NTP TECHNICAL REPORT

ON THE

TOXICOLOGY AND CARCINOGENESIS

STUDIES OF o-NITROTOLUENE

(CAS NO. 88-72-2)

IN F344/N RATS AND B6C3F1 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 B6C3F1 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 B6C3F1 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 B6C3F1 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-YEAR STUDY IN RATS

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 those

of 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: C7H7NO2 Molecular Weight: 137.14

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

NO2

CH3
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 rats

occurred with positive trends in both the core and stopexposure studies and were significantly greater in

exposed 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 gland

fibroadenoma 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 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-exposure males were significantly increased. Cholangiocarcinoma occurred in

three 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 of

females; the incidence of testicular interstitial cell adenoma was significantly decreased in 5,000 ppm stopexposure males.

2-YEAR STUDY IN MICE

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 females

were 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 to

exposure 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 exposed

groups 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.

GENETIC TOXICOLOGY

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.

CONCLUSIONS

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 increased

incidences 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 rats

based 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 of

the 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 leukemia

occurred 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 B6C3F1 Mice B6C3F1 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 2,000

and 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)

Lymph 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 B6C3F1 Mice B6C3F1 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)

Testis: 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 than control

animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set 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 for Research on

Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies 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 evidence 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 activity. 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 clear 

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 different levels 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 equivocal evidence 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 extend 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 borderline 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 impossible 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 lesion 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 in

male 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 B6C3F1 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 such

mutations, 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 with

feed 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 irradiating

the 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 of

analyzing 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, bulk

measures of urine volume are less precise than metabolite concentrations and thus less useful for mathematical

modeling. 

Dr. Malarkey, the second principal reviewer, agreed with

the 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 in

the 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 agreed

with 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

CHEMICAL AND PHYSICAL PROPERTIES

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 the

chemical formula is the same for all isomers, their chemical and physical properties vary (Table 1).

PRODUCTION, USE, 

AND HUMAN EXPOSURE

o-Nitrotoluene and p-nitrotoluene are important commercial chemicals used to synthesize agricultural and

rubber 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 in

rivers and drinking water (USEPA, 1976); all three isomers of nitrotoluene have been found in wastewater

effluent and atmospheric emissions from industrial

plants (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/m3) for nitrotoluenes (NIOSH, 1997), and the

American Conference of Governmental Industrial

Hygienists (2000) recommended a threshold limit value

of 2 ppm (11 mg/m3) for the 8-hour TWA.

INTRODUCTION

o-NITROTOLUENE

CAS No. 88-72-2

Chemical Formula: C7H7NO2 Molecular Weight: 137.14

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

NO2

CH3
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-spraying

machine operators; machine operators; welders and cutters; and operators of separating, filtering, and clarifying

machines. The latter group accounted for approximately

60% 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. 

ABSORPTION, DISTRIBUTION,

METABOLISM, AND EXCRETION

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 dose

was 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 B6C3F1 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 [14C]-o-nitrotoluenederived radioactivity in feces of mice can be attributed to

contamination 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 first

24 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 metabolites

of 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

TABLE 1

Chemical and Physical Properties of the Nitrotoluenesa

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 (H2O, 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

TABLE 2

Urinary Metabolites of o-, m-, and p-Nitrotoluene in Male Rats and Mice

Administered Gavage Doses of 200 mg/kga

o-Nitrotoluene m-Nitrotoluene p-Nitrotoluene

Ratsb

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%)

Micec

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 into

the 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 is

reabsorbed from the intestine and oxidized to a hydroxylamine by hepatic enzymes. The hydroxylamine is then

conjugated with sulfate by hepatic sulfotransferase. The

unstable 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 are

reabsorbed and further metabolized in the liver to electrophilic compounds which presumably can interact with

DNA. Binding of 2,6-DNT and o-nitrotoluene to rat

hepatic 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. 

Humans

No information on the absorption, distribution, metabolism, or excretion of o-, m-, or p-nitrotoluene in humans

was found in a review of the literature.

TOXICITY

Experimental Animals

Oral LD50 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 

FIGURE 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

FIGURE 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 B6C3F1 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 these

studies, 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

B6C3F1 mice in feed at concentrations from 625 to

10,000 ppm. The estimated daily doses based on measures of feed consumption were similar for each of the

three 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 of

the 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 granular

casts 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 incidences

of 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, as

indicated 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 caused

increases 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.

