s gland +

Adenoma Carcinoma X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder ++++++++++++++++++++++++++++++

Papilloma Squamous cell papilloma, multiple


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear X X X X Lymphoma malignant X 

### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

445566666666666666777777777777 

### Number of Days on Study 686803333666788899000000002222 489632669335655718268888892999

444444444444444444444444444444 

### Carcass ID Number 765387677464433446363355558888 525322836274714536795607941678

### 183 o- Nitrotoluene, NTP TR 504



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of o -Nitrotoluene: 2,000 ppm

777777777777777777777777777777 

### Number of Days on Study 223333333333333333333333333333 991113333344444444444444445555

444444444444444444444444444444 T otal 

### Carcass ID Number 898883347734444555556666677778 Tissues/ 903458901421689123680134507890 T umors



### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X X3 Alveolar/bronchiolar carcinoma X 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye 1 Zymbal’s gland + + 3

Adenoma X 1 Carcinoma X 2


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Papilloma X 1 Squamous cell papilloma, multiple X 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X 5 Lymphoma malignant 1

### o-Nitrotoluene, NTP TR 504 184



### T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Clitoral Gland: Adenoma Overall rate a 12/59 (20%) 9/57 (16%) 3/54 (6%) 3/53 (6%) Adjusted rate b 21.5% 17.0% 6.2% 6.3% Terminal rate c 11/46 (24%) 9/45 (20%) 3/36 (8%) 3/32 (9%) First incidence (days) 726 729 (T) 729 (T) 729 (T) Poly-3 test d P=0.005N P=0.366N P=0.025N P=0.027N



### Clitoral Gland: Carcinoma Overall rate 2/59 (3%) 4/57 (7%) 3/54 (6%) 0/53 (0%) Adjusted rate 3.6% 7.5% 6.2% 0.0% Terminal rate 1/46 (2%) 2/45 (4%) 2/36 (6%) 0/32 (0%) First incidence (days) 719 505 706 —e Poly-3 test P=0.229N P=0.319 P=0.434 P=0.275N



### Clitoral Gland: Adenoma or Carcinoma Overall rate 14/59 (24%) 13/57 (23%) 6/54 (11%) 3/53 (6%) Adjusted rate 25.0% 24.3% 12.4% 6.3% Terminal rate 12/46 (26%) 11/45 (24%) 5/36 (14%) 3/32 (9%) First incidence (days) 719 505 706 729 (T) Poly-3 test P=0.003N P=0.550N P=0.083N P=0.009N


### Liver: Hepatocellular Adenoma Overall rate 1/60 (2%) 0/59 (0%) 1/60 (2%) 6/60 (10%) Adjusted rate 1.8% 0.0% 1.9% 11.2% Terminal rate 1/47 (2%) 0/47 (0%) 1/39 (3%) 4/33 (12%) First incidence (days) 729 (T) — 729 (T) 687 Poly-3 test P=0.005 P=0.507N P=0.748 P=0.048


### Lung: Alveolar/bronchiolar Adenoma Overall rate 1/60 (2%) 2/60 (3%) 0/60 (0%) 3/60 (5%) Adjusted rate 1.8% 3.6% 0.0% 5.6% Terminal rate 1/47 (2%) 1/47 (2%) 0/39 (0%) 3/33 (9%) First incidence (days) 729 (T) 705 — 729 (T) Poly-3 test P=0.258 P=0.493 P=0.511N P=0.283


### Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 1/60 (2%) 2/60 (3%) 0/60 (0%) 4/60 (7%) Adjusted rate 1.8% 3.6% 0.0% 7.5% Terminal rate 1/47 (2%) 1/47 (2%) 0/39 (0%) 4/33 (12%) First incidence (days) 729 (T) 705 — 729 (T) Poly-3 test P=0.126 P=0.493 P=0.511N P=0.161


### Mammary Gland: Fibroadenoma Overall rate 23/60 (38%) 47/60 (78%) 52/60 (87%) 56/60 (93%) Adjusted rate 40.0% 82.0% 91.7% 96.2% Terminal rate 18/47 (38%) 39/47 (83%) 36/39 (92%) 33/33 (100%) First incidence (days) 620 505 525 464 Poly-3 test P<0.001 P<0.001 P<0.001 P<0.001


### Mammary Gland: Fibroadenoma or Carcinoma Overall rate 23/60 (38%) 47/60 (78%) 52/60 (87%) 57/60 (95%) f Adjusted rate 40.0% 82.0% 91.7% 97.1% Terminal rate 18/47 (38%) 39/47 (83%) 36/39 (92%) 33/33 (100%) First incidence (days) 620 505 525 464 Poly-3 test P<0.001 P<0.001 P<0.001 P<0.001

### 185 o- Nitrotoluene, NTP TR 504



### T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Pituitary Gland (Pars Distalis): Adenoma Overall rate 19/59 (32%) 32/60 (53%) 26/60 (43%) 20/60 (33%) Adjusted rate 33.8% 55.7% 47.4% 36.2% Terminal rate 16/46 (35%) 25/47 (53%) 19/39 (49%) 10/33 (30%) First incidence 691 531 636 569 Poly-3 test P=0.484N P=0.014 P=0.100 P=0.473


### Skin (Subcutaneous Tissue): Fibroma Overall rate 3/60 (5%) 3/60 (5%) 18/60 (30%) 20/60 (33%) Adjusted rate 5.3% 5.4% 32.7% 36.9% Terminal rate 3/47 (6%) 3/47 (6%) 15/39 (39%) 14/33 (42%) First incidence (days) 729 (T) 729 (T) 432 663 Poly-3 test P<0.001 P=0.652 P<0.001 P<0.001


### Skin (Subcutaneous Tissue): Fibrosarcoma Overall rate 0/60 (0%) 0/60 (0%) 4/60 (7%) 3/60 (5%) Adjusted rate 0.0% 0.0% 7.3% 5.6% Terminal rate 0/47 (0%) 0/47 (0%) 2/39 (5%) 3/33 (9%) First incidence (days) — — 569 729 (T) Poly-3 test P=0.018 —g P=0.056 P=0.109


### Skin (Subcutaneous Tissue): Fibroma or Fibrosarcoma Overall rate 3/60 (5%) 3/60 (5%) 21/60 (35%) 22/60 (37%) Adjusted rate 5.3% 5.4% 37.6% 40.6% Terminal rate 3/47 (6%) 3/47 (6%) 16/39 (41%) 16/33 (49%) First incidence (days) 729 (T) 729 (T) 432 663 Poly-3 test P<0.001 P=0.652 P<0.001 P<0.001


### Thyroid Gland (C-Cell): Adenoma Overall rate 9/60 (15%) 6/60 (10%) 3/60 (5%) 5/60 (8%) Adjusted rate 15.8% 10.6% 5.4% 9.3% Terminal rate 9/47 (19%) 5/47 (11%) 0/39 (0%) 3/33 (9%) First incidence (days) 729 (T) 495 370 663 Poly-3 test P=0.109N P=0.296N P=0.068N P=0.227N


### Thyroid Gland (C-Cell): Carcinoma Overall rate 1/60 (2%) 3/60 (5%) 3/60 (5%) 1/60 (2%) Adjusted rate 1.8% 5.4% 5.6% 1.9% Terminal rate 1/47 (2%) 3/47 (6%) 3/39 (8%) 0/33 (0%) First incidence (days) 729 (T) 729 (T) 729 (T) 708 Poly-3 test P=0.556 P=0.299 P=0.286 P=0.746


### Thyroid Gland (C-Cell): Adenoma or Carcinoma Overall rate 10/60 (17%) 9/60 (15%) 6/60 (10%) 6/60 (10%) Adjusted rate 17.6% 16.0% 10.8% 11.1% Terminal rate 10/47 (21%) 8/47 (17%) 3/39 (8%) 3/33 (9%) First incidence (days) 729 (T) 495 370 663 Poly-3 test P=0.142N P=0.508N P=0.226N P=0.244N


### Uterus: Stromal Polyp Overall rate 11/60 (18%) 7/60 (12%) 15/60 (25%) 16/60 (27%) Adjusted rate 19.3% 12.5% 27.5% 29.2% Terminal rate 11/47 (23%) 6/47 (13%) 13/39 (33%) 9/33 (27%) First incidence (days) 729 (T) 582 523 586 Poly-3 test P=0.043 P=0.229N P=0.212 P=0.159

### o-Nitrotoluene, NTP TR 504 186



### T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Uterus: Stromal Polyp or Stromal Sarcoma Overall rate 11/60 (18%) 8/60 (13%) 15/60 (25%) 17/60 (28%) Adjusted rate 19.3% 14.2% 27.5% 31.0% Terminal rate 11/47 (23%) 7/47 (15%) 13/39 (33%) 10/33 (30%) First incidence (days) 729 (T) 582 523 586 Poly-3 test P=0.032 P=0.318N P=0.212 P=0.113


### Zymbal’s Gland: Adenoma or Carcinoma Overall rate 2/60 (3%) 1/60 (2%) 0/60 (0%) 3/60 (5%) Adjusted rate 3.5% 1.8% 0.0% 5.6% Terminal rate 1/47 (2%) 1/47 (2%) 0/39 (0%) 2/33 (6%) First incidence (days) 719 729 (T) — 632 Poly-3 test P=0.388 P=0.507N P=0.251N P=0.474


### All Organs: Mononuclear Cell Leukemia Overall rate 21/60 (35%) 6/60 (10%) 4/60 (7%) 5/60 (8%) Adjusted rate 35.6% 10.6% 7.3% 9.1% Terminal rate 11/47 (23%) 3/47 (6%) 1/39 (3%) 1/33 (3%) First incidence (days) 453 429 523 488 Poly-3 test P=0.001N P=0.001N P=0.001N P=0.001N


### All Organs: Benign Neoplasms Overall rate 47/60 (78%) 57/60 (95%) 59/60 (98%) 59/60 (98%) Adjusted rate 81.5% 96.3% 99.2% 99.5% Terminal rate 41/47 (87%) 45/47 (96%) 39/39 (100%) 33/33 (100%) First incidence (days) 620 495 370 464 Poly-3 test P<0.001 P=0.008 P<0.001 P<0.001


### All Organs: Malignant Neoplasms Overall rate 24/60 (40%) 16/60 (27%) 18/60 (30%) 20/60 (33%) Adjusted rate 40.6% 27.9% 32.1% 35.5% Terminal rate 13/47 (28%) 12/47 (26%) 11/39 (28%) 10/33 (30%) First incidence (days) 453 429 523 488 Poly-3 test P=0.369N P=0.104N P=0.224N P=0.353N


### All Organs: Benign or Malignant Neoplasms Overall rate 54/60 (90%) 58/60 (97%) 60/60 (100%) 60/60 (100%) Adjusted rate 91.3% 96.7% 100.0% 100.0% Terminal rate 42/47 (89%) 45/47 (96%) 39/39 (100%) 33/33 (100%) First incidence (days) 453 429 370 464 Poly-3 test P=0.003 P=0.195 P=0.026 P=0.026

(T)Terminal sacrifice a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for c litoral gland, liver, lung, pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals th at do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal group f A single adenoma occurred in an animal that also had a fibroadenoma. g Value of statistic cannot be computed.

### 187 o- Nitrotoluene, NTP TR 504



### T ABLE 

### B4a Historical Incidence of Subcutaneous Skin Neoplasms in Control Female F344/N Rats

Incidence in Controls Study Fibroma Fibrosarcoma Fibroma or Fibrosarcoma

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 1/100 0/100 1/100
p,p N -Dichlorodiphenyl sulfone (feed) 0/50 1/50 1/50 Indium phosphide (inhalation) 3/50 0/50 3/50 60-Hz Magnetic fields (whole body exposure) 4/100 1/100 5/100 Methacrylonitrile (gavage) 0/50 0/50 0/50 Naphthalene (inhalation) 0/49 0/49 0/49

o -Nitrotoluene (feed) 3/60 0/60 3/60
p-Nitrotoluene (feed) 0/50 1/50 1/50 Riddelliine (gavage) 1/50 1/50 2/50 Sodium nitrite (drinking water) 1/50 0/50 1/50 Vanadium pentoxide (inhalation) 1/50 0/50 1/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 14/659 (2.1%) 4/659 (0.6%) 18/659 (2.7%) Mean ± standard deviation 2.0% ± 2.1% 0.6% ± 0.9% 2.6% ± 2.1% Range 0%-6% 0%-2% 0%-6%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 1/50 0/50 1/50 2,2-Bis(bromomethyl)-1,3-propanediol 1/50 0/50 1/50 Butyl benzyl phthalate 1/50 0/50 1/50 D&C Yellow No. 11 1/50 0/50 1/50 Emodin 2/50 0/50 2/50

o-Nitroanisole 0/50 0/50 0/50
p-Nitrobenzoic acid 2/50 0/50 2/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 15/1,001 (1.5%) 4/1,001 (0.4%) 19/1,001 (1.9%) Mean ± standard deviation 1.5% ± 1.6% 0.4% ± 0.8% 1.9% ± 2.0% Range 0%-4% 0%-2% 0%-6%

a Data as of January 18, 2001 b Data as of December 21, 1999

### o-Nitrotoluene, NTP TR 504 188



### T ABLE 

### B4b Historical Incidence of Mammary Gland Neoplasms in Control Female F344/N Rats

Incidence in Controls Study Fibroadenoma Carcinoma Fibroadenoma or Carcinoma

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 53/100 0/100 53/100
p,p N -Dichlorodiphenyl sulfone (feed) 15/50 1/50 15/50 Indium phosphide (inhalation) 20/50 0/50 20/50 60-Hz Magnetic fields (whole body exposure) 56/100 2/100 58/100 Methacrylonitrile (gavage) 21/50 3/50 23/50 Naphthalene (inhalation) 17/49 3/49 18/49

o -Nitrotoluene (feed) 23/60 0/60 23/60
p-Nitrotoluene (feed) 14/50 1/50 14/50 Riddelliine (gavage) 28/50 2/50 30/50 Sodium nitrite (drinking water) 21/50 1/50 22/50 Vanadium pentoxide (inhalation) 16/50 2/50 18/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 284/659 (43.1%) 15/659 (2.3%) 294/659 (44.6%) Mean ± standard deviation 41.1% ± 10.1% 2.6% ± 2.2% 42.9% ± 10.7% Range 28%-56% 0% ± 6% 28%-60%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 12/50 0/50 12/50 2,2-Bis(bromomethyl)-1,3-propanediol 25/50 4/50 27/50 Butyl benzyl phthalate 28/50 2/50 29/50 D&C Yellow No. 11 21/50 4/50 24/50 Emodin 23/50 0/50 23/50

o-Nitroanisole 17/50 2/50 18/50
p-Nitrobenzoic acid 22/50 2/50 24/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 431/1,001 (43.1%) 32/1,001 (3.2%) 453/1,001 (45.3%) Mean ± standard deviation 43.1% ± 10.7% 3.2% ± 2.6% 45.3% ± 11.4% Range 24%-60% 0%-8% 24%-62%

a Data as of January 18, 2001 b Data as of December 21, 1999

### 189 o- Nitrotoluene, NTP TR 504



### T ABLE 

### B4c Historical Incidence of Hepatocellular Adenoma in Control Female F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 0/100
p,p N -Dichlorodiphenyl sulfone (feed) 0/50 Indium phosphide (inhalation) 0/50 60-Hz Magnetic fields (whole body exposure) 0/100 Methacrylonitrile (gavage) 1/50 Naphthalene (inhalation) 0/49

o -Nitrotoluene (feed) 1/60
p-Nitrotoluene (feed) 0/50 Riddelliine (gavage) 1/50 Sodium nitrite (drinking water) 0/50 Vanadium pentoxide (inhalation) 1/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 4/659 (0.6%) Mean ± standard deviation 0.7% ± 1.0% Range 0%-2%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 1/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/50 Butyl benzyl phthalate 0/50 D&C Yellow No. 11 0/50 Emodin 0/49

o-Nitroanisole 0/50
p-Nitrobenzoic acid 2/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 4/1,000 (0.4%) Mean ± standard deviation 0.4% ± 1.1% Range 0%-4%

a Data as of January 18, 2001 b Data as of December 21, 1999

### o-Nitrotoluene, NTP TR 504 190



### T ABLE 

### B4d Historical Incidence of Mononuclear Cell Leukemia in Control Female F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Citral (feed) 24/100
p,p N -Dichlorodiphenyl sulfone (feed) 8/50 Indium phosphide (inhalation) 14/50 60-Hz Magnetic fields (whole body exposure) 20/100 Methacrylonitrile (gavage) 21/50 Naphthalene (inhalation) 16/49

o -Nitrotoluene (feed) 21/60
p-Nitrotoluene (feed) 13/50 Riddelliine (gavage) 12/50 Sodium nitrite (drinking water) 15/50 Vanadium pentoxide (inhalation) 21/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 185/659 (28.1%) Mean ± standard deviation 29.1% ± 8.4% Range 16%-42%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 9/50 2,2-Bis(bromomethyl)-1,3-propanediol 15/50 Butyl benzyl phthalate 21/50 D&C Yellow No. 11 16/50 Emodin 14/50

o-Nitroanisole 14/50
p-Nitrobenzoic acid 17/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 293/1,001 (29.3%) Mean ± standard deviation 29.3% ± 7.6% Range 16%-42%

a Data as of January 18, 2001; includes data for lymphocytic, monocytic, and undifferentiated leukemia b Data as of December 21, 1999; includes data for lymphocytic, monocytic, and undifferentiated leukemia

### 191 o- Nitrotoluene, NTP TR 504



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of o-Nitrotoluene a



### 0 ppm 625 ppm 1,250 ppm 2,000 ppm

Disposition Summary Animals initially in study 60 60 60 60 Early deaths

Moribund 10 11 19 22 Natural deaths 3 2 2 5 Survivors

Died last week of study 2 1 Terminal sacrifice 45 46 39 33

Animals examined microscopically 60 60 60 60



### Alimentary System Intestine large, colon (58) (59) (60) (59)

Crypt, diverticulum 1 (2%) Intestine large, rectum (60) (60) (60) (58)

Fibrosis 1 (2%) Intestine large, cecum (58) (59) (60) (59)

Edema 1 (2%) 1 (2%) Liver (60) (59) (60) (60)

Angiectasis 2 (3%) 3 (5%) 4 (7%) 4 (7%) Basophilic focus 51 (85%) 56 (95%) 60 (100%) 54 (90%) Clear cell focus 16 (27%) 30 (51%) 28 (47%) 33 (55%) Degeneration, cystic 1 (2%) 2 (3%) Eosinophilic focus 5 (8%) 12 (20%) 25 (42%) 32 (53%) Hematopoietic cell proliferation 2 (3%) 1 (2%) 3 (5%) Hemorrhage 1 (2%) 1 (2%) Hepatodiaphragmatic nodule 9 (15%) 13 (22%) 10 (17%) 7 (12%) Infiltration cellular, mixed cell 15 (25%) 11 (19%) 6 (10%) 12 (20%) Mixed cell focus 6 (10%) 9 (15%) 11 (18%) 28 (47%) Necrosis, focal 1 (2%) 3 (5%) 5 (8%) Regeneration, diffuse 1 (2%) Regeneration, focal 1 (2%) 2 (3%) Bile duct, hyperplasia 7 (12%) 2 (3%) Centrilobular, necrosis 3 (5%) 2 (3%) 2 (3%) Hepatocyte, vacuolization cytoplasmic 4 (7%) 2 (3%) 1 (2%) Kupffer cell, pigmentation 3 (5%) Mesentery (9) (1) (3) (3)

Accessory spleen 2 (22%) 1 (33%) 1 (33%) Fat, necrosis 7 (78%) 1 (100%) 2 (67%) Pancreas (60) (59) (60) (60)

Atrophy 13 (22%) 10 (17%) 7 (12%) 4 (7%) Acinus, hyperplasia, focal 1 (2%) 1 (2%) 2 (3%) 2 (3%) Salivary glands (60) (60) (60) (60)

Atrophy 2 (3%) 3 (5%) 9 (15%) 48 (80%) Stomach, forestomach (59) (59) (60) (60)

Erosion 1 (2%) 1 (2%) Perforation 1 (2%) Ulcer 1 (2%) 1 (2%) 1 (2%) Epithelium, hyperplasia 5 (8%) 3 (5%) 1 (2%) Stomach, glandular (60) (59) (60) (60)

Erosion 1 (2%) 1 (2%) 1 (2%) 3 (5%) Ulcer 2 (3%) 1 (2%) 1 (2%) Tooth (2)

Inflammation, chronic 1 (50%) Malformation 2 (100%)

a Number of animals examined microscopically at the site and the number of animals with lesion

### o-Nitrotoluene, NTP TR 504 192

Cardiovascular System Heart (60) (60) (60) (60)

Cardiomyopathy 22 (37%) 16 (27%) 13 (22%) 13 (22%) Thrombosis 1 (2%) Myocardium, fibrosis 1 (2%) 1 (2%) 1 (2%)



### Endocrine System Adrenal cortex (60) (60) (60) (59)

Accessory adrenal cortical nodule 8 (13%) 10 (17%) 8 (13%) 10 (17%) Degeneration, fatty 13 (22%) 11 (18%) 17 (28%) 11 (19%) Hyperplasia, diffuse 3 (5%) 1 (2%) 1 (2%) Hyperplasia, focal 9 (15%) 6 (10%) 14 (23%) 15 (25%) Hypertrophy, focal 6 (10%) 7 (12%) 6 (10%) 7 (12%) Necrosis 1 (2%) Adrenal medulla (60) (60) (60) (58)

Hyperplasia 8 (13%) 2 (3%) 3 (5%) 3 (5%) Pituitary gland (59) (60) (60) (60)

Pars distalis, angiectasis 6 (10%) 5 (8%) 4 (7%) 4 (7%) Pars distalis, cyst 22 (37%) 20 (33%) 22 (37%) 24 (40%) Pars distalis, hyperplasia, focal 12 (20%) 6 (10%) 12 (20%) 9 (15%) Pars intermedia, angiectasis 2 (3%) 1 (2%) Pars intermedia, cyst 3 (5%) 1 (2%) 1 (2%) 1 (2%) Thyroid gland (60) (60) (60) (60)

Ultimobranchial cyst 1 (2%) 1 (2%) 1 (2%) C-cell, hyperplasia 22 (37%) 24 (40%) 22 (37%) 9 (15%) Follicle, cyst 2 (3%) 1 (2%) 1 (2%) Follicular cell, hyperplasia 1 (2%)



### General Body System None



### Genital System Clitoral gland (59) (57) (54) (53)

Atrophy 1 (2%) 3 (5%) 6 (11%) 25 (47%) Cyst 2 (3%) 4 (7%) 4 (7%) 2 (4%) Hyperplasia 5 (8%) 1 (2%) 4 (7%) 1 (2%) Inflammation, chronic 4 (7%) 4 (7%) 6 (11%) 5 (9%) Ovary (60) (59) (60) (60)

Angiectasis 2 (3%) Cyst 9 (15%) 8 (14%) 11 (18%) 6 (10%) Uterus (60) (59) (60) (60)

Angiectasis 1 (2%) Cyst 1 (2%) Hydrometra 7 (12%) 6 (10%) 1 (2%) 3 (5%) Hyperplasia, cystic 8 (13%) 6 (10%) 9 (15%) 8 (13%) Inflammation, chronic 2 (3%) 1 (2%) 1 (2%) Epithelium, hyperplasia 1 (2%) 

### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

### 193 o- Nitrotoluene, NTP TR 504

Hematopoietic System Bone marrow (60) (60) (60) (60)

Hyperplasia 2 (3%) 7 (12%) 15 (25%) 24 (40%) Infiltration cellular, histiocyte 2 (3%) 1 (2%) 1 (2%) Myelofibrosis 1 (2%) 2 (3%) Lymph node (8) (4) (4) (4)

Mediastinal, hemorrhage 1 (13%) 1 (25%) 1 (25%) 3 (75%) Mediastinal, pigmentation 6 (75%) 1 (25%) 1 (25%) 3 (75%) Renal, hemorrhage 1 (25%) Renal, pigmentation 1 (25%) Lymph node, mandibular (60) (60) (59) (59)

Ectasia 1 (2%) 3 (5%) 4 (7%) Hemorrhage 4 (7%) 4 (7%) 1 (2%) 4 (7%) Hyperplasia, lymphoid 3 (5%) 5 (8%) 6 (10%) 15 (25%) Pigmentation 31 (52%) 33 (55%) 30 (51%) 30 (51%) Lymph node, mesenteric (59) (58) (59) (59)

Hemorrhage 5 (8%) 3 (5%) 4 (7%) 5 (8%) Hyperplasia, lymphoid 1 (2%) Pigmentation 4 (7%) 1 (2%) 3 (5%) Spleen (60) (59) (60) (59)

Congestion 1 (2%) Fibrosis 1 (2%) 1 (2%) Hematopoietic cell proliferation 22 (37%) 38 (64%) 48 (80%) 48 (81%) Necrosis 2 (3%) Pigmentation 36 (60%) 44 (75%) 43 (72%) 46 (78%) Lymphoid follicle, atrophy 1 (2%) 1 (2%) Thymus (57) (55) (58) (58)

Cyst 1 (2%) 2 (3%) 2 (3%)



### Integumentary System Mammary gland (60) (60) (59) (60)

Dilatation 40 (67%) 30 (50%) 29 (49%) 24 (40%) Galactocele 1 (2%) Hyperplasia 14 (23%) 36 (60%) 30 (51%) 19 (32%) Skin (59) (60) (60) (60)

Angiectasis 1 (2%) Ulcer 1 (2%) Subcutaneous tissue, edema 1 (2%)



### Musculoskeletal System Bone (60) (60) (60) (60)

Cranium, osteopetrosis 2 (3%) 6 (10%) 6 (10%) 10 (17%) Femur, osteopetrosis 8 (13%) 9 (15%) 13 (22%) 16 (27%)



### Nervous System Brain (60) (60) (60) (60)

Compression 6 (10%) 10 (17%) 5 (8%) 3 (5%) Hydrocephalus 2 (3%) 4 (7%) 3 (5%) 

### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

### o-Nitrotoluene, NTP TR 504 194

Respiratory System Lung (60) (60) (60) (60)

Hemorrhage 2 (3%) 2 (3%) 2 (3%) 1 (2%) Infiltration cellular, histiocyte 52 (87%) 54 (90%) 53 (88%) 55 (92%) Alveolar epithelium, hyperplasia 6 (10%) 14 (23%) 16 (27%) 9 (15%) Alveolar epithelium, hyperplasia, atypical 1 (2%) Nose (60) (60) (60) (60)

Foreign body 2 (3%) 7 (12%) Inflammation, chronic 2 (3%) 1 (2%) 2 (3%) 8 (13%) Respiratory epithelium, hyperplasia 2 (3%) 3 (5%) Respiratory epithelium, metaplasia, squamous 1 (2%) 1 (2%)



### Special Senses System Eye (3) (3) (2) (1)

Cataract 1 (33%) 2 (67%) 1 (50%) 1 (100%) Hemorrhage 1 (33%) Inflammation, chronic 1 (33%) 1 (33%) Retina, degeneration 2 (67%) 2 (67%) 1 (50%) 1 (100%)



### Urinary System Kidney (60) (59) (60) (60)

Cyst 1 (2%) 1 (2%) Hydronephrosis 1 (2%) Infarct 2 (3%) 1 (2%) Inflammation, chronic 2 (3%) 1 (2%) 1 (2%) 1 (2%) Nephropathy 53 (88%) 45 (76%) 48 (80%) 52 (87%) Renal tubule, cytoplasmic alteration 1 (2%) 1 (2%) Renal tubule, degeneration, hyaline 9 (15%) 10 (17%) 4 (7%) 11 (18%) Renal tubule, necrosis 2 (3%) Renal tubule, pigmentation 9 (15%) 10 (17%) 7 (12%) 17 (28%) Transitional epithelium, hyperplasia 1 (2%) 1 (2%) 1 (2%) Urinary bladder (60) (59) (60) (60)

Transitional epithelium, hyperplasia 1 (2%) 

### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

### 195



# APPENDIX C SUMMARY OF LESIONS IN MALE MICE IN THE 2-YEAR FEED STUDY OF o-NITROTOLUENE

T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 T ABLE 

### C4a Historical Incidence of Hemangiosarcoma (All Sites) in Control Male B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 T ABLE 

### C4b Historical Incidence of Large Intestine (Cecum) Carcinoma in Control Male B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 T ABLE 

### C4c Historical Incidence of Hepatocellular Neoplasms in Control Male B6C3F 1 

### Mice . . . . . . . . 230 T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

### o-Nitrotoluene, NTP TR 504 196



### T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene a



### 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Disposition Summary Animals initially in study 60 60 60 60 Early deaths

Moribund 3 5 21 31 Natural deaths 5 21 39 29 Survivors

Terminal sacrifice 52 34

Animals examined microscopically 60 60 60 60



### Alimentary System Gallbladder (54) (42) (31) (35) Intestine large, colon (56) (52) (46) (54)

Muscularis, serosa, hemangiosarcoma 1 (2%) Intestine large, cecum (56) (49) (36) (44)

Carcinoma 12 (24%) 9 (25%) Intestine small, duodenum (55) (45) (40) (42)

Hemangiosarcoma 1 (2%) Intestine small, jejunum (56) (43) (35) (40)

Adenocarcinoma 1 (2%) Carcinoma 1 (2%) Intestine small, ileum (55) (40) (35) (39)

Carcinoma 1 (3%) Liver (60) (59) (57) (60)

Hemangiosarcoma 1 (2%) 1 (2%) 1 (2%) Hemangiosarcoma, multiple 3 (5%) Hemangiosarcoma, metastatic, skeletal muscle 1 (2%) Hepatoblastoma 1 (2%) 1 (2%) Hepatocellular carcinoma 9 (15%) 9 (15%) 4 (7%) 2 (3%) Hepatocellular carcinoma, multiple 3 (5%) 7 (12%) 1 (2%) Hepatocellular adenoma 15 (25%) 12 (20%) 1 (2%) Hepatocellular adenoma, multiple 3 (5%) 6 (10%) 2 (4%) Histiocytic sarcoma 1 (2%) Mesentery (12) (12) (42) (38)

Carcinoma, metastatic, intestine large, cecum 1 (8%) 1 (2%) Hemangiosarcoma 8 (67%) 30 (71%) 24 (63%) Hemangiosarcoma, multiple 8 (19%) 14 (37%) Hemangiosarcoma, metastatic, skeletal muscle 1 (2%) Oral mucosa (1)

Pharyngeal, squamous cell carcinoma 1 (100%) Pancreas (59) (59) (59) (58)

Hemangiosarcoma 1 (2%) Acinus, hemangiosarcoma 1 (2%) Salivary glands (60) (60) (60) (60) Stomach, forestomach (60) (60) (58) (60)

Squamous cell papilloma 2 (3%) Stomach, glandular (57) (54) (53) (55)



### Cardiovascular System Heart (60) (60) (60) (60)

Hemangiosarcoma 2 (3%) 2 (3%)

### 197 o- Nitrotoluene, NTP TR 504



### T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Endocrine System Adrenal cortex (60) (60) (58) (60)

Adenoma 1 (2%) Capsule, adenoma 1 (2%) Pituitary gland (53) (50) (50) (46)

Pars distalis, adenoma 1 (2%) Thyroid gland (59) (59) (59) (59)

C-cell, carcinoma 1 (2%) Follicular cell, adenoma 1 (2%)



### General Body System Tissue NOS (2) (2) (3)

Hemangiosarcoma 1 (33%) Pelvic, hemangiosarcoma 1 (50%) 1 (33%) Thoracic, sarcoma 1 (50%) Thoracic, sarcoma, multiple 1 (50%)



### Genital System Coagulating gland (2) (1)

Hemangiosarcoma 1 (50%) Preputial gland (60) (59) (59) (60)

Histiocytic sarcoma 1 (2%) Prostate (60) (59) (57) (58)

Hemangiosarcoma 4 (7%) Hemangiosarcoma, metastatic, mesentery 1 (2%) Testes (60) (60) (60) (60)

Interstitial cell, adenoma 1 (2%)



### Hematopoietic System Bone marrow (60) (60) (60) (60)

Hemangiosarcoma 1 (2%) Mast cell tumor malignant 1 (2%) Lymph node (3) (4) (7) (1)

Inguinal, hemangiosarcoma, metastatic,

skeletal muscle 1 (100%) Mediastinal, hemangiosarcoma, metastatic, mesentery 1 (14%) Mediastinal, sarcoma, metastatic, tissue NOS 1 (33%) Pancreatic, histiocytic sarcoma 1 (25%) Lymph node, mandibular (59) (55) (50) (54)

Hemangioma 1 (2%) Lymph node, mesenteric (60) (55) (56) (53)

Carcinoma, metastatic, intestine large, cecum 1 (2%) 3 (5%) Hemangiosarcoma, metastatic, spleen 1 (2%) Histiocytic sarcoma 1 (2%) Spleen (60) (60) (58) (60)

Hemangioma 1 (2%) Hemangiosarcoma 2 (3%) 3 (5%) Histiocytic sarcoma 1 (2%) Thymus (57) (52) (45) (54)

### o-Nitrotoluene, NTP TR 504 198



### T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Integumentary System Skin (60) (60) (60) (60)

Sebaceous gland, carcinoma 1 (2%) Subcutaneous tissue, fibrosarcoma 1 (2%) Subcutaneous tissue, hemangioma 1 (2%) Subcutaneous tissue, hemangiosarcoma 4 (7%) 8 (13%) 12 (20%) Subcutaneous tissue, hemangiosarcoma, multiple 8 (13%)



### Musculoskeletal System Bone (60) (60) (60) (60)

Cranium, osteoma 1 (2%) Skeletal muscle (1) (6) (35) (47)

Hemangiosarcoma 6 (100%) 25 (71%) 19 (40%) Hemangiosarcoma, multiple 8 (23%) 26 (55%)



### Nervous System Brain (60) (60) (60) (60)



### Respiratory System Lung (60) (60) (60) (60)

Alveolar/bronchiolar adenoma 9 (15%) 6 (10%) 4 (7%) Alveolar/bronchiolar carcinoma 3 (5%) 1 (2%) 2 (3%) Alveolar/bronchiolar carcinoma, multiple 2 (3%) Carcinoma, metastatic, intestine large, cecum 1 (2%) Hemangiosarcoma, metastatic, mesentery 3 (5%) 1 (2%) Hemangiosarcoma, metastatic, skin 3 (5%) 4 (7%) Hemangiosarcoma, metastatic, uncertain primary site 1 (2%) 1 (2%) 2 (3%) Hemangiosarcoma, metastatic, skeletal muscle 2 (3%) 8 (13%) Hepatocellular carcinoma, metastatic, liver 2 (3%) 1 (2%) Histiocytic sarcoma 1 (2%) Mediastinum, hemangiosarcoma 4 (7%) Nose (60) (60) (60) (60)



### Special Senses System Ear (1)

External ear, neural crest tumor, malignant 1 (100%) Harderian gland (5) (3) (3)

Adenoma 4 (80%) 1 (33%) 1 (33%) Carcinoma 1 (20%) 1 (33%) 1 (33%)



### Urinary System Kidney (58) (59) (58) (60)

Hemangiosarcoma, metastatic, mesentery 1 (2%) Histiocytic sarcoma 1 (2%) Capsule, hemangiosarcoma 1 (2%) Urinary bladder (59) (59) (59) (60)

Hemangiosarcoma 1 (2%)

### 199 o- Nitrotoluene, NTP TR 504



### T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Systemic Lesions Multiple organs b (60) (60) (60) (60)

Histiocytic sarcoma 1 (2%) 1 (2%) Lymphoma malignant 3 (5%) 1 (2%)



### Neoplasm Summary Total animals with primary neoplasms c 45 50 59 60

Total primary neoplasms 69 92 117 117 Total animals with benign neoplasms 30 24 8 2

Total benign neoplasms 36 28 9 2 Total animals with malignant neoplasms 26 41 59 60

Total malignant neoplasms 33 64 108 115 Total animals with metastatic neoplasms 1 5 13 17

Total metastatic neoplasms 2 6 18 17 Total animals with malignant neoplasms of uncertain primary site 1 1 2

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms

### o-Nitrotoluene, NTP TR 504 200

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Gallbladder + + + A M M A A ++++++++++++++++++++++ Intestine large, colon + + + A + A A A ++++++++++++++++++++++ Intestine large, rectum + + + A + A A A + + + + + + + + + + + + + + + + + + + +M+ Intestine large, cecum + + + A + A A A ++++++++++++++++++++++ Intestine small, duodenum + + A A + A A A ++++++++++++++++++++++

Hemangiosarcoma Intestine small, jejunum + + + A + A A A ++++++++++++++++++++++

Adenocarcinoma X Carcinoma Intestine small, ileum + + A A + A A A ++++++++++++++++++++++ Liver ++++++++++++++++++++++++++++++

Hemangiosarcoma X Hepatoblastoma X Hepatocellular carcinoma X X X X X X X Hepatocellular carcinoma, multiple X X X Hepatocellular adenoma X X X X X X X Hepatocellular adenoma, multiple X Mesentery + + + + + + Pancreas +++++A++++++++++++++++++++++++ Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular + + + A + A + A ++++++++++++++++++++++ Tooth + + + + + + +


### Cardiovascular System Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++

Adenoma X Capsule, adenoma X Adrenal medulla ++++++++++++++++++++++++++++++ Islets, pancreatic +++++A++++++++++++++++++++++++ Parathyroid gland + + + + + + + + + + + + + + +M+ + + + + + + +M+ + + + + Pituitary gland I + + + M + + + + +M+M+ + + +M+ + + + + + + + + + + +

Pars distalis, adenoma Thyroid gland +++++++A++++++++++++++++++++++

Follicular cell, adenoma X


### General Body System Tissue NOS + +

Thoracic, sarcoma X Thoracic, sarcoma, multiple X

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

255666777777777777777777777777 

### Number of Days on Study 699134112222222222222222222223 326879099999999999999999999990

000000000000000000000000000000 

### Carcass ID Number 251134250000011222233344444550 439813341348956127924704569275

### 201 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Gallbladder + + + + + + + + + + + + + + + + + + + + + + + I + + + + + + 54 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 55 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 55

Hemangiosarcoma X 1 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56

Adenocarcinoma 1 Carcinoma X 1 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 55 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma 1 Hepatoblastoma 1 Hepatocellular carcinoma X X 9 Hepatocellular carcinoma, multiple 3 Hepatocellular adenoma X X X X X X X X 15 Hepatocellular adenoma, multiple X X 3 Mesentery + + + + + + 12 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Tooth + + + + + + + 14


### Cardiovascular System Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma 1 Capsule, adenoma 1 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Parathyroid gland + + + + + + + M M + + + + + + + + + M + + + + + + + + + + + 55 Pituitary gland + + + + + + + + + + + + + + + M + + + + + + M + + + + + + + 53

Pars distalis, adenoma X 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Follicular cell, adenoma 1


### General Body System Tissue NOS 2

Thoracic, sarcoma 1 Thoracic, sarcoma, multiple 1 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 000000000000000000000000011111

000000000000000000000000000000 T otal 

### Carcass ID Number 001111112222333334444555603555 Tissues/ 670123470568356891278568020019 T umors

### o-Nitrotoluene, NTP TR 504 202

Genital System Epididymis ++++++++++++++++++++++++++++++ Preputial gland ++++++++++++++++++++++++++++++

Histiocytic sarcoma Prostate ++++++++++++++++++++++++++++++ Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++

Mast cell tumor malignant Lymph node + +

Mediastinal, sarcoma, metastatic,

tissue NOS X Lymph node, mandibular M +++++++++++++++++++++++++++++ Lymph node, mesenteric ++++++++++++++++++++++++++++++

Hemangiosarcoma, metastatic, spleen X Spleen ++++++++++++++++++++++++++++++

Hemangioma Hemangiosarcoma X X Thymus + + + M M +++++++++++++++++++++++++


### Integumentary System Mammary gland MMMMMM + MMMMMMMMMMMMMMMMMMMMMMM Skin ++++++++++++++++++++++++++++++


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle +


### Nervous System Brain ++++++++++++++++++++++++++++++ Peripheral nerve + Spinal cord + +


### Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X X X X Alveolar/bronchiolar carcinoma X X Alveolar/bronchiolar carcinoma,

multiple X X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++ 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

255666777777777777777777777777 

### Number of Days on Study 699134112222222222222222222223 326879099999999999999999999990

000000000000000000000000000000 

### Carcass ID Number 251134250000011222233344444550 439813341348956127924704569275

### 203 o- Nitrotoluene, NTP TR 504

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma X 1 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Mast cell tumor malignant X 1 Lymph node + 3

Mediastinal, sarcoma, metastatic,

tissue NOS 1 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma, metastatic, spleen 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangioma X 1 Hemangiosarcoma 2 Thymus + + + + + + + + + I + + + + + + + + + + + + + + + + + + + + 57


### Integumentary System Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Peripheral nerve 1 Spinal cord 2


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X X X X X 9 Alveolar/bronchiolar carcinoma X 3 Alveolar/bronchiolar carcinoma,

multiple 2 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 000000000000000000000000011111

000000000000000000000000000000 T otal 

### Carcass ID Number 001111112222333334444555603555 Tissues/ 670123470568356891278568020019 T umors

### o-Nitrotoluene, NTP TR 504 204

Special Senses System Ear +

External ear, neural crest tumor, malignant X Harderian gland +

Adenoma X Carcinoma


### Urinary System Kidney + + + A + A ++++++++++++++++++++++++ Urinary bladder +++++A++++++++++++++++++++++++


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Histiocytic sarcoma Lymphoma malignant X X 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

255666777777777777777777777777 

### Number of Days on Study 699134112222222222222222222223 326879099999999999999999999990

000000000000000000000000000000 

### Carcass ID Number 251134250000011222233344444550 439813341348956127924704569275

### 205 o- Nitrotoluene, NTP TR 504

Special Senses System Ear 1

External ear, neural crest tumor, malignant 1 Harderian gland + + + + 5

Adenoma X X X 4 Carcinoma X 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma X 1 Lymphoma malignant X 3 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 000000000000000000000000011111

000000000000000000000000000000 T otal 

### Carcass ID Number 001111112222333334444555603555 Tissues/ 670123470568356891278568020019 T umors

### o-Nitrotoluene, NTP TR 504 206

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Gallbladder AAAA + + + AAAM+ + + AMAAAA + A + + AA ++++ Intestine large, colon + + + A + A + M + A A + + M + A +++++++++A++++ Intestine large, rectum + + + A + + + + + +M+ +MA + + + + + + A + + + A + + + + Intestine large, cecum + + + A + A A + + A + + + +AA+AA+ + + +AAA+ + + +

Carcinoma X X X X X X X X X Intestine small, duodenum + A A A + A + A A + A + + + A A + A A + + A + + A A + + M + Intestine small, jejunum + A A A + A A A + A A + + AAAAAA+ +A+ +AA ++++ Intestine small, ileum + A A A + A + AAAA+ +AAAAAA+ +A+AAA+ +M+

Carcinoma Liver + + + A ++++++++++++++++++++++++++

Hemangiosarcoma X Hemangiosarcoma, multiple X X Hepatoblastoma X Hepatocellular carcinoma X X X X X X Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X X X X Hepatocellular adenoma, multiple X Histiocytic sarcoma X Mesentery + + + + + +

Carcinoma, metastatic,

intestine large, cecum X Hemangiosarcoma X X X Oral mucosa +

Pharyngeal, squamous cell carcinoma X Pancreas + + + A ++++++++++++++++++++++++++ Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++ Stomach, glandular + + + + + + +A+A+ + + + + + +A+ + + + +AAA+ + + + Tooth + +


### Cardiovascular System Heart ++++++++++++++++++++++++++++++

Hemangiosarcoma X


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++ Islets, pancreatic + + + A ++++++++++++++++++++++++++ Parathyroid gland + + M +++++++++++++++++++++++++++ Pituitary gland + + + +M+ + + + + + + + + I + +M+M+M+ + + + + + + + Thyroid gland ++++++++++++++++++++++++++++++

C-cell, carcinoma X 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

444445555555666666666677777777 

### Number of Days on Study 566682445779033445889911222222 103956259252403115680007689999

111000000001000000110100000000 

### Carcass ID Number 102889676790867796109178866788 930700376681690118057439474213

### 207 o- Nitrotoluene, NTP TR 504



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 222222222233333333333333333333 999999999900000000000000111111

001111111100000000000111000111 T otal 

### Carcass ID Number 890000111166677789999011789001 Tissues/ 542789237812558922359456486061 T umors



### Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Gallbladder + + + + M + + + + + + + + + + + + + + + + + + + + + + + + + 42 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 52 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 54 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 49

Carcinoma X X X 12 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 45 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 43 Intestine small, ileum + + + + + + + + + + + + + M + + + + + + + + + + + + + + + + 40

Carcinoma X 1 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Hemangiosarcoma 1 Hemangiosarcoma, multiple X 3 Hepatoblastoma 1 Hepatocellular carcinoma X X X 9 Hepatocellular carcinoma, multiple X X X X X X 7 Hepatocellular adenoma X X X X X X X 12 Hepatocellular adenoma, multiple X X X X X 6 Histiocytic sarcoma 1 Mesentery + + + + + + 12

Carcinoma, metastatic,

intestine large, cecum 1 Hemangiosarcoma X X X X X 8 Oral mucosa 1

Pharyngeal, squamous cell carcinoma 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 54 Tooth + + + + + + + 9


### Cardiovascular System Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma X 2


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Parathyroid gland M + + + M + + + + + + + + + + + + M + + + + M + + + + M + + 54 Pituitary gland + M M + + + + + + + + + + + + + + + + + + M + M M + + + + + 50 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + I + + + + + + + 59

C-cell, carcinoma 1

### o-Nitrotoluene, NTP TR 504 208

General Body System None


### Genital System Epididymis ++++++++++++++++++++++++++++++ Preputial gland + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + Prostate ++++++++++++++++++++++I+++++++ Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++

Interstitial cell, adenoma


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++

Hemangiosarcoma Lymph node ++ +

Pancreatic, histiocytic sarcoma X Lymph node, mandibular + + M + + + M + + + + + + + + + + + + +M+ + + + +M+ + + Lymph node, mesenteric + + + A + + + M + + + + +M+ + + + + + + + + + + +M+ + +

Carcinoma, metastatic,

intestine large, cecum X Histiocytic sarcoma X Spleen ++++++++++++++++++++++++++++++

Hemangiosarcoma X X Histiocytic sarcoma X Thymus + + + + + + + + + + + + +M+ + +M+ + + + + +M+ + + + +


### Integumentary System Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM Skin ++++++++++++++++++++++++++++++

Sebaceous gland, carcinoma Subcutaneous tissue, hemangioma Subcutaneous tissue, hemangiosarcoma X X X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++

Cranium, osteoma Skeletal muscle + + + + +

Hemangiosarcoma X X X X X


### Nervous System Brain ++++++++++++++++++++++++++++++ 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

444445555555666666666677777777 

### Number of Days on Study 566682445779033445889911222222 103956259252403115680007689999

111000000001000000110100000000 

### Carcass ID Number 102889676790867796109178866788 930700376681690118057439474213

### 209 o- Nitrotoluene, NTP TR 504

General Body System None


### Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Interstitial cell, adenoma X 1


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma X 1 Lymph node + 4

Pancreatic, histiocytic sarcoma 1 Lymph node, mandibular + + + + + + + + + + + + + + + + + + M + + + + + + + + + + + 55 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + M 55

Carcinoma, metastatic,

intestine large, cecum 1 Histiocytic sarcoma 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma X 3 Histiocytic sarcoma 1 Thymus + M + + I + + + + + + + + I + + + + + + I + + M + + + + + + 52


### Integumentary System Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Sebaceous gland, carcinoma X 1 Subcutaneous tissue, hemangioma X 1 Subcutaneous tissue, hemangiosarcoma X 4


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Cranium, osteoma X 1 Skeletal muscle + 6

Hemangiosarcoma X 6


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 222222222233333333333333333333 999999999900000000000000111111

001111111100000000000111000111 T otal 

### Carcass ID Number 890000111166677789999011789001 Tissues/ 542789237812558922359456486061 T umors

### o-Nitrotoluene, NTP TR 504 210

Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X X X X Alveolar/bronchiolar carcinoma Carcinoma, metastatic,

intestine large, cecum X Hemangiosarcoma, metastatic,

uncertain primary site X Hepatocellular carcinoma, metastatic,

liver X Histiocytic sarcoma X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye Harderian gland +

Adenoma Carcinoma X Zymbal’s gland +


### Urinary System Kidney + + + A ++++++++++++++++++++++++++

Histiocytic sarcoma X Urinary bladder + + A +++++++++++++++++++++++++++


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Histiocytic sarcoma X 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

444445555555666666666677777777 

### Number of Days on Study 566682445779033445889911222222 103956259252403115680007689999

111000000001000000110100000000 

### Carcass ID Number 102889676790867796109178866788 930700376681690118057439474213

### 211 o- Nitrotoluene, NTP TR 504

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X X 6 Alveolar/bronchiolar carcinoma X 1 Carcinoma, metastatic,

intestine large, cecum 1 Hemangiosarcoma, metastatic,

uncertain primary site 1 Hepatocellular carcinoma, metastatic,

liver X 2 Histiocytic sarcoma 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye + 1 Harderian gland + + 3

Adenoma X 1 Carcinoma 1 Zymbal’s gland + 2


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Histiocytic sarcoma 1 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Histiocytic sarcoma 1 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 222222222233333333333333333333 999999999900000000000000111111

001111111100000000000111000111 T otal 

### Carcass ID Number 890000111166677789999011789001 Tissues/ 542789237812558922359456486061 T umors

### o-Nitrotoluene, NTP TR 504 212

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Gallbladder A + A A + + + + + +AA+ +A+ + + + +AAA+A+AA+A Intestine large, colon A + + + A + + + + +AA+ +A+ + + + +A+ + +A+ +AA+

Muscularis, serosa, hemangiosarcoma Intestine large, rectum + + + +A+ + + + +AA+ +A+ + + + +A+ + +A+AAA+ Intestine large, cecum + + + A A A + + + +AA+ +A+ +A+ +AAA+A+ AAAA

Carcinoma X X X Intestine small, duodenum A + + A + + + + + +AA+ +A+ + + + +A+A+A+AA+A Intestine small, jejunum A + AAAA + + + +AA+ +A+ + + + +AAA+A+ +AAA Intestine small, ileum A + + A A A + + + +AA+ +A+ + + + +AAA+A+ AAAA Liver A ++++++++++A++++++++A+++++++++

Hemangiosarcoma Hemangiosarcoma, metastatic,

skeletal muscle X Hepatocellular carcinoma Hepatocellular carcinoma, multiple Hepatocellular adenoma X Hepatocellular adenoma, multiple Mesentery +++++ ++ +++++ +++++++ ++

Carcinoma, metastatic,

intestine large, cecum X Hemangiosarcoma X X X X XXXX XXXXX XX Hemangiosarcoma, multiple X X X X Hemangiosarcoma, metastatic,

skeletal muscle Pancreas +++++++++++A++++++++++++++++++

Acinus, hemangiosarcoma X Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach +++++++++++A++++++++++++++++++ Stomach, glandular ++++++++++A+++A+++++A+A+A+A+++ Tooth +


### Cardiovascular System Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex + + + + + + + + + + +A+ + + + + + + + + + + + + + + + +M Adrenal medulla + + + + + + I + + + +A+ + + + + + + +M+ + + + + + + +M Islets, pancreatic +++++++++++A++++++++++++++++++ Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + +M Pituitary gland M + + + I + + + +M+ + + I + + + + + + + +M+M+ + + + + Thyroid gland ++++++++++++++++++++++++++++++


### General Body System Tissue NOS +

Pelvic, hemangiosarcoma X 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

233444444444444444455555555555 

### Number of Days on Study 706112355666778999901122223333 035093411679065226746702361349

111111111111111111111111111111 

### Carcass ID Number 527754766448454363763256352244 747119509100448836644731252653

### 213 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Gallbladder A + A M + A + + M A A A A + M + A + A A + + A + + A + + + A 31 Intestine large, colon + + + + + A + + + + + A + + + + + + + + A + + + + A + + + A 46

Muscularis, serosa, hemangiosarcoma X 1 Intestine large, rectum + + + + + A + + + + + A + + + + + + + + A + + + + A + + + A 46 Intestine large, cecum A + + M + + + + + + A A + + A + + + A A A + + + + A + + + + 36

Carcinoma X X X X X X 9 Intestine small, duodenum A + + + + A + + + + + A + + A + + + A A A + + + + A + + + A 40 Intestine small, jejunum A + + + + A + + + + A A + + A + + + A A A + + + + A + + + A 35 Intestine small, ileum A + + + + A + + + + A A + + A + + + A A A + + + + A + + + A 35 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57

Hemangiosarcoma X 1 Hemangiosarcoma, metastatic,

skeletal muscle 1 Hepatocellular carcinoma X X X X 4 Hepatocellular carcinoma, multiple X 1 Hepatocellular adenoma 1 Hepatocellular adenoma, multiple X X 2 Mesentery + + + + + + + + + + + + + + + + + + + + + 42

Carcinoma, metastatic,

intestine large, cecum 1 Hemangiosarcoma X X X X X X X X X X X X X X X 30 Hemangiosarcoma, multiple XXX X 8 Hemangiosarcoma, metastatic,

skeletal muscle X 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Acinus, hemangiosarcoma 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + I + + + + 58 Stomach, glandular + + + + + + + + + + + A + + + + + + + + + + + + + + + + + + 53 Tooth + 2


### Cardiovascular System Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Parathyroid gland + + + + + + + + + + + + + + + + + + M + + + M + + + + + + + 57 Pituitary gland + + + + + + + + + + + + + + + + + + + + M + + + + + + M I M 50 Thyroid gland + + + + + + + + + + M + + + + + + + + + + + + + + + + + + + 59


### General Body System Tissue NOS + 2

Pelvic, hemangiosarcoma 1 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

555555555555555555555555556667 

### Number of Days on Study 444455555666666666677889991240 048834667003466799922880269456

111111111111111111111111111111 T otal 

### Carcass ID Number 555763223356234736627453727764 Tissues/ 986081585705907832733629219462 T umors

### o-Nitrotoluene, NTP TR 504 214

Genital System Coagulating gland + +

Hemangiosarcoma X Epididymis ++++++++++++++++++++++++++++++ Preputial gland ++++++++++++++++++++++++++++++ Prostate ++++++++++++++++++++++++++++++

Hemangiosarcoma X Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + + +

Mediastinal, hemangiosarcoma,

metastatic, mesentery Lymph node, mandibular + + M + + + + + + + + + + +M+ + +M+A+ + +M+ + +M+

Hemangioma Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + MM + + + + + + + + +

Carcinoma, metastatic,

intestine large, cecum X X Spleen +++++++++++A++A+++++++++++++++ Thymus M + + + + + + + + + + + + I + +M+ + IM+ +M+ + + I + +


### Integumentary System Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM Skin ++++++++++++++++++++++++++++++

Subcutaneous tissue, fibrosarcoma X Subcutaneous tissue, hemangiosarcoma


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle + + + + + + + ++ +++

Hemangiosarcoma X X X X X X X X X Hemangiosarcoma, multiple X X


### Nervous System Brain ++++++++++++++++++++++++++++++ Peripheral nerve + + Spinal cord + + 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

233444444444444444455555555555 

### Number of Days on Study 706112355666778999901122223333 035093411679065226746702361349

111111111111111111111111111111 

### Carcass ID Number 527754766448454363763256352244 747119509100448836644731252653

### 215 o- Nitrotoluene, NTP TR 504

Genital System Coagulating gland 2

Hemangiosarcoma 1 Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + + + + + + M + + + + + + + + + + + 59 Prostate + + + + + + + + + + + + + + + + M + + + + + + + I + + + + M 57

Hemangiosarcoma X X X 4 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + 7

Mediastinal, hemangiosarcoma,

metastatic, mesentery X 1 Lymph node, mandibular M + + M + + + + + + + + + + M + + + + + + + + + + M + + + + 50

Hemangioma X 1 Lymph node, mesenteric + M M + + + + + + + + + + + + + + + + + + + + + + + + + + + 56

Carcinoma, metastatic,

intestine large, cecum X 3 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Thymus + M + + I + + + + I + + + + M + M + + + I + + M + + + M + + 45


### Integumentary System Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Subcutaneous tissue, fibrosarcoma 1 Subcutaneous tissue, hemangiosarcoma X X X X X X X X 8


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle + + + + + + + + + + + + + + + + + + + + + + + 35

Hemangiosarcoma X X X X X X X X X X X X X X X X 25 Hemangiosarcoma, multiple X X X X X X 8


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Peripheral nerve + 3 Spinal cord + 3 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

555555555555555555555555556667 

### Number of Days on Study 444455555666666666677889991240 048834667003466799922880269456

111111111111111111111111111111 T otal 

### Carcass ID Number 555763223356234736627453727764 Tissues/ 986081585705907832733629219462 T umors

### o-Nitrotoluene, NTP TR 504 216

Respiratory System Lung ++++++++++++++++++++++++++++++

Alveolar/bronchiolar adenoma X X X Alveolar/bronchiolar carcinoma Hemangiosarcoma, metastatic,

mesentery Hemangiosarcoma, metastatic, skin Hemangiosarcoma, metastatic,

uncertain primary site X Hemangiosarcoma, metastatic,

skeletal muscle X Hepatocellular carcinoma, metastatic,

liver Nose ++++++++++++++++++++++++++++++ Pleura + Trachea ++++++++++++++++++++++++++++++


### Special Senses System Eye + Harderian gland

Adenoma Carcinoma


### Urinary System Kidney ++++++++++++++A+++++A+++++++++

Hemangiosarcoma, metastatic,

mesentery X Urethra + Urinary bladder + + + + + + + + + + + + + +M+ + + + + + + + + + + + + + +

Hemangiosarcoma


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++

Lymphoma malignant X 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

233444444444444444455555555555 

### Number of Days on Study 706112355666778999901122223333 035093411679065226746702361349

111111111111111111111111111111 

### Carcass ID Number 527754766448454363763256352244 747119509100448836644731252653

### 217 o- Nitrotoluene, NTP TR 504

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Alveolar/bronchiolar adenoma X 4 Alveolar/bronchiolar carcinoma X X 2 Hemangiosarcoma, metastatic,

mesentery X X X 3 Hemangiosarcoma, metastatic, skin X X X 3 Hemangiosarcoma, metastatic,

uncertain primary site 1 Hemangiosarcoma, metastatic,

skeletal muscle X 2 Hepatocellular carcinoma, metastatic,

liver X 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Pleura 1 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Eye 1 Harderian gland + + + 3

Adenoma X 1 Carcinoma X 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58

Hemangiosarcoma, metastatic,

mesentery 1 Urethra 1 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Hemangiosarcoma X 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Lymphoma malignant 1 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

555555555555555555555555556667 

### Number of Days on Study 444455555666666666677889991240 048834667003466799922880269456

111111111111111111111111111111 T otal 

### Carcass ID Number 555763223356234736627453727764 Tissues/ 986081585705907832733629219462 T umors

### o-Nitrotoluene, NTP TR 504 218

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Gallbladder + + A + + + +MAAM+ + + +M+ + A + + + AAM+ A + + + Intestine large, colon + + + + + + + +AA+ + + + +A+ + + + + + + + + + + + + + Intestine large, rectum ++++++++A++++++A++++++++++++++ Intestine large, cecum + A + + + + + +AA+ + + + +A+ +A+ + + +A+ + + + + + Intestine small, duodenum + A A + + + +AAA+ + + + +A+ +A+ + +A+A+A+ + + Intestine small, jejunum + A + + + + +AAA+ + + + +A+ +A+ + +AAA+A+ + + Intestine small, ileum + A + + + +MAAA + + + + + A + + A + + + A + A + A + + + Liver ++++++++++++++++++++++++++++++

Hepatocellular carcinoma X Mesentery + + + + + +++++++++ ++

Hemangiosarcoma X X X X X X X X Hemangiosarcoma, multiple X X X X XXXX Pancreas ++++++++++++++++++++++++++A+++

Hemangiosarcoma X Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++

Squamous cell papilloma X Stomach, glandular + A +++++++++++++A++++++++++A+++


### Cardiovascular System Heart ++++++++++++++++++++++++++++++

Hemangiosarcoma X


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++ Islets, pancreatic ++++++++++++++++++++++++++++++ Parathyroid gland + + M + + + + + + + + + + + +M+ + + + + + + + I + + + + + Pituitary gland + + + + + + + I + + I +M+ +M+ + I + + + I + + + + + + + Thyroid gland +++++++++++++++I++++++++++++++


### General Body System Tissue NOS + + +

Hemangiosarcoma X Pelvic, hemangiosarcoma X


### Genital System Coagulating gland Epididymis ++++++++++++++++++++++++++++++ Preputial gland ++++++++++++++++++++++++++++++ Prostate + + + + + +M+ + + + + + + + + + + + + + + + + +M+ + + +

Hemangiosarcoma, metastatic,

mesentery Seminal vesicle ++++++++++++++++++++++++++++++ Testes ++++++++++++++++++++++++++++++ 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

222333333333333333333333333333 

### Number of Days on Study 799012345556666677777778899999 757226950485888812455560611122

122222222221122212211212122211 

### Carcass ID Number 900131232329901381089282802399 017357733143958231384260521768

### 219 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Gallbladder + I A A A M + + A + + A + A + + + A A + + + + + M + A + A M 35 Intestine large, colon + + + + + A + + + + + + + + + + + + A + + + + + + + + + + A 54 Intestine large, rectum + + + + + A + + A + + + + + + + + + A + + + + + + + + + + A 54 Intestine large, cecum + + A A + A + + A + + A + A + + + + A + + + + + + + A + A A 44 Intestine small, duodenum A + A A + A + + + + + A + A + + + + A + + + + + + + A + + + 42 Intestine small, jejunum A + + A + A + + A + + A + A + + + A A + + + + + + + A + + A 40 Intestine small, ileum A + A + + A + + A + + A + A ++++A+++++++ A A A A 39 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular carcinoma X 2 Mesentery + + + + + + + + + + + + + + + + + + + + + + 38

Hemangiosarcoma X X X X X X X X X X X X X X X X 24 Hemangiosarcoma, multiple X X X X X X 14 Pancreas + + + + + + + + A + + + + + + + + + + + + + + + + + + + + + 58

Hemangiosarcoma 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Squamous cell papilloma X 2 Stomach, glandular A + A + + + + + + + + + + + + + + + + + + + + + + + + + + + 55


### Cardiovascular System Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma X 2


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Islets, pancreatic + + + + + + + + A + + + + + + + + + + + + + + + + + + + + + 59 Parathyroid gland + + + M + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Pituitary gland I + + M + + + + M + I + + + + + + M I + + + + + + + + M M + 46 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### General Body System Tissue NOS 3

Hemangiosarcoma 1 Pelvic, hemangiosarcoma 1


### Genital System Coagulating gland + 1 Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58

Hemangiosarcoma, metastatic,

mesentery X 1 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o- Nitrotoluene: 5,000 ppm

333334444444444444444444444444 

### Number of Days on Study 999990000000000001111122224456 234461335668889992444402270902

221212122221221222112212222122 T otal 

### Carcass ID Number 108081813028229133891290430913 Tissues/ 441025420869597949756819066208 T umors

### o-Nitrotoluene, NTP TR 504 220

Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node

Inguinal, hemangiosarcoma,

metastatic, skeletal muscle Lymph node, mandibular + + + M + + + M + + + M + M + M ++++++++++++++ Lymph node, mesenteric + M + + + + +M+ + + + + + + + + + + + + +M+ + + + + + + Spleen ++++++++++++++++++++++++++++++ Thymus + + + + + + + + + + I + + + + + + + + + + + + + + + +M+ +


### Integumentary System Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM Skin ++++++++++++++++++++++++++++++

Subcutaneous tissue, hemangiosarcoma X X X X X Subcutaneous tissue, hemangiosarcoma, multiple X X X X X X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle +++++ ++++++ ++ +++++ ++ ++

Hemangiosarcoma XXXXX X XXXX XX Hemangiosarcoma, multiple X X X X X X X X


### Nervous System Brain ++++++++++++++++++++++++++++++ Peripheral nerve + + + + + + Spinal cord + + + + + +


### Respiratory System Lung ++++++++++++++++++++++++++++++

Hemangiosarcoma, metastatic,

mesentery X Hemangiosarcoma, metastatic, skin X X X Hemangiosarcoma, metastatic,

uncertain primary site X Hemangiosarcoma, metastatic,

skeletal muscle X X X X Mediastinum, hemangiosarcoma X Nose ++++++++++++++++++++++++++++++ Trachea ++++++++++++++++++++++++++++++


### Special Senses System Lacrimal gland + 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

222333333333333333333333333333 

### Number of Days on Study 799012345556666677777778899999 757226950485888812455560611122

122222222221122212211212122211 

### Carcass ID Number 900131232329901381089282802399 017357733143958231384260521768

### 221 o- Nitrotoluene, NTP TR 504

Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + 1

Inguinal, hemangiosarcoma,

metastatic, skeletal muscle X 1 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + M 54 Lymph node, mesenteric + + + + + + + M M + + + + + + + + + + + + + + + + + + + M M 53 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Thymus + + + + + + + + + + + + + + + + + + + M + + + + + + + M M I 54


### Integumentary System Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Subcutaneous tissue, hemangiosarcoma X X X X X X X 12 Subcutaneous tissue, hemangiosarcoma, multiple X X 8


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle + + + + + + + + + + + + + + + + + + + + + + + + + 47

Hemangiosarcoma X X X X X X X 19 Hemangiosarcoma, multiple X X X X X X X X X X X X X X X X X X 26


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Peripheral nerve + + + + + + 12 Spinal cord + + + + + + 12


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hemangiosarcoma, metastatic,

mesentery 1 Hemangiosarcoma, metastatic, skin X 4 Hemangiosarcoma, metastatic,

uncertain primary site PX 2 Hemangiosarcoma, metastatic,

skeletal muscle X X X X 8 Mediastinum, hemangiosarcoma X X X 4 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Special Senses System Lacrimal gland 1 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

333334444444444444444444444444 

### Number of Days on Study 999990000000000001111122224456 234461335668889992444402270902

221212122221221222112212222122 T otal 

### Carcass ID Number 108081813028229133891290430913 Tissues/ 441025420869597949756819066208 T umors

### o-Nitrotoluene, NTP TR 504 222

Urinary System Kidney ++++++++++++++++++++++++++++++

Capsule, hemangiosarcoma X Urethra + Urinary bladder ++++++++++++++++++++++++++++++


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++ 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

222333333333333333333333333333 

### Number of Days on Study 799012345556666677777778899999 757226950485888812455560611122

122222222221122212211212122211 

### Carcass ID Number 900131232329901381089282802399 017357733143958231384260521768

### 223 o- Nitrotoluene, NTP TR 504

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Capsule, hemangiosarcoma 1 Urethra + 2 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 

### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

333334444444444444444444444444 

### Number of Days on Study 999990000000000001111122224456 234461335668889992444402270902

221212122221221222112212222122 T otal 

### Carcass ID Number 108081813028229133891290430913 Tissues/ 441025420869597949756819066208 T umors

### o-Nitrotoluene, NTP TR 504 224



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Harderian Gland: Adenoma Overall rate a 4/60 (7%) 1/60 (2%) 1/60 (2%) 0/60 (0%) Adjusted rate b 7.0% 2.0% 4.2% 0.0% Terminal rate c 4/52 (8%) 1/34 (3%) 0/0 0/0 First incidence (days) 729 (T) 729 (T) 645 —e Poly-3 test d P=0.196N P=0.225N P=0.509N P=0.483N



### Harderian Gland: Adenoma or Carcinoma Overall rate 5/60 (8%) 2/60 (3%) 2/60 (3%) 0/60 (0%) Adjusted rate 8.8% 4.0% 8.2% 0.0% Terminal rate 5/52 (10%) 1/34 (3%) 0/0 0/0 First incidence (days) 729 (T) 641 560 — Poly-3 test P=0.298N P=0.274N P=0.617N P=0.443N


### Large Intestine (Cecum): Carcinoma Overall rate 0/60 (0%) 12/60 (20%) 9/60 (15%) 0/60 (0%) Adjusted rate 0.0% 22.7% 31.6% 0.0% Terminal rate 0/52 (0%) 4/34 (12%) 0/0 0/0 First incidence (days) — 485 270 — Poly-3 test P<0.001 P<0.001 P<0.001 — f



### Liver: Hemangiosarcoma Overall rate 1/60 (2%) 4/59 (7%) 1/57 (2%) 0/60 (0%) Adjusted rate 1.8% 8.0% 4.3% 0.0% Terminal rate 1/52 (2%) 1/34 (3%) 0/0 0/0 First incidence (days) 729 (T) 641 588 — Poly-3 test P=0.257 P=0.142 P=0.542 P=0.721N


### Liver: Hepatocellular Adenoma Overall rate 18/60 (30%) 18/59 (31%) 3/57 (5%) 0/60 (0%) Adjusted rate 31.4% 35.0% 12.3% 0.0% Terminal rate 17/52 (33%) 14/34 (41%) 0/0 0/0 First incidence (days) 637 451 470 — Poly-3 test P=0.118N P=0.421 P=0.087N P=0.183N


### Liver: Hepatocellular Carcinoma Overall rate 12/60 (20%) 16/59 (27%) 5/57 (9%) 2/60 (3%) Adjusted rate 20.5% 31.4% 20.1% 18.5% Terminal rate 7/52 (14%) 10/34 (29%) 0/0 0/0 First incidence (days) 596 451 556 350 Poly-3 test P=0.354 P=0.138 P=0.587N P=0.562N


### Liver: Hepatocellular Adenoma or Carcinoma Overall rate 27/60 (45%) 28/59 (47%) 7/57 (12%) 2/60 (3%) Adjusted rate 46.1% 53.7% 26.7% 18.5% Terminal rate 22/52 (42%) 19/34 (56%) 0/0 0/0 First incidence (days) 596 451 470 350 Poly-3 test P=0.149N P=0.271 P=0.093N P=0.198N


### Liver: Hepatocellular Carcinoma or Hepatoblastoma Overall rate 13/60 (22%) 17/59 (29%) 5/57 (9%) 2/60 (3%) Adjusted rate 22.2% 33.4% 20.1% 18.5% Terminal rate 7/52 (14%) 10/34 (29%) 0/0 0/0 First incidence (days) 596 451 556 350 Poly-3 test P=0.399 P=0.136 P=0.526N P=0.531N

### 225 o- Nitrotoluene, NTP TR 504



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Liver: Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastoma Overall rate 28/60 (47%) 29/59 (49%) 7/57 (12%) 2/60 (3%) Adjusted rate 47.8% 55.6% 26.7% 18.5% Terminal rate 22/52 (42%) 19/34 (56%) 0/0 0/0 First incidence (days) 596 451 470 350 Poly-3 test P=0.125N P=0.262 P=0.073N P=0.181N


### Lung: Alveolar/bronchiolar Adenoma Overall rate 9/60 (15%) 6/60 (10%) 4/60 (7%) 0/60 (0%) Adjusted rate 15.8% 11.8% 15.4% 0.0% Terminal rate 9/52 (17%) 3/34 (9%) 0/0 0/0 First incidence (days) 729 (T) 469 434 — Poly-3 test P=0.323N P=0.372N P=0.595N P=0.332N


### Lung: Alveolar/bronchiolar Carcinoma Overall rate 5/60 (8%) 1/60 (2%) 2/60 (3%) 0/60 (0%) Adjusted rate 8.7% 2.0% 8.1% 0.0% Terminal rate 4/52 (8%) 1/34 (3%) 0/0 0/0 First incidence (days) 649 729 (T) 544 — Poly-3 test P=0.219N P=0.140N P=0.614N P=0.444N


### Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 14/60 (23%) 7/60 (12%) 6/60 (10%) 0/60 (0%) Adjusted rate 24.4% 13.7% 22.1% 0.0% Terminal rate 13/52 (25%) 4/34 (12%) 0/0 0/0 First incidence (days) 649 469 434 — Poly-3 test P=0.136N P=0.121N P=0.513N P=0.241N


### Lung: Hemangiosarcoma Overall rate 0/60 (0%) 0/60 (0%) 0/60 (0%) 4/60 (7%) Adjusted rate 0.0% 0.0% 0.0% 32.1% Terminal rate 0/52 (0%) 0/34 (0%) 0/0 0/0 First incidence (days) — — — 358 Poly-3 test P=0.014 — — P=0.006


### Mesentery: Hemangiosarcoma Overall rate 0/60 (0%) 8/60 (13%) 38/60 (63%) 38/60 (63%) Adjusted rate 0.0% 15.8% 83.0% 92.8% Terminal rate 0/52 (0%) 5/34 (15%) 0/0 0/0 First incidence (days) — 572 410 297 Poly-3 test P<0.001 P=0.002 P<0.001 P<0.001


### Prostate Gland: Hemangiosarcoma Overall rate 0/60 (0%) 0/59 (0%) 4/57 (7%) 0/58 (0%) Adjusted rate 0.0% 0.0% 16.8% 0.0% Terminal rate 0/52 (0%) 0/34 (0%) 0/0 0/0 First incidence (days) — — 476 — Poly-3 test P=0.029 — P=0.011 —


### Skeletal Muscle: Hemangiosarcoma Overall rate 0/60 (0%) 6/60 (10%) 33/60 (55%) 45/60 (75%) Adjusted rate 0.0% 11.7% 79.1% 95.3% Terminal rate 0/52 (0%) 1/34 (3%) 0/0 0/0 First incidence (days) — 542 365 277 Poly-3 test P<0.001 P=0.011 P<0.001 P<0.001

### o-Nitrotoluene, NTP TR 504 226



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Skin (Subcutaneous Tissue): Hemangiosarcoma Overall rate 0/60 (0%) 4/60 (7%) 8/60 (13%) 20/60 (33%) Adjusted rate 0.0% 8.0% 29.4% 77.0% Terminal rate 0/52 (0%) 2/34 (6%) 0/0 0/0 First incidence (days) — 542 544 326 Poly-3 test P<0.001 P=0.047 P<0.001 P<0.001


### Spleen: Hemangiosarcoma Overall rate 2/60 (3%) 3/60 (5%) 0/58 (0%) 0/60 (0%) Adjusted rate 3.5% 6.0% 0.0% 0.0% Terminal rate 1/52 (2%) 1/34 (3%) 0/0 0/0 First incidence (days) 710 641 — — Poly-3 test P=0.589N P=0.441 P=0.462N P=0.602N


### All Organs: Hemangiosarcoma Overall rate 4/60 (7%) 17/60 (28%) 55/60 (92%) 60/60 (100%) Adjusted rate 7.0% 32.7% 97.9% 100.0% Terminal rate 3/52 (6%) 9/34 (27%) 0/0 0/0 First incidence (days) 710 542 365 277 Poly-3 test P<0.001 P<0.001 P<0.001 P<0.001


### All Organs: Hemangioma or Hemangiosarcoma Overall rate 5/60 (8%) 18/60 (30%) 55/60 (92%) 60/60 (100%) Adjusted rate 8.8% 34.7% 97.9% 100.0% Terminal rate 4/52 (8%) 10/34 (29%) 0/0 0/0 First incidence (days) 710 542 365 277 Poly-3 test P<0.001 P<0.001 P<0.001 P<0.001


### All Organs: Malignant Lymphoma Overall rate 3/60 (5%) 0/60 (0%) 1/60 (2%) 0/60 (0%) Adjusted rate 5.3% 0.0% 4.1% 0.0% Terminal rate 3/52 (6%) 0/34 (0%) 0/0 0/0 First incidence (days) 729 (T) — 434 — Poly-3 test P=0.220N P=0.146N P=0.614N P=0.533N


### All Organs: Benign Neoplasms Overall rate 30/60 (50%) 24/60 (40%) 8/60 (13%) 2/60 (3%) Adjusted rate 52.3% 45.3% 28.6% 18.6% Terminal rate 29/52 (56%) 17/34 (50%) 0/0 0/0 First incidence (days) 637 451 434 391 Poly-3 test P=0.016N P=0.288N P=0.040N P=0.137N


### All Organs: Malignant Neoplasms Overall rate 26/60 (43%) 41/60 (68%) 59/60 (98%) 60/60 (100%) Adjusted rate 44.4% 71.0% 99.9% 100.0% Terminal rate 20/52 (39%) 19/34 (56%) 0/0 0/0 First incidence (days) 596 451 270 277 Poly-3 test P<0.001 P=0.002 P<0.001 P<0.001

### 227 o- Nitrotoluene, NTP TR 504



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

All Organs: Benign or Malignant Neoplasms Overall rate 45/60 (75%) 50/60 (83%) 59/60 (98%) 60/60 (100%) Adjusted rate 76.8% 85.5% 99.9% 100.0% Terminal rate 39/52 (75%) 27/34 (79%) 0/0 0/0 First incidence (days) 596 451 270 277 Poly-3 test P<0.001 P=0.164 P<0.001 P<0.001

(T)Terminal sacrifice a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for l iver, lung, prostate gland, and spleen; for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals th at do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal group f Value of statistic cannot be computed.