CARCINOGENICITY

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 of

these 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

TABLE 3

Summary of Selected Treatment-Related Effects in the 13-Week Nitrotoluene Feed Studiesa

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 

TABLE 4

Treatment-Related Lesions in F344/N Rats and B6C3F1 Mice in the 13-Week Feed Studies of o-Nitrotoluenea

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

Male Rats

Pancreatic Isletsb 10 10 10 10 10 10

Cellular Hyperplasiac 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

Hyperplasiae 0 0 0 002

Mesotheliomaf 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

TABLE 4

Treatment-Related Lesions in F344/N Rats and B6C3F1 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.

GENETIC TOXICITY

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. For

example, 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

B6C3F1 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 by

intestinal 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.

STUDY RATIONALE

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

B6C3F1 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 those

studies, 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 hyperplasia

after 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

PROCUREMENT

AND CHARACTERIZATION

OF O-NITROTOLUENE

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 (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, proton

nuclear magnetic resonance, and low- and high-resolution mass spectroscopy. The purity of lot 8056-58-

05RTI was determined by Karl Fischer water analysis

and 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 by

the 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 in

amber glass bottles inside metal cans at room temperature. Stability was monitored during the studies using

gas chromatography. No degradation of the bulk chemical was detected.

PREPARATION AND ANALYSIS

OF DOSE FORMULATIONS

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 dose

formulation 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 when

stored in sealed containers protected from light at temperatures up to 3° C; significant chemical losses due to

volatility were seen in the dose formulations under simulated animal room conditions.

Analyses of the dose formulations of o-nitrotoluene were

conducted 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-YEAR STUDIES

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 B6C3F1 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) July

1996 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 from

each 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/block

match, 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 male

and 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 the

PWG consensus differed from the opinion of the laboratory pathologist, the diagnosis was changed. Final diagnoses for reviewed lesions represent a consensus

between 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 core

study 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 to

the rat urine samples, which were then diluted with trifluoroacetic acid in methanol and water. Triethylamine

was 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-C18 column (Varian, Palo Alto,

CA) using ultraviolet detection at 254 nm (for

o-aminobenzoic acid concentration) or 266 nm. Mouse

urine 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

B6C3F1 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.

TABLE 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

CO2 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 tissues were

examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine

(duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (except male mice), nose, 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.

TABLE 5

Experimental Design and Materials and Methods in the 2-Year Feed Studies of o-Nitrotoluene
32 o-Nitrotoluene, NTP TR 504 

STATISTICAL METHODS

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 natural

causes 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, when

macroscopic 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 of

occurrence (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 otherwise

specified, 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 neoplasm

onset time distributions for a variety of site-specific neoplasms in control F344 rats and B6C3F1 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 of

each 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 of

lower incidences or decreasing trends in lesions are represented as 1-P with the letter N added (e.g., P=0.99 is

presented 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 Variables

Two 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 make

pairwise comparisons, urinary biomarker data were analyzed by Fisher’s least significant difference test (following initial ANOVA) (Miller, 1960). Average severity

values 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 studies

conducted 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 and

decrease 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 animals

on study were fed the NTP-2000 diet. Because the incidence of some neoplastic and nonneoplastic lesions may

be 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 diet

was 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 of

neoplasms 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 concentrations

before and after irradiation. The concentrations of nutrients and their byproducts of irradiated diets were not

substantially different from the same batches before irradiation and nutritionally adequate rodent diets.

QUALITY ASSURANCE METHODS

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 the

NTP Archives, these studies were audited retrospectively by an independent quality assurance contractor.

Separate audits covered completeness and accuracy of

the 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 findings

were reviewed and assessed by NTP staff, and all comments were resolved or otherwise addressed during the

preparation of this Technical Report.