### o-Nitrotoluene, NTP TR 504 228



### T ABLE 

### C4a Historical Incidence of Hemangiosarcoma (All Sites) in Control Male B6C3F 1 

### Mice

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Acrylonitrile (gavage) 3/50 Citral (feed) 3/100
p,p N -Dichlorodiphenyl sulfone (feed) 3/50 Indium phosphide (inhalation) 3/50 60-Hz Magnetic fields (whole body exposure) 6/100 Methacrylonitrile (gavage) 3/49

o -Nitrotoluene (feed) 4/60
p-Nitrotoluene (feed) 1/50 Riddelliine (gavage) 3/50 Sodium nitrite (drinking water) 7/50 Vanadium pentoxide (inhalation) 1/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 37/659 (5.6%) Mean ± standard deviation 5.8% ± 3.2% Range 2%-14%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 2/50 2,2-Bis(bromomethyl)-1,3-propanediol 2/50
t- Butylhydroquinone 7/50 Emodin 3/50

o-Nitroanisole 3/50
p-Nitrobenzoic acid 6/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 53/952 (5.6%) Mean ± standard deviation 5.6% ± 3.5% Range 2%-14%

a Data as of December 22, 2000 b Data as of December 23, 1999

### 229 o- Nitrotoluene, NTP TR 504



### T ABLE 

### C4b Historical Incidence of Large Intestine (Cecum) Carcinoma in Control Male B6C3F 1 

### Mice

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a

Acrylonitrile (gavage) 0/50 Citral (feed) 0/100
p,p N -Dichlorodiphenyl sulfone (feed) 0/50 Indium phosphide (inhalation) 0/50 60-Hz Magnetic fields (whole body exposure) 0/100 Methacrylonitrile (gavage) 0/49

o -Nitrotoluene (feed) 0/60
p-Nitrotoluene (feed) 1/50 Riddelliine (gavage) 0/50 Sodium nitrite (drinking water) 0/50 Vanadium pentoxide (inhalation) 0/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 1/659 (0.2%) Mean ± standard deviation 0.2% ± 0.6% Range 0%-2%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 0/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/50
t- Butylhydroquinone 0/50 Emodin 0/50

o-Nitroanisole 0/50
p-Nitrobenzoic acid 0/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total 0/952

a Data as of December 22, 2000 b Data as of December 23, 1999

### o-Nitrotoluene, NTP TR 504 230



### T ABLE 

### C4c Historical Incidence of Hepatocellular Neoplasms in Control Male B6C3F 1 

### Mice

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence in Controls Given NTP-2000 Diet a

Acrylonitrile (gavage) 23/50 14/50 32/50 Citral (feed) 20/100 13/100 28/100
p,p N -Dichlorodiphenyl sulfone (feed) 6/50 9/50 15/50 Indium phosphide (inhalation) 17/50 11/50 26/50 60-Hz Magnetic fields (whole body exposure) 30/100 19/100 46/100 Methacrylonitrile (gavage) 17/49 13/49 24/49

o -Nitrotoluene (feed) 18/60 12/60 27/60
p-Nitrotoluene (feed) 14/50 8/50 20/50 Riddelliine (gavage) 16/50 23/50 36/50 Sodium nitrite (drinking water) 19/50 9/50 24/50 Vanadium pentoxide (inhalation) 15/50 14/50 26/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 195/659 (29.6%) 145/659 (22.0%) 304/659 (46.1%) Mean ± standard deviation 30.4% ± 8.9% 23.1% ± 9.0% 47.8% ± 12.9% Range 12%-46% 13%-46% 28%-72%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 10/50 12/50 22/50 2,2-Bis(bromomethyl)-1,3-propanediol 18/49 11/49 27/49
t- Butylhydroquinone 28/50 8/50 31/50 Emodin 9/50 20/50 28/50

o-Nitroanisole 14/50 7/50 21/50
p-Nitrobenzoic acid 17/50 8/50 22/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 363/950 (38.2%) 194/950 (20.4%) 493/950 (51.9%) Mean ± standard deviation 38.2% ± 10.7% 20.4% ± 6.7% 51.9% ± 7.9% Range 18%-60% 10%-40% 40%-68%

a Data as of December 22, 2000 b Data as of December 23, 1999

### 231 o- Nitrotoluene, NTP TR 504



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of o- Nitrotoluene a



### 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Disposition Summary Animals initially in study 60 60 60 60 Early deaths

Moribund 3 5 21 31 Natural deaths 5 21 39 29 Survivors

Terminal sacrifice 52 34

Animals examined microscopically 60 60 60 60



### Alimentary System Gallbladder (54) (42) (31) (35)

Fibrosis 1 (2%) Intestine large, cecum (56) (49) (36) (44)

Epithelium, degeneration, mucoid 1 (2%) Intestine small, jejunum (56) (43) (35) (40)

Inflammation, focal 1 (2%) Ulcer 1 (2%) Epithelium, degeneration, cystic, focal 1 (2%) Epithelium, hyperplasia, focal, adenomatous 1 (2%) Peyer’s patch, hyperplasia, lymphoid 1 (2%) Peyer’s patch, inflammation, chronic 1 (2%) Liver (60) (59) (57) (60)

Amyloid deposition 1 (2%) Angiectasis 1 (2%) 2 (4%) 1 (2%) Basophilic focus 6 (10%) 4 (7%) Clear cell focus 3 (5%) 2 (3%) Congestion, focal 2 (3%) 1 (2%) Eosinophilic focus 3 (5%) 14 (24%) 1 (2%) 1 (2%) Fibrosis, focal 1 (2%) 1 (2%) Infiltration cellular, mixed cell 46 (77%) 40 (68%) 24 (42%) 29 (48%) Inflammation, chronic, focal 1 (2%) 1 (2%) Inflammation, focal, suppurative 1 (2%) Mineralization, focal 1 (2%) Mixed cell focus 2 (3%) Necrosis 1 (2%) 15 (25%) 27 (47%) 30 (50%) Artery, angiectasis 1 (2%) Artery, inflammation, chronic 1 (2%) Hepatocyte, fatty change, diffuse 2 (3%) Hepatocyte, necrosis, focal 2 (3%) Hepatocyte, syncytial alteration, focal 16 (27%) 26 (44%) 43 (75%) 39 (65%) Hepatocyte, tension lipidosis 1 (2%) Hepatocyte, vacuolization cytoplasmic, diffuse 10 (17%) 4 (7%) Hepatocyte, vacuolization cytoplasmic, focal 4 (7%) 5 (8%) 2 (4%) 1 (2%) Hepatocyte, periportal, atrophy 1 (2%) Hepatocyte, centrilobular, depletion glycogen 1 (2%) 3 (5%) Hepatocyte, centrilobular, fatty change 1 (2%) 2 (3%) 5 (9%) 1 (2%) Hepatocyte, midzonal, fatty change 1 (2%) Oval cell, hyperplasia 2 (3%)

a Number of animals examined microscopically at the site and the number of animals with lesion

### o-Nitrotoluene, NTP TR 504 232

Alimentary System (continued) Mesentery (12) (12) (42) (38)

Inflammation, chronic 3 (25%) 2 (17%) 2 (5%) Inflammation, chronic, focal 1 (2%) Artery, inflammation, chronic 3 (25%) 1 (8%) Fat, necrosis 6 (50%) 1 (8%) 1 (2%) Pancreas (59) (59) (59) (58)

Amyloid deposition 1 (2%) Acinus, atrophy, focal 1 (2%) 1 (2%) 1 (2%) Duct, cyst 1 (2%) 1 (2%) Duct, inflammation, chronic, focal 1 (2%) Stomach, forestomach (60) (60) (58) (60)

Inflammation, focal 1 (2%) 3 (5%) 1 (2%) Necrosis, focal 1 (2%) Ulcer 1 (2%) 3 (5%) 1 (2%) Epithelium, cyst 1 (2%) Epithelium, hyperplasia 2 (3%) 5 (8%) 6 (10%) 3 (5%) Stomach, glandular (57) (54) (53) (55)

Erosion 2 (4%) Inflammation, chronic, focal 1 (2%) Inflammation, focal 1 (2%) 1 (2%) Pigmentation, focal 1 (2%) Tooth (14) (9) (2)

Malformation 5 (36%) Peridontal tissue, inflammation, chronic 10 (71%) 9 (100%) 1 (50%)



### Cardiovascular System Heart (60) (60) (60) (60)

Infiltration cellular, mixed cell 1 (2%) 3 (5%) 1 (2%) Inflammation, chronic, focal 1 (2%) 4 (7%) 1 (2%) Mineralization 2 (3%) 1 (2%) Thrombosis 1 (2%)



### Endocrine System Adrenal cortex (60) (60) (58) (60)

Accessory adrenal cortical nodule 1 (2%) Cyst 2 (3%) Cytoplasmic alteration, focal 7 (12%) 2 (3%) Fibrosis 1 (2%) Hypertrophy, focal 1 (2%) Inflammation, chronic, focal 1 (2%) Parathyroid gland (55) (54) (57) (56)

Amyloid deposition 1 (2%) Cyst 1 (2%) Pituitary gland (53) (50) (50) (46)

Pars distalis, cyst 2 (4%) 1 (2%) Pars distalis, hyperplasia, focal 1 (2%) Rathke’s cleft, cyst 1 (2%) Thyroid gland (59) (59) (59) (59)

Degeneration, cystic, focal 10 (17%) 11 (19%) 3 (5%) Follicular cell, hyperplasia 1 (2%) 

### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

### 233 o- Nitrotoluene, NTP TR 504

General Body System Tissue NOS (2) (2) (3)

Pelvic, inflammation, chronic, focal 1 (33%)



### Genital System Coagulating gland (2) (1)

Inflammation, chronic 1 (50%) 1 (100%) Epididymis (60) (60) (60) (60)

Inflammation, chronic 1 (2%) Preputial gland (60) (59) (59) (60)

Degeneration, cystic 28 (47%) 27 (46%) 16 (27%) 1 (2%) Inflammation, chronic 1 (2%) 5 (8%) 1 (2%) Prostate (60) (59) (57) (58)

Inflammation, chronic 1 (2%) 7 (12%) 4 (7%) Seminal vesicle (60) (60) (60) (60)

Congestion 1 (2%) 1 (2%) Dilatation 2 (3%) 1 (2%) 1 (2%) Inflammation, chronic 1 (2%) Inflammation, suppurative 1 (2%)



### Hematopoietic System Bone marrow (60) (60) (60) (60)

Depletion cellular 1 (2%) Hemorrhage, focal 2 (3%) 1 (2%) Hyperplasia 3 (5%) 2 (3%) Thrombosis 1 (2%) Erythroid cell, depletion cellular 1 (2%) Lymph node (3) (4) (7) (1)

Iliac, hematopoietic cell proliferation 1 (14%) Iliac, hyperplasia 1 (14%) Mediastinal, hemorrhage 1 (33%) 1 (25%) 3 (43%) Mediastinal, hyperplasia 1 (25%) 1 (14%) Mediastinal, pigmentation 1 (25%) Pancreatic, hemorrhage 1 (25%) Lymph node, mandibular (59) (55) (50) (54)

Hyperplasia, lymphoid 1 (2%) Lymph node, mesenteric (60) (55) (56) (53)

Hematopoietic cell proliferation 1 (2%) Hemorrhage 1 (2%) 2 (4%) Hyperplasia 1 (2%) Hyperplasia, lymphoid 1 (2%) Spleen (60) (60) (58) (60)

Amyloid deposition 1 (2%) Depletion cellular 1 (2%) 7 (12%) 2 (3%) Hematopoietic cell proliferation 13 (22%) 24 (40%) 49 (84%) 60 (100%) Hyperplasia, lymphoid 2 (3%) 3 (5%) Pigmentation, focal 1 (2%) Thymus (57) (52) (45) (54)

Atrophy 3 (6%) 3 (7%) 6 (11%) Cyst 4 (7%) 3 (6%) 1 (2%) Hyperplasia, lymphoid 1 (2%) Epithelial cell, hyperplasia 1 (2%) 

### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

### o-Nitrotoluene, NTP TR 504 234

Integumentary System Skin (60) (60) (60) (60)

Ulcer, chronic, focal 1 (2%) Artery, subcutaneous tissue, inflammation, chronic 2 (3%) Prepuce, subcutaneous tissue, inflammation,

suppurative 1 (2%) Subcutaneous tissue, edema 3 (5%) 14 (23%) 22 (37%) Subcutaneous tissue, hemorrhage, focal 1 (2%) 2 (3%) Subcutaneous tissue, inflammation, chronic, focal 1 (2%) 1 (2%) 2 (3%) Subcutaneous tissue, necrosis 1 (2%)



### Musculoskeletal System Bone (60) (60) (60) (60)

Fracture 1 (2%) Skeletal muscle (1) (6) (35) (47)

Hemorrhage, focal 2 (6%) 2 (4%)



### Nervous System None



### Respiratory System Lung (60) (60) (60) (60)

Congestion 1 (2%) 1 (2%) 1 (2%) Cyst 1 (2%) Hemorrhage 1 (2%) 1 (2%) 5 (8%) Hyperplasia, histiocytic 2 (3%) 1 (2%) Infiltration cellular, polymorphonuclear 1 (2%) Infiltration cellular, mixed cell 4 (7%) 9 (15%) 3 (5%) Inflammation, chronic, focal 3 (5%) Alveolar epithelium, hyperplasia 1 (2%) 1 (2%) 1 (2%) 1 (2%) Bronchus, glands, cyst 1 (2%) Nose (60) (60) (60) (60)

Mucosa, glands, dilatation, focal 1 (2%) Nasolacrimal duct, inflammation 1 (2%) Olfactory epithelium, degeneration 36 (60%) 60 (100%) 60 (100%) Sinus, foreign body 1 (2%) Sinus, inflammation, chronic, suppurative 1 (2%)



### Special Senses System Eye (1) (1)

Atrophy 1 (100%) Inflammation, chronic 1 (100%) Cornea, necrosis, focal 1 (100%) 

### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

### 235 o- Nitrotoluene, NTP TR 504

Urinary System Kidney (58) (59) (58) (60)

Amyloid deposition 1 (2%) Atrophy 1 (2%) 1 (2%) Atrophy, focal 2 (3%) Congestion 5 (9%) 3 (5%) Cyst 2 (3%) 4 (7%) 1 (2%) 1 (2%) Fibrosis, focal 2 (3%) Hyperplasia, lymphoid 1 (2%) 3 (5%) Infarct 1 (2%) Infiltration cellular, mixed cell 1 (2%) Inflammation, chronic 1 (2%) 1 (2%) Inflammation, chronic, focal 1 (2%) Inflammation, focal, suppurative 1 (2%) Inflammation, suppurative 1 (2%) 3 (5%) Metaplasia, focal, osseous 1 (2%) Nephropathy 56 (97%) 53 (90%) 34 (59%) 18 (30%) Artery, inflammation, chronic 2 (3%) Papilla, necrosis 1 (2%) Pelvis, dilatation 2 (3%) Renal tubule, accumulation, hyaline droplet 1 (2%) 2 (3%) 5 (9%) 3 (5%) Renal tubule, dilatation 1 (2%) 4 (7%) Renal tubule, necrosis 1 (2%) 2 (3%) Renal tubule, pigmentation 1 (2%) 6 (10%) 32 (55%) 35 (58%) Renal tubule, vacuolization cytoplasmic 1 (2%) 1 (2%) Urethra (1) (2)

Inflammation, focal 1 (100%) 2 (100%) Urinary bladder (59) (59) (59) (60)

Calculus, microscopic observation only 1 (2%) Inflammation, chronic 1 (2%) Transitional epithelium, hyperplasia 1 (2%) 

### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

### o-Nitrotoluene, NTP TR 504 236

### 237



# APPENDIX D SUMMARY OF LESIONS IN FEMALE MICE IN THE 2-YEAR FEED STUDY OF o-NITROTOLUENE

T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 T ABLE 

### D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 T ABLE 

### D4a Historical Incidence of Hemangiosarcoma (All Sites) in Control Female B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 T ABLE 

### D4b Historical Incidence of Hepatocellular Neoplasms in Control Female B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

### o-Nitrotoluene, NTP TR 504 238



### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluene a



### 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Disposition Summary Animals initially in study 60 60 60 60 Early deaths

Moribund 4 6 7 22 Natural deaths 4 8 6 33 Survivors

Terminal sacrifice 52 46 47 5

Animals examined microscopically 60 60 60 60



### Alimentary System Esophagus (60) (60) (59) (57) Gallbladder (54) (53) (54) (31) Intestine large, colon (56) (55) (57) (41)

Hemangiosarcoma, metastatic, mesentery 1 (2%) Intestine large, cecum (56) (53) (54) (32)

Carcinoma 1 (2%) 4 (7%) 3 (9%) Sarcoma 1 (2%) Intestine small, jejunum (56) (53) (56) (34)

Carcinoma 1 (3%) Intestine small, ileum (57) (53) (56) (34) Liver (60) (59) (59) (60)

Cholangioma 1 (2%) Hemangiosarcoma 1 (2%) Hemangiosarcoma, metastatic, skin 1 (2%) Hepatocellular carcinoma 2 (3%) 3 (5%) 4 (7%) 9 (15%) Hepatocellular carcinoma, multiple 1 (2%) 2 (3%) 7 (12%) Hepatocellular adenoma 7 (12%) 5 (8%) 16 (27%) 9 (15%) Hepatocellular adenoma, multiple 3 (5%) 20 (33%) Hepatocholangiocarcinoma 1 (2%) Histiocytic sarcoma 2 (3%) 1 (2%) Osteosarcoma, metastatic, bone 1 (2%) Sarcoma, metastatic, uncertain primary site 1 (2%) Mesentery (11) (9) (9) (34)

Carcinoma, metastatic, uncertain primary site 1 (11%) Carcinoma, metastatic, intestine large, cecum 1 (11%) Hemangiosarcoma 25 (74%) Hemangiosarcoma, multiple 7 (21%) Histiocytic sarcoma 1 (11%) Sarcoma 1 (3%) Sarcoma, multiple 1 (11%) Sarcoma, metastatic, skin 1 (11%) Sarcoma, metastatic, skeletal muscle 1 (11%) Pancreas (60) (57) (58) (57)

Hemangiosarcoma, metastatic, mesentery 1 (2%) Sarcoma 1 (2%) Sarcoma, metastatic, mesentery 1 (2%) Salivary glands (59) (59) (58) (49)

Histiocytic sarcoma 1 (2%) Stomach, forestomach (60) (58) (58) (59)

Hemangioma 1 (2%) Squamous cell papilloma 1 (2%) 2 (3%) 2 (3%) 1 (2%) Stomach, glandular (58) (57) (57) (52)

Sarcoma 1 (2%)

### 239 o- Nitrotoluene, NTP TR 504

Cardiovascular System Heart (60) (60) (59) (58)

Carcinoma, metastatic, uncertain primary site 1 (2%)



### Endocrine System Adrenal cortex (59) (59) (58) (60)

Capsule, adenoma 1 (2%) Adrenal medulla (59) (57) (58) (59)

Pheochromocytoma benign 1 (2%) Islets, pancreatic (60) (57) (60) (56)

Carcinoma 1 (2%) Pituitary gland (58) (58) (53) (53)

Adenoma 1 (2%) Pars distalis, adenoma 2 (3%) 4 (7%) 3 (6%) Pars intermedia, adenoma 1 (2%) Thyroid gland (60) (59) (58) (53)

Follicular cell, adenoma 1 (2%)



### General Body System Tissue NOS (1) (3) (3)

Abdominal, hemangiosarcoma 1 (33%) Abdominal, hemangiosarcoma, multiple 1 (33%) Thoracic, sarcoma 1 (33%)



### Genital System Ovary (59) (56) (56) (56)

Carcinoma 1 (2%) Cystadenoma 1 (2%) 2 (4%) Hemangiosarcoma 1 (2%) Histiocytic sarcoma 1 (2%) Luteoma 2 (3%) 1 (2%) 1 (2%) Teratoma malignant 1 (2%) Tubulostromal adenoma 1 (2%) Oviduct (1) Uterus (60) (59) (60) (59)

Hemangioma 1 (2%) 2 (3%) Hemangiosarcoma 1 (2%) 2 (3%) Histiocytic sarcoma 1 (2%) 1 (2%) Leiomyoma 1 (2%) Leiomyosarcoma 1 (2%) Cervix, hemangiosarcoma 1 (2%) Endometrium, carcinoma 1 (2%) Endometrium, polyp stromal 2 (3%) 2 (3%) 2 (3%) Vagina (1)

Carcinoma, metastatic, uncertain primary site 1 (100%)



### Hematopoietic System Bone marrow (60) (58) (60) (60)

Carcinoma, metastatic, uncertain primary site 1 (2%) Histiocytic sarcoma 1 (2%) 1 (2%) 

### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

### o-Nitrotoluene, NTP TR 504 240

Hematopoietic System (continued) Lymph node (6) (3) (10) (11)

Iliac, sarcoma, metastatic, skin 1 (9%) Inguinal, sarcoma, metastatic, skin 1 (10%) Mediastinal, sarcoma, metastatic, uncertain primary site 1 (9%) Pancreatic, sarcoma 1 (10%) Renal, hemangiosarcoma, metastatic, mesentery 1 (9%) Renal, sarcoma, metastatic, skin 1 (10%) 1 (9%) Lymph node, mandibular (58) (55) (55) (48)

Histiocytic sarcoma 1 (2%) Lymph node, mesenteric (53) (58) (57) (57)

Histiocytic sarcoma 1 (2%) 1 (2%) Sarcoma 1 (2%) Sarcoma, metastatic, mesentery 1 (2%) Spleen (59) (57) (58) (57)

Histiocytic sarcoma 1 (2%) Thymus (57) (56) (55) (44)

Thymoma malignant 1 (2%)



### Integumentary System Skin (60) (60) (60) (60)

Basal cell carcinoma 1 (2%) Basosquamous tumor malignant 1 (2%) Subcutaneous tissue, carcinoma, multiple 1 (2%) Subcutaneous tissue, hemangiosarcoma 2 (3%) 16 (27%) Subcutaneous tissue, hemangiosarcoma, multiple 3 (5%) Subcutaneous tissue, sarcoma 1 (2%) 1 (2%) 5 (8%) 2 (3%) Subcutaneous tissue, sarcoma, multiple 1 (2%)



### Musculoskeletal System Bone (60) (60) (60) (60)

Osteosarcoma 1 (2%) Femur, osteosarcoma 1 (2%) Skeletal muscle (2) (3) (3) (18)

Carcinoma, metastatic, uncertain primary site 1 (33%) Hemangioma 1 (50%) Hemangiosarcoma 14 (78%) Hemangiosarcoma, multiple 2 (11%) Sarcoma 1 (33%) 2 (67%) 1 (6%) Sarcoma, multiple 1 (33%) 1 (6%)



### Nervous System None



### Respiratory System Lung (60) (60) (59) (57)

Alveolar/bronchiolar adenoma 2 (3%) 3 (5%) 4 (7%) 4 (7%) Alveolar/bronchiolar carcinoma 3 (5%) 1 (2%) Alveolar/bronchiolar carcinoma, multiple 2 (3%) 2 (4%) Carcinoma, metastatic, harderian gland 1 (2%) 1 (2%) Carcinoma, metastatic, uncertain primary site 1 (2%) Hemangiosarcoma, metastatic, skin 1 (2%) 

### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

### 241 o- Nitrotoluene, NTP TR 504

Respiratory System (continued) Lung (continued) (60) (60) (59) (57)

Hemangiosarcoma, metastatic, uncertain primary site 1 (2%) Hemangiosarcoma, metastatic, skeletal muscle 2 (4%) Hepatocellular carcinoma, metastatic, liver 1 (2%) 1 (2%) Histiocytic sarcoma 1 (2%) 1 (2%) Osteosarcoma, metastatic, bone 1 (2%) 1 (2%) Sarcoma, metastatic, mesentery 1 (2%) Sarcoma, metastatic, skeletal muscle 1 (2%) Teratoma malignant, metastatic, ovary 1 (2%) Mediastinum, alveolar/bronchiolar carcinoma,

metastatic, lung 1 (2%) Mediastinum, carcinoma 1 (2%) Mediastinum, carcinoma, metastatic, harderian gland 1 (2%) Mediastinum, histiocytic sarcoma 1 (2%) Mediastinum, sarcoma, metastatic,

uncertain primary site 1 (2%) Mediastinum, sarcoma, metastatic, skeletal muscle 1 (2%) Nose (60) (60) (59) (57)



### Special Senses System Harderian gland (5) (1) (4) (4)

Adenoma 4 (80%) 1 (25%) 2 (50%) Carcinoma 1 (20%) 1 (100%) 3 (75%) 2 (50%)



### Urinary System Kidney (59) (56) (58) (59)

Carcinoma 1 (2%) Histiocytic sarcoma 1 (2%) Urinary bladder (59) (54) (57) (54)

Hemangiosarcoma 1 (2%)



### Systemic Lesions Multiple organs b (60) (60) (60) (60)

Histiocytic sarcoma 2 (3%) 1 (2%) Lymphoma malignant 8 (13%) 7 (12%) 5 (8%) 3 (5%)



### Neoplasm Summary Total animals with primary neoplasms c 33 37 44 56

Total primary neoplasms 42 51 76 146 Total animals with benign neoplasms 21 20 26 33

Total benign neoplasms 26 22 35 38 Total animals with malignant neoplasms 16 23 27 55

Total malignant neoplasms 16 29 41 108 Total animals with metastatic neoplasms 4 6 12

Total metastatic neoplasms 12 11 16 Total animals with malignant neoplasms of uncertain primary site 1 1 2

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms 

### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

### o-Nitrotoluene, NTP TR 504 242

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Gallbladder A A + A + + + A + + + + + + + + + +M+ + + + + + + + + + + Intestine large, colon + A + A + M + A ++++++++++++++++++++++ Intestine large, rectum + A + A + + + A ++++++++++++++++++++++ Intestine large, cecum A A + A + + + A ++++++++++++++++++++++ Intestine small, duodenum A A + A + + + A ++++++++++++++++++++++ Intestine small, jejunum A A + A + + + A ++++++++++++++++++++++ Intestine small, ileum + A + A + + + A ++++++++++++++++++++++ Liver ++++++++++++++++++++++++++++++ Hepatocellular carcinoma X X Hepatocellular adenoma XX X Mesentery + + + + Oral mucosa + Pancreas ++++++++++++++++++++++++++++++ Salivary glands ++++++++++++++++++++++++++++++ Stomach, forestomach ++++++++++++++++++++++++++++++ Hemangioma X Squamous cell papilloma X Stomach, glandular + A + A ++++++++++++++++++++++++++ Tooth + +


### Cardiovascular System Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex +++++++A++++++++++++++++++++++ Capsule, adenoma Adrenal medulla +++++++A++++++++++++++++++++++ Islets, pancreatic ++++++++++++++++++++++++++++++ Parathyroid gland + + + + +M+ + + + + + + + + + + + + + + + + + + + + + + + Pituitary gland + A ++++++++++++++++++++++++++++ Adenoma X Pars distalis, adenoma X Thyroid gland ++++++++++++++++++++++++++++++


### General Body System Tissue NOS +


### Genital System Clitoral gland + + + + + + + + + + + + + + +M+ + + + I + + + + + + + + + Ovary +++++++++++++I++++++++++++++++ Cystadenoma X Luteoma X Oviduct Uterus ++++++++++++++++++++++++++++++ Hemangioma Endometrium, polyp stromal X

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

234557777777777777777777777777 

### Number of Days on Study 968691123333333333333333333333 923220051111111111111222222222

222222222222222222222222222222 

### Carcass ID Number 899864584444555566666444455555 695835111234678901245678902345

### 243 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Gallbladder + + + + + I + + + + + + + + + + + + + + + + + + + + + + + + 54 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Hepatocellular carcinoma 2 Hepatocellular adenoma X X X X 7 Mesentery + + + + + + + 11 Oral mucosa 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Salivary glands + + + + M + + + + + + + + + + + + + + + + + + + + + + + + + 59 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Hemangioma 1 Squamous cell papilloma 1 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Tooth + + + + + + 8


### Cardiovascular System Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Capsule, adenoma X 1 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Parathyroid gland + + + + + + + + + + + + + M + + + + + + + + M + + + + + + + 57 Pituitary gland + + + + + M + + + + + + + + + + + + + + + + + + + + + + + + 58 Adenoma 1 Pars distalis, adenoma X 2 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### General Body System Tissue NOS 1


### Genital System Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Cystadenoma 1 Luteoma X 2 Oviduct + 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Hemangioma X 1 Endometrium, polyp stromal X 2 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 222233333333333333555555555555

222222222222222223222222222222 T otal 

### Carcass ID Number 888866667777779990777788899999 Tissues/ 234567890123456780678907901234 T umors

### o-Nitrotoluene, NTP TR 504 244

Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node + + Lymph node, mandibular ++++++++++++++++++++++++++++I+ Lymph node, mesenteric M + + + + + + + + + +M+ + + + + + + + + + + + + + + + +M Sarcoma Spleen + + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + Thymus + + M +++++++++++++++++++++++++++


### Integumentary System Mammary gland ++++++++++++++++++++++++++++++ Skin ++++++++++++++++++++++++++++++ Subcutaneous tissue, sarcoma X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle + Hemangioma


### Nervous System Brain ++++++++++++++++++++++++++++++


### Respiratory System Lung ++++++++++++++++++++++++++++++ Alveolar/bronchiolar adenoma X Alveolar/bronchiolar carcinoma X X Nose ++++++++++++++++++++++++++++++ Pleura + Trachea ++++++++++++++++++++++++++++++


### Special Senses System Harderian gland A + + + Adenoma X X Carcinoma X Zymbal’s gland


### Urinary System Kidney + + + A ++++++++++++++++++++++++++ Urinary bladder + A ++++++++++++++++++++++++++++


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++ Lymphoma malignant X X X 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

234557777777777777777777777777 

### Number of Days on Study 968691123333333333333333333333 923220051111111111111222222222

222222222222222222222222222222 

### Carcass ID Number 899864584444555566666444455555 695835111234678901245678902345

### 245 o- Nitrotoluene, NTP TR 504

Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + + 6 Lymph node, mandibular + + + + M + + + + + + + + + + + + + + + + + + + + + + + + + 58 Lymph node, mesenteric + + + + + + + + + + + + + + + + + I I + + + + M + + + M + + 53 Sarcoma X 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Thymus + + + + + + + + + + + + + + + + + M + + + + + + + + + I + + 57


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Subcutaneous tissue, sarcoma 1


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle + 2 Hemangioma X 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60


### Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Alveolar/bronchiolar adenoma X 2 Alveolar/bronchiolar carcinoma X 3 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Pleura 1 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + M + + + + 59


### Special Senses System Harderian gland + + 5 Adenoma X X 4 Carcinoma 1 Zymbal’s gland + 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymphoma malignant X X X X X 8 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 0 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 222233333333333333555555555555

222222222222222223222222222222 T otal 

### Carcass ID Number 888866667777779990777788899999 Tissues/ 234567890123456780678907901234 T umors

### o-Nitrotoluene, NTP TR 504 246

Alimentary System Esophagus ++++++++++++++++++++++++++++++ Gallbladder A + A + + A + M A + + + +A+ + + + + + + + + + + +M+ + + Intestine large, colon A + A + + A + A A +++++++++++++++++++++ Intestine large, rectum A + A ++++A++++++++++++++++++++++ Intestine large, cecum A + A + + A + A A A + + + A ++++++++++++++++ Carcinoma Intestine small, duodenum A + A + + + +AA+ + + +A+ + + + + + + + + + + + + + + + Intestine small, jejunum A + A + + A + A A A + + + A ++++++++++++++++ Intestine small, ileum A + A + + A + A A A + + + A ++++++++++++++++ Liver +++++++A++++++++++++++++++++++ Hepatocellular carcinoma Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X Histiocytic sarcoma X X Osteosarcoma, metastatic, bone X Mesentery + + + + + + Carcinoma, metastatic,

uncertain primary site X Histiocytic sarcoma X Pancreas A + + + + + +AA+ + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + +M+ + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach A +++++++A+++++++++++++++++++++ Squamous cell papilloma X X Stomach, glandular A + A +++++A+++++++++++++++++++++ Tooth +


### Cardiovascular System Blood vessel + Heart ++++++++++++++++++++++++++++++ Carcinoma, metastatic,

uncertain primary site X


### Endocrine System Adrenal cortex ++++++++A+++++++++++++++++++++ Adrenal medulla + + + + + + + + AM+ + + + + + + + + + + + I + + + + + + + Pheochromocytoma benign Islets, pancreatic A + + + + + +AA+ + + + + + + + + + + + + + + + + + + + + Carcinoma Parathyroid gland + + M + + + + + + + + + + M + + M + + + + + + + MM + + + + Pituitary gland + + I + + + + +M+ + + + + + + + + + + + + + + + + + + + + Pars distalis, adenoma X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + +M+ + + + + Follicular cell, adenoma


### General Body System None 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

233456666667777777777777777777 

### Number of Days on Study 614950145671223333333333333333 623738248092131112222222222223

333333333333333333333333333333 

### Carcass ID Number 355443100320400000014444455551 993986281297042356901234512456

### 247 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 55 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 Carcinoma X 1 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 55 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Hepatocellular carcinoma X X X 3 Hepatocellular carcinoma, multiple 1 Hepatocellular adenoma X X X 5 Histiocytic sarcoma 2 Osteosarcoma, metastatic, bone 1 Mesentery + + + 9 Carcinoma, metastatic, 1

uncertain primary site Histiocytic sarcoma 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Squamous cell papilloma 2 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Tooth + + + 4


### Cardiovascular System Blood vessel + 2 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Carcinoma, metastatic,

uncertain primary site 1


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Pheochromocytoma benign X 1 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Carcinoma X 1 Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 55 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Pars distalis, adenoma X X X 4 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Follicular cell, adenoma X 1


### General Body System None 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333335555555555555555555555

333333333333333333333333333333 T otal 

### Carcass ID Number 111222231111222223333334455556 Tissues/ 789067801345123451345786706780 T umors

### o-Nitrotoluene, NTP TR 504 248

Genital System Clitoral gland + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + + Ovary A +++++++A++++A++++++++++++++++ Carcinoma X Hemangiosarcoma X Histiocytic sarcoma X Tubulostromal adenoma Uterus A +++++++++++++++++++++++++++++ Hemangioma X Hemangiosarcoma Histiocytic sarcoma X Leiomyoma Endometrium, polyp stromal X X Vagina + Carcinoma, metastatic,

uncertain primary site X


### Hematopoietic System Bone marrow A +++++++A+++++++++++++++++++++ Carcinoma, metastatic,

uncertain primary site X Histiocytic sarcoma X Lymph node + Lymph node, mandibular A + + + + + +MAM+ + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric A +++++++A+++++++++++++++++++++ Histiocytic sarcoma X Spleen A + A + + + + +M+ + + + + + + + + + + + + + + + + + + + + Thymus + + + M + + + +M+ + + + + + + + I + + + + + + + + + + + +


### Integumentary System Mammary gland +++++++++I++++++++++++++++++++ Skin ++++++++++++++++++++++++++++++ Basosquamous tumor malignant X Subcutaneous tissue, carcinoma, multiple X Subcutaneous tissue, sarcoma


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Osteosarcoma X Skeletal muscle + + + Carcinoma, metastatic,

uncertain primary site X Sarcoma X


### Nervous System Brain ++++++++++++++++++++++++++++++ Peripheral nerve + + Spinal cord + + 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

233456666667777777777777777777 

### Number of Days on Study 614950145671223333333333333333 623738248092131112222222222223

333333333333333333333333333333 

### Carcass ID Number 355443100320400000014444455551 993986281297042356901234512456

### 249 o- Nitrotoluene, NTP TR 504

Genital System Clitoral gland + + + + I + + + + + + + + + + + + + + + + + + + + + M + + + 57 Ovary + + + M + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Carcinoma 1 Hemangiosarcoma 1 Histiocytic sarcoma 1 Tubulostromal adenoma X 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Hemangioma X 2 Hemangiosarcoma X 1 Histiocytic sarcoma 1 Leiomyoma X 1 Endometrium, polyp stromal 2 Vagina 1 Carcinoma, metastatic,

uncertain primary site 1


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Carcinoma, metastatic,

uncertain primary site 1 Histiocytic sarcoma 1 Lymph node + +3 Lymph node, mandibular + + + + + + + + + M + + + + + + + + + + + + + + + + + + + + 55 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Histiocytic sarcoma 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Thymus + + + + + + + + + + + + + + + + M + + + + + + + + + + + + + 56


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Basosquamous tumor malignant 1 Subcutaneous tissue, carcinoma, multiple 1 Subcutaneous tissue, sarcoma X 1


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Osteosarcoma 1 Skeletal muscle 3 Carcinoma, metastatic,

uncertain primary site 1 Sarcoma 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Peripheral nerve 2 Spinal cord 2 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333335555555555555555555555

333333333333333333333333333333 T otal 

### Carcass ID Number 111222231111222223333334455556 Tissues/ 789067801345123451345786706780 T umors

### o-Nitrotoluene, NTP TR 504 250

Respiratory System Lung ++++++++++++++++++++++++++++++ Alveolar/bronchiolar adenoma X Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma,

multiple X X Carcinoma, metastatic, harderian gland X Carcinoma, metastatic,

uncertain primary site X Hepatocellular carcinoma, metastatic,

liver X Histiocytic sarcoma X Osteosarcoma, metastatic, bone X Mediastinum, alveolar/bronchiolar

carcinoma, metastatic, lung X Mediastinum, carcinoma X Mediastinum, carcinoma, metastatic,

harderian gland X Nose ++++++++++++++++++++++++++++++ Trachea + + + + + + + + + + + + + + + + + + + + + + + +M+ + + + +


### Special Senses System Harderian gland + Carcinoma X


### Urinary System Kidney A + A + + + +AA+ + + + + + + + + + + + + + + + + + + + + Carcinoma X Histiocytic sarcoma X Urinary bladder + + A + + + + AMA + + +M+ + + + + + + + + + + + + + + +


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++ Histiocytic sarcoma X X Lymphoma malignant X X X X 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

233456666667777777777777777777 

### Number of Days on Study 614950145671223333333333333333 623738248092131112222222222223

333333333333333333333333333333 

### Carcass ID Number 355443100320400000014444455551 993986281297042356901234512456

### 251 o- Nitrotoluene, NTP TR 504

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Alveolar/bronchiolar adenoma X X 3 Alveolar/bronchiolar carcinoma X 1 Alveolar/bronchiolar carcinoma,

multiple 2 Carcinoma, metastatic, harderian gland 1 Carcinoma, metastatic,

uncertain primary site 1 Hepatocellular carcinoma, metastatic,

liver 1 Histiocytic sarcoma 1 Osteosarcoma, metastatic, bone 1 Mediastinum, alveolar/bronchiolar

carcinoma, metastatic, lung 1 Mediastinum, carcinoma 1 Mediastinum, carcinoma, metastatic,

harderian gland 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Special Senses System Harderian gland 1 Carcinoma 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Carcinoma 1 Histiocytic sarcoma 1 Urinary bladder + + + + + + + + + + + + + + + + M + + + + + + + + + + + + + 54


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Histiocytic sarcoma 2 Lymphoma malignant X X X 7 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 1,250 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 333333335555555555555555555555

333333333333333333333333333333 T otal 

### Carcass ID Number 111222231111222223333334455556 Tissues/ 789067801345123451345786706780 T umors

### o-Nitrotoluene, NTP TR 504 252

Alimentary System Esophagus + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + + Gallbladder + A + + A + + + A A A + + + +M+ + + + + + + + + + + + + + Intestine large, colon + + + + + + + +AAA+ + + + + + + + + + + + + + + + + + + Intestine large, rectum + A + + + + + + +AA+ + + + + + + + + + + + + + + + + + + Intestine large, cecum + A + M + + + +AAA+ + + + + + + + + + + + + + + + + + + Carcinoma X X X Sarcoma X Intestine small, duodenum + + + + + + + +AAA+ + + + + + + + + + + + + + + + + + + Intestine small, jejunum + A + + + + + +AAA+ + + + + + + + + + + + + + + + + + + Intestine small, ileum + A + + + + + +AAA+ + + + + + + + + + + + + + + + + + + Liver ++++++++A+++++++++++++++++++++ Hemangiosarcoma Hepatocellular carcinoma X X X Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X X X X X Hepatocellular adenoma, multiple X X Histiocytic sarcoma X Sarcoma, metastatic, uncertain primary site X Mesentery + + + + + + Carcinoma, metastatic,

intestine large, cecum X Sarcoma, multiple X Sarcoma, metastatic, skin X Sarcoma, metastatic, skeletal muscle X Pancreas ++++++++A+A+++++++++++++++++++ Sarcoma X Salivary glands + + + + +M+ + + +M+ + + + + + + + + + + + + + + + + + + Histiocytic sarcoma X Stomach, forestomach ++++++++A+A+++++++++++++++++++ Squamous cell papilloma X X Stomach, glandular + + + + + + + +AAA+ + + + + + + + + + + + + + + + + + + Sarcoma X


### Cardiovascular System Blood vessel + Heart + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + +


### Endocrine System Adrenal cortex + + + + +M+ +A+ + + + + + + + + + + + + + + + + + + + + Adrenal medulla + + + + +M+ +A+ + + + + + + + + + + + + + + + + + + + + Islets, pancreatic ++++++++++++++++++++++++++++++ Parathyroid gland + + + + + + + + + + MM + + + + + + + + + + + + + + + + + + Pituitary gland + + + + + + + +A+M+ + + + + + + + + + + + + + + + + + + Pars distalis, adenoma X X Pars intermedia, adenoma Thyroid gland + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + + 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

566666667777777777777777777777 

### Number of Days on Study 512677790001133333333333333333 384112301190511111111111122222

433334433343333333334444433333 

### Carcass ID Number 167891266807766699990000177777 345259083520367912346789012467

### 253 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 54 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 54 Carcinoma X 4 Sarcoma 1 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 56 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Hemangiosarcoma X 1 Hepatocellular carcinoma X 4 Hepatocellular carcinoma, multiple X 2 Hepatocellular adenoma X X X X X X X X X X 16 Hepatocellular adenoma, multiple X 3 Histiocytic sarcoma 1 Sarcoma, metastatic, uncertain primary site 1 Mesentery + + + 9 Carcinoma, metastatic,

intestine large, cecum 1 Sarcoma, multiple 1 Sarcoma, metastatic, skin 1 Sarcoma, metastatic, skeletal muscle 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Sarcoma 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Histiocytic sarcoma 1 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Squamous cell papilloma 2 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Sarcoma 1


### Cardiovascular System Blood vessel 1 Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + 57 Pituitary gland + + + +M+ + + + + + + MMMM + + + + + + + + + + + + + + 53 Pars distalis, adenoma X 3 Pars intermedia, adenoma X 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + I + + + + + 58 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o- Nitrotoluene: 2,500 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 222222222223333333333333555555

333444444443333333333334333444 T otal 

### Carcass ID Number 778000011118888888999990666111 Tissues/ 890134512451346789067890125678 T umors

### o-Nitrotoluene, NTP TR 504 254

General Body System Tissue NOS +


### Genital System Clitoral gland + + + + + + + + + +M+ + + + + + I +M+ + + + + + + + + + Ovary + + + + + + + +A+ + + + + + + +M+ + + +M+ + + + + + + Cystadenoma Luteoma X Teratoma malignant X Uterus ++++++++++++++++++++++++++++++ Histiocytic sarcoma X Leiomyosarcoma Endometrium, carcinoma X Endometrium, polyp stromal X


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Histiocytic sarcoma X Lymph node + + + + + + Inguinal, sarcoma, metastatic, skin X Pancreatic, sarcoma X Renal, sarcoma, metastatic, skin X Lymph node, mandibular + + + + +M+ + + +M+ + + + + + + + + + + + + + + +M+ + Histiocytic sarcoma X Lymph node, mesenteric ++++++++A+A++++++++ I ++++++++++ Histiocytic sarcoma X Spleen ++++++++A+A+++++++++++++++++++ Histiocytic sarcoma X Thymus M M + + + + + +A IM+ + + + + + + + + + + + + + + + + + + Thymoma malignant


### Integumentary System Mammary gland ++++++++++++++++++++++++++++++ Skin ++++++++++++++++++++++++++++++ Subcutaneous tissue, hemangiosarcoma X X Subcutaneous tissue, sarcoma X X X X X Subcutaneous tissue, sarcoma, multiple X


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Femur, osteosarcoma X Skeletal muscle + + + Sarcoma X X Sarcoma, multiple X


### Nervous System Brain + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + + Peripheral nerve + + Spinal cord + + 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

566666667777777777777777777777 

### Number of Days on Study 512677790001133333333333333333 384112301190511111111111122222

433334433343333333334444433333 

### Carcass ID Number 167891266807766699990000177777 345259083520367912346789012467

### 255 o- Nitrotoluene, NTP TR 504

General Body System Tissue NOS ++ 3


### Genital System Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Ovary + + + + + + + + M + + + + + + + + + + + + + + + + + + + + + 56 Cystadenoma X X 2 Luteoma 1 Teratoma malignant 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Histiocytic sarcoma 1 Leiomyosarcoma X 1 Endometrium, carcinoma 1 Endometrium, polyp stromal X 2


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Histiocytic sarcoma 1 Lymph node + + + + 10 Inguinal, sarcoma, metastatic, skin 1 Pancreatic, sarcoma 1 Renal, sarcoma, metastatic, skin 1 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + I M + + + + + 55 Histiocytic sarcoma 1 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Histiocytic sarcoma 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Histiocytic sarcoma 1 Thymus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 55 Thymoma malignant X 1


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Subcutaneous tissue, hemangiosarcoma 2 Subcutaneous tissue, sarcoma 5 Subcutaneous tissue, sarcoma, multiple 1


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Femur, osteosarcoma 1 Skeletal muscle 3 Sarcoma 2 Sarcoma, multiple 1


### Nervous System Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Peripheral nerve 2 Spinal cord 2 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o-Nitrotoluene: 2,500 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 222222222223333333333333555555

333444444443333333333334333444 T otal 

### Carcass ID Number 778000011118888888999990666111 Tissues/ 890134512451346789067890125678 T umors

### o-Nitrotoluene, NTP TR 504 256

Respiratory System Lung + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + + Alveolar/bronchiolar adenoma X X Histiocytic sarcoma X Osteosarcoma, metastatic, bone X Sarcoma, metastatic, skeletal muscle X Teratoma malignant, metastatic, ovary X Mediastinum, histiocytic sarcoma X Mediastinum, sarcoma, metastatic,

uncertain primary site X Mediastinum, sarcoma, metastatic,

skeletal muscle X Nose ++++++++A+++++++++++++++++++++ Trachea + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + +


### Special Senses System Harderian gland + Adenoma Carcinoma X Lacrimal gland


### Urinary System Kidney ++++++++A+A+++++++++++++++++++ Urinary bladder + + + + + + + +M+M+ + + + + + + + + + + + + + + + + + +


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++ Histiocytic sarcoma X Lymphoma malignant X X X 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 2,500 ppm