GENETIC TOXICOLOGY

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, and

increases 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 animals

based 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 growing

out 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 tissue

sites (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 vitro

genetic 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 tests

are 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 is

important 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

RATS

Survival

Estimates of 2-year survival probabilities for male and

female 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, the

differences 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 exposed

females 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 

TABLE 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 evaluationa 10 0 0 0 10 10

Accidental deathsa 0 030 00

Moribund 18 35 48 53 39 57

Natural deaths 3 7 6 7 10 3

Animals surviving to study termination 39b 18 3 0 11 0

Percent probability of survival at end of studyc 65 30 5 0 18 0

Mean survival (days)d 698 647 590 508 612 483

Survival analysise P<0.001 P<0.001 P<0.001 P<0.001

Survival analysisf 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 47g 47b

39 33

Percent probability of survival at end of study 78 78 65 55

Mean survival (days) 712 706 696 697

Survival analysise 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 life 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

FIGURE 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 

TABLE 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

16a 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

TABLE 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

16a 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 

FIGURE 4

Growth Curves for Male and Female Rats 

Exposed to o-Nitrotoluene in Feed for 2 Years
o-Nitrotoluene, NTP TR 504 41

TABLE 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 Analyses

This section describes the statistically significant or biologically noteworthy changes in the incidences of

mononuclear 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 and

nonneoplastic lesions, individual animal tumor diagnoses, statistical analyses of primary neoplasms that

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

animal 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 arise

from 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). 

TABLE 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 Mesotheliomaa

Overall rateb 2/60 (3%) 20/60 (33%) 29/60 (48%) 44/60 (73%) 44/60 (73%) 54/60 (90%)

Adjusted ratec 3.7% 40.6% 62.4% 87.1% 80.3% 95.1%

Terminal rated 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 teste P<0.001 P<0.001 P<0.001 P<0.001

Poly-3 testf 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%), range 

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 group incidence 

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for 

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 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 10, A4b, and

B4a). 

Fibromas occurred in the subcutis of the skin of the

torso, 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 

TABLE 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, Multiplea 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 Fibrosarcomae

Overall ratef 5/60 (8%) 47/60 (78%) 55/60 (92%) 59/60 (98%) 47/60 (78%) 53/60 (88%)k

Adjusted rateg 9.3% 86.3% 98.7% 99.8% 89.0% 97.8%

Terminal rateh 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 testi P<0.001 P<0.001 P<0.001 P<0.001

Poly-3 testj 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 testi 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%), range 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%-10% 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 group incidence 

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for

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 and

females 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 occurred

with 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 fibrous

elements varied greatly among the neoplasms. Epithelial cells were usually uniform and arranged in a single layer (Plate 7); a few fibroadenomas had small areas

of 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 with

an increase in the number of alveoli separated by collagenous stroma.
46 o-Nitrotoluene, NTP TR 504 

TABLE 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

Hyperplasiaa 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 rated 0/60 (0%) 7/60 (12%) 10/60 (17%) 2/60 (3%) 13/60 (22%) 20/60 (33%)

Adjusted ratee 0.0% 15.6% 26.2% 9.0% 31.2% 61.1%

Terminal ratef 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 testg 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%) 56/60k (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 testg P<0.001 P<0.001 P<0.001 P<0.001

Carcinomal 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%), range 

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 group incidence 

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for 

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 of

the 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-exposure

males 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-07

diet (Tables 12, A4d, and B4c). Hepatocellular adenomas consisted of nodules of hepatocytes that compressed

adjacent hepatic parenchyma and lacked the normal lobular and sinusoidal pattern (Plate 8). Hepatocellular carcinomas consisted of solid sheets of hepatocytes or

trabeculae 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, and

cholangiocarcinomas 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 biliary

epithelium (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 of

hepatocellular 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 potential

preneoplastic 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 in

a 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 

TABLE 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

Vacuolizationa 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)

Hepatocholangiocarcinomac 0 1 0 10 0

Cholangiocarcinomac 0 0 0 0 0 3*

Hepatocellular Adenoma, Multiple 0 1 0 0 1 1

Hepatocellular Adenoma (includes multiple)d

Overall ratee 2/60 (3%) 3/60 (5%) 3/60 (5%) 7/60 (12%) 3/60 (5%) 4/60 (7%)

Adjusted ratef 3.7% 6.8% 8.4% 27.1% 7.6% 18.4%

Terminal rateg 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 testi 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 Carcinomaj

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 testi 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: 23/1,002 

(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 group incidence

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for 

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

TABLE 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
TABLE 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, Hyperplasiaa 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 rated 1/60 (2%) 5/60 (8%) 1/60 (2%) 2/60 (3%) 3/60 (5%) 8/60 (13%)

Adjusted ratee 1.9% 11.2% 2.9% 8.7% 7.6% 33.0%

Terminal ratef 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 testg 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 Carcinomai

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 testg 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 carcinomas

were solid or papillary, obliterated the normal pulmonary architecture, and sometimes invaded adjacent

structures. 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

TABLE 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 group incidence

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for 

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 

TABLE 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 Organsa

Overall rateb 0/60 (0%) 3/60 (5%) 0/60 (0%) 2/60 (3%) 0/60 (0%) 3/60 (5%)

Adjusted ratec 0.0% 6.7% 0.0% 8.7% 0.0% 14.0%

Terminal rated 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 teste P=0.158 P=0.090 —h P=0.112

Poly-3 testf P=0.025 — P=0.034

Hemangioma or Hemangiosarcoma, All Organsi

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 teste P=0.232 P=0.243 P=0.654 P=0.254

Poly-3 testf 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 study 

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 group incidence 

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for 

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 greater

and 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 in

leukemia 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. 

TABLE 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 Leukemiaa

Overall rateb 30/60 (50%) 21/60 (35%) 3/60 (5%) 3/60 (5%) 13/60 (22%) 1/60 (2%)

Adjusted ratec 52.2% 42.8% 8.5% 12.7% 31.1% 5.0%

Terminal rated 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 teste P<0.001N P=0.214N P<0.001N P=0.003N

Poly-3 testf P<0.001N P=0.027N P<0.001N

Female

Mononuclear Cell Leukemiag

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 teste 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 group incidence

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for 

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 resulted

primarily 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 this

group 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. The

decreased 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, occurring

with 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 characterized

by 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, with

few to no spermatogenic cells remaining. Mineralization was associated with tubular degeneration. In

some tubules, cellular debris and multinucleated giant

cells 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 were

atypical; 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

TABLE 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

Epididymisa 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 ratee 55/60 (92%) 53/60 (88%) 51/60 (85%) 46/60 (77%) 50/60 (83%) 27/60 (45%)

Adjusted ratef 94.3% 93.7% 96.5% 92.2% 92.6% 74.7%

Terminal rateg 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 testi 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 feed 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 group incidence

are the P values corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for 

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 the

spleen 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 droplet

degeneration 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 characterized

by 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 a

reduction 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

TABLE 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

Kidneya 10 10 10

Renal Tubule, Degeneration, Hyalineb 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 

TABLE 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

MICE

Survival

Estimates of 2-year survival probabilities for male and

female 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 in

urine 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. 

TABLE 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 studya 87 57 0 0

Mean survival (days)b 712 674 523 386

Survival analysisc 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 

FIGURE 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

FIGURE 6

Growth Curves for Male and Female Mice 

Exposed to o-Nitrotoluene in Feed for 2 Years
62 o-Nitrotoluene, NTP TR 504 

TABLE 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

TABLE 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 Analyses

This 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 at

least 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 (all

routes) 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 B6C3F1 mouse, hemangiosarcomas can

be multicentric, and often arise primarily in the spleen

and liver (Ward et al., 1999).
o-Nitrotoluene, NTP TR 504 65

TABLE 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, Multiplea 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 Organsb

Overall ratec 4/60 (7%) 17/60 (28%) 55/60 (92%) 60/60 (100%)

Adjusted rated 7.0% 32.7% 97.9% 100.0%

Terminal ratee 3/52 (6%) 9/34 (27%) 0/0 0/0 

First incidence (days) 710 542 365 277

Poly-3 testf 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 Organsg

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 mortality 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 carcinoma

of 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, they

were 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) or

NIH-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) or

invaded 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 not

apparent, 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).

TABLE 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

Carcinomaa

Overall rateb 0/60 (0%) 12/60 (20%) 9/60 (15%) 0/60 (0%)

Adjusted ratec 0.0% 22.7% 31.6% 0.0% 

Terminal rated 0/52 (0%) 4/34 (12%) 0/0 0/0 

First incidence (days) —e 485 270 —

Poly-3 testf 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 mortality 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 less

frequently 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 B6C3F1

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 little

or no compression of the surrounding normal hepatocytes, although some degree of compression was present

in some larger foci. Adenomas were discrete masses with

distinct 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 distinguishing

feature of carcinomas was the presence of abnormal patterns of growth. The most common abnormal growth

pattern 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 change

was 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, and

D5). 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 necrotic

hepatocytes that were associated with mixed inflammatory cells.
68 o-Nitrotoluene, NTP TR 504 