566666667777777777777777777777 

### Number of Days on Study 512677790001133333333333333333 384112301190511111111111122222

433334433343333333334444433333 

### Carcass ID Number 167891266807766699990000177777 345259083520367912346789012467

### 257 o- Nitrotoluene, NTP TR 504

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Alveolar/bronchiolar adenoma X X 4 Histiocytic sarcoma 1 Osteosarcoma, metastatic, bone 1 Sarcoma, metastatic, skeletal muscle 1 Teratoma malignant, metastatic, ovary 1 Mediastinum, histiocytic sarcoma 1 Mediastinum, sarcoma, metastatic,

uncertain primary site 1 Mediastinum, sarcoma, metastatic,

skeletal muscle 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59


### Special Senses System Harderian gland + + + 4 Adenoma X 1 Carcinoma X X 3 Lacrimal gland + 1


### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + M + + + 57


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Histiocytic sarcoma 1 Lymphoma malignant X X 5 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o-Nitrotoluene: 2,500 ppm

777777777777777777777777777777 

### Number of Days on Study 333333333333333333333333333333 222222222223333333333333555555

333444444443333333333334333444 T otal 

### Carcass ID Number 778000011118888888999990666111 Tissues/ 890134512451346789067890125678 T umors

### o-Nitrotoluene, NTP TR 504 258

Alimentary System Esophagus + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + + Gallbladder + + + +AAA+ + +M+AAA+ + + +A+A+ + + + AAAA Intestine large, colon + + + + +A+ + + +A+AA+ + + + +A+A+ +A+ +AAA Hemangiosarcoma, metastatic,

mesentery Intestine large, rectum +++++A++++A+A++++++A+A+++++A++ Intestine large, cecum + + + +AA+ +A+A+AA+ + + + +AAA+ +A+ AAAA Carcinoma X Intestine small, duodenum + + + + +A+ +A+A+AA+ + + + +AAA+ +A+ AAAA Intestine small, jejunum + + + +AA+ + + +A+AA+ + + + +AAA+ +A+ AAAA Carcinoma Intestine small, ileum + + + +AA+ + + +A+AA+ + + + +AAA+ +A+ AAAA Liver ++++++++++++++++++++++++++++++ Cholangioma Hemangiosarcoma, metastatic, skin Hepatocellular carcinoma X X X X X X X Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X X X X X X Hepatocellular adenoma, multiple X XXX Hepatocholangiocarcinoma Mesentery + + + + + + +++++ + + ++ Hemangiosarcoma X X X X X X X X XX Hemangiosarcoma, multiple X X X Sarcoma X Pancreas ++++++++++A+++++++++++++++++++ Hemangiosarcoma, metastatic,

mesentery X Sarcoma, metastatic, mesentery X Salivary glands + + + + + + + + + + M + + M + + + + + M + M + + + + + MM + Stomach, forestomach ++++++++++++++++++++++++++++++ Squamous cell papilloma Stomach, glandular + + + +AA+ + + +A+ + + + + + + + + + + + + + + + + + + Tooth


### Cardiovascular System Blood vessel Heart ++++++++++++++++++++++++++++++


### Endocrine System Adrenal cortex ++++++++++++++++++++++++++++++ Adrenal medulla ++++++++++++++++++++++++++++++ Islets, pancreatic ++++++++++A+++++++++++++++A+++ Parathyroid gland + + + + + +M+ + +M+ + + + + + + +M+M+ + + +M+M+ Pituitary gland + + + + + + +M+ +M+ + + + + + + +M+ + + +M+ + + + + Thyroid gland + + + + + +M+ + +M+ +M+ + + + + + +M+ + + + + + + + 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

444445555555555555556666666666 

### Number of Days on Study 004480124666778888990001112234 394487628779591338075890194900

444444444444444444444444444444 

### Carcass ID Number 364523646256372232257324865532 171066144955043845721913097334

### 259 o- Nitrotoluene, NTP TR 504

Alimentary System Esophagus + + A + + + + + + + + + + + + + + + + M + + + + + + + + + + 57 Gallbladder AAAA + AAAAAA + AMA + + + + M A + + A + + + + + + 31 Intestine large, colon A + A + + + + + + A A + A + A + + + + A A + + A + + + + + + 41 Hemangiosarcoma, metastatic,

mesentery X 1 Intestine large, rectum A + A + + + + + + + A + A + A + + + + A A + + A + + + + + + 46 Intestine large, cecum AAAA+AAAA+A+A+A + + + + A A + + A + + + + + + 32 Carcinoma X X 3 Intestine small, duodenum AAAA+ +AA+AA+A+A + + + + A A + + A + + + + + + 34 Intestine small, jejunum AAAA+ +AA+AA+A+A + + + + A A + + A + + + + + + 34 Carcinoma X 1 Intestine small, ileum AAAA+ +AA+A+AA+A + + + + A A + + A + + + + + + 34 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Cholangioma X 1 Hemangiosarcoma, metastatic, skin X 1 Hepatocellular carcinoma X X9 Hepatocellular carcinoma, multiple X X X X X X 7 Hepatocellular adenoma X X 9 Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X 20 Hepatocholangiocarcinoma X 1 Mesentery + + + + + + + + + + + + + + + + + + + 34 Hemangiosarcoma X X X X X X X X X X X X X X X 25 Hemangiosarcoma, multiple X X X X 7 Sarcoma 1 Pancreas + + A + + + + + + + + + + + M + + + + + + + + + + + + + + + 57 Hemangiosarcoma, metastatic,

mesentery 1 Sarcoma, metastatic, mesentery 1 Salivary glands + M M + + + + M + + M + + + + + + + + M + + + + + + + + + + 49 Stomach, forestomach + + + + + + + + + + + + + + A + + + + + + + + + + + + + + + 59 Squamous cell papilloma X 1 Stomach, glandular + + A + + + + + + A A + + + A + + + + A + + + + + + + + + + 52 Tooth ++ 2


### Cardiovascular System Blood vessel + 1 Heart + + M + + + + + + + + + + + + + + + + M + + + + + + + + + + 58


### Endocrine System Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Adrenal medulla + + + + + + + + + M + + + + + + + + + + + + + + + + + + + + 59 Islets, pancreatic + + A + + + + + + + + + + + M + + + + + + + + + + + + + + + 56 Parathyroid gland + + M + + + + + + + + M + + + + + + + M + + + + + + + + M + 50 Pituitary gland + + M + + I + + M + + + + + + + + + + + + + + + + + + + + + 53 Thyroid gland + + M + + + + A + + + + + + + + + + + M + + + + + + + + + + 53 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

666666666666666666667777777777 

### Number of Days on Study 445566667777888899990112222222 378911891579566800362002599999

444444444444444444444444444444 T otal 

### Carcass ID Number 735245353465477467644566347777 Tissues/ 886288512784902539602902763567 T umors

### o-Nitrotoluene, NTP TR 504 260

General Body System Tissue NOS + + Abdominal, hemangiosarcoma X Abdominal, hemangiosarcoma, multiple X Thoracic, sarcoma


### Genital System Clitoral gland ++++++++++++++++++++++++++++++ Ovary + + + + + + + + + + + + + +M+ + + + + + + + + + + +M+ + Luteoma Uterus ++++++++++++++++++++++++++++++ Hemangiosarcoma X Cervix, hemangiosarcoma X


### Hematopoietic System Bone marrow ++++++++++++++++++++++++++++++ Lymph node ++++ ++ Iliac, sarcoma, metastatic, skin Mediastinal, sarcoma, metastatic,

uncertain primary site X Renal, hemangiosarcoma, metastatic,

mesentery X Renal, sarcoma, metastatic, skin Lymph node, mandibular + + + + + + + + + + M + MM + + M + + M + M + + + + + + M + Lymph node, mesenteric ++++++++++A+++++++++++++++++++ Sarcoma, metastatic, mesentery X Spleen ++++++++++A+++++++++++++++++++ Thymus + + + + + M I + + + A + + MM + + M + I I M + + + + + + + M


### Integumentary System Mammary gland ++++++++++A+++++++++++++++++++ Skin ++++++++++++++++++++++++++++++ Basal cell carcinoma Subcutaneous tissue, hemangiosarcoma X X X X Subcutaneous tissue, hemangiosarcoma, multiple Subcutaneous tissue, sarcoma


### Musculoskeletal System Bone ++++++++++++++++++++++++++++++ Skeletal muscle + + + + + + + + + Hemangiosarcoma X X X X X X X Hemangiosarcoma, multiple X Sarcoma Sarcoma, multiple X 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

444445555555555555556666666666 

### Number of Days on Study 004480124666778888990001112234 394487628779591338075890194900

444444444444444444444444444444 

### Carcass ID Number 364523646256372232257324865532 171066144955043845721913097334

### 261 o- Nitrotoluene, NTP TR 504

General Body System Tissue NOS + 3 Abdominal, hemangiosarcoma 1 Abdominal, hemangiosarcoma, multiple 1 Thoracic, sarcoma X 1


### Genital System Clitoral gland + + + I M + + + + + + + + + + + + + + + + + + + + + + + + + 58 Ovary + + + + + + + + + + A + + + M + + + + + + + + + + + + + + + 56 Luteoma X 1 Uterus + + + + + + + + + + + + + + A + + + + + + + + + + + + + + + 59 Hemangiosarcoma X 2 Cervix, hemangiosarcoma 1


### Hematopoietic System Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + + + 11 Iliac, sarcoma, metastatic, skin X 1 Mediastinal, sarcoma, metastatic,

uncertain primary site 1 Renal, hemangiosarcoma, metastatic,

mesentery 1 Renal, sarcoma, metastatic, skin X 1 Lymph node, mandibular + M M + + + + M + + M + + + + + + + + A + + + + + + + + + + 48 Lymph node, mesenteric + + M + + + + + + + + + + + A + + + + + + + + + + + + + + + 57 Sarcoma, metastatic, mesentery 1 Spleen + + + + + + + + + + M + + + A + + + + + + + + + + + + + + + 57 Thymus + M M + + + + + + + M + M + + + + + + M + + + I + + + + + + 44


### Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Basal cell carcinoma X 1 Subcutaneous tissue, hemangiosarcoma X X X X X X X X X X X X 16 Subcutaneous tissue, hemangiosarcoma, multiple X X X 3 Subcutaneous tissue, sarcoma XX 2


### Musculoskeletal System Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Skeletal muscle + + + + + + + + + 18 Hemangiosarcoma X X X X X X X 14 Hemangiosarcoma, multiple X 2 Sarcoma X 1 Sarcoma, multiple 1 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

666666666666666666667777777777 

### Number of Days on Study 445566667777888899990112222222 378911891579566800362002599999

444444444444444444444444444444 T otal 

### Carcass ID Number 735245353465477467644566347777 Tissues/ 886288512784902539602902763567 T umors

### o-Nitrotoluene, NTP TR 504 262

Nervous System Brain + + + + + + + + + +M+ + + + + + + + + + + + + + + + + + + Peripheral nerve + + + Spinal cord + + +


### Respiratory System Lung ++++++++++++++++++++++++++++++ Alveolar/bronchiolar adenoma X X Alveolar/bronchiolar carcinoma,

multiple X Carcinoma, metastatic, harderian gland Hemangiosarcoma, metastatic, skin Hemangiosarcoma, metastatic,

uncertain primary site Hemangiosarcoma, metastatic,

skeletal muscle X X Hepatocellular carcinoma, metastatic,

liver Sarcoma, metastatic, mesentery Nose + + + + + + + + + +M+ + + + + + + + + +M+ + + + + + + + Trachea + + + + + + + + + +M+ +M+ + + + + + +M+ + + + + + + +


### Special Senses System Harderian gland + + Adenoma X Carcinoma X


### Urinary System Kidney ++++++++++++++++++++++++++++++ Urinary bladder + + + +A+ + + + +A+ + + + + + I + + + + + + + + +M+ + Hemangiosarcoma X


### Systemic Lesions Multiple organs ++++++++++++++++++++++++++++++ Lymphoma malignant 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

444445555555555555556666666666 

### Number of Days on Study 004480124666778888990001112234 394487628779591338075890194900

444444444444444444444444444444 

### Carcass ID Number 364523646256372232257324865532 171066144955043845721913097334

### 263 o- Nitrotoluene, NTP TR 504

Nervous System Brain + + M + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Peripheral nerve 3 Spinal cord 3


### Respiratory System Lung + M M + + + + + + + + + + + + + + + + M + + + + + + + + + + 57 Alveolar/bronchiolar adenoma X X 4 Alveolar/bronchiolar carcinoma,

multiple X 2 Carcinoma, metastatic, harderian gland X 1 Hemangiosarcoma, metastatic, skin X 1 Hemangiosarcoma, metastatic,

uncertain primary site X 1 Hemangiosarcoma, metastatic,

skeletal muscle 2 Hepatocellular carcinoma, metastatic,

liver X 1 Sarcoma, metastatic, mesentery X 1 Nose + + M + + + + + + + + + + + + + + + + + + + + + + + + + + + 57 Trachea + + M + + + + + + + + + + + + + + + + M + + + + + + + + + + 55


### Special Senses System Harderian gland ++ 4 Adenoma X 2 Carcinoma X 2


### Urinary System Kidney + + + + + + + + + + + + + + A + + + + + + + + + + + + + + + 59 Urinary bladder + + + + + + + + + + M + + + A + + + + + + + + + + + + + + + 54 Hemangiosarcoma 1


### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymphoma malignant X X X 3 

### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of o -Nitrotoluene: 5,000 ppm

666666666666666666667777777777 

### Number of Days on Study 445566667777888899990112222222 378911891579566800362002599999

444444444444444444444444444444 T otal 

### Carcass ID Number 735245353465477467644566347777 Tissues/ 886288512784902539602902763567 T umors

### o-Nitrotoluene, NTP TR 504 264



### T ABLE 

### D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Harderian Gland: Adenoma Overall rate a 4/60 (7%) 0/60 (0%) 1/60 (2%) 2/60 (3%) Adjusted rate b 7.1% 0.0% 1.8% 5.0% Terminal rate c 4/52 (8%) 0/46 (0%) 1/47 (2%) 1/5 (20%) First incidence (days) 729 (T) —e 729 (T) 630 Poly-3 test d P=0.378N P=0.067N P=0.175N P=0.501N



### Harderian Gland: Carcinoma Overall rate 1/60 (2%) 1/60 (2%) 3/60 (5%) 2/60 (3%) Adjusted rate 1.8% 1.8% 5.2% 5.0% Terminal rate 1/52 (2%) 0/46 (0%) 2/47 (4%) 1/5 (20%) First incidence (days) 729 (T) 644 672 548 Poly-3 test P=0.190 P=0.752 P=0.313 P=0.388



### Harderian Gland: Adenoma or Carcinoma Overall rate 5/60 (8%) 1/60 (2%) 4/60 (7%) 4/60 (7%) Adjusted rate 8.9% 1.8% 7.0% 9.8% Terminal rate 5/52 (10%) 0/46 (0%) 3/47 (6%) 2/5 (40%) First incidence (days) 729 (T) 644 672 548 Poly-3 test P=0.397 P=0.112N P=0.488N P=0.575


### Large Intestine (Cecum): Carcinoma Overall rate 0/60 (0%) 1/60 (2%) 4/60 (7%) 3/60 (5%) Adjusted rate 0.0% 1.9% 7.0% 7.4% Terminal rate 0/52 (0%) 1/46 (2%) 3/47 (6%) 1/5 (20%) First incidence (days) — 729 (T) 715 619 Poly-3 test P=0.024 P=0.492 P=0.064 P=0.072


### Liver: Hepatocellular Adenoma Overall rate 7/60 (12%) 5/59 (8%) 19/59 (32%) 29/60 (48%) Adjusted rate 12.4% 9.4% 33.5% 62.0% Terminal rate 7/52 (14%) 5/46 (11%) 17/47 (36%) 5/5 (100%) First incidence (days) 729 (T) 729 (T) 673 567 Poly-3 test P<0.001 P=0.419N P=0.006 P<0.001


### Liver: Hepatocellular Carcinoma Overall rate 2/60 (3%) 4/59 (7%) 6/59 (10%) 16/60 (27%) Adjusted rate 3.5% 7.5% 10.6% 36.2% Terminal rate 1/52 (2%) 4/46 (9%) 4/47 (9%) 1/5 (20%) First incidence (days) 362 729 (T) 661 567 Poly-3 test P<0.001 P=0.307 P=0.132 P<0.001


### Liver: Hepatocellular Adenoma or Carcinoma Overall rate 9/60 (15%) 9/59 (15%) 24/59 (41%) 39/60 (65%) Adjusted rate 15.7% 16.9% 42.1% 79.1% Terminal rate 8/52 (15%) 9/46 (20%) 20/47 (43%) 5/5 (100%) First incidence (days) 362 729 (T) 661 567 Poly-3 test P<0.001 P=0.538 P<0.001 P<0.001


### Lung: Alveolar/bronchiolar Adenoma Overall rate 2/60 (3%) 3/60 (5%) 4/59 (7%) 4/57 (7%) Adjusted rate 3.6% 5.6% 7.1% 10.3% Terminal rate 2/52 (4%) 3/46 (7%) 3/47 (6%) 0/5 (0%) First incidence (days) 729 (T) 729 (T) 701 516 Poly-3 test P=0.126 P=0.481 P=0.339 P=0.190

### 265 o- Nitrotoluene, NTP TR 504



### T ABLE 

### D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Lung: Alveolar/bronchiolar Carcinoma Overall rate 3/60 (5%) 3/60 (5%) 0/59 (0%) 2/57 (4%) Adjusted rate 5.3% 5.5% 0.0% 5.2% Terminal rate 3/52 (6%) 2/46 (4%) 0/47 (0%) 0/5 (0%) First incidence (days) 729 (T) 553 — 488 Poly-3 test P=0.358N P=0.647 P=0.120N P=0.666N


### Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 5/60 (8%) 6/60 (10%) 4/59 (7%) 6/57 (11%) Adjusted rate 8.9% 11.0% 7.1% 15.1% Terminal rate 5/52 (10%) 5/46 (11%) 3/47 (6%) 0/5 (0%) First incidence (days) 729 (T) 553 701 488 Poly-3 test P=0.293 P=0.478 P=0.499N P=0.273


### Mesentery: Hemangiosarcoma Overall rate 0/60 (0%) 0/60 (0%) 0/60 (0%) 32/60 (53%) Adjusted rate 0.0% 0.0% 0.0% 65.5% Terminal rate 0/52 (0%) 0/46 (0%) 0/47 (0%) 4/5 (80%) First incidence (days) — — — 507 Poly-3 test P<0.001 —f — P<0.001


### Pituitary Gland (Pars Distalis or Unspecified Site): Adenoma Overall rate 3/58 (5%) 4/58 (7%) 3/53 (6%) 0/53 (0%) Adjusted rate 5.4% 7.5% 6.0% 0.0% Terminal rate 2/51 (4%) 3/46 (7%) 3/42 (7%) 0/5 (0%) First incidence (days) 299 644 729 (T) — Poly-3 test P=0.201N P=0.474 P=0.611 P=0.221N


### Skeletal Muscle: Hemangiosarcoma Overall rate 0/60 (0%) 0/60 (0%) 0/60 (0%) 16/60 (27%) Adjusted rate 0.0% 0.0% 0.0% 35.6% Terminal rate 0/52 (0%) 0/46 (0%) 0/47 (0%) 0/5 (0%) First incidence (days) — — — 444 Poly-3 test P<0.001 — — P<0.001



### Skeletal Muscle: Sarcoma Overall rate 0/60 (0%) 1/60 (2%) 3/60 (5%) 2/60 (3%) Adjusted rate 0.0% 1.9% 5.2% 5.0% Terminal rate 0/52 (0%) 1/46 (2%) 0/47 (0%) 0/5 (0%) First incidence (days) — 729 (T) 553 583 Poly-3 test P=0.074 P=0.492 P=0.125 P=0.173


### Skin (Subcutaneous Tissue): Hemangiosarcoma Overall rate 0/60 (0%) 0/60 (0%) 2/60 (3%) 19/60 (32%) Adjusted rate 0.0% 0.0% 3.5% 43.2% Terminal rate 0/52 (0%) 0/46 (0%) 1/47 (2%) 2/5 (40%) First incidence (days) — — 624 409 Poly-3 test P<0.001 — P=0.243 P<0.001


### Skin (Subcutaneous Tissue): Sarcoma Overall rate 1/60 (2%) 1/60 (2%) 6/60 (10%) 2/60 (3%) Adjusted rate 1.8% 1.9% 10.4% 5.0% Terminal rate 0/52 (0%) 1/46 (2%) 2/47 (4%) 0/5 (0%) First incidence (days) 710 729 (T) 661 696 Poly-3 test P=0.112 P=0.751 P=0.062 P=0.384

### o-Nitrotoluene, NTP TR 504 266



### T ABLE 

### D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Uterus: Hemangiosarcoma Overall rate 0/60 (0%) 1/60 (2%) 0/60 (0%) 3/60 (5%) Adjusted rate 0.0% 1.9% 0.0% 7.3% Terminal rate 0/52 (0%) 1/46 (2%) 0/47 (0%) 0/5 (0%) First incidence (days) — 729 (T) — 522 Poly-3 test P=0.033 P=0.492 — P=0.074


### All Organs: Hemangioma Overall rate 3/60 (5%) 2/60 (3%) 0/60 (0%) 0/60 (0%) Adjusted rate 5.3% 3.7% 0.0% 0.0% Terminal rate 3/52 (6%) 2/46 (4%) 0/47 (0%) 0/5 (0%) First incidence (days) 729 (T) 729 (T) — — Poly-3 test P=0.043N P=0.519N P=0.117N P=0.195N


### All Organs: Hemangiosarcoma Overall rate 0/60 (0%) 2/60 (3%) 3/60 (5%) 50/60 (83%) Adjusted rate 0.0% 3.6% 5.2% 90.2% Terminal rate 0/52 (0%) 1/46 (2%) 2/47 (4%) 4/5 (80%) First incidence (days) — 343 624 409 Poly-3 test P<0.001 P=0.232 P=0.124 P<0.001


### All Organs: Hemangioma or Hemangiosarcoma Overall rate 3/60 (5%) 4/60 (7%) 3/60 (5%) 50/60 (83%) Adjusted rate 5.3% 7.3% 5.2% 90.2% Terminal rate 3/52 (6%) 3/46 (7%) 2/47 (4%) 4/5 (80%) First incidence (days) 729 (T) 343 624 409 Poly-3 test P<0.001 P=0.486 P=0.651N P<0.001


### All Organs: Malignant Lymphoma Overall rate 8/60 (13%) 7/60 (12%) 5/60 (8%) 3/60 (5%) Adjusted rate 14.2% 12.6% 8.7% 7.4% Terminal rate 7/52 (14%) 5/46 (11%) 3/47 (6%) 0/5 (0%) First incidence (days) 725 266 671 661 Poly-3 test P=0.148N P=0.514N P=0.266N P=0.244N


### All Organs: Benign Neoplasms Overall rate 21/60 (35%) 20/60 (33%) 26/60 (43%) 33/60 (55%) Adjusted rate 36.4% 36.8% 45.1% 68.5% Terminal rate 18/52 (35%) 19/46 (41%) 23/47 (49%) 5/5 (100%) First incidence (days) 299 644 673 516 Poly-3 test P<0.001 P=0.557 P=0.221 P<0.001


### All Organs: Malignant Neoplasms Overall rate 16/60 (27%) 23/60 (38%) 27/60 (45%) 55/60 (92%) Adjusted rate 27.9% 39.3% 45.2% 96.1% Terminal rate 13/52 (25%) 14/46 (30%) 15/47 (32%) 5/5 (100%) First incidence (days) 362 266 553 409 Poly-3 test P<0.001 P=0.136 P=0.039 P<0.001

### 267 o- Nitrotoluene, NTP TR 504



### T ABLE 

### D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

All Organs: Benign or Malignant Neoplasms Overall rate 33/60 (55%) 37/60 (62%) 44/60 (73%) 56/60 (93%) Adjusted rate 56.2% 63.2% 73.3% 97.6% Terminal rate 28/52 (54%) 28/46 (61%) 31/47 (66%) 5/5 (100%) First incidence (days) 299 266 553 409 Poly-3 test P<0.001 P=0.281 P=0.038 P<0.001

(T)Terminal sacrifice a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for l iver, lung, and pituitary gland; for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals th at do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal group f Value of statistic cannot be computed.

### o-Nitrotoluene, NTP TR 504 268



### T ABLE 

### D4a Historical Incidence of Hemangiosarcoma (All Sites) in Control Female B6C3F 1 

### Mice

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Diet a Acrylonitrile (gavage) 4/50 Citral (feed) 0/99
p,p N -Dichlorodiphenyl sulfone (feed) 0/50 Indium phosphide (inhalation) 3/50 60-Hz Magnetic fields (whole body exposure) 2/100 Methacrylonitrile (gavage) 2/50

o -Nitrotoluene (feed) 0/60
p-Nitrotoluene (feed) 1/50 Riddelline (gavage) 0/50 Sodium nitrite (drinking water) 1/50 Vanadium pentoxide (inhalation) 2/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 15/659 (2.3%) Mean ± standard deviation 2.6% ± 2.7% Range 0%-8%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 0/50 2,2-Bis(bromomethyl)-1,3-propanediol 0/52
t- Butylhydroquinone 2/51 Emodin 1/50

o-Nitroanisole 1/50
p-Nitrobenzoic acid 4/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 31/953 (3.3%) Mean ± standard deviation 3.3% ± 2.4% Range 0%-8%

a Data as of December 22, 2000 b Data as of December 23, 1999

### 269 o- Nitrotoluene, NTP TR 504



### T ABLE 

### D4b Historical Incidence of Hepatocellular Neoplasms in Control Female B6C3F 1 

### Mice

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence in Controls Given NTP-2000 Diet a Acrylonitrile (gavage) 14/50 7/50 20/50 Citral (feed) 8/99 4/99 12/99
p,p N -Dichlorodiphenyl sulfone (feed) 4/50 3/50 6/50 Indium phosphide (inhalation) 12/50 6/50 18/50 60-Hz Magnetic fields (whole body exposure) 17/98 6/98 22/98 Methacrylonitrile (gavage) 9/50 2/50 10/50

o -Nitrotoluene (feed) 7/60 2/60 9/60
p-Nitrotoluene (feed) 6/49 3/49 8/49 Riddelliine (gavage) 9/49 8/49 16/49 Sodium nitrite (drinking water) 9/50 2/50 10/50 Vanadium pentoxide (inhalation) 6/50 6/50 12/50



### Overall Historical Incidence in Controls Given NTP-2000 Diet

Total (%) 101/655 (15.4%) 49/655 (7.5%) 143/655 (21.8%) Mean ± standard deviation 16.0% ± 6.3% 8.0% ± 4.7% 22.8% ± 9.6% Range 8%-28% 3%-16% 12%-40%



### Historical Incidence in Feed Controls Given NIH-07 Diet at Southern Research Institute b

Benzyl acetate 6/50 10/50 14/50 2,2-Bis(bromomethyl)-1,3-propanediol 16/51 5/51 20/51
t- Butylhydroquinone 9/51 8/51 17/51 Emodin 9/50 4/50 13/50

o-Nitroanisole 14/50 5/50 17/50
p-Nitrobenzoic acid 11/50 4/50 15/50



### Overall Historical Incidence in Feed Controls Given NIH-07 Diet

Total (%) 218/951 (22.9%) 104/951 (10.9%) 292/951 (30.7%) Mean ± standard deviation 22.9% ± 9.9% 10.9% ± 4.6% 30.7% ± 10.2% Range 12%-50% 4%-20% 12%-56%

a Data as of December 22, 2000 b Data as of December 23, 1999

### o-Nitrotoluene, NTP TR 504 270



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of o- Nitrotoluene a



### 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Disposition Summary Animals initially in study 60 60 60 60 Early deaths

Moribund 4 6 7 22 Natural deaths 4 8 6 33 Survivors

Terminal sacrifice 52 46 47 5

Animals examined microscopically 60 60 60 60



### Alimentary System Gallbladder (54) (53) (54) (31)

Cyst 1 (2%) 1 (2%) Intestine large, cecum (56) (53) (54) (32)

Edema 1 (2%) Inflammation, chronic 1 (3%) Intestine small, jejunum (56) (53) (56) (34)

Peyer’s patch, hyperplasia, lymphoid 1 (2%) 1 (2%) Peyer’s patch, inflammation, chronic, focal, suppurative 1 (2%) Liver (60) (59) (59) (60)

Angiectasis 1 (2%) 3 (5%) Basophilic focus 1 (2%) 6 (10%) 2 (3%) 6 (10%) Clear cell focus 1 (2%) 2 (3%) 4 (7%) Congestion, focal 1 (2%) 2 (3%) 1 (2%) Cyst 1 (2%) Eosinophilic focus 2 (3%) 3 (5%) 6 (10%) 28 (47%) Hyperplasia, focal, lymphoid 2 (3%) 5 (8%) 2 (3%) 1 (2%) Infiltration cellular, mixed cell 45 (75%) 38 (64%) 37 (63%) 43 (72%) Inflammation, focal 1 (2%) Mixed cell focus 1 (2%) 3 (5%) Necrosis 3 (5%) 2 (3%) 13 (22%) Tension lipidosis 1 (2%) 1 (2%) Bile duct, cyst 1 (2%) Hepatocyte, fatty change 1 (2%) Hepatocyte, fatty change, diffuse 2 (3%) Hepatocyte, necrosis, focal 6 (10%) Hepatocyte, tension lipidosis 1 (2%) Hepatocyte, vacuolization cytoplasmic, diffuse 2 (3%) Hepatocyte, vacuolization cytoplasmic, focal 1 (2%) 2 (3%) 2 (3%) 9 (15%) Hepatocyte, periportal, depletion glycogen 1 (2%) Hepatocyte, centrilobular, fatty change 2 (3%) 2 (3%) Hepatocyte, centrilobular, necrosis 1 (2%) Oval cell, hyperplasia 4 (7%) Mesentery (11) (9) (9) (34)

Hemorrhage 1 (11%) Inflammation, chronic 1 (9%) 2 (6%) Artery, inflammation, chronic 1 (11%) Fat, necrosis 6 (55%) 5 (56%) 4 (44%) Pancreas (60) (57) (58) (57)

Acinus, atrophy, diffuse 2 (4%) 1 (2%) 1 (2%) Acinus, atrophy, focal 1 (2%) 1 (2%) Duct, cyst 2 (3%) 1 (2%) Duct, inflammation, chronic, focal 2 (3%)

a Number of animals examined microscopically at the site and the number of animals with lesion

### 271 o- Nitrotoluene, NTP TR 504



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Alimentary System (continued) Stomach, forestomach (60) (58) (58) (59)

Diverticulum 1 (2%) Edema 1 (2%) Inflammation, focal 1 (2%) 2 (3%) 1 (2%) Ulcer 1 (2%) 1 (2%) 1 (2%) Epithelium, hyperplasia 1 (2%) 4 (7%) 6 (10%) 1 (2%) Stomach, glandular (58) (57) (57) (52)

Erosion 1 (2%) Inflammation, focal 2 (4%) Ulcer 1 (2%) 2 (4%) 1 (2%) Glands, degeneration, cystic, focal 2 (4%) Tooth (8) (4) (2)

Peridontal tissue, inflammation, chronic 8 (100%) 4 (100%) 2 (100%)



### Cardiovascular System Blood vessel (2) (1) (1)

Aorta, mineralization 1 (50%) 1 (100%) Heart (60) (60) (59) (58)

Angiectasis 1 (2%) Infiltration cellular, mixed cell 1 (2%) 1 (2%) Inflammation, chronic, focal 2 (3%)



### Endocrine System Adrenal cortex (59) (59) (58) (60)

Accessory adrenal cortical nodule 2 (3%) 1 (2%) Cyst 2 (3%) Cytoplasmic alteration, focal 1 (2%) Hyperplasia, focal 1 (2%) Adrenal medulla (59) (57) (58) (59)

Hyperplasia 2 (3%) Islets, pancreatic (60) (57) (60) (56)

Atrophy, diffuse 1 (2%) Parathyroid gland (57) (55) (57) (50)

Cyst 1 (2%) 1 (2%) 1 (2%) Pituitary gland (58) (58) (53) (53)

Pars distalis, cyst 1 (2%) Pars distalis, cytoplasmic alteration, focal 1 (2%) 4 (7%) 3 (6%) Pars distalis, hyperplasia, focal 1 (2%) 1 (2%) 2 (4%) Thyroid gland (60) (59) (58) (53)

Degeneration, cystic, focal 14 (23%) 16 (27%) 16 (28%) 13 (25%) Hyperplasia, lymphoid 1 (2%) Inflammation, chronic, focal 2 (3%) 1 (2%) Follicle, cyst 1 (2%) 2 (4%) Follicular cell, hyperplasia 1 (2%) 1 (2%)

### o-Nitrotoluene, NTP TR 504 272



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

General Body System Tissue NOS (1) (3) (3)

Abdominal, cyst 1 (33%)



### Genital System Clitoral gland (58) (57) (57) (58)

Degeneration, cystic 11 (19%) 4 (7%) 5 (9%) 2 (3%) Inflammation, chronic 3 (5%) 2 (4%) Pigmentation 3 (5%) 2 (4%) 1 (2%) Ovary (59) (56) (56) (56)

Angiectasis 3 (5%) 2 (4%) 1 (2%) Cyst 11 (19%) 16 (29%) 12 (21%) 11 (20%) Cyst, multiple 1 (2%) 2 (4%) 1 (2%) Hemorrhage 1 (2%) 2 (4%) 1 (2%) Hyperplasia, tubular 1 (2%) Mineralization 1 (2%) Interstitial cell, hyperplasia 2 (4%) Periovarian tissue, cyst 1 (2%) Periovarian tissue, inflammation, chronic 1 (2%) Uterus (60) (59) (60) (59)

Angiectasis 1 (2%) 2 (3%) 1 (2%) Angiectasis, focal 1 (2%) Atrophy 1 (2%) Congestion 1 (2%) Cyst 1 (2%) 1 (2%) 3 (5%) Hemorrhage 1 (2%) 1 (2%) 1 (2%) Hydrometra 24 (40%) 24 (41%) 22 (37%) 15 (25%) Hyperplasia, focal, histiocytic 1 (2%) Inflammation, suppurative 2 (3%) 1 (2%) 1 (2%) 2 (3%) Pigmentation, focal 1 (2%) Endometrium, hyperplasia, cystic 54 (90%) 51 (86%) 53 (88%) 37 (63%)



### Hematopoietic System Bone marrow (60) (58) (60) (60)

Hemorrhage, focal 1 (2%) Hyperplasia 1 (2%) 2 (3%) 2 (3%) Inflammation, chronic, focal 1 (2%) Myelofibrosis 2 (3%) Myelofibrosis, focal 1 (2%) Lymph node (6) (3) (10) (11)

Iliac, hematopoietic cell proliferation 1 (9%) Iliac, hemorrhage 1 (10%) Iliac, hyperplasia 1 (10%) Iliac, hyperplasia, lymphoid 2 (20%) Inguinal, hyperplasia 1 (9%) Mediastinal, hematopoietic cell proliferation 1 (9%) Mediastinal, hemorrhage 3 (27%) Mediastinal, hyperplasia 1 (9%) Mediastinal, hyperplasia, lymphoid 2 (18%) Pancreatic, hyperplasia, lymphoid 1 (10%) Renal, hemorrhage 1 (10%) Renal, hyperplasia 2 (20%) 1 (9%) Renal, hyperplasia, lymphoid 1 (10%)

### 273 o- Nitrotoluene, NTP TR 504



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Hematopoietic System (continued) Lymph node, mandibular (58) (55) (55) (48)

Hyperplasia 1 (2%) Hyperplasia, lymphoid 1 (2%) 1 (2%) 1 (2%) Lymph node, mesenteric (53) (58) (57) (57)

Hemorrhage 1 (2%) 1 (2%) Hyperplasia 2 (4%) 1 (2%) 2 (4%) 4 (7%) Hyperplasia, lymphoid 6 (11%) 2 (3%) 2 (4%) 2 (4%) Hyperplasia, mast cell 1 (2%) Spleen (59) (57) (58) (57)

Congestion 2 (3%) Depletion cellular 1 (2%) Fibrosis 1 (2%) Hematopoietic cell proliferation 11 (19%) 19 (33%) 21 (36%) 54 (95%) Hyperplasia, lymphoid 8 (14%) 5 (9%) 4 (7%) 2 (4%) Pigmentation, focal 1 (2%) Thymus (57) (56) (55) (44)

Angiectasis 1 (2%) 1 (2%) 1 (2%) Atrophy 1 (2%) 1 (2%) Cyst 1 (2%) Hyperplasia, lymphoid 5 (9%) 2 (4%) 1 (2%)



### Integumentary System Mammary gland (60) (59) (60) (59)

Ectasia 1 (2%) 1 (2%) Skin (60) (60) (60) (60)

Subcutaneous tissue, angiectasis, focal 1 (2%) Subcutaneous tissue, cyst 1 (2%) Subcutaneous tissue, edema 1 (2%) 2 (3%) 4 (7%) Subcutaneous tissue, hemorrhage, focal 1 (2%) Subcutaneous tissue, inflammation, chronic, focal 2 (3%) 1 (2%)



### Musculoskeletal System Bone (60) (60) (60) (60)

Callus 1 (2%) Fibrous osteodystrophy 1 (2%) Hyperostosis 2 (3%) Femur, callus 1 (2%) Maxilla, hyperostosis, focal 1 (2%)



### Nervous System Brain (60) (60) (59) (58)

Atrophy, focal 1 (2%) 1 (2%) Compression, focal 1 (2%)

### o-Nitrotoluene, NTP TR 504 274



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Respiratory System Lung (60) (60) (59) (57)

Congestion 1 (2%) 2 (3%) 1 (2%) 1 (2%) Edema, focal 1 (2%) Hemorrhage 2 (3%) 1 (2%) 1 (2%) 2 (4%) Hyperplasia, focal, histiocytic 2 (3%) Hyperplasia, histiocytic 1 (2%) Hyperplasia, lymphoid 1 (2%) 1 (2%) 2 (3%) 1 (2%) Infiltration cellular, mixed cell 9 (16%) Metaplasia, osseous 1 (2%) Alveolar epithelium, hyperplasia 1 (2%) Artery, mediastinum, inflammation, chronic 1 (2%) Bronchus, glands, cyst 1 (2%) Nose (60) (60) (59) (57)

Hemorrhage 1 (2%) Mucosa, glands, dilatation, focal 3 (5%) 1 (2%) Olfactory epithelium, degeneration 28 (47%) 59 (100%) 57 (100%)



### Special Senses System None



### Urinary System Kidney (59) (56) (58) (59)

Atrophy, focal 2 (3%) Congestion 2 (3%) 3 (5%) 3 (5%) 1 (2%) Cyst 2 (3%) 2 (3%) 1 (2%) Hydronephrosis 1 (2%) Hyperplasia, lymphoid 4 (7%) 1 (2%) Infiltration cellular, mixed cell 1 (2%) Inflammation, suppurative 1 (2%) Mineralization, focal 1 (2%) Nephropathy 9 (15%) 14 (25%) 10 (17%) 11 (19%) Pigmentation 1 (2%) Artery, inflammation, chronic 1 (2%) Bilateral, atrophy, focal 1 (2%) Bilateral, fibrosis, focal 1 (2%) Bilateral, inflammation, chronic, focal 1 (2%) Pelvis, dilatation 1 (2%) 1 (2%) Pelvis, hemorrhage 1 (2%) Renal tubule, accumulation, hyaline droplet 1 (2%) 3 (5%) 2 (3%) 10 (17%) Renal tubule, casts protein 6 (10%) 8 (14%) 7 (12%) 6 (10%) Renal tubule, dilatation 1 (2%) Renal tubule, necrosis 1 (2%) Renal tubule, pigmentation 1 (2%) 3 (5%) 35 (59%) Renal tubule, vacuolization cytoplasmic 1 (2%) Urinary bladder (59) (54) (57) (54)

Transitional epithelium, hyperplasia, focal 1 (2%)

### 275



# APPENDIX E GENETIC TOXICOLOGY

S

ALMONELLA TYPHIMURIUM 

### M UTAGENICITY 

### T EST 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 C HINESE 

### H AMSTER 

### O VARY 

### C ELL 

### C YTOGENETICS 

### P ROTOCOLS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 R

AT AND 

### M OUSE 

### B ONE 

### M ARROW 

### M ICRONUCLEUS 

### T EST 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 M OUSE 

### P ERIPHERAL 

### B LOOD 

### M ICRONUCLEUS 

### T EST 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 E VALUATION 

### P ROTOCOL


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 RESULTS


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 T ABLE 

### E1 Mutagenicity of o -Nitrotoluene in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . 279 T ABLE 

### E2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 T ABLE 

### E3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 T ABLE 

### E4 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats Treated with o -Nitrotoluene by Intraperitoneal Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 T ABLE 

### E5 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Mice Treated with o -Nitrotoluene by Intraperitoneal Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 T ABLE 

### E6 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Administration of o -Nitrotoluene in Feed for 13 Weeks . . . . . . . . . . . . . . . . . . . . 285

### 276 o-Nitrotoluene, NTP TR 504



# GENETIC TOXICOLOGY

S

ALMONELLA TYPHIMURIUM 

# M UTAGENICITY 

# T EST 

# P ROTOCOL Testing was performed as reported by Haworth et al. (1983). o-Nitrotoluene was sent to the laboratory as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 37° C. Top agar supplemented with L 

### -histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of

o- nitrotoluene. The high dose was limited by toxicity. All trials were repeated.

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.



# C HINESE 

# H AMSTER 

# O VARY 

# C ELL 

# C YTOGENETICS 

# P ROTOCOLS Testing was performed as reported by Galloway et al. (1987). o-Nitrotoluene was sent to the laboratory as a coded aliquot by Radian Corporation. It was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs), both in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of o- nitrotoluene; the high dose was limited by toxicity. A single flask per dose was used, and all tests were repeated.