TABLE 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 Focusa 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 Carcinomac 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 Carcinomaf

Overall rateg 9/60 (15%) 9/59 (15%) 24/59 (41%) 39/60 (65%) 

Adjusted rateh 15.7% 16.9% 42.1% 79.1% 

Terminal ratei 8/52 (15%) 9/46 (20%) 20/47 (43%) 5/5 (100%)

First incidence (days) 362 729 (T) 661 567 

Poly-3 testj 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 mortality 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 hyaline

droplet 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. 

TABLE 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 Pigmentationa 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, the

thickened 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 obstruction

caused 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. 

GENETIC TOXICOLOGY

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-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 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. 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, three intraperitoneal injections

also yielded negative results, although a small increase

in the frequency of micronucleated polychromatic erythrocytes was observed at all doses tested (Table E5).

o-Nitrotoluene, administered in feed for 13 weeks, did

not increase the frequency of micronucleated normochromatic (mature) erythrocytes in peripheral blood

of female mice. However, a small increase in the frequency of micronucleated normochromatic erythrocytes

was 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.
PLATE 1

Malignant 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; 16x

PLATE 2

Higher magnification of papillary portion of the mesothelioma in Plate 1. 

Note the malignant mesothelial cells covering the stroma. H&E; 80x

PLATE 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; 80x

PLATE 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; 80x

o-Nitrotoluene, NTP TR 504 
PLATE 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; 5x

PLATE 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; 10x

PLATE 7

Higher magnification of the fibroadenoma in Plate 6. Uniform epithelial cells

are arranged in a single layer forming glands and ducts. H&E; 40x

PLATE 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; 80x

o-Nitrotoluene, NTP TR 504 
PLATE 12

Higher magnification of the alveolar/bronchiolar adenoma in Plate 11. Note 

the papillary arrangement of well-differentiated neoplastic cells. H&E; 80x

PLATE 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; 80x

PLATE 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; 80x

PLATE 11

Alveolar/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; 8x

o-Nitrotoluene, NTP TR 504 
PLATE 15

Hemangiosarcoma in the skeletal muscle (thorax) of a male B6C3F1 mouse

exposed to 5,000 ppm o-nitrotoluene in feed for 2 years. Note that the central

portion of the hemangiosarcoma has undergone necrosis, leaving a large bloodfilled cavity surrounded by a rim of neoplastic endothelial cells. H&E; 8x

PLATE 13

Hemangiosarcoma in the mesentery from a male B6C3F1 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; 10x

PLATE 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; 80x

PLATE 14

Higher magnification of Plate 13. Malignant endothelial cells line the

cavernous spaces. Note the nuclear pleomorphism and hyperchromatic nuclei.

H&E; 80x

o-Nitrotoluene, NTP TR 504 
PLATE 19

Carcinoma in the cecum of a female B6C3F1 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; 40x

PLATE 17

Carcinoma in the cecum of a male B6C3F1 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; 10x

PLATE 20

Hepatocellular carcinoma in a female B6C3F1 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; 66x

PLATE 18

Higher magnification of Plate 17. Note the infiltrative nature of the anaplastic

epithelial cells within the cecal wall. H&E; 16x

o-Nitrotoluene, NTP TR 504 
71

This NTP Technical Report continues the reporting of

the comparative biologic effects of the nitrotoluene isomers. The results for the 2-year carcinogenicity studies

of o-nitrotoluene in F344/N rats and B6C3F1 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/or

reproductive 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-year

studies 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 male

rats 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, or

100 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 rats

and 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 the

surface serosal cells of the pleural, peritoneal, and pericardial cavities and from the tunica vaginalis (Attanoos

and 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 not

occur 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 were

observed 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 ppm

group 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 the

epididymis 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 rats

after exposure to 10 other chemicals in 2-year NTP studies: acronycine (NCI, 1978a); cytembena (NTP, 1981);

1,2-dibromoethane (NTP, 1982); 3,3N-dimethoxybenzidine dihydrochloride (NTP, 1990a), 2,3-dibromo1-propanol (NTP, 1993); 3,3N-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 a

similar 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 not

well 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 human

mesotheliomas 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 and

2,000 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 incidences

exceeded the historical control ranges for these neoplasms in control rats given NTP-2000 or NIH-07 diet.