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 25.5 hours with

o- nitrotoluene in supplemented McCoy’s 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 25.5 hours, the medium containing o- nitrotoluene was removed and replaced with fresh mediumplus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shakeoff, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with

o- nitrotoluene, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no o- nitrotoluene. Incubation proceeded for an additional 25.5 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. All slides were scored blind, and those from a single test were read by the same person. Fifty second-divisionmetaphase cells were scored for frequency of SCEs/cell from each dose level. Because significant chemicalinduced cell cycle delay was seen in the trial without S9, incubation time was lengthened 8.5 hours to ensure a sufficient number of scorable (second-division metaphase) cells.

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points (Galloway et al. , 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more doses resulted in a determination that the trial was positive. A statistically significant trend (P<0.005) in the absence of any responses reaching 20% above background led to a call of equivocal.

### o-Nitrotoluene, NTP TR 504 277



### Chromosomal Aberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium with

o- nitrotoluene for 8.5 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with

o- nitrotoluene and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 8.5 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the Abs test was based on the cell cycle information obtained in the SCE test.

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. One hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P # 0.05) difference for one dose point and a significant trend (P # 0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call (Galloway et al. , 1987). Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.



# R

AT AND 

# M OUSE 

# B ONE 

# M ARROW 

# M ICRONUCLEUS 

# T EST 

# P ROTOCOL Preliminary range-finding studies were performed. Factors affecting dose selection included chemical solubility and toxicity and the extent of cell cycle delay induced by o- nitrotoluene exposure. Male F344/N rats were given a single intraperitoneal injection of o- nitrotoluene dissolved in corn oil followed by bone marrow analysis 24 (trial 1) or 48 (trial 2) hours after the injection. The standard three-exposure protocol used for mice is described in detail by Shelby et al. (1993). Male B6C3F 1 

### mice were injected intraperitoneally (three times at 24-hour intervals) with

o- nitrotoluene dissolved in corn oil. The mice were killed 24 hours after the third injection. Vehicle control rats and mice were injected with corn oil only. The positive control animals received injections of cyclophosphamide. Blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained; 2,000 polychromatic erythrocytes (PCEs) were scored in up to five animals per dose group.

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among PCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.



# M OUSE 

# P ERIPHERAL 

# B LOOD 

# M ICRONUCLEUS 

# T EST 

# P ROTOCOL A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 13-week toxicity study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared

### 278 o-Nitrotoluene, NTP TR 504



### and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange dye and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic (mature) erythrocytes (NCEs) in each of 10 animals per dose group.

The results for NCEs were tabulated and analyzed as described for PCEs in the bone marrow micronucleus test. In addition, the percentage of PCEs among the total erythrocyte population in the peripheral blood was scored for each exposure group as a measure of toxicity.



# E VALUATION 

# P ROTOCOL These are the basic guidelines for arriving at an overall assay result for assays performed by the National Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the statistical procedures for data analysis have been described in the preceding protocols. There have been instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity in an assay. In addition to multiple aliquots, the in vitro assays have another variable that must be considered in arriving at an overall test result. In vitro assays are conducted with and without exogenous metabolic activation. Results obtained in the absence of activation are not combined with results obtained in the presence of activation; each testing condition is evaluated separately. The summary table in the Abstract of this Technical Report presents a result that represents a scientific judgement of the overall evidence for activity of the chemical in an assay.



# RESULTS

o-Nitrotoluene (3.0 to 1,000 μg/plate) was not mutagenic in S. typhimurium strain TA98, TA100, TA1535, or TA1537, with or without Aroclor-induced rat or hamster liver S9 (Table E1; Haworth et al. , 1983). Significantly increased SCE frequencies were observed in cultured CHO cells treated with o- nitrotoluene with S9; an equivocal response was seen without S9 (Table E2; Galloway et al. , 1987). Due to o- nitrotoluene-induced cell cycle delay in the trial without S9, an extended culture time was employed to permit accumulation of sufficient second-division metaphase cells for analysis. o-Nitrotoluene did not induce Abs in cultured CHO cells with or without S9 (Table E3; Galloway et al. , 1987). No increases in the frequencies of micronucleated polychromatic erythrocytes were observed in vivo in the bone marrow of male rats or male mice treated with o- nitrotoluene. In male F344/N rats, an acute micronucleus test was performed using two protocols (Table E4). The initial test used a single intraperitoneal injection of o- nitrotoluene followed by bone marrow analysis 24 hours later; in the second test, bone marrow was harvested for analysis 48 hours after a single intraperitoneal injection of o- nitrotoluene. In neither test was a positive response observed. In male mice, a three-intraperitoneal injection protocol also yielded negative results, although a small increase in the frequency of micronucleated PCEs was observed at all doses tested (Table E5). o-Nitrotoluene, administered in feed for 13 weeks, did not increase the frequency of micronucleated NCEs in peripheral blood of female mice. However, a small increase in the frequency of micronucleated NCEs was noted in male mice at the highest dose tested, 10,000 ppm (Table E6). The increase in male mice was sufficient to generate a significant trend test (P=0.003), but because none of the frequencies in individual dose groups were significantly increased over the corresponding control group and because the increase in the frequency of micronuclei in the 10,000 ppm group was small, the test in male mice was judged to be equivocal.

### o-Nitrotoluene, NTP TR 504 279



### T ABLE 

### E1 Mutagenicity of o -Nitrotoluene in Salmonella typhimurium a



### Revertants/Plate b


### Strain Dose -S9 +10% hamster S9 +10% rat S9 (μg/plate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Study performed at SRI International

TA100 0.0 145 ± 1.5 120 ± 4.7 160 ± 11.8 126 ± 9.1 154 ± 16.5 117 ± 7.8 3.0 151 ± 6.1 118 ± 8.5 10.0 142 ± 7.8 105 ± 4.7 149 ± 5.4 133 ± 4.7 150 ± 7.6 130 ± 3.8 33.0 139 ± 12.7 110 ± 7.5 148 ± 15.8 125 ± 0.9 126 ± 12.7 133 ± 7.5 100.0 146 ± 12.4 104 ± 11.2 154 ± 5.5 125 ± 13.9 134 ± 7.4 147 ± 5.3 333.0 45 ± 5.9 c Toxic 156 ± 7.4 125 ± 7.0 111 ± 6.3 114 ± 1.9 666.0 137 ± 5.7 119 ± 2.5 1,000.0 Toxic Toxic

Trial summary Negative Negative Negative Negative Negative Negative Positive control d 321 ± 11.7 424 ± 16.2 2,113 ± 4.8 1,895 ± 83.7 1,055 ± 61.4 900 ± 15.3



### TA1535 0.0 21 ± 3.7 15 ± 1.7 10 ± 1.7 12 ± 2.1 7 ± 2.5 10 ± 1.9 3.0 22 ± 3.7 10 ± 1.5 10.0 22 ± 3.0 11 ± 1.2 7 ± 2.0 6 ± 1.7 9 ± 1.5 10 ± 2.8 33.0 20 ± 5.5 14 ± 1.5 9 ± 3.2 10 ± 2.6 8 ± 2.5 10 ± 2.1 100.0 20 ± 3.9 18 ± 5.2 11 ± 3.5 10 ± 1.5 7 ± 2.3 7 ± 2.9 333.0 2 ± 1.2 c 0 ± 0.0 c 7 ± 0.9 8 ± 0.7 11 ± 1.9 11 ± 1.5 666.0 Toxic 9 ± 3.0 1,000.0 3 ± 1.8 c Toxic

Trial summary Negative Negative Negative Negative Negative Negative Positive control 384 ± 17.9 396 ± 2.3 429 ± 31.8 507 ± 35.4 255 ± 18.4 313 ± 77.0



### TA1537 0.0 8 ± 0.6 5 ± 2.6 5 ± 1.9 8 ± 2.6 13 ± 2.9 6 ± 0.9 3.0 12 ± 3.0 4 ± 0.6 10.0 9 ± 2.6 3 ± 0.7 7 ± 2.5 9 ± 2.3 11 ± 2.5 5 ± 1.3 33.0 9 ± 0.0 4 ± 0.3 6 ± 0.9 6 ± 1.2 9 ± 1.5 6 ± 0.7 100.0 5 ± 0.6 3 ± 0.9 7 ± 1.0 8 ± 2.6 8 ± 0.7 8 ± 0.7 333.0 0 ± 0.0 c Toxic 8 ± 2.6 6 ± 1.5 6 ± 1.5 6 ± 1.0 666.0 Toxic 6 ± 1.3 1,000.0 Toxic 0 ± 0.0 c

Trial summary Negative Negative Negative Negative Negative Negative Positive control 99 ± 3.5 100 ± 18.2 385 ± 63.6 353 ± 32.0 238 ± 4.4 283 ± 14.2



### TA98 0.0 25 ± 0.9 22 ± 2.7 28 ± 1.9 28 ± 4.6 38 ± 6.7 24 ± 2.6 3.0 25 ± 0.6 24 ± 1.9 10.0 24 ± 3.7 16 ± 0.9 22 ± 2.2 35 ± 1.7 28 ± 4.3 29 ± 2.3 33.0 24 ± 3.3 17 ± 1.5 25 ± 4.1 29 ± 1.8 26 ± 4.1 30 ± 0.9 100.0 19 ± 3.5 36 ± 22.3 25 ± 2.1 27 ± 1.3 27 ± 1.2 34 ± 2.7 333.0 Toxic 0 ± 0.0 c 27 ± 0.7 28 ± 2.6 21 ± 3.7 31 ± 3.8 666.0 0 ± 0.0 c 32 ± 0.5 1,000.0 10 ± 5.4 c 0 ± 0.0 c

Trial summary Negative Negative Negative Negative Negative Negative Positive control 830 ± 33.4 760 ± 8.0 1,845 ± 75.2 1,761 ± 147.7 613 ± 12.5 640 ± 8.7

### 280 o-Nitrotoluene, NTP TR 504



### T ABLE 

### E1 Mutagenicity of o -Nitrotoluene in Salmonella typhimurium

Revertants/Plate Strain Dose -S9 +10% hamster S9 +10% rat S9 (μg/plate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Study performed at EG&G Mason Research Institute

TA100 0.0 122 ± 9.1 138 ± 16.3 79 ± 2.6 134 ± 6.2 86 ± 10.1 123 ± 7.9 3.3 135 ± 4.3 127 ± 2.3 149 ± 7.8 10.0 125 ± 3.5 139 ± 5.8 89 ± 3.5 118 ± 7.6 78 ± 3.2 128 ± 3.2 33.0 104 ± 1.2 122 ± 6.9 76 ± 3.7 134 ± 2.5 109 ± 6.2 147 ± 21.7 100.0 113 ± 3.7 121 ± 11.1 89 ± 3.5 135 ± 9.3 118 ± 6.1 142 ± 5.2 333.0 83 ± 1.9 c 132 ± 9.2 c 90 ± 7.3 c 148 ± 3.3 111 ± 7.2 c 115 ± 11.0 666.0 Toxic Toxic Toxic

Trial summary Negative Negative Negative Negative Negative Negative Positive control 2,037 ± 65.0 2,103 ± 44.5 1,741 ± 95.2 1,900 ± 92.3 1,045 ± 81.2 991 ± 44.5



### TA1535 0.0 22 ± 2.0 25 ± 3.8 9 ± 1.3 14 ± 1.7 15 ± 1.5 10 ± 0.6 3.3 26 ± 2.5 15 ± 3.2 10 ± 1.2 10.0 37 ± 3.6 23 ± 3.3 10 ± 1.8 17 ± 2.5 12 ± 2.3 9 ± 1.7 33.0 28 ± 3.2 24 ± 1.7 10 ± 1.9 20 ± 1.5 12 ± 0.6 10 ± 3.3 100.0 28 ± 1.7 22 ± 4.9 8 ± 3.0 16 ± 1.5 13 ± 1.5 19 ± 2.0 333.0 29 ± 2.9 c 23 ± 2.8 c 11 ± 2.0 c 16 ± 2.5 12 ± 3.8 c 12 ± 1.2 666.0 Toxic Toxic Toxic

Trial summary Negative Negative Negative Negative Negative Equivocal Positive control 1,380 ± 77.8 1,320 ± 43.1 74 ± 5.6 128 ± 6.3 110 ± 7.0 108 ± 5.5



### TA1537 0.0 6 ± 0.7 8 ± 1.5 6 ± 0.6 13 ± 1.2 8 ± 1.0 10 ± 1.5 3.3 8 ± 2.1 9 ± 2.1 8 ± 0.9 10.0 5 ± 1.2 5 ± 1.8 6 ± 0.6 12 ± 0.0 8 ± 0.3 6 ± 1.2 33.0 6 ± 1.0 6 ± 1.2 13 ± 0.9 9 ± 0.6 8 ± 1.3 7 ± 2.1 100.0 6 ± 1.3 8 ± 1.3 9 ± 3.1 8 ± 1.2 6 ± 1.2 6 ± 0.9 333.0 7 ± 1.7 c 6 ± 0.3 c 10 ± 2.7 c 11 ± 0.9 5 ± 0.9 c 7 ± 0.9 666.0 Toxic 6 ± 2.1 c 6 ± 0.3 c

Trial summary Negative Negative Negative Negative Negative Negative Positive control 361 ± 85.0 901 ± 105.9 118 ± 12.0 173 ± 7.2 123 ± 2.3 71 ± 4.9



### TA98 0.0 16 ± 1.2 14 ± 2.7 31 ± 1.5 28 ± 1.2 20 ± 2.3 25 ± 0.7 3.3 20 ± 3.8 31 ± 1.9 29 ± 1.2 10.0 20 ± 2.7 16 ± 1.9 29 ± 2.6 31 ± 7.1 22 ± 3.5 27 ± 0.3 33.0 18 ± 3.2 22 ± 1.2 32 ± 3.1 32 ± 1.9 23 ± 1.2 27 ± 5.2 100.0 18 ± 4.4 20 ± 1.2 26 ± 6.4 34 ± 2.3 24 ± 4.0 25 ± 1.9 333.0 14 ± 0.6 c 12 ± 1.8 c 27 ± 2.3 36 ± 3.5 27 ± 3.5 c 27 ± 3.7 666.0 Toxic Toxic Toxic

Trial summary Negative Negative Negative Negative Negative Negative Positive control 1,661 ± 63.7 2,119 ± 51.4 1,189 ± 54.6 1,454 ± 95.4 1,173 ± 31.0 874 ± 29.2

a The detailed protocol and these data are presented by Haworth et al . (1983). b Revertants are presented as mean ± standard error from three plates. c Slight toxicity d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), an d 4-nitro- o -phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.

### o-Nitrotoluene, NTP TR 504 281



### T ABLE 

### E2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by o -Nitrotoluene a



### Total No. of SCEs/ Relative Dose Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/ Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosome b 

### (%)

–S9 Summary: Equivocal

Dimethylsulfoxide c 50 1,035 485 0.46 9.7 25.5

o -Nitrotoluene 117.000 50 1,039 569 0.54 11.4 34.0 d 16.87 176.000 50 1,021 557 0.54 11.1 34.0 d 16.42 218.000 50 1,035 576 0.55 11.5 34.0 d 18.76 282.000 0

P=0.005 e

Mitomycin-C f 0.005 50 1,037 2,236 2.15 44.7 25.5 360.15



### +S9 Summary: Positive

Dimethylsulfoxide 50 1,050 380 0.36 7.6 25.5

o -Nitrotoluene 354.83 50 1,050 499 0.47 10.0 25.5 31.32* 380.95 50 1,050 467 0.44 9.3 25.5 22.90* 423.28 50 1,047 488 0.46 9.8 25.5 28.79*

P=0.001

Cyclophosphamide f 1.50 50 1,049 1,651 1.57 33.0 25.5 334.89

* Positive ( $ 20% increase over solvent control) a Study was performed at Litton Bionetics, Inc. The detailed protocol and these data are presented by Galloway et al . (1987). SCE=sister chromatid exchange; BrdU=bromodeoxyuridine b SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells c Solvent control d Due to cell cycle delay, harvest time was extended to maximize the proportion of second-division metaphase cells available for analysis. e Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose f Positive control

### 282 o-Nitrotoluene, NTP TR 504



### T ABLE 

### E3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by o -Nitrotoluene a



### Dose Total Cells Number Aberrations/ Cells with Compound (μg/mL) Scored of Abberations Cell Aberrations (%)

–S9 Harvest time: 10.5 hours Summary: Negative

Dimethylsulfoxide b 100 3 0.03 3.0

o -Nitrotoluene 200.7 100 2 0.02 2.0 252.5 100 1 0.01 1.0 393.6 100 1 0.01 1.0

P=0.873 c

Mitomycin-C d 0.5 100 20 0.20 16.0



### +S9 Harvest time: 10.5 hours Summary: Negative

Dimethylsulfoxide 100 5 0.05 4.0

o -Nitrotoluene 375.36 100 3 0.03 2.0 398.82 100 9 0.09 8.0 422.28 100 5 0.05 5.0

P=0.168

Cyclophosphamide d 50 100 30 0.30 20.0

a Study was performed at Litton Bionetics, Inc. The detailed protocol and these data are presented by Galloway et al . (1987). b Solvent control c Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose d Positive control

### o-Nitrotoluene, NTP TR 504 283

Trial 1 (24-hour sample) Corn oil d 0 5 0.60 ± 0.19

o-Nitrotoluene 625 5 1.50 ± 0.76 0.0247 1,250 3 0.33 ± 0.33 0.7675 2,500 5 0.80 ± 0.25 0.2964

P=0.609 e

Cyclophosphamide f 25 5 9.80 ± 2.18 0.0000



### Trial 2 (48-hour sample) Corn oil 0 5 1.10 ± 0.19

o-Nitrotoluene 625 5 1.30 ± 0.41 0.3415 2,500 4 0.75 ± 0.25 0.7763

P=0.814

Cyclophosphamide 25 5 13.40 ± 1.76 0.0000

a Study was performed at Integrated Laboratory Systems, Inc. PCE=polychromatic erythrocyte b Mean ± standard error c Pairwise comparison with the vehicle control. Dosed group values are significant at P # 0.008 (trial 1) or P # 0.013 (trial 2); positive control values are significant at P # 0.05 (ILS, 1990). d Vehicle control e Significance of micronucleated PCEs/1,000 PCEs tested by the one-tailed trend test; significant at P # 0.025 (ILS, 1990) f Positive control 

### T ABLE 

### E4 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats Treated with o- Nitrotoluene by Intraperitoneal Injection a



### Number of Rats Compound Dose with Erythrocytes Micronucleated PCEs/ P Value c 

### (mg/kg) Scored 1,000 PCEs b

### 284 o-Nitrotoluene, NTP TR 504



### T ABLE 

### E5 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Mice Treated with o- Nitrotoluene by Intraperitoneal Injection a



### Number of Mice Compound Dose with Erythrocytes Micronucleated PCEs/ P Value c 

### (mg/kg) Scored 1,000 PCEs b

Corn oil d 0 5 0.90 ± 0.10

o-Nitrotoluene 100 5 1.50 ± 0.16 0.1102 200 5 1.30 ± 0.60 0.1968 300 5 1.60 ± 0.37 0.0806 400 5 1.80 ± 0.30 0.0415

P=0.055 e

Cyclophosphamide f 25 5 6.20 ± 1.15 0.0000

a Study was performed at Integrated Laboratory Systems, Inc. The detailed protocol is presented by Shelby et al. (1993). PCE=polychromatic erythrocyte b Mean ± standard error c Pairwise comparison with the vehicle control. Dosed group values are significant at P # 0.006; positive control value is significant at P # 0.05 (ILS, 1990). d Vehicle control e Significance of micronucleated PCEs/1,000 PCEs tested by the one-tailed trend test; significant at P # 0.025 (ILS, 1990) f Positive control

### o-Nitrotoluene, NTP TR 504 285



### T ABLE 

### E6 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Administration of o-Nitrotoluene in Feed for 13 Weeks a



### Number of Mice Concentration with Erythrocytes Micronucleated NCEs/ P Value c 

### PCEs (%) (ppm) Scored 1,000 NCEs b



### Male

0 10 0.70 ± 0.21 1.6 625 10 0.60 ± 0.15 0.6526 1.5 1,250 10 0.90 ± 0.18 0.2397 1.4 2,500 10 0.85 ± 0.24 0.2949 1.3 5,000 10 0.90 ± 0.15 0.2397 1.7 10,000 10 1.40 ± 0.23 0.0153 1.1

P=0.003 d



### Female

0 10 0.40 ± 0.12 1.5 625 10 0.55 ± 0.17 0.2456 1.6 1,250 10 0.15 ± 0.08 0.9342 1.5 2,500 10 0.40 ± 0.16 0.5000 1.6 5,000 10 0.30 ± 0.11 0.7035 1.4 10,000 10 0.30 ± 0.11 0.7035 1.0

P=0.750

a Study was performed at SITEK Research Laboratories, Inc. The detailed protocol is presented by MacGregor et al . (1990). NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte b Mean ± standard error c Pairwise comparison with the controls, significant at P # 0.005 (ILS, 1990) d Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P # 0.025 (ILS, 1990)

### 286 o-Nitrotoluene, NTP TR 504

### 287



# APPENDIX F

o -NITROBENZOIC ACID,

o -NITROBENZYLMERCAPTURIC ACID, AND o -AMINOBENZOIC ACID — BIOMARKERS OF EXPOSURE

I NTRODUCTION


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 M ATERIALS AND 

### M ETHODS


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 RESULTS AND 

### D ISCUSSION


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 T ABLE 

### F1 Urinary Biomarker Data for Rats in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . 290 T ABLE 

### F2 Urinary Biomarker Data for Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . 292

### 288



# o-NITROBENZOIC ACID,

o-NITROBENZYLMERCAPTURIC ACID, AND o-AMINOBENZOIC ACID — BIOMARKERS OF EXPOSURE

I NTRODUCTION Biotransformation studies of o- nitrotoluene in rats indicate only one major route of metabolism: oxidation of the methyl group and further modification of the benzyl alcohol thus formed. A small percentage (less than 2%) of the urinary metabolites resulted from nitro-group reduction (Chism et al. , 1984). To determine how the relative contribution of these pathways may change with exposure concentration and age, three urinary metabolites were followed during the 2-year o- nitrotoluene studies, o- nitrobenzoic acid, o- nitrobenzylmercapturic acid and

o- aminobenzoic acid.



# M ATERIALS AND 

# M ETHODS Urinary metabolites were quantitated by high-performance liquid chromatography using a C 18 

### column. For

o- nitrobenzoic acid and o- nitrobenzylmercapturic acid, the solvent system was A) water:methanol:trifluoroacetic acid (850:150:1) and B) water:methanol:trifluoroacetic acid (500:500:1) at a flow rate of 1 mL per minute; the pH was adjusted to 2.3 with triethylamine. The solvent system program was 50% A:50% B for 1 minute, followed by a linear increase to 23% A:77% B over 19 minutes. Detection was by ultraviolet absorption (266 nm for

o- nitrobenzoic acid and o- nitrobenzylmercapturic acid; 254 nm for o- aminobenzoic acid). The limits of quantitation were 0.0321 mg/mL for o- nitrobenzoic acid, 0.0438 mg/mL for o- nitrobenzylmercapturic acid, and 0.0517 mg/mL for o- aminobenzoic acid.



# RESULTS AND 

# D ISCUSSION Comparisons among the metabolite data were made using the metabolite/creatinine ratio obtained by dividing the metabolite concentration by the creatinine concentration. Creatinine excretion is considered related to muscle mass. Thus, normalizing the metabolite data to creatinine in effect normalizes the metabolite to body weight. This step was considered necessary because comparisons are being made across time as the animals’ weight changes and because males are generally heavier than females. The efficiency of urine collection in a metabolism cage is not 100%, so calculation of total metabolite based on the amount of urine collected has some uncertainty. Basing the comparison on concentrations of creatinine and metabolite in measure aliquots eliminates the need to know the total urine output and the associated uncertainties.

The ratios of o- nitrobenzoic acid to creatinine excreted in urine by rats are shown in Table F1. With only one exception, the ratios for male rats were larger at the 2-week time point than at later time points. In females, by contrast, the difference between the time points was generally not significant. The ratios were generally larger for females than males. The ratios of o- nitrobenzoic acid to creatinine were linear with exposure concentration.

In contrast to the o -nitrobenzoic acid/creatinine ratios, larger ratios of o- nitrobenzylmercapturic acid to creatinine were seen in urine at week 2 compared to later time points in all exposed groups, with one exception (Table F1). The ratios were significantly smaller for females than males. The o- nitrobenzylmercapturic acid/creatinine ratios were linear with exposure concentration.

The ratios of o- aminobenzoic acid to creatinine excreted in urine by control rats were similar to ratios seen in exposed groups of animals in many cases (Table F1). o- Aminobenzoic acid is a product of catabolism of o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 289



### tryptophan (White et al. , 1978) and is a relatively minor metabolite of o- nitrotoluene (Chism et al. , 1984). No further analyses of these data were performed.

The concentrations of o- aminobenzoic acid and o- nitrobenzylmercapturic acid in urine of mice were generally below the limit of quantitation (Table F2). At time points with sufficient data to make determinations, the ratios of urinary o- nitrobenzoic acid to creatinine appeared to increase with increasing exposure concentration. Because of the large standard errors, no statistical analyses were attempted.

There appeared to be a change in metabolism of o- nitrotoluene to o- nitrobenzoic acid in male, but not female, rats after week 2 (Table F1). The relative concentrations of o- nitrobenzoic acid were generally greater for females. There was no evidence for nonlinear metabolism with respect to exposure concentration.

In contrast to o- nitrobenzoic acid formation, there appeared to be a change in metabolism of o- nitrotoluene with respect to formation of o- nitrobenzylmercapturic acid after week 2 in both sexes of rats. Because the first step in the formation of either metabolite is oxidation of the methyl group to a benzyl alcohol, metabolic differences must be either in further oxidation to the carboxylic acid or in formation of conjugates of the alcohol and further reaction with reduced glutathione. The relative concentrations of the mercapturic acid were lower for female rats than for males. The relative concentrations of o- nitrobenzoic acid, by contrast, were higher for females than for males. Again, because the first step in formation of both metabolites is oxidation of the methyl group to a benzylic alcohol, these observations are consistent with a sex-related difference in one of the later metabolic steps that favors formation of the mercapturic acid in males.

### 290 o-Nitrotoluene, NTP TR 504



### T ABLE 

### F1 Urinary Biomarker Data for Rats in the 2-Year Feed Study of o- Nitrotoluene a



### 0 ppm 625 ppm 1,250 ppm 2,000 ppm

Male

n 55 5 5

Volume (mL/24 hours) Week 2 3.2 ± 0.4 4.2 ± 0.6 3.0 ± 0.4 4.2 ± 0.4 Month 3 5.7 ± 1.7 5.0 ± 0.8 3.8 ± 0.4 4.5 ± 0.4 Month 12 3.4 ± 0.3 2.9 ± 0.4 4.5 ± 1.6 3.2 ± 0.6 Month 18 5.6 ± 1.2 3.8 ± 0.3 5.8 ± 1.3 5.2 ± 1.6 Creatinine (mg/dL) Week 2 186 ± 15 210 ± 58 212 ± 23 196 ± 25 Month 3 244 ± 43 280 ± 25 274 ± 36 265 ± 37 Month 12 356 ± 35 401 ± 19 326 ± 36 375 ± 59 Month 18 215 ± 45 266 ± 34 224 ± 12 190 ± 18

o -Nitrobenzoic acid (mg/24 hours) Week 2 0.131 ± 0.017 2.24 ± 0.17 3.66 ± 0.51 7.61 ± 0.66 Month 3 0 ± 0 2.77 ± 0.43 4.65 ± 0.63 6.05 ± 0.42 Month 12 0 ± 0 1.96 ± 0.27 4.05 ± 0.80 5.74 ± 0.37 Month 18 0 ± 0 1.75 ± 0.30 3.67 ± 0.71 7.06 ± 1.96

o -Nitrobenzoic acid/creatinine ratio Week 2 0.0222 ± 0.0010 0.316 ± 0.051 0.595 ± 0.046 0.965 ± 0.075 Month 3 0 ± 0 0.204 ± 0.019* 0.451 ± 0.038* 0.543 ± 0.059* Month 12 0 ± 0 0.173 ± 0.018* 0.315 ± 0.015* 0.534 ± 0.031* Month 18 0 ± 0 0.175 ± 0.011* 0.289 ± 0.021* 0.842 ± 0.129

o -Nitrobenzylmercapturic acid (mg/24 hours) Week 2 0 ± 0 0.739 ± 0.102 1.23 ± 0.06 2.13 ± 0.12 Month 3 0 ± 0 1.12 ± 0.16 1.55 ± 0.28 2.07 ± 0.19 Month 12 0 ± 0 0.628 ± 0.113 1.34 ± 0.31 1.81 ± 0.13 Month 18 0 ± 0 0.337 ± 0.095 0.749 ± 0.119 1.65 ± 0.45

o -Nitrobenzylmercapturic acid/creatinine ratio Week 2 0 ± 0 0.101 ± 0.014 0.206 ± 0.013 0.281 ± 0.018 Month 3 0 ± 0 0.0824 ± 0.0056 0.152 ± 0.015* 0.182 ± 0.014* Month 12 0 ± 0 0.0543 ± 0.0071* 0.103 ± 0.014* 0.164 ± 0.014* Month 18 0 ± 0 0.0299 ± 0.0076* 0.0653 ± 0.0099* 0.163 ± 0.014*

o -Aminobenzoic acid (mg/24 hours) Week 2 0.221 ± 0.139 0.0527 ± 0.0323 0.0349 ± 0.0349 0.258 ± 0.122 Month 3 0.131 ± 0.094 0.304 ± 0.183 0.227 ± 0.092 0.277 ± 0.158 Month 12 0.110 ± 0.049 0.191 ± 0.031 0.241 ± 0.047 0.143 ± 0.059 Month 18 0 ± 0 0 ± 0 0 ± 0 0 ± 0

o -Aminobenzoic acid/creatinine ratio Week 2 0.0291 ± 0.0178 0.00810 ± 0.00532 0.00600 ± 0.00600 0.0321 ± 0.0136 Month 3 0.0115 ± 0.0081 0.0256 ± 0.0135 0.0218 ± 0.0098 0.0269 ± 0.0167 Month 12 0.00935 ± 0.00427 0.0168 ± 0.0031 0.0196 ± 0.0036 0.0128 ± 0.0049 Month 18 0 ± 0 0 ± 0 0 ± 0 0 ± 0

### o-Nitrotoluene, NTP TR 504 291



### T ABLE 

### F1 Urinary Biomarker Data for Rats in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm

Female

n 55 5 5

Volume (mL/24 hours) Week 2 3.4 ± 0.2 4.6 ± 0.5 3.0 ± 0.6 4.6 ± 0.7 Month 3 3.4 ± 0.5 3.3 ± 0.5 3.9 ± 0.8 4.2 ± 0.7 Month 12 3.6 ± 0.4 3.3 ± 0.4 3.6 ± 1.4 2.0 ± 0.5 Month 18 5.5 ± 1.0 4.4 ± 0.8 6.5 ± 0.7 4.9 ± 1.0 Creatinine (mg/dL) Week 2 137 ± 20 146 ± 17 159 ± 14 103 ± 4 Month 3 169 ± 21 206 ± 17 182 ± 8 154 ± 20 Month 12 213 ± 46 247 ± 17 170 ± 25 245 ± 38 Month 18 170 ± 16 186 ± 14 198 ± 8 177 ± 16

o -Nitrobenzoic acid (mg/24 hours) Week 2 0.140 ± 0.030 2.64 ± 0.26 3.95 ± 0.75 7.30 ± 1.06 Month 3 0.0561 ± 0.0161 2.62 ± 0.34 5.57 ± 1.09 7.51 ± 0.97 Month 12 0 ± 0 2.94 ± 0.39 4.39 ± 1.18 3.52 ± 0.40 Month 18 0.0115 ± 0.0115 2.84 ± 0.613 6.30 ± 0.34 6.26 ± 0.93

o -Nitrobenzoic acid/creatinine ratio Week 2 0.0301 ± 0.0035 0.430 ± 0.072 0.861 ± 0.088 • 1.55 ± 0.05 • Month 3 0.00952 ± 0.00255 0.392 ± 0.015 • 0.789 ± 0.065 • 1.26 ± 0.13 • Month 12 0 ± 0 0.369 ± 0.048 • 0.865 ± 0.080 • 0.868 ± 0.087* • Month 18 0.00146 ± 0.00146 0.345 ± 0.030 • 0.510 ± 0.045* • 0.748 ± 0.029*

o -Nitrobenzylmercapturic acid (mg/24 hours) Week 2 0 ± 0 0.216 ± 0.029 0.472 ± 0.100 0.654 ± 0.102 Month 3 0 ± 0 0.107 ± 0.043 0.361 ± 0.094 0.518 ± 0.068 Month 12 0 ± 0 0.0956 ± 0.0299 0.136 ± 0.067 0.209 ± 0.024 Month 18 0.0679 ± 0.0679 0 ± 0 0.176 ± 0.079 0.370 ± 0.122

o -Nitrobenzylmercapturic acid/creatinine ratio Week 2 0 ± 0 0.0347 ± 0.0063 • 0.101 ± 0.010 • 0.138 ± 0.007 • Month 3 0 ± 0 0.0151 ± 0.0064* • 0.0485 ± 0.0047* • 0.0867 ± 0.0085* • Month 12 0 ± 0 0.0113 ± 0.0030* • 0.0344 ± 0.0122* • 0.0519 ± 0.0067* • Month 18 0.0065 ± 0.0064 0 ± 0* • 0.0160 ± 0.0076* • 0.0413 ± 0.0106* •

o -Aminobenzoic acid (mg/24 hours) Week 2 0.120 ± 0.038 0.305 ± 0.208 0.331 ± 0.208 0.362 ± 0.137 Month 3 0 ± 0 0.094 ± 0.057 0.0429 ± 0.0429 0 ± 0 Month 12 0.0214 ± 0.0214 0.176 ± 0.042 0.158 ± 0.075 0.0701 ± 0.0345 Month 18 0 ± 0 0 ± 0 0 ± 0 0 ± 0

o -Aminobenzoic acid/creatinine ratio Week 2 0.0303 ± 0.0100 0.0338 ± 0.0185 0.0687 ± 0.0335 0.0749 ± 0.0226 Month 3 0 ± 0 0.0108 ± 0.0053 0.00464 ± 0.00464 0 ± 0 Month 12 0.00285 ± 0.00285 0.0213 ± 0.0036 0.0368 ± 0.0158 0.0158 ± 0.0080 Month 18 0 ± 0 0 ± 0 0 ± 0 0 ± 0

* Significantly different (P # 0.05) from corresponding 2-week data by ANOVA by Fisher’s least significant difference test

• Significantly different (P # 0.05) from corresponding data for males by ANOVA by Fisher’s least significant difference test a Data are presented as mean ± standard error.

### T ABLE 

### F2 Urinary Biomarker Data for Mice in the 2-Year Feed Study of o-Nitrotoluene a



### 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Male

n

Week 2 3 2 0 3 Month 3 4 5 5 5 Month 12 5 5 5 5 Month 18 5 5 5 0 b

Volume (mL/24 hours) Week 2 0.5 ± 0.3 0.4 ± 0.1 — 0.3 ± 0.1 Month 3 0.6 ± 0.1 0.8 ± 0.2 1.0 ± 0.2 0.9 ± 0.1 Month 12 1.0 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 1.2 ± 0.1 Month 18 1.2 ± 0.2 1.5 ± 0.1 c 0.9 ± 0.2 — Creatinine (mg/dL) Week 2 36.7 ± 8.5 40.5 ± 7.8 — 40.5 ± 5.5 Month 3 39.5 ± 2.0 54.3 ± 12.9 38.1 ± 6.6 38.9 ± 3.0 Month 12 53.2 ± 7.8 58.6 ± 15.5 50.8 ± 2.9 33.2 ± 2.0 Month 18 48.9 ± 6.7 49.5 ± 2.6 c 42.2 ± 4.8 —

o -Nitrobenzoic acid (mg/24 hours) Week 2 0.0164 ± 0.0067 0.452 ± 0.068 — 1.61 ± 0.18 Month 3 0 ± 0 0.378 ± 0.077 1.06 ± 0.04 1.50 ± 0.25 Month 12 0 ± 0 0.161 ± 0.043 0.793 ± 0.103 0.777 ± 0.185 Month 18 0.0134 ± 0.0017 0.418 ± 0.077 0.448 ± 0.066 —

o -Nitrobenzoic acid/creatinine ratio Week 2 0.0950 ± 0.0159 3.24 ± 0.40 — 13.4 ± 2.5 Month 3 0 ± 0 1.05 ± 0.21 1.15 ± 0.17 4.21 ± 0.50 Month 12 0 ± 0 0.365 ± 0.119 0.824 ± 0.250 2.44 ± 0.67 Month 18 0.0282 ± 0.0073 0.840 ± 0.177 0.954 ± 0.279 —

o -Nitrobenzylmercapturic acid (mg/24 hours) Week 2 0 ± 0 0 ± 0 — 0.00132 ± 0.00072 Month 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0.0066 ± 0.0059 0.0230 ± 0.0165 Month 18 0.0605 ± 0.0173 0.0192 ± 0.0148 0 ± 0 —

o -Nitrobenzylmercapturic acid/creatinine ratio Week 2 0 ± 0 0 ± 0 — 0.00124 ± 0.0079 Month 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0.0210 ± 0.0187 0.0372 ± 0.0332 Month 18 0.100 ± 0.025 0.0226 ± 0.0175 0 ± 0 —

o -Aminobenzoic acid (mg/24 hours) Week 2 0 ± 0 0 ± 0 — 0 ± 0 Month 3 0.0123 ± 0.0031 0.00476 ± 0.00426 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 18 0 ± 0 0 ± 0 0 ± 0 —

o -Aminobenzoic acid/creatinine ratio Week 2 0 ± 0 0 ± 0 — 0 ± 0 Month 3 0.0601 ± 0.0140 0.0144 ± 0.0129 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 18 0 ± 0 0 ± 0 0 ± 0 — 

### 292 o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 293



### T ABLE 

### F2 Urinary Biomarker Data for Mice in the 2-Year Feed Study of o- Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm

Female

n

Week 2 2 4 2 2 Month 3 5 5 5 5 Month 12 5 5 5 5 Month 18 5 5 5 5

Volume (mL/24 hours) Week 2 0.4 ± 0.0 0.4 ± 0.1 0.3 ± 0.0 1.4 ± 0.7 Month 3 0.3 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 0.6 ± 0.1 Month 12 0.9 ± 0.0 0.8 ± 0.1 1.0 ± 0.1 0.9 ± 0.1 Month 18 1.0 ± 0.2 1.3 ± 0.3 0.9 ± 0.2 1.2 ± 0.1 Creatinine (mg/dL) Week 2 43.8 ± 3.0 46.5 ± 4.8 40.4 ± 12.1 17.9 ± 8.7 Month 3 88.9 ± 14.3 82.0 ± 9.4 59.1 ± 7.2 54.3 ± 8.0 Month 12 51.4 ± 5.3 48.4 ± 3.6 47.7 ± 7.2 45.7 ± 1.6 Month 18 65.8 ± 8.1 43.4 ± 6.0 58.6 ± 3.1 38.2 ± 3.6

o -Nitrobenzoic acid (mg/24 hours) Week 2 0.0173 ± 0.0019 0.501 ± 0.075 0.840 ± 0.243 2.13 ± 0.03 Month 3 0.00534 ± 0.0020 0.207 ± 0.059 0.346 ± 0.057 1.14 ± 0.24 Month 12 0 ± 0 0.190 ± 0.034 0.370 ± 0.038 0.398 ± 0.078 Month 18 0.0181 ± 0.0046 0.372 ± 0.161 0.542 ± 0.076 0.837 ± 0.115

o -Nitrobenzoic acid/creatinine ratio Week 2 0.0891 ± 0.018 3.02 ± 0.49 5.73 ± 0.09 11.5 ± 0.4 Month 3 0.0190 ± 0.0086 0.815 ± 0.215 1.77 ± 0.51 3.59 ± 0.53 Month 12 0 ± 0 0.386 ± 0.061 0.858 ± 0.166 0.866 ± 0.170 Month 18 0.0275 ± 0.0095 0.730 ± 0.250 0.968 ± 0.203 2.20 ± 0.32

o -Nitrobenzylmercapturic acid (mg/24 hours) Week 2 0 ± 0 0.00324 ± 0.00281 0 ± 0 0 ± 0 Month 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0.0158 ± 0.0142 0.0075 ± 0.0067 Month 18 0 ± 0 0.00284 ± 0.00254 0.0667 ± 0.0364 0 ± 0

o -Nitrobenzylmercapturic acid/creatinine ratio Week 2 0 ± 0 0.0193 ± 0.0168 0 ± 0 0 ± 0 Month 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0.0316 ± 0.0283 0.0317 ± 0.0283 Month 18 0 ± 0 0.0252 ± 0.0226 0.102 ± 0.049 0 ± 0

o -Aminobenzoic acid (mg/24 hours) Week 2 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 3 0.00176 ± 0.00157 0.00227 ± 0.00203 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 18 0 ± 0 0 ± 0 0 ± 0 0 ± 0

o -Aminobenzoic acid/creatinine ratio Week 2 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 3 0.00523 ± 0.00470 0.0149 ± 0.0163 0 ± 0 0 ± 0 Month 12 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Month 18 0 ± 0 0 ± 0 0 ± 0 0 ± 0

a Data are presented as mean ± standard error. Each group consisted of five animals; however, an insufficient sample for complet e analysis was obtained from some animals. Therefore, n=number of animals for which metabolite data were obtained. b No data were available at this time point due to 100% mortality. c n=4

### 294 o-Nitrotoluene, NTP TR 504

### 295



# APPENDIX G ORGAN WEIGHTS AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS

T ABLE 

### G1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats at the 3-Month Interim Evaluation in the 2-Year Feed Study of o -Nitrotoluene . . . . . . . . . 296

### 296 o-Nitrotoluene, NTP TR 504



### T ABLE 

### G1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats at the 3-Month Interim Evaluation in the 2-Year Feed Study of o -Nitrotoluene a



### 0 ppm 2,000 ppm 5,000 ppm (Stop-Exposure) (Stop-Exposure)

n 10 10 10

Necropsy body wt 360 ± 5 319 ± 7** 264 ± 4**

Heart Absolute 0.936 ± 0.023 0.928 ± 0.019 0.928 ± 0.016 Relative 2.595 ± 0.038 2.909 ± 0.026** 3.518 ± 0.057** R. Kidney Absolute 1.162 ± 0.018 1.057 ± 0.026** 1.096 ± 0.022 Relative 3.225 ± 0.034 3.312 ± 0.043 4.153 ± 0.068** Liver Absolute 12.674 ± 0.431 12.860 ± 0.294 15.858 ± 0.248** Relative 35.100 ± 0.801 40.293 ± 0.354** 60.110 ± 0.822** Lung Absolute 1.379 ± 0.017 1.302 ± 0.044 1.205 ± 0.018** Relative 3.829 ± 0.036 4.075 ± 0.089** 4.566 ± 0.046** R. Testis Absolute 1.498 ± 0.030 1.348 ± 0.030* 1.111 ± 0.061** Relative 4.160 ± 0.082 4.232 ± 0.097 4.226 ± 0.257 Thymus Absolute 0.266 ± 0.010 0.219 ± 0.015* 0.234 ± 0.012 Relative 0.739 ± 0.029 0.686 ± 0.047 0.886 ± 0.044*

* Significantly different (P # 0.05) from the control group by Williams’ or Dunnett’s test ** P # 0.01 a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).

### 297



# APPENDIX H CHEMICAL CHARACTERIZATION AND DOSE FORMULATION STUDIES

P ROCUREMENT AND 

### C HARACTERIZATION OF O


### -N ITROTOLUENE 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 P REPARATION AND 

### ANALYSIS OF 

### D OSE 

### F ORMULATIONS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 F IGURE 

### H1 Infrared Absorption Spectrum of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 F IGURE 

### H2 Nuclear Magnetic Resonance Spectrum of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 T ABLE 

### H1 Gas Chromatography Systems Used in the 2-Year Feed Studies of o- Nitrotoluene . . . . . . . 302 T ABLE 

### H2 Preparation and Storage of Dose Formulations in the 2-Year Feed Studies of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

### 298



# CHEMICAL CHARACTERIZATION AND DOSE FORMULATION STUDIES

P ROCUREMENT AND 

# C HARACTERIZATION OF O


# -N ITROTOLUENE

o-Nitrotoluene was obtained from Aldrich Chemical Company (Milwaukee, WI) in one lot (8056-58-05RTI). Identity, purity, and stability analyses were conducted by the analytical chemistry laboratory, Research Triangle Institute (Research Triangle Park, NC), and the study laboratory. Reports on analyses performed in support of the

o- nitrotoluene studies are on file at the National Institute of Environmental Health Sciences.

The chemical, a yellow-green liquid, was identified as o- nitrotoluene by infrared, ultraviolet/visible, proton nuclear magnetic resonance (NMR), and low- and high-resolution mass spectroscopy by the analytical chemistry laboratory and by infrared and NMR spectroscopy by the study laboratory. All spectra were consistent with the literature spectra ( Sadtler, 1979; Aldrich, 1981; Handbook of Proton NMR Spectra and Data, 1985; NIST Standard Reference Database ) and with the structure of o- nitrotoluene. The infrared and NMR spectra are presented in Figures H1 and H2. The observed boiling point of 222° C and density of 1.154 at 22° C were in agreement with literature values ( Merck Index , 1996).

The purity of lot 8056-58-05RTI was determined by Karl Fischer water analysis and gas chromatography by systems A and B (Table H1). The study laboratory performed gas chromatography using system C with naphthalene added as an internal standard.

Karl Fischer analysis indicated 0.29% water. Gas chromatography using system A indicated one major peak, one impurity peak with an area of 0.11% of the total integrated area, and five minor peaks, each with an area of less than 0.1%. System B indicated one major peak and six minor peaks, each accounting for less than 0.1% of the total integrated area. Gas chromatography by system C indicated one major peak and no impurities. The overall purity of lot 8056-58-05RTI was determined to be greater than 99%.

Accelerated stability studies of the bulk chemical were performed by the analytical chemistry laboratory, using gas chromatography by system D. These studies indicated that o- nitrotoluene was stable as a bulk chemical for at least 2 weeks when stored protected from light at temperatures up to 25° C. To ensure stability, the bulk chemical was stored in amber glass bottles inside metal cans at room temperature. Stability was monitored during the studies with gas chromatography (system C). No degradation of the bulk chemical was detected.



# P REPARATION AND 

# ANALYSIS OF 

# D OSE 

# F ORMULATIONS The dose formulations were prepared every 2 weeks by mixing o- nitrotoluene with feed (Table H2). Nonirradiated NTP-2000 feed was used through June 26, 1996; irradiated NTP-2000 feed was used thereafter. A premix was prepared by hand and then blended with additional feed in a Patterson-Kelly twin-shell blender for 15 minutes using an intensifier bar for the initial 5 minutes. Formulations were stored in doubled opaque plastic bags inside plastic buckets at approximately 5° C, protected from light and with minimal headspace, for 35 days.

Homogeneity studies of 625 and 5,000 ppm dose formulations in nonirradiated NTP-2000 feed were performed with gas chromatography by the analytical chemistry laboratory (system E) and by the study laboratory (system F). Stability studies of the 625 ppm dose formulation in nonirradiated NTP-2000 feed were performed by the analytical chemistry laboratory with gas chromatography by system E. Homogeneity was confirmed, and stability was confirmed for 36 days for dose formulations stored in sealed containers, protected from light at temperatures up to 3° C. Samples stored at room temperature under simulated dosing conditions, open to air and light, were not stable. Losses were shown to be due to volatility. Due to the volatile losses during formulation, the study laboratory prepared dose formulations at concentrations up to 115% of the target concentrations. Dose o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 299



### formulations were replaced in animal room feeders on a 2-day, 2-day, 3-day schedule. After the change to irradiated feed, the study laboratory performed additional homogeneity analyses of the 625, 2,000, and 5,000 ppm dose formulations with gas chromatography (system F) to compare the homogeneity of dose formulations prepared with nonirradiated feed to that of dose formulations prepared with irradiated feed. The homogeneity of all samples was confirmed.

Analyses of the dose formulations of o- nitrotoluene were conducted by the study laboratory every 8 to 12 weeks using gas chromatography by system F. Because of the expected loss of o- nitrotoluene during mixing, the NTP permitted the use of dose formulations within the range of 90% to 115% of the target concentrations. Of the dose formulations analyzed for rats, 202 of 210 had concentrations that were 90% to 115% of the target concentrations; the concentrations in animal room samples analyzed after 3 days of use in the feeders for rats ranged from 75% to 94% of the target concentrations. Of the dose formulations analyzed for mice, all 63 were within 90% to 115% of the target concentrations; concentrations in animal room samples analyzed after 3 days of use in the feeders for mice ranged from 53% to 81% of the target concentrations. Of the eight dose formulations for rats that were not within 90% to 115% of the target concentration, seven were remixed and were found to be within the specified range; one was discarded without being remixed because a sufficient amount of dose formulation at this concentration was available for dosing.

### 300 o-Nitrotoluene, NTP TR 504



### F IGURE 

### H1 Infrared Absorption Spectrum of o- Nitrotoluene

### o-Nitrotoluene, NTP TR 504 301



### F IGURE 

### H2 Nuclear Magnetic Resonance Spectrum of o-Nitrotoluene

### System A Flame ionization



### System B Flame ionization



### System C Flame ionization



### System D Flame ionization



### System E Flame ionization



### System F Flame ionization SE-30, 30 m × 0.25 mm, 0.25-μm film (J&W Scientific, Folsom, CA)

DB-17, 30 m × 0.25 mm, 0.25-μm film (J&W Scientific)

Rtx5, 30 m × 0.53 mm, 1.0-μm film (Restek, Bellefonte, PA)

SE-30, 30 m × 0.25 mm, 0.25-μm film (J&W Scientific)

SE-30, 30 m × 0.25 mm, 0.25-μm film (J&W Scientific)

Rtx5, 30 m × 0.53 mm, 1.0-μm film (Restek) Helium at 1 mL/minute

Nitrogen at 1 mL/minute

Helium at approximately 12 mL/minute

Helium at 1 mL/minute

Nitrogen at 1 mL/minute

Helium at approximately 12 mL/minute 50° C for 5 minutes, then 10° C/minute to 250° C, held for 15 minutes

40° C for 5 minutes, then 10° C/minute to 220° C, held for 17 minutes

Isothermal at 130° C

Isothermal at 130° C for 7 minutes

120° C for 7 minutes, then 30° C/minute to 300° C, held for 7 minutes

110° C for 10 minutes, then 70° C/minute to 230° C, held for 10 minutes



### T ABLE 

### H2 Preparation and Storage of Dose Formulations in the 2-Year Feed Studies of o-Nitrotoluene

Preparation A premix of feed and o- nitrotoluene was prepared, then layered into the remaining feed and blended in a Patterson-Kelly twin-shell blender with the intensifier bar on for 5 minutes and off for 10 minutes. The dose formulations were prepared every 2 weeks.



### Chemical Lot Number 8056-58-05RTI


### Maximum Storage Time 35 days


### Storage Conditions Stored in doubled opaque plastic bags inside plastic buckets at approximately 5° C, protected from light and with minimal headsp ace


### Study Laboratory Southern Research Institute (Birmingham, AL) 

### 302



### T ABLE 

### H1 Gas Chromatography Systems Used in the 2-Year Feed Studies of o-Nitrotoluene a



### Detection Column Carrier Gas Oven Temperature

System Program 

### o-Nitrotoluene, NTP TR 504

a All gas chromatographs were manufactured by Hewlett Packard (Palo Alto, CA).

### o-Nitrotoluene, NTP TR 504 303



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o- Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration a 

### from Target (ppm) (ppm) (%)

Rats

February 7-8, 1996 February 7-9, 1996 625 669 +7 625 678 +8 625 677 +8 625 677 +8 625 664 +6 625 718 +15 1,250 1,330 +6 1,250 1,340 +7 1,250 1,310 +5 1,250 1,320 +6 1,250 1,310 +5 1,250 1,260 +1 2,000 2,100 +5 2,000 2,110 +6 2,000 2,090 +5 2,000 2,140 +7 2,000 2,110 +6 2,000 1,950 –2 2,000 1,980 –1 2,000 1,970 –1 2,000 1,970 –1 2,000 1,990 0 5,000 5,310 +6 5,000 5,040 +1 5,000 4,970 –1 5,000 4,970 –1

February 26-27, 1996 b 625 585 –6 1,250 1,030 –18 2,000 1,500 –25 5,000 3,970 –21

### 304 o-Nitrotoluene, NTP TR 504



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o- Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration from Target (ppm) (ppm) (%)

Rats (continued)

April 17-18, 1996 April 18-19, 1996 625 627 0 625 621 –1 625 620 –1 625 625 0 625 620 –1 625 632 +1 1,250 1,280 +2 1,250 1,270 +2 1,250 1,280 +2 1,250 1,250 0 1,250 1,280 +2 1,250 1,270 +2 2,000 2,020 +1 2,000 2,000 0 2,000 2,010 +1 2,000 1,980 –1 2,000 1,950 –2 2,000 2,000 0 2,000 2,110 +6 2,000 2,040 +2 2,000 2,050 +3 2,000 2,020 +1 5,000 5,030 +1 5,000 4,910 –2 5,000 4,930 –1 5,000 4,930 –1

June 26-27, 1996 June 26-29, 1996 625 605 –3 625 638 +2 625 616 –1 625 659 +5 625 634 +1 625 664 +6 625 600 –4 1,250 1,310 +5 1,250 1,310 +5 1,250 1,300 +4 1,250 1,290 +3 1,250 1,290 +3 1,250 1,280 +2 2,000 2,020 +1 2,000 1,780 c –11 2,000 1,990 0 2,000 1,980 –1 2,000 1,970 –1 2,000 1,930 –3 2,000 1,910 –4

July 5, 1996 July 9-11, 1996 2,000 1,860 d –7

### o-Nitrotoluene, NTP TR 504 305



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o -Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration from Target (ppm) (ppm) (%)

Rats (continued)

September 4-5, 1996 September 4-6, 1996 625 518 c –17 625 565 –10 625 546 c –13 625 577 –8 625 584 –7 625 582 –7 1,250 1,160 –7 1,250 1,170 –6 1,250 1,180 –6 1,250 1,180 –6 1,250 1,150 –8 1,250 1,130 –10 2,000 1,930 –3 2,000 1,900 –5 2,000 1,930 –3 2,000 1,890 –5 2,000 2,000 0 2,000 1,990 0

September 9, 1996 September 9, 1996 625 604 d –3 625 616 d –1

October 30, 1996 October 30-November 1, 1996 625 614 –2 625 638 +2 625 613 –2 625 616 –1 625 619 –1 625 600 –4 1,250 1,220 –2 1,250 1,220 –2 1,250 1,200 –4 1,250 1,220 –2 1,250 1,250 0 1,250 1,230 –2 2,000 1,910 –4 2,000 1,880 –6 2,000 1,990 0 2,000 1,970 –1 2,000 1,990 0 2,000 2,030 +2

November 21, 1996 b 625 533 –15 1,250 976 –22 2,000 1,510 –24

### 306 o-Nitrotoluene, NTP TR 504



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o -Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration from Target (ppm) (ppm) (%)

Rats (continued)

January 8-9, 1997 January 8-10, 1997 625 622 0 625 631 +1 625 643 +3 625 641 +3 625 642 +3 625 645 +3 1,250 1,250 0 1,250 1,260 +1 1,250 1,250 0 1,250 1,280 +2 1,250 1,230 –2 1,250 1,250 0 2,000 1,940 –3 2,000 1,980 –1 2,000 1,960 –2 2,000 2,000 0 2,000 1,960 –2 2,000 1,990 0

April 1-2, 1997 April 2-4, 1997 625 582 –7 625 605 –3 625 603 –4 625 604 –3 625 580 –7 625 595 –5 1,250 1,200 –4 1,250 1,250 0 1,250 1,270 +2 1,250 1,300 +4 1,250 1,250 0 1,250 1,280 +2 2,000 2,050 +3 2,000 2,050 +3 2,000 1,960 –2 2,000 1,970 –1 2,000 1,950 –2 2,000 1,920 –4

### o-Nitrotoluene, NTP TR 504 307



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o -Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration from Target (ppm) (ppm) (%)

Rats (continued)

May 28-29, 1997 May 29-30, 1997 625 572 –8 625 558 c –11 625 546 c –13 625 574 –8 625 566 –9 625 565 –10 1,250 1,180 –6 1,250 1,180 –6 1,250 1,220 –2 1,250 1,230 –2 1,250 1,220 –2 1,250 1,210 –3 2,000 1,940 –3 2,000 1,980 –1 2,000 1,990 0 2,000 1,980 –1 2,000 2,040 +2 2,000 1,980 –1

June 2, 1997 June 3, 1997 625 709 d +13 625 716 d +15

June 23-24, 1997 b 625 571 –9 1,250 1,080 –14 2,000 1,760 –12

August 20-21, 1997 August 20-22, 1997 625 654 +5 625 676 +8 625 668 +7 625 639 +2 625 642 +3 625 642 +3 1,250 1,330 +6 1,250 1,410 +13 1,250 1,400 +12 1,250 1,410 +13 1,250 1,440 +15 1,250 1,340 +7 2,000 2,010 +1 2,000 2,150 +8 2,000 2,120 +6 2,000 2,230 +12 2,000 2,160 +8 2,000 2,170 +9

### 308 o-Nitrotoluene, NTP TR 504



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o -Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration from Target (ppm) (ppm) (%)

Rats (continued)

October 30, 1997 October 30-November 1, 1997 625 650 +4 625 647 +4 625 634 +1 625 652 +4 625 645 +3 625 651 +4 1,250 1,280 +2 1,250 1,320 +6 1,250 1,290 +3 1,250 1,320 +6 1,250 1,300 +4 2,000 2,750 c +38 2,000 2,330 c +17 2,000 2,240 +12 2,000 2,220 +11

November 7, 1997 November 7, 1997 2,000 2,080 d +4 2,000 2,240 d +12

January 8, 1998 January 8-10, 1998 625 619 –1 625 621 –1 625 622 0 625 653 +4 625 634 +1 625 611 –2 1,250 623 e –50 1,250 1,260 +1 1,250 1,220 –2 1,250 1,230 –2 1,250 1,210 –3 2,000 1,870 –6 2,000 1,910 –4 2,000 1,910 –4 2,000 1,930 –3

February 3, 1998 b 625 539 –14 1,250 1,030 –18 2,000 1,770 –11



### Mice

February 7-8, 1996 February 7-9, 1996 1,250 1,340 +7 1,250 1,320 +6 2,500 2,430 –3 2,500 2,470 –1 5,000 4,960 –1 5,000 4,950 –1

March 6, 1996 b 1,250 730 –42 2,500 1,330 –47 5,000 2,910 –42

### o-Nitrotoluene, NTP TR 504 309



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o -Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration from Target (ppm) (ppm) (%)

Mice (continued)

April 17-18, 1996 April 18-19, 1996 1,250 1,200 –4 1,250 1,210 –3 2,500 2,580 +3 2,500 2,480 –1 5,000 4,860 –3 5,000 4,900 –2

June 26-27, 1996 June 26-29, 1996 1,250 1,240 –1 1,250 1,260 +1 2,500 2,440 –2 2,500 2,430 –3 5,000 4,760 –5 5,000 4,850 –3 5,000 4,880 –2

September 5, 1996 September 5-6, 1996 1,250 1,200 –4 1,250 1,170 –6 2,500 2,490 0 2,500 2,490 0 5,000 4,980 0 5,000 4,990 0

September 30-October 1, 1996 b 1,250 859 –31 2,500 1,880 –25 5,000 3,600 –28

October 30, 1996 October 30-November 1, 1996 1,250 1,260 +1 1,250 1,300 +4 2,500 2,530 +1 2,500 2,520 +1 5,000 5,040 +1 5,000 5,040 +1

January 8-9, 1997 January 8-10, 1997 1,250 1,290 +3 1,250 1,280 +2 2,500 2,530 +1 2,500 2,480 –1 5,000 5,050 +1 5,000 5,130 +3

April 2, 1997 April 2-4, 1997 1,250 1,280 +2 1,250 1,300 +4 2,500 2,490 0 2,500 2,540 +2 5,000 4,960 –1 5,000 4,900 –2

### 310 o-Nitrotoluene, NTP TR 504



### T ABLE 

### H3 Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies of o -Nitrotoluene

Target Determined Difference Date Prepared Date Analyzed Concentration Concentration from Target (ppm) (ppm) (%)

Mice (continued)

May 28-29, 1997 May 29-30, 1997 1,250 1,260 +1 1,250 1,250 0 2,500 2,570 +3 2,500 2,560 +2 5,000 5,010 0 5,000 4,990 0

June 23-24, 1997 b 1,250 969 –22 2,500 1,890 –24 5,000 3,770 –25

August 20-21, 1997 August 20-22, 1997 1,250 1,400 +12 1,250 1,400 +12 2,500 2,660 +6 2,500 2,710 +8 5,000 5,130 +3 5,000 5,140 +3

October 30, 1997 October 30-November 1, 1997 1,250 1,290 +3 1,250 1,360 +9 2,500 2,750 +10 5,000 5,500 +10

January 8, 1998 January 8-10, 1998 1,250 1,180 –6 1,250 1,230 –2 2,500 2,430 –3 5,000 5,160 +3

February 3, 1998 b 1,250 888 –29 2,500 1,630 –35 5,000 4,060 –19

a Results of duplicate analyses b Animal room samples c Remixed; not used in study d Results of remix e Sample was discarded and not remixed because there was sufficient dose formulation available for dosing at the 1,250 ppm concentration.

### 311



# APPENDIX I FEED AND COMPOUND CONSUMPTION IN THE 2-YEAR FEED STUDIES OF o -NITROTOLUENE

T ABLE 

### I1 Feed and Compound Consumption by Male Rats in the 2-Year Feed Study of o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 T ABLE 

### I2 Feed and Compound Consumption by Female Rats in the 2-Year Feed Study of o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 T ABLE 

### I3 Feed and Compound Consumption by Male Mice in the 2-Year Feed Study of o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 T ABLE 

### I4 Feed and Compound Consumption by Female Mice in the 2-Year Feed Study of o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

### 312 o-Nitrotoluene, NTP TR 504

4 18.1 239 18.4 237 48 18.5 235 99 17.9 228 157 8 17.5 303 17.0 299 36 16.8 295 71 16.1 282 114 12 17.3 344 17.0 337 32 17.1 327 65 16.5 315 105 16 17.3 370 16.8 363 29 16.7 350 60 16.3 329 99 20 17.1 391 15.9 378 26 15.9 366 54 16.0 350 91 24 16.3 401 16.8 385 27 16.3 372 55 16.8 357 94 28 17.7 410 18.2 399 28 18.0 384 59 16.9 363 93 32 16.5 422 16.5 406 25 16.6 392 53 16.5 371 89 36 16.4 429 15.9 413 24 16.0 399 50 15.9 377 84 40 16.4 432 16.5 414 25 16.1 400 50 16.4 378 87 44 16.3 434 16.5 416 25 15.9 400 50 16.2 378 86 48 16.6 433 16.4 417 25 16.4 402 51 16.3 378 86 52 16.9 436 16.4 419 24 16.5 404 51 16.3 383 85 56 16.0 428 16.1 415 24 15.3 398 48 15.8 377 84 60 17.6 439 17.1 423 25 15.6 403 48 16.0 380 84 64 17.3 443 16.9 427 25 16.3 409 50 16.7 388 86 68 16.9 447 16.8 432 24 16.3 417 49 15.6 394 79 72 15.9 446 16.2 433 23 16.1 418 48 15.6 396 79 76 14.9 435 15.7 424 23 15.1 409 46 16.0 393 82 80 15.9 433 15.3 419 23 15.6 405 48 16.7 386 87 84 16.7 430 17.1 423 25 16.5 407 51 16.4 374 88 88 16.2 434 15.4 420 23 14.7 411 45 92 15.1 428 15.7 425 23 14.5 408 45 96 15.2 425 16.0 424 24 13.4 395 42 100 15.5 424 14.9 421 22 16.0 408 49 104 14.6 425 13.5 415 20

Mean for weeks 4-13 17.6 295 17.5 291 39 17.5 285 78 16.8 275 125 14-52 16.8 416 16.6 401 26 16.4 387 53 16.4 367 89 53-104 16.0 434 15.9 423 23 15.5 407 47 16.1 386 84 

### T ABLE 

### I1 Feed and Compound Consumption by Male Rats in the 2-Year Feed Study of o -Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm Feed Body Feed Body Dose Feed Body Dose Feed Body Dose (g/day) a 

### Weight (g/day) Weight (mg/kg) b 

### (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) Week (g) (g) (g) (g)

### o-Nitrotoluene, NTP TR 504 313

4 18.0 230 156 16.2 211 384 8 16.2 285 114 14.1 247 286 12 16.4 316 104 14.5 263 276 16 16.4 341 15.8 279 20 16.7 359 16.6 297 24 16.5 371 16.1 305 28 17.5 381 17.4 312 32 16.2 390 16.2 321 36 16.0 398 15.7 328 40 16.2 400 15.8 329 44 16.4 403 15.7 331 48 16.2 402 16.1 334 52 16.9 408 16.2 336 56 16.1 402 15.5 329 60 17.3 411 16.6 342 64 17.0 417 16.0 346 68 16.4 422 15.8 356 72 15.9 421 16.0 364 76 15.8 415 16.0 367 80 15.3 408 15.5 360 84 15.7 403 16.8 368 88 16.2 413 92 15.0 408 96 14.2 412 100 14.2 412 104 15.4 404

Mean for weeks 4-13 16.9 277 125 14.9 240 315 14-52 16.5 385 16.2 317 53-104 15.7 412 16.0 354

a Grams of feed consumed per animal per day b Milligrams of o -nitrotoluene consumed per kilogram body weight per day 

### T ABLE 

### I1 Feed and Compound Consumption by Male Rats in the 2-Year Feed Study of o -Nitrotoluene

2,000 ppm (Stop-Exposure) 5,000 ppm (Stop-Exposure) Feed Body Dose Feed Body Dose (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) Week (g) (g)

### 314 o-Nitrotoluene, NTP TR 504

4 11.3 145 11.5 144 50 12.0 145 103 11.5 143 161 8 9.9 171 9.9 168 37 10.2 170 75 9.8 168 117 12 10.2 185 10.0 180 35 10.2 182 70 10.1 181 112 16 10.1 195 10.3 193 33 10.5 193 68 10.3 193 106 20 10.2 203 9.6 200 30 10.1 200 63 9.9 196 101 24 10.0 207 10.2 204 31 10.4 204 64 10.3 201 102 28 10.7 212 10.9 210 33 11.1 209 67 10.4 206 101 32 10.3 221 10.4 217 30 10.5 216 61 10.2 213 96 36 9.8 225 10.0 221 28 10.1 219 57 9.8 214 92 40 10.0 227 10.1 225 28 10.3 224 58 10.0 216 93 44 10.2 231 10.2 228 28 10.6 227 58 10.3 220 93 48 10.5 236 10.4 232 28 10.7 231 58 10.2 222 91 52 10.7 239 10.6 235 28 11.0 235 59 10.5 224 94 56 11.3 244 11.4 241 30 11.1 241 58 10.9 227 96 60 11.6 254 11.0 247 28 11.1 247 56 10.8 230 94 64 11.1 258 11.3 256 28 11.5 251 57 11.0 234 94 68 11.7 266 11.8 263 28 12.0 259 58 10.8 241 89 72 11.9 271 12.2 267 29 12.0 267 56 11.5 243 95 76 12.2 276 12.2 271 28 11.4 268 53 11.4 243 94 80 12.0 281 11.8 274 27 11.6 272 53 11.2 247 91 84 12.0 284 12.4 275 28 12.6 272 58 11.3 247 91 88 11.7 284 12.5 278 28 12.0 274 55 11.3 250 91 92 11.9 290 12.2 285 27 11.9 282 53 10.9 257 85 96 11.7 291 11.8 287 26 11.7 287 51 11.3 264 86 100 11.9 294 11.7 291 25 11.8 293 50 11.3 273 83 104 11.5 299 12.1 302 25 12.3 298 52 11.7 275 85

Mean for weeks 4-13 10.5 167 10.5 164 40 10.8 166 83 10.5 164 130 14-52 10.3 220 10.3 217 30 10.5 216 61 10.2 210 97 53-104 11.7 276 11.9 272 27 11.8 270 55 11.2 248 90

a Grams of feed consumed per animal per day b Milligrams of o -nitrotoluene consumed per kilogram body weight per day 

### T ABLE 

### I2 Feed and Compound Consumption by Female Rats in the 2-Year Feed Study of o -Nitrotoluene

0 ppm 625 ppm 1,250 ppm 2,000 ppm Feed Body Feed Body Dose Feed Body Dose Feed Body Dose (g/day) a 

### Weight (g/day) Weight (mg/kg) b 

### (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) Week (g) (g) (g) (g)

### o-Nitrotoluene, NTP TR 504 315

4 5.1 24.0 4.7 23.4 250 4.5 22.6 493 4.2 21.4 979 8 4.1 27.7 4.0 27.1 184 3.9 26.2 367 3.6 25.1 722 12 4.6 30.6 4.4 29.0 191 4.3 28.3 376 4.2 26.7 794 16 4.7 34.2 4.6 32.3 179 4.5 30.9 367 4.0 28.3 700 20 4.5 37.3 4.4 35.9 154 4.2 33.8 314 3.8 29.7 636 24 4.9 38.7 4.9 36.4 168 4.7 34.3 341 4.0 30.0 664 28 5.1 40.5 5.1 38.4 165 4.8 35.6 337 4.2 30.8 674 32 5.0 41.9 5.3 39.9 165 5.1 36.8 344 4.2 31.1 673 36 4.9 43.6 4.9 41.0 148 4.8 37.7 318 4.0 31.7 632 40 5.2 44.7 5.2 42.1 153 5.1 38.3 333 4.3 31.9 669 44 5.2 44.8 5.1 42.7 149 5.0 39.0 319 4.2 32.2 646 48 5.0 45.8 5.2 43.2 152 5.4 39.4 341 4.6 32.8 701 56 5.3 47.1 5.3 44.4 150 5.4 40.2 335 3.8 32.4 594 60 5.3 47.0 5.3 44.0 150 5.4 40.1 336 64 5.3 46.7 5.5 43.4 159 5.1 40.4 318 68 5.4 47.7 5.4 45.0 151 5.3 40.7 326 72 5.5 47.3 5.5 44.6 154 5.3 41.0 325 76 5.8 47.6 5.7 44.2 160 5.2 39.5 326 80 5.6 48.0 5.9 44.8 166 5.0 38.9 322 84 5.7 46.3 5.9 43.4 170 5.6 37.3 378 88 5.4 46.5 5.7 43.6 163 92 5.0 46.5 5.2 42.1 154 96 5.3 46.6 5.2 43.1 151 100 5.1 46.8 5.1 43.7 146 104 5.4 46.5 5.4 42.6 159

Mean for weeks 4-13 4.6 27.4 4.4 26.5 208 4.2 25.7 412 4.0 24.4 832 14-52 4.9 41.3 5.0 39.1 159 4.8 36.2 335 4.1 30.9 666 53-104 5.4 47.0 5.5 43.8 156 5.5 38.6 364 3.8 32.4 594

a Grams of feed consumed per animal per day b Milligrams of o -nitrotoluene consumed per kilogram body weight per day 

### T ABLE 

### I3 Feed and Compound Consumption by Male Mice in the 2-Year Feed Study of o -Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm Feed Body Feed Body Dose Feed Body Dose Feed Body Dose (g/day) a 

### Weight (g/day) Weight (mg/kg) b 

### (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) Week (g) (g) (g) (g)

### 316 o-Nitrotoluene, NTP TR 504

4 3.7 20.6 3.8 21.1 226 3.7 20.4 449 3.3 19.4 857 8 3.4 22.4 3.8 23.2 202 3.6 23.3 388 3.2 22.1 731 12 3.8 25.7 3.9 25.5 193 3.9 25.1 386 3.5 23.2 761 16 4.0 27.8 4.1 27.4 188 4.0 26.8 374 3.7 24.3 758 20 3.7 29.4 3.8 29.3 162 3.9 29.0 336 3.5 25.4 697 24 4.3 31.8 4.5 32.2 177 4.2 30.1 353 3.9 26.4 733 28 4.3 33.2 3.9 33.6 145 4.1 32.0 323 3.8 27.1 704 32 4.6 35.4 4.5 35.8 159 4.7 33.6 348 4.1 28.0 727 36 4.2 37.3 4.1 36.7 141 4.4 35.0 313 3.8 29.0 647 40 4.5 38.1 4.6 37.7 152 4.4 35.9 306 3.9 29.5 666 44 4.4 38.1 4.2 38.6 135 4.3 37.0 288 3.8 30.5 616 48 4.3 39.3 4.4 38.8 143 4.3 37.5 290 3.8 30.4 628 52 4.7 40.4 5.0 40.7 154 4.8 38.1 315 4.6 30.6 747 56 4.2 41.8 4.4 42.6 130 4.2 38.8 269 3.7 31.5 592 60 5.0 42.4 4.9 42.5 143 4.8 39.3 304 4.2 31.6 673 64 4.6 43.7 4.8 43.5 138 4.4 39.9 277 4.1 32.2 644 68 4.3 45.2 4.8 44.4 136 4.5 41.3 274 4.4 33.4 662 72 4.6 44.5 4.8 44.4 135 4.5 41.2 270 4.3 33.3 643 76 4.5 44.8 4.7 43.9 134 4.4 40.8 268 4.4 33.8 653 80 4.2 44.8 4.5 44.2 127 4.4 40.9 268 4.1 33.6 613 84 4.8 43.8 4.7 42.2 139 4.5 38.7 293 4.5 32.8 687 88 5.2 44.7 5.3 43.1 153 5.1 39.3 325 5.0 33.4 745 92 4.3 45.1 4.6 43.9 130 4.5 39.9 283 4.8 33.0 722 96 4.0 45.0 4.5 45.3 123 4.1 39.7 260 5.4 32.7 832 100 4.4 46.2 4.8 47.4 126 4.9 41.3 296 6.0 32.3 934 104 5.1 46.6 5.2 46.9 139 5.5 41.2 334

Mean for weeks 4-13 3.6 22.9 3.8 23.3 207 3.7 22.9 408 3.4 21.6 783 14-52 4.3 35.1 4.3 35.1 156 4.3 33.5 325 3.9 28.1 692 53-104 4.5 44.5 4.8 44.2 135 4.6 40.2 286 4.6 32.8 700

a Grams of feed consumed per animal per day b Milligrams of o -nitrotoluene consumed per kilogram body weight per day 

### T ABLE 

### I4 Feed and Compound Consumption by Female Mice in the 2-Year Feed Study of o -Nitrotoluene

0 ppm 1,250 ppm 2,500 ppm 5,000 ppm Feed Body Feed Body Dose Feed Body Dose Feed Body Dose (g/day) a 

### Weight (g/day) Weight (mg/kg) b 

### (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) Week (g) (g) (g) (g)

### 317



# APPENDIX J INGREDIENTS, NUTRIENT COMPOSITION, AND CONTAMINANT LEVELS IN NTP-2000 RAT AND MOUSE RATION

T ABLE 

### J1 Ingredients of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 T ABLE 

### J2 Vitamins and Minerals in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . 318 T ABLE 

### J3 Nutrient Composition of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . 319 T ABLE 

### J4 Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

### 318 o-Nitrotoluene, NTP TR 504



### T ABLE 

### J1 Ingredients of NTP-2000 Rat and Mouse Ration

Ingredients Percent by Weight

Ground hard winter wheat 22.26 Ground #2 yellow shelled corn 22.18 Wheat middlings 15.0 Oat hulls 8.5 Alfalfa meal (dehydrated, 17% protein) 7.5 Purified cellulose 5.5 Soybean meal (49% protein) 5.0 Fish meal (60% protein) 4.0 Corn oil (without preservatives) 3.0 Soy oil (without preservatives) 3.0 Dried brewer’s yeast 1.0 Calcium carbonate (USP) 0.9 Vitamin premix a 0.5 Mineral premixb 0.5 Calcium phosphate, dibasic (USP) 0.4 Sodium chloride 0.3 Choline chloride (70% choline) 0.26 Methionine 0.2

a Wheat middlings as carrier b Calcium carbonate as carrier



### T ABLE 

### J2 Vitamins and Minerals in NTP-2000 Rat and Mouse Ration a



### Amount Source

Vitamins A 4,000 IU Stabilized vitamin A palmitate or acetate D 1,000 IU D-activated animal sterol K 1.0 mg Menadione sodium bisulfite complex


### " -Tocopheryl acetate 100 IU Niacin 23 mg Folic acid 1.1 mg d

-Pantothenic acid 10 mg d -Calcium pantothenate Riboflavin 3.3 mg Thiamine 4 mg Thiamine mononitrate B 12 52 μg Pyridoxine 6.3 mg Pyridoxine hydrochloride Biotin 0.2 mg d -Biotin



### Minerals Magnesium 514 mg Magnesium oxide Iron 35 mg Iron sulfate Zinc 12 mg Zinc oxide Manganese 10 mg Manganese oxide Copper 2.0 mg Copper sulfate Iodine 0.2 mg Calcium iodate Chromium 0.2 mg Chromium acetate

a Per kg of finished product

### o-Nitrotoluene, NTP TR 504 319



### T ABLE 

### J3 Nutrient Composition of NTP-2000 Rat and Mouse Ration

Mean ± Standard Nutrient Deviation Range Number of Samples

Protein (% by weight) 13.6 ± 0.55 12.6 – 14.7 24 Crude fat (% by weight) 8.1 ± 0.32 7.5 – 9.0 24 Crude fiber (% by weight) 9.7 ± 0.64 8.4 – 11.1 24 Ash (% by weight) 5.1 ± 0.29 4.6 – 5.9 24


### Amino Acids (% of total diet) Arginine 0.731 ± 0.050 0.670 – 0.800 8 Cystine 0.224 ± 0.012 0.210 – 0.240 8 Glycine 0.684 ± 0.041 0.620 – 0.740 8 Histidine 0.333 ± 0.018 0.310 – 0.350 8 Isoleucine 0.524 ± 0.046 0.430 – 0.590 8 Leucine 1.061 ± 0.061 0.960 – 1.130 8 Lysine 0.708 ± 0.056 0.620 – 0.790 8 Methionine 0.401 ± 0.035 0.350 – 0.460 8 Phenylalanine 0.598 ± 0.036 0.540 – 0.640 8 Threonine 0.501 ± 0.051 0.430 – 0.590 8 Tryptophan 0.126 ± 0.014 0.110 – 0.150 8 Tyrosine 0.390 ± 0.056 0.280 – 0.460 8 Valine 0.640 ± 0.049 0.550 – 0.690 8


### Essential Fatty Acids (% of total diet) Linoleic 3.97 ± 0.284 3.59 – 4.54 8 Linolenic 0.30 ± 0.042 0.21 – 0.35 8



### Vitamins Vitamin A (IU/kg) 4,699 ± 1,320 2,570 – 8,140 24 Vitamin D (IU/kg) 1,000 a



### " -Tocopherol (ppm) 82.2 ± 14.08 62.2 – 107.0 8 Thiamine (ppm) b 8.7 ± 1.24 6.6 – 11.7 24 Riboflavin (ppm) 5.6 ± 1.12 4.20 – 7.70 8 Niacin (ppm) 74.3 ± 5.94 66.4 – 85.8 8 Pantothenic acid (ppm) 22.5 ± 3.96 17.4 – 29.1 8 Pyridoxine (ppm) b 9.04 ± 2.37 6.4 – 12.4 8 Folic acid (ppm) 1.64 ± 0.38 1.26 – 2.32 8 Biotin (ppm) 0.333 ± 0.15 0.225 – 0.704 8 Vitamin B 12 (ppb) 68.7 ± 63.0 18.3 – 174.0 8 Choline (ppm) b 3,155 ± 325 2,700 – 3,790 8



### Minerals Calcium (%) 0.986 ± 0.053 0.884 – 1.080 24 Phosphorus (%) 0.575 ± 0.029 0.487 – 0.616 24 Potassium (%) 0.659 ± 0.022 0.627 – 0.691 8 Chloride (%) 0.357 ± 0.027 0.300 – 0.392 8 Sodium (%) 0.189 ± 0.019 0.160 – 0.212 8 Magnesium (%) 0.199 ± 0.009 0.185 – 0.213 8 Sulfur (%) 0.178 ± 0.021 0.153 – 0.209 8 Iron (ppm) 160 ± 14.7 135 – 177 8 Manganese (ppm) 50.3 ± 4.82 42.1 – 56.0 8 Zinc (ppm) 50.7 ± 6.59 43.3 – 61.1 8 Copper (ppm) 6.29 ± 0.828 5.08 – 7.59 8 Iodine (ppm) 0.461 ± 0.187 0.233 – 0.843 8 Chromium (ppm) 0.724 ± 0.529 0.330 – 2.000 8 Cobalt (ppm) 0.45 ± 0.628 0.20 – 2.0 8

a From formulation b As hydrochloride (thiamine and pyridoxine) or chloride (choline)

### 320 o-Nitrotoluene, NTP TR 504



### T ABLE 

### J4 Contaminant Levels in NTP-2000 Rat and Mouse Ration a



### Mean ± Standard Deviation b 

### Range Number of Samples

Contaminants Arsenic (ppm) 0.24 ± 0.096 0.10 – 0.50 24 Cadmium (ppm) 0.05 ± 0.011 0.04 – 0.09 24 Lead (ppm) 0.10 ± 0.052 0.06 – 0.28 24 Mercury (ppm) <0.02 24 Selenium (ppm) 0.16 ± 0.029 0.12 – 0.24 24 Aflatoxins (ppb) <5.00 24 Nitrate nitrogen (ppm) c 14.7 ± 5.69 9.04 – 33.6 24 Nitrite nitrogen (ppm) c 0.72 ± 0.40 0.40 – 2.00 24 BHA (ppm) d 1.1 ± 0.54 0.01 – 3.37 24 BHT (ppm) d 1.0 ± 0.37 0.01 – 2.29 24 Aerobic plate count (CFU/g) e 88,956 ± 100,661 15 – 260,000 8 Coliform (MPN/g) f 134 ± 212.8 9 – 510 5 Escherichia coli (MPN/g) <10 24 Salmonella (MPN/g) Negative 24 Total nitrosoamines (ppb) g 5.1 ± 2.29 2.7 – 12.6 24 N

-Nitrosodimethylamine (ppb) g 2.3 ± 1.54 0.9 – 5.7 24 N

-Nitrosopyrrolidine (ppb) g 2.8 ± 1.75 1.0 – 8.7 24


### Pesticides (ppm)



### " -BHC <0.01 24 

### $ -BHC <0.02 24 

### ( -BHC <0.01 24 

### * -BHC <0.01 24 Heptachlor <0.01 24 Aldrin <0.01 24 Heptachlor epoxide <0.01 24 DDE <0.01 24 DDD <0.01 24 DDT <0.01 24 HCB <0.01 24 Mirex <0.01 24 Methoxychlor <0.05 24 Dieldrin <0.01 24 Endrin <0.01 24 Telodrin <0.01 24 Chlordane <0.05 24 Toxaphene <0.10 24 Estimated PCBs <0.20 24

### o-Nitrotoluene, NTP TR 504 321



### T ABLE 

### J4 Contaminant Levels in NTP-2000 Rat and Mouse Ration

Mean ± Standard Deviation Range Number of Samples

Pesticides (ppm) (continued) Ronnel <0.01 24 Ethion <0.02 24 Trithion <0.05 24 Diazinon <0.10 24 Methyl chlorpyrifos 0.093 ± 0.084 0.010 – 0.300 20 Methyl parathion <0.02 24 Ethyl parathion <0.02 24 Malathion 0.224 ± 0.482 0.020 – 2.430 24 Endosulfan I <0.01 24 Endosulfan II <0.01 24 Endosulfan sulfate <0.03 24

a CFU=colony-forming units; MPN=most probable number; BHC=hexachlorocyclohexane or benzene hexachloride b For values less than the limit of detection, the detection limit is given as the mean. c Sources of contamination: alfalfa, grains, and fish meal d Sources of contamination: soy oil and fish meal e Includes three irradiated and five nonirradiated samples. Microbial counts for remaining samples were below the detection limit. f Nonirradiated samples. Microbial counts for irradiated samples were below the detection limit. g All values were corrected for percent recovery.

### 322 o-Nitrotoluene, NTP TR 504

### 323



# APPENDIX K SENTINEL ANIMAL PROGRAM

M ETHODS


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 RESULTS


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

### 324



# SENTINEL ANIMAL PROGRAM

M ETHODS Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel animals come from the same production source and weanling groups as the animals used for the studies of chemical compounds.

Serum samples were collected from randomly selected rats and mice during the 2-year studies. Blood from each animal was collected and allowed to clot, and the serum was separated. The samples were processed appropriately and sent to Microbiological Associates, Inc. (Rockville, MD), for determination of antibody titers. The laboratory serology methods and viral agents for which testing was performed are tabulated below; the times at which blood was collected during the studies are also listed. At 18 months, live mice were shipped to MA BioServices (Rockville, MD) for evaluation of bacterial profile and viral serology according to NIEHS Advisory Number 19.

Method and Test Time of Analysis 

# R

ATS
ELISA Mycoplasma arthritidis Study termination Mycoplasma pulmonis Study termination PVM (pneumonia virus of mice) 6, 12, and 18 months, study termination RCV/SDA

(rat coronavirus/sialodacryoadenitis virus) 6, 12, and 18 months, study termination Sendai 6, 12, and 18 months, study termination

Immunofluorescence Assay M. arthritidis Study termination Parvovirus Study termination RCV/SDA 6 months

Hemagglutination Inhibition H-1 (Toolan’s H-1 virus) 6, 12, and 18 months KRV (Kilham rat virus) 6, 12, and 18 months o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 325



# M ICE Bacterial Assays Oral 18 months Fecal 18 months

ELISA Ectromelia virus 6, 12, and 18 months, study termination EDIM (epizootic diarrhea of infant mice) 6, 12, and 18 months, study termination GDVII (mouse encephalomyelitis virus) 6, 12, and 18 months, study termination LCM (lymphocytic choriomeningitis virus) 6, 12, and 18 months, study termination Mouse adenoma virus-FL 6, 12, and 18 months, study termination MHV (mouse hepatitis virus) 6, 12, and 18 months, study termination M. arthritidis 18 months and study termination M. pulmonis 18 months and study termination PVM 6, 12, and 18 months, study termination Reovirus 3 6, 12, and 18 months, study termination Sendai 6, 12, and 18 months, study termination

Immunofluorescence Assay Helicobacter hepaticus 18 months Mouse adenoma virus-FL 18 months MCMV (mouse cytomegalovirus) 18 months and study termination M. arthritidis Study termination Parvovirus Study termination Reovirus 3 18 months

Hemagglutination Inhibition K (papovavirus) 6, 12, and 18 months MVM (minute virus of mice) 6, 12, and 18 months Polyoma virus 6, 12, and 18 months



# RESULTS For the 2-year study in rats, all serology tests were negative. Bacterial profiles of sentinel mice shipped live to the rodent disease diagnostic laboratory at 20 months indicated Enterococcus faecalis in five males and five females and Klebsiella oxytoca in five males. These had no impact on the study results. These mice were subjected to comprehensive health evaluations, including histologic evaluation of liver sections by special stains for Helicobacter infections. Helicobacter spp. were not isolated from any of these mice. Two mice had positive titers for M. arthritidis at study termination. Further evaluation of the samples positive for M. arthritidis by immunoblot and Western blot procedures indicated that the positive titers may have been due to cross reaction with antibodies of nonpathogenic Mycoplasma or other agents. Only sporadic samples were positive and there were no clinical findings or histopathologic changes of M. arthritidis infection in animals with positive titers. Accordingly, M. arthritidis -positive titers were considered false positives.

### 326 o-Nitrotoluene, NTP TR 504

### 327



# APPENDIX L COMPARATIVE METABOLISM STUDIES OF o-NITROTOLUENE

I NTRODUCTION


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 M ATERIALS AND 

### M ETHODS


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 RESULTS AND 

### D ISCUSSION


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 REFERENCES


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 T ABLE 

### L1 Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F 1 

### Mice after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 334 T ABLE 

### L2 Cumulative Urinary Excretion of Radioactivity by F344/N Rats after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 334 F IGURE 

### L1 HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 335 F IGURE 

### L2 HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene and Incubated with $ -Glucuronidase/Sulfatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 F IGURE 

### L3 HPLC Radiochromatograms of Urine Collected from B6C3F 1 

### Mice 0 to 24 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 337 T ABLE 

### L3 Urinary Metabolite Profile for F344/N Rats and Male B6C3F 1 

### Mice after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 338 T ABLE 

### L4 Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F 1 

### Mice after a Single Gavage Dose of 2 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . 339 T ABLE 

### L5 Urinary Metabolite Profile for F344/N Rats and Male B6C3F 1 

### Mice after a Single Gavage Dose of 2 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . 340 T ABLE 

### L6 Cumulative Excretion of Radioactivity by Male F344/N Rats Administered 200 mg/kg [ 14 

### C]- o -Nitrotoluene in the 14-Day Gavage Study . . . . . . . . . . . . . 341 T ABLE 

### L7 Urinary Metabolite Profile for Male F344/N Rats Administered 200 mg/kg [ 14 

### C]- o -Nitrotoluene in the 14-Day Gavage Study . . . . . . . . . . . . . 342 T ABLE 

### L8 Urinary Metabolite Profile for Male F344/N Rats Pretreated with Buthionine Sulfoxamine after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 T ABLE 

### L9 Urinary Metabolite Profile for Male F344/N Rats Pretreated with Pentachlorophenol after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . 343 T ABLE 

### L10 Plasma Concentration of o-Nitrotoluene in Male F344/N Rats after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 344 F IGURE 

### L4 Isolation of Globin and Binding of o- Nitrotoluene to Globin in F344/N Rats 72 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 345

### 328



# COMPARATIVE METABOLISM STUDIES OF o-NITROTOLUENE

I NTRODUCTION Studies were conducted in male and female F344/N rats and male B6C3F1 mice to determine and compare the metabolism and excretion of [ 14 

### C]- o- nitrotoluene following the administration of single doses by gavage; similar studies were conducted in male F344/N rats following 14 days of repeated gavage dosing. These studies were conducted by Research Triangle Institute (1995).



# M ATERIALS AND 

# M ETHODS Nonradiolabeled o- nitrotoluene was obtained from Aldrich Chemical Company (St. Louis, MO) in two lots (08506MV and 04328DG). Radiolabeled [ 14 

### C]- o- nitrotoluene, uniformly ring-labeled, was obtained from Chemsyn Science Laboratories (Lenexa, KS; lot CSL-91-331-50-27; 15 mCi/mmol, 0.50 mCi/mL) and Du Pont NEN Research Products (Boston, MA; lot 3048-101; 8.99 mCi/mmol, 1.03 mCi/mL).

The identity of the nonradiolabeled o- nitrotoluene was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Radiochemical purity of the [ 14 

### C]- o- nitrotoluene was determined to be 98% or greater by the study laboratory using high-performance liquid chromatography (HPLC), a radioactivity detector with a scintillator flow cell, and ultraviolet detection at 254 nm.

Male and female F344/N rats and male B6C3F 1 

### mice were obtained from Charles River Laboratories, Inc. (Raleigh, NC). The animals were quarantined for at least 1 week before the beginning of each study. Animals were approximately 9 to 14 (male rats), 9 to 12 (female rats), or 9 to 11 (male mice) weeks old at dosing. Animals were housed in polycarbonate cages. The day before dosing, animals in single-dose studies were transferred to individual glass metabolism cages, which provided for separate collection of urine and feces. Rats in the repeat dosing study were housed in polycarbonate cages until the day before administration of the radiolabeled dose, after which they were transferred to glass metabolism cages. Animals received certified Purina Rodent Chow #5002 and tap water ad libitum.

The four male F344/N rats in the pharmacokinetic study were implanted with indwelling jugular cannulae to allow serial blood sampling. Rats were anesthetized with an intraperitoneal injection of 60 mg ketamine:xylazine (7:1)/kg body weight before cannulation. The indwelling cannula design was similar to that of Harms and Ojeda (1974) as modified by McKenna and Bieri (1984). Following implantation of the cannulae, rats were returned to metabolism cages to recover for one day prior to dosing.

At the end of each study, rats were anesthetized with an intraperitoneal injection of 60 mg ketamine:xylazine (7:1)/kg body weight before final blood collection. Rats not used for the pharmacokinetic study were sacrificed by an intracardiac injection of 300 mg sodium pentobarbital/kg body weight. Pharmacokinetic study rats were sacrificed by asphyxiation with CO 2 

### . Mice were anesthetized with an intraperitoneal injection of 60 mg/kg sodium pentobarbital prior to final blood collection, and they were sacrificed by cervical dislocation.

Gavage dosing formulations used in the initial 200 mg/kg rat and mouse studies contained 10 to 100 μCi radiolabel, an appropriate amount of unlabeled o- nitrotoluene, and Emulphor EL-620 to yield a dose volume of 5 mL/kg. The doses for the hemoglobin binding study in male and female rats dosed with 200 mg/kg [ 14 

### C]- o- nitrotoluene contained 275 to 300 μCi radiolabel and an appropriate amount of unlabeled o- nitrotoluene in the same vehicle. Dosing solutions were prepared one day prior to use and stored at –20° C in the dark. Subsequent gavage dose formulations were prepared in Emulphor:ethanol:water (1:1:8). These formulations contained 14 to 21 μCi (rats) or 1.7 to 1.9 μCi (mice) of radiolabel, an appropriate amount of unlabeled

o- nitrotoluene, and sufficient vehicle for a single dose volume of 5 mL/kg (rats) or 0.5 mL (mice). o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 329



### Oral doses of [ 14 

### C]- o- nitrotoluene were administered to animals by intragastric gavage at 2 or 200 mg/kg. Doses were delivered using a ball-tipped gavage needle that was wiped clean of excess dose formulation before the filled dosing apparatus was weighed. After dosing, the needle was wiped clean with a Kimwipe ® 

### , and the empty dosing apparatus was reweighed; the Kimwipe ® 

### was placed into a vial containing 10 mL of scintillation cocktail to determine the quantity of residual radiolabel on the gavage needle. Each dose was calculated as the difference between the weights of the filled and empty dosing apparatus, less the amount found on the wipes. To measure the concentration of [ 14 

### C]- o- nitrotoluene in the dose formulation, two weighed aliquots were taken before, two after, and one in the midst of dosing a series of animals.



## Determination of Excretion and Urinary Metabolites of [ 14 

## C]- o-Nitrotoluene in Rats and Mice Groups of three or four male or female rats or male mice were administered single gavage doses of 2 or 200 mg [ 14 

### C]- o- nitrotoluene/kg body weight in Emulphor. For the initial 200 mg/kg studies, radioactivity was measured in urine and feces collected 4, 8, 24, 48, and 72 hours after dosing. In subsequent studies, urine or urine and feces were collected 24, 48, and 72 hours after dosing. Urine and feces were collected separately into round-bottom flasks cooled with dry ice. Samples were stored at –20° C, protected from light, until analysis.

To determine radioactivity, aliquots of urine and plasma were added to vials containing scintillation cocktail (Ultima Gold® 

### , Packard Instrument Company, Meriden, CT). Samples of feces and blood were digested in 2 mL Soluene ® 

### -350 (Packard Instrument Company). After digestion, samples requiring bleaching were decolorized with perchloric acid/hydrogen peroxide before the addition of scintillation cocktail.

To isolate, purify, and characterize urinary metabolites, composite samples of urine from animals administered 200 mg/kg [ 14 

### C]- o- nitrotoluene by gavage were made by mixing 20% of each sample collected. Samples from male and female rats and male mice were pooled separately. The pooled samples were centrifuged, decanted, and filtered through a Millex HV 0.45-μm filter (Millipore Corporation, Bedford, MA). Concentrations of radiolabel in the pooled samples were determined by liquid scintillation counting. In groups where metabolite profiles were measured, individual animal urine samples were analyzed separately to obtain group mean and standard deviation estimates.

Urinary metabolite profiles were obtained using HPLC with a Supelco LC-18 DB analytical column (Supelco, Inc., Bellefonte, PA). Metabolites were eluted using a linear gradient, changing from 5% to 90% acetonitrile in 5 mM sodium phosphate buffer (pH 4.5) over 30 minutes; the flow rate was 2 mL/minute. Metabolites eluting from the column were detected using both ultraviolet absorption (254 nm) and flow-through radioactivity detectors.

Glucuronide and sulfate metabolite conjugates were determined by incubating aliquots of urine with glucuronidase/sulfatase (Sigma Chemical Co., St. Louis, MO); 1.5 μL of enzyme solution and 6 μL of 1 M ammonium acetate were added to 45 μL of the pooled urine samples. The mixture was incubated for 24 hours at 37° C. Controls were prepared with 1,4-saccharolactone, an inhibitor of $ -glucuronidase, and with heatdeactivated enzyme or no enzyme. Aliquots were taken before and after incubation to determine recovery based on radioactivity. Recovery was greater than 90% in all cases. Alternatively, urine samples were incubated for 18 hours in TRIZMA ® 

### buffer with sulfatase (Sigma Chemical Co.). These profiles were then compared with the HPLC profiles of the same samples prior to incubation with glucuronidase/sulfatase or sulfatase.

The other metabolites were tentatively identified by coelution with authentic standards using HPLC. Upon reevaluation of the metabolite profiles in urine collected from rats dosed with o- nitrotoluene having a higher specific activity, some of the original assignments appeared questionable. In these cases, four metabolite peaks, labeled C, D, E, and F, were collected individually as they eluted from the HPLC column, concentrated, and freed of phosphate buffer by use of a C 18 

### Sep Pak (Waters, Milford, MA) prior to analysis by gas chromatography/mass spectrometry (GC/MS). GC/MS was not effective in characterizing metabolite E; accordingly, additional urine

### 330



### samples were prepared, and metabolite E was subsequently analyzed using thermospray liquid chromatography/mass spectrometry.

In separate studies of male rats administered single gavage doses of [ 14 

### C]- o- nitrotoluene, the urinary profile was assessed following pretreatment with buthionine sulfoximine (an inhibitor of glutathione synthesis) or pentachlorophenol (an inhibitor of O-sulfation). Specifically, three male rats were provided drinking water containing 30 mM buthionine sulfoximine for 6 days before and until 72 hours after a single gavage dose of 200 mg/kg [ 14 

### C]- o- nitrotoluene. Alternatively, three male rats were injected intraperitoneally with pentachlorophenol 45 minutes before gavage administration of 200 mg/kg [ 14 

### C]- o- nitrotoluene. In a third study, the effect of repeat gavage dosing on the metabolism and excretion of [ 14 

### C]- o- nitrotoluene was measured in three male rats. The rats were administered nonradiolabeled doses of 200 mg o- nitrotoluene daily for 11 days; on day 12, the dose was radiolabeled, and after dosing, the animals were placed in metabolism cages to facilitate collection of urine and feces. The rats received single gavage doses of nonradiolabeled o- nitrotoluene (200 mg/kg) on days 13 and 14 and were sacrificed on day 15.



## Determination of Plasma Concentration of o-Nitrotoluene Samples were collected from four rats 15 and 30 minutes and 1, 2, 4, 8, and 24 hours after a single gavage dose of 200 mg/kg o- nitrotoluene. Blood was sampled for liquid scintillation analysis and plasma was prepared by centrifugation. Plasma aliquots were analyzed for total radioactivity and the remainder was extracted to analyze for concentration of o- nitrotoluene. For extraction, the samples were placed in 1 mL vials and 200 μL n-butyl chloride was added to each vial. The vials were capped and mixed on an end-over-end rotator for 30 minutes and then centrifuged. The supernatant was transferred to a 200 μL low-volume insert for GC analysis. To construct a standard curve, seven samples of plasma containing 223 to 26,700 ng o- nitrotoluene/g plasma were prepared and duplicate samples were extracted as above. The samples were analyzed using a DB5 column (J&W Scientific, Folsom, CA) in an isothermal oven at 75° C with a flame ionization detector at 300° C. Each sample was analyzed twice, and the reported concentrations are the average of the two determinations.



## Determination of the Role of Sulfation in the Conjugation of 2-Nitrobenzyl Alcohol with Glutathione 2-Nitrobenzyl alcohol was incubated with rat liver cytosol in the presence or absence of a 3 N -phosphoadenosine5 N -phosphosulfate (PAPS)-generating system. The incubation mixture contained 1 mL rat liver cytosol, 10 mM [ 3 

### H]-glutathione, 6 μmol magnesium chloride, and phosphate buffer (pH 7.4). Adenosine triphosphate and sodium sulfate (10 μmol each) were added as cofactors for the PAPS-generating system. The mixtures were incubated for 1 hour at 37° C, with 2-nitrobenzyl alcohol (1 mM) added after 30 minutes; incubations were stopped by the addition of 1 mL methanol. The samples were centrifuged and the supernatants were analyzed by HPLC.



## Determination of the Binding of o-Nitrotoluene Equivalents to Hemoglobin Blood was collected via cardiac puncture into heparinized syringes from four anesthetized male rats that had been administered 200 mg/kg o- nitrotoluene. Plasma was prepared by centrifugation and was stored at –20° C. The erythrocytes were washed three times with an equal volume of 0.9% saline, then lysed by the addition of an equal volume of water. Approximately 1 mL of lysate was mixed with 6 mL of 50 mM hydrogen chloride in isopropanol and centrifuged. The supernatant was decanted, 3 mL of ethyl acetate was added, and the mixture was centrifuged to sediment the precipitated protein pellet containing globin. The pellet was washed first with ethyl acetate, then with n -pentane, resedimenting the pellet by centrifugation as before. The pellet was dried under a gentle stream of nitrogen and then in vacuo overnight. Protein content was determined by the method of Lowry et al. (1951), and radiochemical content was measured as before. The calculated pmol-equivalents/mg protein were monitored before and after continuous (Soxhlet) extraction (ethyl acetate, 2 days), and changed less than 10%. o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 331



# RESULTS AND 

# D ISCUSSION 

## Excretion and Urinary Metabolites of [ 14 

## C]- o-Nitrotoluene in Rats and Mice Oral doses of 200 mg/kg o- nitrotoluene were excreted mainly in urine, with approximately 86% (male rats), 92% (female rats), and 66% (male mice) of the dose excreted in the first 24 hours after dosing (Table L1). Fecal elimination accounted for 3% to 4% of the dose in both male and female rats and approximately 9% in male mice. Total recoveries of radiolabel for male and female rats were approximately 106%, while those of male mice were approximately 87%. In another study using a higher specific activity dose, the values for the excretion of radioactivity were similar to those of the previous investigation (Table L2).

The profiles of metabolites found in urine collected after a single gavage dose of 200 mg/kg o- nitrotoluene were determined by HPLC analysis of pooled urine samples (Figures L1 to L3). The relative amounts of these metabolites found upon analysis of individual urine samples are presented in Table L3. At least eight metabolites are present in the HPLC radiochromatograms of urine collections from male and female rats, and only two major metabolites are present in male mice; o- nitrotoluene, which elutes at about 25 minutes, was not detected in these samples. The HPLC radiochromatograms indicate the presence of several polar and nonpolar metabolites in rats and two polar metabolites in mice. The HPLC radiochromatograms of urine from F/344N rats after incubation with glucuronidase/sulfatase are shown in Figure L2. The most pronounced change in the profile of metabolites is the loss of the peak B at retention time 5.3 minutes giving rise to peak G; upon incubation with purified sulfatase, peak B remained, confirming it is a glucuronide rather than a sulfate conjugate. The identity of peak G was confirmed by coelution with the authentic standard, 2-nitrobenzyl alcohol. The HPLC radiochromatogram of male B6C3F 1 

### mouse urine after incubation with glucuronidase/sulfatase is shown in Figure L3. Again, metabolite B was hydrolyzed to yield peak G by incubation with glucuronidase, but not with sulfatase. The major metabolites excreted in the urine of male F/344N rats were 2-nitrobenzoic acid (peak A, 21.0% of the dose), 2-nitrobenzyl glucuronide (B, 16.6%), 2-aminobenzyl alcohol (D, 18.2%), S-(2-nitrobenzyl)-N-acetylcysteine ( o- nitrobenzylmercapturic acid) (F, 10.4%), 2-nitrobenzyl alcohol (G, 1.8%), and o- toluidine (H, 1.3%). Female rats produced a similar profile of major metabolites, except the production of 2-aminobenzyl alcohol and S-(2-nitrobenzyl)-N-acetylcysteine was significantly less than that of male rats. Male mice produced only two major urinary metabolites, 2-nitrobenzoic acid (38.2%) and 2-nitrobenzyl glucuronide (23.9%). Thermospray mass spectral analysis of isolated metabolite E shows that while its retention time was the same as that of the synthetic standard S-(2-nitrobenzyl)glutathione, its mass spectrum does not match that of the synthesized compound.

Excretion of radioactivity following a 2 mg/kg gavage dose of o- nitrotoluene was similar to that found after a 200 mg/kg dose, with about 98% and 60% of the dose excreted in the urine of rats and mice, respectively, in the first 24 hours after dosing (Table L4). The metabolite profiles (Table L5) are similar to those seen after the 200 mg/kg dose, except that rats excreted a greater proportion of the dose as 2-nitrobenzoic acid and 2-nitrobenzyl glucuronide after the lower dose. The sex-dependent variance in metabolite profile remains at the 2 mg/kg dose level, with female rats excreting significantly less 2-aminobenzyl alcohol and o- nitrobenzylmercapturic acid than male rats, but more nitrobenzoic acid. Male mice again excreted primarily 2-nitrobenzoic acid and 2-nitrobenzyl glucuronide in urine, but a small amount of 2-aminobenzyl alcohol (4%) was also detected.

In rats administered 200 mg/kg o- nitrotoluene for 11 days, followed by a 200 mg/kg radiolabeled dose on day 12 and nonradiolabeled doses on days 13 and 14, excretion of radioactivity during the 72 hours following the radiolabeled dose was similar to that found after a single dose of o- nitrotoluene (Table L6). There was a 50% decrease in the amount of o- nitrobenzylmercapturic acid in urine (Table L7).

Rats treated with buthionine sulfoximine, an inhibitor of glutathione synthesis, before administration of 200 mg/kg

o- nitrotoluene excreted significantly less of the dose in urine (57% in 24 hours; Table L8) than nonpretreated rats (83% in 24 hours; Table L2). The amount of o- nitrobenzylmercapturic excreted was about halved, and the amount of 2-nitrobenzyl alcohol excreted tripled relative to rats that were not pretreated. Rats pretreated with pentachlorophenol, an inhibitor of O-sulfation, also excreted significantly less of the dose (52%) in the urine than

### 332



### nonpretreated rats (Table L9). The amount of o- nitrobenzylmercapturic acid excreted decreased from 9.9% of the dose to 1.5%. These data suggest nitrobenzyl alcohol is converted to an alkylating species by O-sulfation, and that glutathione may serve in a protective role by conjugation with that reactive intermediate. The urinary excretion of 2-nitrobenzyl glucuronide was also decreased by pentachlorophenol pretreatment (8% of the dose compared to 15% for nonpretreated animals); 2-aminobenzyl alcohol decreased from 17% of the dose in nonpretreated rats to 4% in the rats receiving pentachlorophenol. The decrease in the glucuronide may have resulted from competition of pentachlorophenol for glucuronidation. The resulting decreases in its biliary excretion, nitro group reduction, and deconjugation by gut microflora may have led to the observed decrease in the urinary excretion of aminobenzyl alcohol.

Roughly twice as much S-(2-nitrobenzyl)-N-acetylcysteine was detected in the urine of male rats than in the urine of female rats. None of this mercapturate was detected in the urine of male mice. In previous studies conducted by the National Institute of Environmental Health Sciences, only male rats showed hepatotoxicity when exposed to

o- nitrotoluene in feed (NTP, 1992). Rickert et al. (1984) found o- nitrotoluene binds covalently to hepatic macromolecules to a greater extent than the other nitrotoluene isomers. In addition, o- nitrotoluene binds only when its concentration is greater than 40 pmol o- nitrotoluene equivalents per mg macromolecule (type and molecular weight of macromolecule not specified). This binding was inhibited by sulfotransferase inhibitors. These findings, along with the results of the present studies, suggest that there may be a threshold production of reactive intermediates, above which the protective nucleophile glutathione may be depleted, and alkylation of DNA and other crucial macromolecules occurs. Rickert et al. (1984) suggested 2-aminobenzyl sulfate was the

o- nitrotoluene metabolite responsible for binding covalently to hepatic macromolecules. The electrophilic 2-nitrobenzyl sulfate, if formed, could react with glutathione to produce S-(2-nitrobenzyl) glutathione and the corresponding mercapturate. The mercapturate present in the urine of male rats is consistent with the production of a reactive intermediate and may rationalize the toxicities peculiar to that species and gender.



## Blood Plasma Concentration of o-Nitrotoluene

o-Nitrotoluene concentrations in plasma from male rats peaked at nearly 10,000 ng/g plasma 15 to 60 minutes after a single gavage dose of 200 mg/kg (Table L10). The concentration in plasma rapidly decreased through 24 hours after dosing, and concentrations at 24 hours were below the limit of detection. The half-life of o- nitrotoluene in plasma was about 1.5 hours.



## Role of Sulfation in the Conjugation of 2-Nitrobenzyl Alcohol with Glutathione No evidence for the formation of glutathione conjugates with 2-nitrobenzyl alcohol was found by HPLC analysisof incubation mixtures of 2-nitrobenzyl alcohol with glutathione in rat liver cytosol with or without a PAPSgenerating system (data not presented).



## Binding of o-Nitrotoluene Equivalents to Hemoglobin An abbreviated look at excretion rates and routes in the globin binding study indicated this study was consistent with the earlier 200 mg/kg excretion study (data not presented). Of the total radioactivity in blood 72 hours after dosing, 89% was associated with red blood cells (Figure L4), a much higher value than would be predicted from a simple estimation of hematocrit (approximately 42%). Of the radioactivity in the red blood cells, approximately 40% was associated with the isolated, washed protein pellet. This pellet was subjected to continuous (Soxhlet) extraction to ensure that only covalently bound o- nitrotoluene equivalents remained. There were 26.0 pmolequivalents/mg globin for male rats and 29.9 pmol-equivalents/mg globin for female rats. These data do not indicate a marked sex-related difference in globin binding of o- nitrotoluene equivalents and, contrary to the metabolite profiles, do not suggest a differential production of alkylating species by male and female rats. o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 333



# REFERENCES Harms, P.G., and Ojeda, S.R. (1974). A rapid and simple procedure for chronic cannulation of the rat jugular vein. J. Appl. Physiol. 36, 391-392.

Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

McKenna, M.C., and Bieri, J.G. (1984). Multilayer cannula for long-term infusion of unrestrained rats. Lab. Anim. Sci. 34, 308-310.

National Toxicology Program (NTP) (1992). NTP Technical Report on Toxicity Studies of o-, m- , and
p-Nitrotoluenes (CAS Nos. 88-72-2, 99-08-1, 99-99-0) Administered in Dosed Feed to F344/N Rats and B6C3F 1 

### Mice. Toxicity Report Series No. 23. NIH Publication No. 93-3346. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

Research Triangle Institute (RTI) (1995). Comparative Metabolism and Excretion of [ 14 

### C]ortho-Nitrotoluene (ONT) and [ 14 

### C]ortho-Toluidine (OTL) in Rats and Mice. Project Report No. 06/13, NIEHS Contract No. N01-ES-15329. Research Triangle Park, NC.

Rickert, D.E., Long, R.M., Dyroff, M.C., and Kedderis, G.L. (1984). Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer 344 rats. Chem. Biol. Interact. 52, 131-139.

### 334 o-Nitrotoluene, NTP TR 504

Male 0 to 24 82.5 ± 5.5 24 to 48 89.0 ± 3.5 48 to 72 89.6 ± 3.4



### Female 0 to 24 83.9 ± 6.9 24 to 48 90.8 ± 2.3 48 to 72 91.5 ± 2.0

a n=3 for each collection period 

### T ABLE 

### L2 Cumulative Urinary Excretion of Radioactivity by F344/N Rats after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene a



### Time (hours Percent of Dose after dosing) Recovered Rats

Male 4 7.3 ± 5.2 — b 7.3 ± 5.2 8 34.6 ± 8.9 0.1 ± 0.0 34.7 ± 8.9 24 85.9 ± 7.5 1.9 ± 1.0 87.8 ± 7.9 48 99.4 ± 4.2 3.3 ± 0.6 103 ± 4 72 102 ± 3 3.5 ± 0.6 106 ± 2

Female 4 9.4 ± 7.2 — 9.4 ± 7.2 8 33.4 ± 9.3 0.0 ± 0.1 33.4 ± 9.3 24 91.9 ± 7.4 2.0 ± 0.9 93.9 ± 6.6 48 101 ± 5 3.0 ± 1.3 104 ± 4 72 103 ± 4 3.2 ± 1.4 106 ± 3



### Mice

Male 4 12.1 ± 17.4 — 12.1 ± 17.4 8 38.8 ± 6.1 0.5 ± 0.6 39.3 ± 5.7 24 66.3 ± 12.6 1.1 ± 0.8 67.4 ± 12.2 48 74.1 ± 13.7 6.6 ± 4.6 80.7 ± 12.8 72 78.0 ± 13.4 8.6 ± 5.1 86.6 ± 14.6

a n=4 at each time point; data are presented as cumulative percentage of dose recovered (mean ± standard deviation). b Not detected 

### T ABLE 

### L1 Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F 1 

### Mice after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o -Nitrotoluene a



### Time (hours after dosing) Urine Feces Total

### o-Nitrotoluene, NTP TR 504 335



### F IGURE 

### L1 HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o- Nitrotoluene Metabolites: A=2-nitrobenzoic acid; B=2-nitrobenzyl glucuronide; C=unknown; D=2-aminobenzyl alcohol; E=unknown; F=S-(2-nitrobenzyl)-N-acetylcysteine; G=2-nitrobenzyl alcohol; H= o- toluidine

### 336 o-Nitrotoluene, NTP TR 504



### F IGURE 

### L2 HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene and Incubated with $ -Glucuronidase/Sulfatase Metabolites: A=2-nitrobenzoic acid; B=2-nitrobenzyl glucuronide; C=unknown; D=2-aminobenzyl alcohol; E=unknown; F=S-(2-nitrobenzyl)-N-acetylcysteine; G=2-nitrobenzyl alcohol; H= o- toluidine

### o-Nitrotoluene, NTP TR 504 337



### F IGURE 

### L3 HPLC Radiochromatograms of Urine Collected from B6C3F 1 

### Mice 0 to 24 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene Metabolites: A=2-nitrobenzoic acid; B=2-nitrobenzyl glucuronide; G=2-nitrobenzyl alcohol

### 338 o-Nitrotoluene, NTP TR 504TABLEL3Urinary Metabolite Profile for F344/N Rats and Male B6C3F1Mice after a Single Gavage Dose of 200 mg/kg [14C]-o-NitrotolueneTime (hours 2-Nitrobenzoic 2-Nitrobenzyl 2-Aminobenzyl S-(2-Nitrobenzyl)- 2-Nitrobenzylafter dosing) acid glucuronide Caalcohol EaN-acetylcysteine alcoholo-ToluidineRatsbMale24 19.07 ± 3.20 15.34 ± 1.98 17.01 ± 2.31 3.98 ± 0.36 9.88 ± 1.23 1.77 ± 0.75 1.07 ± 0.7148 1.90 ± 1.20 1.29 ± 0.47 0.19d1.16 ± 0.42 0.30 ± 0.07e0.51 ± 0.12 0.23dTotalc20.97 ± 2.03 16.64 ± 2.16 0.19d18.17 ± 1.89 4.18 ± 0.48 10.38 ± 1.32 1.77 ± 0.75 1.30 ± 0.79Female24 21.84 ± 2.81 22.09 ± 1.66 2.75 ± 0.27 7.89 ± 0.86 2.43 ± 0.42 3.74 ± 0.35 0.77 ± 0.47 1.38 ± 0.6748 2.06 ± 1.63 1.65 ± 0.94 0.39d0.65 ± 0.45 0.22 ± 0.12 0.34 ± 0.21 0.14 ± 0.08 0.06 ± 0.05Total 23.90 ± 3.43 23.75 ± 1.21 3.14 ± 0.17 8.54 ± 0.61 2.65 ± 0.40 4.08 ± 0.22 0.91 ± 0.41 1.44 ± 0.62MicefMale24 38.2 23.9 —g—— —— —aMetabolite not identifiedbn=3 at each time point; data are presented as percentage of dose recovered (mean ± standard deviation).cMean of individual animal cumulative urinary excretion from 0 to 48 hours.dn=1; standard deviation not calculated because of an insufficient number of samplesen=2fUrine was pooled from four animals; data are presented as percentage of dose recovered.gMetabolite not detected

### o-Nitrotoluene, NTP TR 504 339



### T ABLE 

### L4 Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F 1 

### Mice after a Single Gavage Dose of 2 mg/kg [ 14 

### C]- o -Nitrotoluene a



### Time (hours after dosing) Urine Feces Total

Rats

Male 0 to 24 98.2 ± 5.2 4.93 ± 1.37 103 ± 4 24 to 48 104 ± 4 5.31 ± 1.39 110 ± 3 48 to 72 b 106 ± 3 5.54 ± 1.46 112 ± 2

Female 0 to 24 97.1 ± 13.5 2.73 ± 0.92 99.9 ± 13.3 24 to 48 104 ± 18 3.16 ± 0.78 107 ± 17 48 to 72 b 108 ± 20 3.39 ± 0.84 113 ± 20



### Mice

Male 0 to 24 60.4 ± 18.9 15.8 ± 2.4 76.2 ± 19.6 24 to 48 69.0 ± 17.6 22.5 ± 2.0 91.5 ± 15.8 48 to 72 b 84.9 ± 8.2 23.2 ± 2.2 108 ± 7

a n=3 for each collection period; data are presented as cumulative percentage of dose recovered (mean ± standard deviation). b Cage rinse included for this collection period

### 340 o-Nitrotoluene, NTP TR 504TABLEL5Urinary Metabolite Profile for F344/N Rats and Male B6C3F1Mice after a Single Gavage Dose of 2 mg/kg [14C]-o-NitrotolueneTime (hours 2-Nitrobenzoic 2-Nitrobenzyl 2-Aminobenzyl S-(2-Nitrobenzyl)- 2-Nitrobenzylafter dosing) acid glucuronide Caalcohol EaN-acetylcysteine alcoholo-ToluidineRatsbMale24 30.6 ± 4.3 28.0 ± 5.9 2.6 ± 0.4 11.0 ± 4.7 2.4 ± 0.1 12.4 ± 1.2 1.9 ± 1.2 —cFemale24 43.9 ± 2.6 26.5 ± 6.7 2.5 ± 0.6 4.4 ± 0.7 1.2 ± 0.4 4.9 ± 0.7 1.0 ± 0.4 —MicedMale24 20.1 27.9 0.0 4.0 0.0 0.0 0.0 —48 3.5 3.5 0.0 0.0 0.0 0.0 0.0 —aMetabolite not identifiedbn=3 at each time point; data are presented as percentage of dose recovered (mean ± standard deviation).cNot detecteddUrine was pooled from three mice at each time point; data are presented as percentage of dose recovered.

### o-Nitrotoluene, NTP TR 504 341



### T ABLE 

### L6 Cumulative Excretion of Radioactivity by Male F344/N Rats Administered 200 mg/kg [ 14 

### C]- o -Nitrotoluene in the 14-Day Gavage Study a



### Time (hours after dosing Urine Feces Total with radiolabel)

0 to 24 77.7 ± 12.5 10.2 ± 13.7 87.9 ± 1.3 24 to 48 84.7 ± 16.0 10.7 ± 13.6 95.5 ± 2.2 48 to 72 b 87.4 ± 15.9 10.9 ± 13.6 98.4 ± 2.8

a n=3 for each collection period. Radiolabeled dose was administered on day 12; data are presented as cumulative percentage of d ose recovered (mean ± standard deviation). b Cage rinse included for this collection period

### 342 o-Nitrotoluene, NTP TR 504TABLEL7Urinary Metabolite Profile for Male F344/N Rats Administered 200 mg/kg [14C]-o-Nitrotoluene in the 14-Day Gavage StudyaTime (hoursafter dosing 2-Nitrobenzoic 2-Nitrobenzyl 2-Aminobenzyl S-(2-Nitrobenzyl)- 2-Nitrobenzylwith radiolabel) acid glucuronide Cbalcohol EbN-acetylcysteine alcoholo-Toluidine24 19.7 23.1 0.0 12.0 3.1 4.8 1.3 0.848 2.3 1.9 0.0 1.0 0.4 0.5 0.1 0.0Total 22.1 25.0 0.0 12.9 3.6 5.3 1.4 0.8aRadiolabeled dose was administered on day 12. Urine was pooled from three rats at each time point; data are presented as percentage of dose recovered.bMetabolite not identifiedTABLEL8Urinary Metabolite Profile for Male F344/N Rats Pretreated with Buthionine Sulfoxamineafter a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluenea2-Nitro- 2-Nitro- 2-Amino- S-(2-Nitro- 2-Nitro-Time (hours benzoic benzyl Cbbenzyl Ebbenzyl)-N- benzylo-Toluidine % Excretedcafter dosing) acid glucuronide alcohol acetylcysteine alcohol0 to 24 13.0 12.9 0.0 10.0 3.8 4.7 5.8 0.6 57.2 ± 6.324 to 48 2.6 2.5 0.0 1.4 0.5 0.7 0.3 0.0 65.7 ± 4.448 to 72 0.4 0.4 0.0 0.2 0.1 0.1 0.0 0.0 67.2 ± 5.0Total 16.0 15.8 0.0 11.5 4.4 5.5 6.2 0.6aUrine was pooled from three rats for each collection period; data are presented as percentage of dose recovered.bMetabolite not identifiedcCumulative percentage of the dose recovered as all metabolites excreted in the indicated time period (mean ± standard deviation)

### o-Nitrotoluene, NTP TR 504 343TABLEL9Urinary Metabolite Profile for Male F344/N Rats Pretreated with Pentachlorophenolafter a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluenea2-Nitro- 2-Nitro- 2-Amino- S-(2-Nitro- 2-Nitro-Time (hours benzoic benzyl Cbbenzyl Ebbenzyl)-N- benzylo-Toluidine % Excretedcafter dosing) acid glucuronide alcohol acetylcysteine alcohol0 to 4 1.25 1.34 0.00 0.18 0.11 0.19 0.91 0.00 4.2 ± 2.04 to 8 4.45 2.73 0.00 1.07 0.60 0.40 0.51 0.21 13.0 ± 1.70 to 24 21.52 7.79 2.06 4.08 1.96 1.50 2.43 0.83 51.6 ± 8.5aUrine was pooled from three rats for each collection period; data are presented as percentage of dose recovered.bMetabolite not identifiedcCumulative percentage of the dose recovered as all metabolites excreted in the indicated time period (mean ± standard deviation)

### 344 o-Nitrotoluene, NTP TR 504



### T ABLE 

### L10 Plasma Concentration of o-Nitrotoluene in Male F344/N Rats after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene a



### Time (hours after dosing) Rat 1 Rat 2 Rat 3 Rat 4 Mean ± Standard Deviation

0.25 8,239 10,928 8,738 7,811 8,929 ± 1,385 0.5 9,647 9,600 8,931 8,943 9,280 ± 397 1 9,091 6,942 9,183 10,249 8,866 ± 1,386 2 8,126 5,227 4,179 7,970 5,200 ± 3,610 4 2,807 2,431 1,257 2,765 2,315 ± 725 8 429 365 347 267 352 ± 67 24 <LD b <LD <LD <LD <LD

a Data are presented as ng o- nitrotoluene/g plasma; values are the average of two determinations. b LD (limit of detection) was approximately 50 ng/g plasma.

### o-Nitrotoluene, NTP TR 504 345



### F IGURE 

### L4 Isolation of Globin and Binding of o-Nitrotoluene to Globin in F344/N Rats 72 Hours after a Single Gavage Dose of 200 mg/kg [ 14 

### C]- o-Nitrotoluene Values are percent of initial blood radioactivity

### 346 o-Nitrotoluene, NTP TR 504

### 347



# APPENDIX M GENETIC ALTERATIONS IN ras , p53 , AND $ -CATENIN GENES IN HEMANGIOSARCOMAS OF B6C3F 1 

# MICE FOLLOWING EXPOSURE TO o-NITROTOLUENE FOR 2 YEARS

Robert C. Sills, Hue-Hua L. Hong, Thai Vu Ton,

June K. Dunnick, and Theodora R. Devereux

National Institute of Environmental Health Sciences Research Triangle Park, North Carolina

I NTRODUCTION


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 M ATERIALS AND 

### M ETHODS


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 RESULTS


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 D ISCUSSION


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 REFERENCES


### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 T ABLE 

### M1 Summary of Immunohistochemistry for p53 and $ -Catenin Protein Accumulation in Spontaneous Hemangiosarcomas in Control B6C3F 1 

### Mice in Selected NTP Studies . . . 355 T ABLE 

### M2 Summary of Immunohistochemistry for p53 and $ -Catenin Protein Accumulation in Hemangiosarcomas in B6C3F 1 

### Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . 355 T ABLE 

### M3 Summary of ras , p53, and $ -Catenin Mutations in Hemangiosarcomas from B6C3F 1 

### Mice in the 2-Year Feed Study of o- Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . 356

### 348



# GENETIC ALTERATIONS IN ras , p53 , AND $ -CATENIN GENES IN HEMANGIOSARCOMAS OF B6C3F 1 

# MICE FOLLOWING EXPOSURE TO o-NITROTOLUENE FOR 2 YEARS

I NTRODUCTION In the 2-year o- nitrotoluene mouse study, there were significant increases in the incidences of hemangiosarcomas, cecal carcinomas, and hepatocellular neoplasms. The focus of this study was to evaluate spontaneous and

o- nitrotoluene-induced hemangiosarcomas for mutations in cancer genes important in the pathogenesis of human cancer. A representative number of hemangiosarcomas from the 2-year o- nitrotoluene mouse study and spontaneous hemangiosarcomas from the NTP Archives were available for analysis.

In evaluating potential hazards of chemical exposure to humans, it is important to assess how the chemical acts at the molecular level, i.e., through a direct genotoxic mechanism or via indirect mechanisms such as promotion of spontaneous DNA damage. In the past, the patterns of mutations in proto-oncogenes such as ras and in tumor suppressor genes such as p53 have helped in the understanding of tumorigenesis (Harris, 1993; Maronpot et al.
, 1995). For example, in some neoplasms, the profiles of activating mutations in ras genes or inactivating mutations in the p53 gene are specific for particular chemicals and differ from those detected in spontaneous neoplasms (Trukhanova et al. , 1998; Sills et al. , 1999). Recently, genetic alterations in the $ -catenin gene were identified in human and rodent neoplasms (De La Coste et al. , 1998; Mirabelli-Primdahl et al. , 1999; Anna et al. , 2000). $ -catenin is a multifunctional protein involved in two important biological processes: cell-cell adhesion and signal transduction (Behrens, 1999). In this study, hemangiosarcomas from B6C3F 1 

### mice exposed to o- nitrotoluene in feed were examined for genetic alterations in ras , p53 and $ -catenin genes, genes shown to be altered in human neoplasms.



# M ATERIALS AND 

# M ETHODS Hemangiosarcomas: Fifteen subcutaneous, mesentery, and skeletal muscle hemangiosarcomas from mice exposed to 1,250, 2,500, or 5,000 ppm o- nitrotoluene and 13 subcutaneous hemangiosarcomas from control male and female B6C3F 1 

### mice from previous NTP studies were used. Because this neoplasm is rarely found in control mice, spontaneous hemangiosarcomas were obtained from several studies (Table M1). Hemangiosarcomas from the

o- nitrotoluene study were selected based on the samples available in the frozen tissue archives. The frozen samples were not used in this study; however, the paraffin-embedded samples that matched the frozen samples were used for immunohistochemistry and mutation analysis.

Immunohistochemistry, p53 and $ -Catenin: o-Nitrotoluene-induced and spontaneous hemangiosarcomas were first screened for p53 and $

- catenin protein expression. Wild-type p53 protein has a short half-life but, when mutated, can be detected in the nucleus of neoplastic cells. Wild-type $

- catenin protein expression is quickly degraded in normal cells, but when a genetic alteration (point mutation, deletion, or splicing intron) occurs in the $

- catenin gene, increased expression can be detected in the cell membrane, cytoplasm, or nucleus of neoplastic cells. Hemangiosarcomas were screened for p53 and $

- catenin protein by immunohistochemical analysis using the avidin-biotin-peroxidase detection system (Hsu et al. , 1981; Hegi et al. , 1993; Rao et al. , 1996; Devereux et al.
, 1999; Vectastain Rabbit Elite Kit, Vector Laboratories, Burlingame, CA). Because mutant p53 has a much longer half-life than wild-type p53 , immunohistochemistry primarily detects the mutated protein. Immunohistochemical staining for expression of mutant p53 protein was performed as previously described (Sills et al. , 1995; Trukhanova et al. , 1998). A 1:300 dilution of the primary polyclonal rabbit antibody CM-5 (Vector Laboratories), which detects accumulation of the mutant p53 protein in rodents, was used. Normal rabbit serum (Vector Laboratories) was used as the negative control in place of the primary antibody. Tissue specimens from a o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 349



### p53 transgenic mouse (mutation in codon 135 of p53 ) served as the positive control. Localization of $ -catenin expression was investigated using a polyclonal goat anti- $ -catenin antibody at a dilution of 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA). Nonimmune goat IgG (Jackson Immunoresearch Labs, West Grove, PA) at equivalent conditions in place of the primary antibody was used as the negative control. The positive control for $

- catenin protein was a mouse hepatoblastoma where a $

- catenin point mutation was confirmed by direct sequencing.

DNA Isolation: The DNA isolation procedure has been described previously (Marmur, 1961; Devereux et al.
, 1993). DNA was isolated from paraffin-embedded sections containing hemangiosarcomas.

DNA Amplification: DNA was amplified by the polymerase chain reaction (PCR) (Saiki et al. , 1988; Sills et al.
, 1995); details of the use of nested primers for K- and H- ras have been described previously (Devereux et al. , 1991, 1993). Touch-down PCR was performed for exons 5 through 8 of the p53 gene; PCR/sequencing primers and annealing temperature profile of the cycles have been described previously (Trukhanova et al. , 1998). Positive controls for K- ras and H- ras or p53 mutations and no-DNA controls were run with all sets of reactions. DNA amplification by PCR and details of the use of nested primers for $

- catenin have been described previously (Devereux et al. , 1999; Anna et al. , 2000).

Single-Strand Conformational Analysis: For analysis of K- ras and p53 mutations, non-radioactive single-strand conformational polymorphism (SSCP) analysis was performed. A mixture of 5 μL of PCR product, 0.6 μL of 1 M methylmercury hydroxide, 1 μL of 15% (w/v) Ficoll (molecular weight 400,000) loading buffer containing 0.25% bromophenol blue and 0.25% xylene cyanol, and 13.4 μL of 1X tris-borate EDTA (TBE) buffer (Novex, San Diego, CA) was prepared to yield a total volume of 20 μL (Hongyo et al. , 1993). This mixture was denatured at 85° C for 5 minutes prior to loading on a 20% polyacrylamide TBE gel (Novex). The buffer chamber was filled with 1.5X TBE buffer. The gel was run at 300 volts at 10° and 13° C for K- ras exon 1, at 8° C for exon 2, and at 9° C for p53 until the xylene cyanol light blue dye reached the bottom of the gel. Positive controls for K- ras mutations and one undenatured DNA control (without methylmercury hydroxide and no heat) were run with unknown samples. Gels were stained with ethidium bromide. For analysis of $ -catenin, SSCP analysis was carried out on PCR products of exon 2 (corresponds to exon 3 in humans) of the mouse $ -catenin gene (De La Coste et al. , 1998), which contains the glycogen serine-threonine kinase-3 $ -targeted phosphorylation sites within residues 33 through 45. The sequences of the intronic PCR primers flanking the borders of exon 2 were: BCAT-IF, 5 N -TACAGGTAGCATTTTCAGTTCAC-3 N and BCAT-2R, 5 N -TAGCTTCCAAACACAAATGC-3 N (De La Coste et al. , 1998). Inner primers were BCAT-7F, 5 N -TAACATACTCTGTTTTTACAGCTG-3 N , and BCAT-8R, 5 N -ACATCTTCTTCCTCAGGGTTG-3 N . For DNA from fixed sections, nested PCR was used with primers 1F X 2R for the outer reactions and both 1F X 8R and 7F X 8R for the inner reactions. [ 33 

### P]-dATP was incorporated into the inner PCR reactions for SSCP analysis, and two gel conditions were used to detect mutations: 6% acrylamide gels with 10% glycerol were electrophoresed at 40 watts for 6 hours at 5° C, and 0.5X Mutations Detection Enhancement gels (AT Biochem, Malvern, PA) were electrophoresed at 3 watts for 17 hours at room temperature. DNA from normal tissue and no-DNA controls were included with all amplification experiments to confirm no cross-contamination of PCR products.

Cycle Sequencing: To identify mutations, samples were sequenced with a cycle sequencing kit (US Biochemical, Cleveland, OH) that incorporates [ "
- 33 

### P]-dideoxynucleotide (ddNTP) terminators (A,C,G,T) into the sequencing products. Prior to sequencing, PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The sequencing primers for K- ras were 5 N -TCGTACTCATCCACAAAGTG-3 N and 5 N -ACCTGTCTCTTGGAT-3 N for exons 1 and 2 respectively; for H- ras exon 2, the sequencing primer was 5 N -TGTGCGCATGTACTG-3 N . The sequencing primers for p53 have been described previously (Trukhanova et al. , 1998). The sequencing reactions were stopped by adding formamide-dye stop solution (Amersham, Cleveland, OH) and were heat denatured at 70° C for 10 minutes prior to electrophoresis on an 8% acrylamide gel containing 8M urea. To identify $ -catenin point mutations, small deletions, or splicing introns, samples with altered bands on SSCP gels were reamplified. Other deletions were identified by excision and boiling of the altered band, followed by a fresh amplification. The amplified bands were gel-purified on Qiagen columns

### 350



### (QIAGEN) before sequencing with a [ 33 

### P]-Thermo-Sequenase kit (Amersham). The amplification primers also served as sequencing primers. Mutation identification was confirmed with at least two amplification reactions from original DNA.



# RESULTS Spontaneous hemangiosarcomas from control mice lacked p53 protein expression (Table M1), and these neoplasms were not screened for p53 mutations. All of the hemangiosarcomas from B6C3F 1 

### mice administered o- nitrotoluene in feed exhibited positive immunohistochemical staining of p53 (Table M2). Hemangiosarcomas originating from skeletal muscle, mesentery, and subcutaneous tissues from exposed mice were examined for genetic alterations in the K- ras , H- ras , p53 and $

- catenin genes. Mutations in at least one of these genes were identified in 13 of 15 (87%) of the o- nitrotoluene-induced hemangiosarcomas, and missense mutations in p53 exons 5 through 8 were detected in 11 of 15 (73%) of these lesions (Table M3). Mutations in p53 were identified at codons 190, 195, 200, 205, 210, 220, 235, 241, 243, 250, 263 and 264. Of the 15 p53 mutations identified, six were G-to-A transitions, three were A-to-G transitions, three were C-to-T transitions, and the other three were various base pair alterations. Four hemangiosarcomas from the 5,000 ppm group exhibited double mutations in p53 ; one of these hemangiosarcomas also had a $ -catenin mutation, while another had a CTA mutation at codon 61 of K- ras

.

The lack of p53 mutations in some of the hemangiosarcomas that were positive by immunohistochemical methods for the p53 protein may be due to mutations outside the exons 5 to 8 evaluated, or to changes in the expression of other genes that influence the expression of p53 . Mutations in ras genes or the $ -catenin gene were not detected in five of the o- nitrotoluene-induced hemangiosarcomas that had p53 mutations.

Seven of 15 (47%) hemangiosarcomas from mice exposed to o- nitrotoluene had splice site mutations at the beginning of exon 2 or deletions in the $

- catenin gene (Table M3). Two o- nitrotoluene-induced hemangiosarcomas had $

- catenin genetic alterations but not p53 or ras mutations. There was a generally good correlation (six of seven; 86%) between hemangiosarcomas that had membrane staining for the $

- catenin protein and genetic alterations in the $

- catenin gene (Tables M2 and M3).



# D ISCUSSION This is the first report in which spontaneous subcutaneous hemangiosarcomas have been screened for both p53 and $ -catenin protein expression. The lack of p53 and $ -catenin protein expression in spontaneous hemangiosarcomas provides a basis for comparison with carcinogen-induced hemangiosarcomas. The current findings are consistent with those of Duddy et al. (1999a) where p53 protein expression was not detected in spontaneous hemangiosarcomas in B6C3F 1 

### mice. Similar to the current study, low frequencies of ras mutations were detected in spontaneous or thiazolidinedione-induced hemangiosarcomas in the B6C3F 1 

### mouse (Duddy et al. , 1999b). In contrast, a high frequency of the signature K- ras codon 13 G-to-C transversion was detected in 1,3-butadieneinduced hemangiosarcomas (Hong et al. , 2000). These studies suggest there may be chemical-specific mutation profiles or pathways in hemangiosarcoma formation.

Similar spectra of p53 mutations in mice have been identified in hemangiosarcomas following 1,3-butadiene exposure (Hong et al. , 2000) and o- nitrotoluene exposure (this study). Although most of the in vitro genotoxicity tests were negative, the current in vivo mutation data suggest o- nitrotoluene is metabolized to genotoxic intermediates. o-Nitrotoluene has a number of potentially active metabolites that could account for the mutation profile observed in these tumors.

Further evidence that mutation of p53 plays an important role in o- nitrotoluene-induced hemangiosarcoma formation is provided by tumorigenesis findings in p53 knockout mice. Hemangiosarcomas have been found in six of 56 (11%) untreated p53 heterozygous (+/–) mice and in 10 of 60 (17%) untreated p53 homozygous (–/–) knockout mice. Multiple hemangiosarcomas of the liver were found in 14 of 17 (82%) p53 heterozygous mice o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 351



### exposed to low concentrations of dimethylnitrosamine (0.0005%) in drinking water (Harvey et al. , 1993). The p53 mutation data from the current study, 1,3-butadiene studies, and studies with p53 knockout mice collectively provide growing evidence that genetic alterations in the p53 gene contribute to induction of hemangiosarcomas and that these chemicals target p53 in tissues in which hemangiosarcomas arise.

$ -Catenin protein was detected in 47% of the o- nitrotoluene-induced hemangiosarcomas but not in the spontaneous neoplasms, suggesting the Wnt-signaling pathway may be involved in the carcinogenesis process of a subset of hemangiosarcomas. $ -Catenin protein accumulation and upregulation of the Wnt-signaling pathway have been shown following mutations in either the adenomatous polyposis coli (APC) or $ -catenin gene (Behrens, 1999). The immunohistochemical localization of $ -catenin protein at the cell membrane but not in the nucleus of hemangiosarcoma cells was similar to previous findings for chemical-induced mouse hepatocellular neoplasms (Devereux et al. , 1999). Because $ -catenin plays a pivotal role in cell adhesion through participation in the cadherin/catenin complex of adherence junctions, changes in the expression and structure of $ -catenin may lead to loss of adhesiveness and may promote invasiveness and/or metastasis. Consistent with this hypothesis, Gamallo et al. (1999) demonstrated that endometrial carcinomas with membrane expression of $ -catenin had a poor prognosis. Alternatively, immunohistochemistry may not be the most sensitive measure of signal transduction, and perhaps only a small increase in $ -catenin accumulation is necessary for increased nuclear translocation and transcriptional activation. These hypotheses are now being tested.

Activating mutations in the ras gene and inactivating mutations in the p53 gene have been detected in human hemangiosarcomas. For example, in liver hemangiosarcomas from factory workers exposed to vinyl chloride, the N

6

### -ethenoadduct has been postulated to lead to specific A-to-T transversions detected in the p53 tumor suppressor gene (Marion et al. , 1991; Hollstein et al. , 1994). On the other hand, G-to-A transversions at the second base of codon 13 of the c-ki- ras -2 gene in human hemangiosarcomas associated with vinyl chloride exposure have been shown to be consistent with the known miscoding properties of N 2 

### , 3-ethenoguanine and 3, N 4 

### -ethenocytosine (La and Swenberg, 1996). The finding of chemical-associated mouse hemangiosarcomas with p53 and $ -catenin mutations suggests these neoplasms are good models for the human equivalent. Moreover, the human and mouse neoplasms may have similar carcinogenic pathways with implications for human risk from exposure to this chemical.



# REFERENCES Anna, C.H., Sills, R.C., Foley, J.F., Stockton, P.S., Ton, T., and Devereux, T.R. (2000). $ -catenin mutations and protein accumulation in all hepatoblastomas examined from B6C3F 1 

### mice treated with anthraquinone or oxazepam. Cancer Res. 60 , 2864-2868.

Behrens, J. (1999). Cadherins and catenins: Role in signal transduction and tumor progression. Cancer Metastasis Rev. 18 , 15-30.

De La Coste, A., Romagnolo, B., Billuart, P., Renard, C., Buendia, M., Soubrane, O., Fabre, M., Chelly, J., Beldjord, C., Kahn, A., and Perret, C. (1998). Somatic mutations of $ -catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc. Natl. Acad. Sci. U.S.A. 95 , 8847-8851.

Devereux, T.R., Anderson, M.W., and Belinsky, S.A. (1991). Role of ras protooncogene activation in the formation of spontaneous and nitrosamine-induced lung tumors in the resistant C3H mouse. Carcinogenesis 12
, 299-303.

Devereux, T.R., Foley, J.F., Maronpot, R.R., Kari, F., and Anderson, M.W. (1993). Ras proto-oncogene activation in liver and lung tumors from B6C3F 1 

### mice exposed chronically to methylene chloride. Carcinogenesis 14
, 795-801.

### 352



### Devereux, T.R., Anna, C.H., Foley, J.F., White, C.M., Sills, R.C., and Barrrett, J.C. (1999). Mutation of $ -catenin is an early event in chemically induced mouse hepatocellular carcinogenesis. Oncogene 18 , 4726-4733.

Duddy, S.K., Parker, R.F., Bleavins, M.R., Gough, A.W., Rowse, P.E., Gorospe, S., Dethloff, L.A., and de la Iglesia, F.A. (1999a). p53 is not inactivated in B6C3F 1 

### mouse vascular tumors arising spontaneously or associated with long-term administration of the thiazolidinedione troglitazone. Toxicol. Appl. Pharmacol. 156
, 106-112.

Duddy, S.K., Gorospe, S.M., Bleavins, M.R., and de la Iglesia, F.A. (1999b). Spontaneous and thiazolidinedioneinduced B6C3F 1 

### mouse hemangiosarcomas exhibit low ras oncogene mutation frequencies. Toxicol. Appl. Pharmacol. 160 , 133-140.

Gamallo, C., Palacios, J., Moreno, G., de mora, J., Suarez, A., and Armas, A. (1999). $ -Catenin expression pattern in stage I and II ovarian carcinomas. Am. J. Pathol. 155 , 527-536.

Harris, C.C. (1993). p53 : at the crossroads of molecular carcinogenesis and risk assessment. Science 262
, 1980-1981.

Harvey, M., McArthur, M.J., Montgomery, C.A., Jr., Butel, J S., Bradley, A., and Donehower, L.A. (1993). Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice. Nature Genetics 5 , 225-229.

Hegi, M.E., Soderkvist, P., Foley, J.F., Schoonhoven, R., Swenberg, J.A., Kari, F., Maronpot, R., Anderson, M.W., and Wiseman, R.W. (1993). Characterization of p53 mutations in methylene chloride-induced lung tumors from B6C3F 1 

### mice. Carcinogenesis 14 , 803-810.

Hollstein, M., Marion, M.J., Lehman, T., Welsh, J., Harris, C.C., Martel-Planche, G., Kusters, I., and Montesano, R. (1994). p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride-exposed factory workers. Carcinogenesis 15 , 1-3.

Hong, H.L., Devereux, T.R., Melnick, R.L., Moomaw, C.R., Boorman, G.A., and Sills, R.C. (2000). Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F 1 

### mice exposed to 1,3-butadiene for 2 years. Toxicol. Pathol. 28 , 529-534.

Hongyo, T., Buzard, G.S., Calvert, R.J., and Weghorst, C.M. (1993). “Cold SSCP”: a simple, rapid and nonradioactive method for optimized single-strand conformation polymorphism analyses. Nucleic Acids Res. 21
, 3637-3642.

Hsu, S.M., Raine, L., and Fanger, H. (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29 , 577-580.

La, D.K., and Swenberg, J.A. (1996). DNA adducts: Biological markers of exposure and potential applications to risk assessment. Mutat. Res. 365 , 129-146.

Marion, M.J., Froment, O., and Trepo, C. (1991). Activation of Ki-ras gene by point mutation in human liver angiosarcoma associated with vinyl chloride exposure. Mol. Carcinog. 4 , 450-454.

Marmur, J. (1961). A procedure for isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3
, 208-218. o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 353



### Maronpot, R.R., Fox, T., Malarkey, D., and Goldsworthy, T. (1995). Mutations in the ras proto-oncogene: Clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology 101 , 125-156.

Mirabelli-Primdahl, L., Gryfe, R., Kim, H., Millar, A., Luceri, C., Dale, D., Holowaty, E., Bapat, B., Gallinger, S., and Redston, M. (1999). $ -Catenin mutations are specific for colorectal carcinomas with microsatellite instability but occur in endometrial carcinomas irrespective of mutator pathway. Cancer Res. 59 , 3346-3351.

National Toxicology Program (NTP) (1990). Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No. 7681-49-4) in F344/N Rats and B6C3F 1 

### Mice (Drinking Water Studies). Technical Report Series No. 393. NIH Publication No. 91-2848. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1994). Toxicology and Carcinogenesis Studies of Ozone (CAS No. 10028-15-6/64091-91-4) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 440. NIH Publication No. 95-3371. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1996). Toxicology and Carcinogenesis Studies of Nickel Oxide (CAS No. 1313-99-1) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 451. NIH Publication No. 96-3367. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1997). Toxicology and Carcinogenesis Studies of t- Butylhydroquinone (CAS No. 1948-33-0) in F344/N Rats and B6C3F 1 

### Mice (Feed Studies). Technical Report Series No. 459. NIH Publication No. 97-3375. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1998a). Toxicology and Carcinogenesis Studies of Chloroprene (CAS No. 126-99-8) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 467. NIH Publication No. 98-3957. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1998b). Toxicology and Carcinogenesis Studies of Cobalt Sulfate Heptahydrate (CAS No. 10026-24-1) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 471. NIH Publication No. 98-3961. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1998c). Toxicology and Carcinogenesis Studies of 1-Chloro-2-propanol (Technical Grade) (CAS No. 127-00-4) in F344/N Rats and B6C3F 1 

### Mice (Drinking Water Studies). Technical Report Series No. 477. NIH Publication No. 98-3967. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1998d). Toxicology and Carcinogenesis Studies of Oxazepam (CAS No. 604-75-1) in F344/N Rats (Feed Studies). Technical Report Series No. 468. NIH Publication No. 99-3958. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (1999). Toxicology and Carcinogenesis Studies of Glutaraldehyde (CAS No. 111-30-8) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 490. NIH Publication No. 490. NIH Publication No. 99-3980. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

### 354



### National Toxicology Program (NTP) (2000a). Toxicology and Carcinogenesis Studies of Pyridine (CAS No. 110-86-1) in F344/N Rats, Wistar Rats, and B6C3F 1 

### Mice (Drinking Water Studies). Technical Report Series No. 470. NIH Publication No. 00-3960. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (2000b). Toxicology and Carcinogenesis Studies of Primidone (CAS No. 125-33-7) in F344/N Rats and B6C3F 1 

### Mice (Feed Studies). Technical Report Series No. 476. NIH Publication No. 00-3966. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (2001a). Toxicology and Carcinogenesis Studies of Sodium Nitrite (CAS No. 7632-00-0) in F344/N Rats and B6C3F 1 

### Mice (Drinking Water Studies). Technical Report Series No. 495. NIH Publication No. 01-3954. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

National Toxicology Program (NTP) (2001b). Toxicology and Carcinogenesis Studies of Indium Phosphide (CAS No. 22398-80-7) in F344/N Rats and B6C3F 1 

### Mice (Inhalation Studies). Technical Report Series No. 499. NIH Publication No. 01-4433. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

Rao, G.N., Collins, B.J., Giles, H.D., Heath, J.E., Foley, J.F., May, R.D., and Buckley, L.A. (1996). Carcinogenicity of 2 N ,3 N -dideoxycytidine in mice. Cancer Res. 56 , 4666-4672.

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 , 487-490.

Sills, R.C., Hong, H.L., Greenwell, A., Herbert, R.A., Boorman, G.A., and Devereux, T.R. (1995). Increased frequency of K-ras mutations in lung neoplasms from female B6C3F 1 

### mice exposed to ozone for 24 or 30 months. Carcinogenesis 16 , 1623-1628.

Sills, R.C., Boorman, G.A., Neal, J.E., Hong, H.L., and Devereux, T.R. (1999). Mutations in ras genes in experimental tumours of rodents. In The Use of Short- and Medium-Term Tests for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation (D.B. McGregor, J.M. Rice, and S. Venitt, Eds.), pp. 55-86. International Agency for Research on Cancer, Lyon, France.

Trukhanova, L.S., Hong, H.L., Sills, R.C., Bowser, A.D., Gaul, B., Boorman, G.A., Turusov, V.S., Devereux, T.R., and Dixon, D. (1998). Predominant p53 G to A transition mutation and enhanced cell proliferation in uterine sarcomas of CBA mice treated with 1,2-dimethylhydrazine. Toxicol. Pathol. 26 , 367-374. o-Nitrotoluene, NTP TR 504

### o-Nitrotoluene, NTP TR 504 355



### T ABLE 

### M1 Summary of Immunohistochemistry for p53 and $ -Catenin Protein Accumulation in Spontaneous Hemangiosarcomas in Control B6C3F 1 

### Mice in Selected NTP Studies

Study a 

### Technical p53 $ -Catenin Report Number

Sodium fluoride 393 — b

— Chloroprene 467 — — Cobalt sulfate 471 — — 1-Chloro-2-propanol 477 — — Oxazepam 468 — —
t-Butylhydroquinone 459 — — Pyridine 470 — — Sodium nitrite 495 — — Ozone 440 — — Primidone 476 — — Indium phosphide 499 — — Nickel oxide 451 — — Glutaraldehyde 490 — —

a NTP study from which subcutaneous hemangiosarcoma was obtained from a control mouse; each row represents a single hemangiosarco ma. b No positive immunohistochemical response was observed



### T ABLE 

### M2 Summary of Immunohistochemistry for p53 and $ -Catenin Protein Accumulation in Hemangiosarcomas in B6C3F 1 

### Mice in the 2-Year Feed Study of o -Nitrotoluene

Exposure Concentration a 

### p53 $ -Catenin (ppm)

1,250 +b

—c 1,250 + + 2,500 + — 2,500 + — 5,000 + — 5,000 + + 5,000 + + 5,000 + + 5,000 + — 5,000 + — 5,000 + — 5,000 + — 5,000 + + 5,000 + + 5,000 + +

a Samples match in sequence with Table M3 samples; each row represents a single hemangiosarcoma. b A positive immunohistochemical response was observed c No positive immunohistochemical response was observed

### 356 o-Nitrotoluene, NTP TR 504TABLEM3Summary ofras,p53, and$-Catenin Mutations in Hemangiosarcomas from B6C3F1Mice in the 2-Year Feed Study ofo-NitrotolueneaExposure Concentration Tissue K-rasH-ras p53$-Catenin(ppm) Codon 12 Codon 13 Codon 61 Codon 61 Exon 5 Exon 6 Exon 7 Exon 81,250 Skeletal Muscle —b— — — — Codon 190 — — —CAT to CGT(His to Arg)1,250 Mesentery — — — — — — — — Splicing Intron2,500 Skeletal Muscle — — — — — Codon 195 — — —GAA to GGA(Glu to Gly)2,500 Skeletal Muscle — — — — — — — — —5,000 Skeletal Muscle — — — — — — Codon 243 — Splicing IntronATG to ATA(Met to Ile)5,000 Skeletal Muscle — — — — — Codon 220 — — Deletions 25-36CCC to TCC(Pro to Ser)5,000 Skeletal Muscle — — — — — Codon 200 Codon 241 — Deletions 5-6CCC to CTC GGG to GAG Codon 28(Pro to Leu) (Gly to Glu) CAG to CGG(Gln to Arg)5,000 Skeletal Muscle — — — — — — Codon 235 — Splicing IntronTGT to TTT(Cys to Phe)5,000 Skeletal Muscle — — — — — Codon 210 — Codon 263 —CGC to TGC GGA to AGA(Arg to Cys) (Gly to Arg)5,000 Subcutaneous Tissues — — — — — Codon 210 — Codon 263 —CGC to AGC GGA to AGA(Arg to Ser) (Gly to Arg)

### o-Nitrotoluene, NTP TR 504 357TABLEM3Summary ofras,p53, and$-Catenin Mutations in Hemangiosarcomas from B6C3F1Mice in the 2-Year Feed Study ofo-NitrotolueneExposure Concentration Tissue K-rasH-ras p53$-Catenin(ppm) Codon 12 Codon 13 Codon 61 Codon 61 Exon 5 Exon 6 Exon 7 Exon 85,000 Subcutaneous Tissues — — — — — — — — —5,000 Subcutaneous Tissues — — CAA to CTA — — Codon 205 Codon 250 — —(Gln to Leu) GAC to GGC ACC to TCC(Asp to Gly) (Thr to Ser)5,000 Subcutaneous Tissues — — — — — — — Codon 263 —GGA to AGA(Gly to Arg)5,000 Subcutaneous Tissues — — — — — — — Codon 264 Splicing IntronCGG to CAG(Arg to Glu)5,000 Subcutaneous Tissues — — — — — — — — Splicing IntronTotal combinedmutations 1/15 (7%) 0/15 (0%) 11/15 (73%) 7/15 (47%)Hemangiomascontainingat least onemutation 13/15 (87%)aHis=histidine; Arg=arginine; Glu=glutamic acid; Gly=glycine; Met=methionine; Ile=isoleucine; Pro=proline; Ser=serine; Leu=leucine; Cys=cysteine; Phe=phenylalanine;Asp=aspartic acid; Thr=threonine; Gln=glutaminebNo mutation identified

### 358 o-Nitrotoluene, NTP TR 504Acetaminophen 394 Acetonitrile 447 Acrylonitrile 506 Agar 230 Allyl Glycidyl Ether 376 Allyl Isothiocyanate 234 Allyl Isovalerate 253 1-Amino-2,4-Dibromoanthraquinone 383 2-Amino-4-Nitrophenol 339 2-Amino-5-Nitrophenol 334 11-Aminoundecanoic Acid 216 dl-Amphetamine Sulfate 387 Ampicillin Trihydrate 318 Asbestos, Amosite (Hamsters) 249 Asbestos, Amosite (Rats) 279 Asbestos, Chrysotile (Hamsters) 246 Asbestos, Chrysotile (Rats) 295 Asbestos, Crocidolite 280 Asbestos, Tremolite 277

L -Ascorbic Acid 247 AZT and AZT/ " -Interferon A/D 469 Barium Chloride Dihydrate 432 Benzaldehyde 378 Benzene 289 Benzethonium Chloride 438 Benzofuran 370 Benzyl Acetate (Gavage) 250 Benzyl Acetate (Feed) 431 Benzyl Alcohol 343

o -Benzyl- p -Chlorophenol (Gavage) 424

o -Benzyl- p -Chlorophenol (Mouse Skin) 444 2-Biphenylamine Hydrochloride 233 2,2-Bis(Bromomethyl)-1,3-Propanediol 452 Bis(2-Chloro-1-Methylethyl) Ether 239 Bisphenol A 215 Boric Acid 324 Bromodichloromethane 321 Bromoethane 363 1,3-Butadiene 288 1,3-Butadiene 434
t-Butyl Alcohol 436 Butyl Benzyl Phthalate 213 Butyl Benzyl Phthalate 458

n -Butyl Chloride 312
t-Butylhydroquinone 459
( -Butyrolactone 406 Caprolactam 214 d

-Carvone 381 Chloral Hydrate 502 Chlorinated and Chloraminated Water 392 Chlorendic Acid 304 Chlorinated Paraffins: C 23 , 43% Chlorine 305 Chlorinated Paraffins: C 12 , 60% Chlorine 308 Chlorinated Trisodium Phosphate 294 2-Chloroacetophenone 379
p -Chloroaniline Hydrochloride 351 CS2 377 Chlorobenzene 261 Chlorodibromomethane 282 Chloroethane 346 2-Chloroethanol 275 3-Chloro-2-Methylpropene 300 Chloroprene 467 1-Chloro-2-Propanol 477 Chlorpheniramine Maleate 317 C.I. Acid Orange 3 335 C.I. Acid Orange 10 211 C.I. Acid Red 14 220 C.I. Acid Red 114 405 C.I. Basic Red 9 Monohydrochloride 285 C.I. Direct Blue 15 397 C.I. Direct Blue 218 430 C.I. Disperse Blue 1 299 C.I. Disperse Yellow 3 222 C.I. Pigment Red 3 407 C.I. Pigment Red 23 411 C.I. Solvent Yellow 14 226 Cobalt Sulfate Heptahydrate 471 Coconut Oil Acid Diethanolamine Condensate 479 Codeine 455 Comparative Initiation/Promotion Studies (Mouse Skin) 441 Corn Oil, Safflower Oil, and Tricaprylin 426 Coumarin 422 Cytembena 207 D&C Red No. 9 225 D&C Yellow No. 11 463 Decabromodiphenyl Oxide 309 Diallyl Phthalate (Mice) 242 Diallyl Phthalate (Rats) 284 4,4 ́-Diamino-2,2 ́-Stilbenedisulfonic Acid, Disodium Salt 412 2,4-Diaminophenol Dihydrochloride 401 1,2-Dibromo-3-Chloropropane 206 1,2-Dibromoethane 210 2,3-Dibromo-1-Propanol 400 1,2-Dichlorobenzene ( o -Dichlorobenzene) 255 1,4-Dichlorobenzene ( p -Dichlorobenzene) 319
p,p  ́-Dichlorodiphenyl sulfone 501 2,4-Dichlorophenol 353 2,6-Dichloro- p -Phenylenediamine 219 1,2-Dichloropropane 263 1,3-Dichloropropene (Telone II) 269 Dichlorvos 342 Dietary Restriction 460 Diethanolamine 478 Di(2-Ethylhexyl) Adipate 212 Di(2-Ethylhexyl) Phthalate 217 Diethyl Phthalate 429 Diglycidyl Resorcinol Ether 257 3,4-Dihydrocoumarin 423 1,2-Dihydro-2,2,4-Trimethylquinoline (Monomer) 456 Dimethoxane 354 3,3 ́-Dimethoxybenzidine Dihydrochloride 372 N,N-Dimethylaniline 360 3,3 ́-Dimethylbenzidine Dihydrochloride 390 Dimethyl Hydrogen Phosphite 287 Dimethyl Methylphosphonate 323 Dimethyl Morpholinophosphoramidate 298 Dimethylvinyl Chloride 316 Diphenhydramine Hydrochloride 355 5,5-Diphenylhydantoin 404 Emodin 493 Ephedrine Sulfate 307 Epinephrine Hydrochloride 380 1,2-Epoxybutane 329 Erythromycin Stearate 338 Ethyl Acrylate 259 Ethylbenzene 466 Ethylene Glycol 413 

## National Toxicology Program Technical Reports Printed as of May 2002 The Environmental Health Information Service (EHIS) maintains the library of NTP Technical Reports in electronic and print form at. To gain access to these reports, contact EHIS online at http://ehis.niehs.nih.gov or call 800-315-3010 or 919-541-3841.



### Chemical TR No. Chemical TR No.Ethylene Glycol Monobutyl Ether 484 Ethylene Oxide 326 Ethylene Thiourea 388 Eugenol 223 FD&C Yellow No. 6 208 Fumonisin B 1 496 Furan 402 Furfural 382 Furfuryl Alcohol 482 Furosemide 356 Gallium Arsenide 492 Geranyl Acetate 252 Glutaraldehyde 490 Glycidol 374 Guar Gum 229 Gum Arabic 227 HC Blue 1 271 HC Blue 2 293 HC Red 3 281 HC Yellow 4 419 Hexachlorocyclopentadiene 437 Hexachloroethane 361 4-Hexylresorcinol 330 Hydrochlorothiazide 357 Hydroquinone 366 8-Hydroxyquinoline 276 Indium Phosphide 499 Iodinated Glycerol 340 Isobutene 487 Isobutyl Nitrite 448 Isobutyraldehyde 472 Isophorone 291 Isoprene 486 Lauric Acid Diethanolamine Condensate 480 d

-Limonene 347 Locust Bean Gum 221 60-Hz Magnetic Fields 488 Magnetic Field Promotion 489 Malonaldehyde, Sodium Salt 331 Manganese Sulfate Monohydrate 428

D -Mannitol 236 Marine Diesel Fuel and JP-5 Navy Fuel 310 Melamine 245 2-Mercaptobenzothiazole 332 Mercuric Chloride 408 Methacrylonitrile 497 8-Methoxypsoralen 359

" -Methylbenzyl Alcohol 369 Methyl Bromide 385 Methyl Carbamate 328 Methyldopa Sesquihydrate 348 Methylene Chloride 306 4,4 ́-Methylenedianiline Dihydrochloride 248 Methyleugenol 491 Methyl Methacrylate 314 N-Methylolacrylamide 352 Methylphenidate Hydrochloride 439 Mirex 313 Molybdenum Trioxide 462 Monochloroacetic Acid 396 Monuron 266 Nalidixic Acid 368 Naphthalene (Mice) 410 Naphthalene (Rats) 500 Nickel (II) Oxide 451 Nickel Sulfate Hexahydrate 454 Nickel Subsulfide 453
p -Nitroaniline 418

o -Nitroanisole 416
p -Nitrobenzoic Acid 442 Nitrofurantoin 341 Nitrofurazone 337 Nitromethane 461
p -Nitrophenol 417

o -Nitrotoluene 504
p -Nitrotoluene 498 Ochratoxin A 358 Oleic Acid Diethanolamine Condensate 481 Oxazepam (Mice) 443 Oxazepam (Rats) 468 Oxymetholone 485 Oxytetracycline Hydrochloride 315 Ozone and Ozone/NNK 440 Penicillin VK 336 Pentachloroanisole 414 Pentachloroethane 232 Pentachloronitrobenzene 325 Pentachlorophenol, Purified 483 Pentachlorophenol, Technical Grade 349 Pentaerythritol Tetranitrate 365 Phenolphthalein 465 Phenylbutazone 367 Phenylephrine Hydrochloride 322 N-Phenyl-2-Naphthylamine 333

o -Phenylphenol 301 Polybrominated Biphenyl Mixture (Firemaster FF-1) (Gavage) 244 Polybrominated Biphenyl Mixture (Firemaster FF-1) (Feed) 398 Polysorbate 80 (Glycol) 415 Polyvinyl Alcohol 474 Primidone 476 Probenecid 395 Promethazine Hydrochloride 425 Propylene 272 1,2-Propylene Oxide 267 Propyl Gallate 240 Pyridine 470 Quercetin 409 Resorcinol 403 Rhodamine 6G 364 Rotenone 320 Roxarsone 345 Salicylazosulfapyridine 457 Scopolamine Hydrobromide Trihydrate 445 Sodium Azide 389 Sodium Fluoride 393 Sodium Nitrite 495 Sodium Xylenesulfonate 464 Stannous Chloride 231 Succinic Anhydride 373 Talc 421 Tara Gum 224 2,3,7,8-Tetrachlorodibenzo- p -Dioxin (Dermal) 201 2,3,7,8-Tetrachlorodibenzo- p -Dioxin (Gavage) 209 1,1,1,2-Tetrachloroethane 237 Tetrachloroethylene 311 Tetracycline Hydrochloride 344 Tetrafluoroethylene 450 1-Trans-Delta 9 -Tetrahydrocannabinol 446 Tetrahydrofuran 475 Tetrakis(Hydroxymethyl)Phosphonium Sulfate 296 Tetrakis(Hydroxymethyl)Phosphonium Chloride 296 Tetranitromethane 386 Theophylline 473 4,4-Thiobis(6- t-Butyl- m -Cresol) 435 Titanocene Dichloride 399 Toluene 371 2,4- & 2,6-Toluene Diisocyanate 251

o -Toluidine Hydrochloride 153 Triamterene 420 Tribromomethane 350 Trichloroethylene 243 Trichloroethylene 273 

### Chemical TR No. Chemical TR No.1,2,3-Trichloropropane 384 Tricresyl Phosphate 433 Triethanolamine 449 Tris(2-Chloroethyl) Phosphate 391 Tris(2-Ethylhexyl) Phosphate 274 Turmeric Oleoresin (Curcumin) 427 4-Vinylcyclohexene 303 4-Vinyl-1-Cyclohexene Diepoxide 362 Vinylidene Chloride 228 Vinyl Toluene 375 Xylenes (Mixed) 327 2,6-Xylidine 278 Zearalenone 235 Ziram 238 

### Chemical TR No. Chemical TR No.