Subcutaneous skin fibrosarcomas and mammary gland

fibroadenomas in male and female rats were clear evidence of a carcinogenic response because the effects

were 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 the

2,000 ppm core study group exceeded the historical control ranges in control rats given NTP-2000 or NIH-07

diet. 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 as

mesotheliomas. 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 and

5,000 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 or

hemangiosarcoma (combined) were significantly increased in 5,000 ppm stop-exposure male rats. The

presence 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 cell

leukemia 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 fill

the 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 mononuclear

cell 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 in

male 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 of

hemangiosarcoma 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 hemangiosarcomas

occurred primarily in the skeletal muscle, skin (subcutaneous tissue), and/or mesentery. This was considered to

be clear evidence of a carcinogenic effect because the

neoplasms 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 B6C3F1 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 hemangiosarcomas

from control mice from other NTP studies were examined for genetic alterations (Appendix M). The purpose

of these genetic toxicity studies was to compare the

spectrum 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 of

the 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 the

cecum in male and female mice. None of the approximately 500 other chemicals evaluated by the NTP have

caused carcinomas of the cecum in mice. The occurrence of cecum carcinomas in the current study was

considered clear evidence of a carcinogenic effect

because 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 to

the 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 and

5,000 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 benign

and 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 from

glutathione 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 weeks

and 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, the

animals 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 an

aromatic 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 [14C]-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 that

oxidation 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 504 TABLE 25 Comparison of Selected Neoplasms in the 2-Year Feed Studies of p-Nitrotoluene and o-Nitrotoluene p-Nitrotoluenea o-Nitrotoluene 0 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 Rats Average Daily Dose (mg/kg) 0 55 110 240 0 25 50 90 125 315 Survival 31/50 38/50 38/50 40/50 39/60 18/60 3/60 0/60 11/60 0/60 Mesothelium Malignant Mesotheliomab 5/50 2/50 1/50 4/50 2/60 20/60 29/60 44/60 44/60 54/60 Skin (Subcutaneous) Lipoma 0/50 0/50 0/50 0/50 0/60 4/60 13/60 13/60 10/60 12/60 Fibroma 1/50 2/50 7/50 1/50 5/60 46/60 52/60 59/60 45/60 52/60 Fibrosarcoma 0/50 2/50 7/50 1/50 0/60 7/60 17/60 20/60 8/60 12/60 Fibroma or Fibrosarcoma 1/50 2/50 9/50 1/50 5/60 47/60 55/60 59/60 47/60 53/60 Mammary Gland Fibroadenoma 0/50 0/50 0/50 0/50 0/60 7/60 10/60 2/60 13/60 20/60 Liver Hepatocellular Adenoma 0/50 0/50 0/50 0/50 2/60 3/60 3/60 7/60 3/60 4/60 Hepatocellular Adenoma or Carcinoma 1/50 0/50 0/50 0/50 3/60 3/60 3/60 8/60 3/60 6/60 Cholangiocarcinoma 0/50 0/50 0/50 0/50 0/60 0/60 0/60 0/60 0/60 3/60 Hepatocholangiocarcinoma 0/50 0/50 0/50 0/50 0/60 1/60 0/60 1/60 0/60 0/60 LungAlveolar/bronchiolar Adenoma 1/50 0/50 2/50 2/50 1/60 5/60 1/60 2/60 3/60 8/60 Alveolar/bronchiolar Adenoma or Carcinoma 1/50 1/50 2/50 3/50 2/60 5/60 1/60 2/60 3/60 11/60 a NTP, 2002 b Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver and lung; for other tissues, denominator is number of animals necropsied.
o-Nitrotoluene, NTP TR 504 77 TABLE 25 Comparison of Selected Neoplasms in the 2-Year Feed Studies of p-Nitrotoluene and o-Nitrotoluene p-Nitrotoluene o-Nitrotoluene 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm 0 ppm 625 ppm 1,250 ppm 2,000 ppm Female Rats Average Daily Dose (mg/kg) 0 60 125 265 0 30 60 100 Survival 39/50 37/50 39/50 41/50 47/60 47/60 39/60 33/60 Skin (Subcutaneous) Fibroma 0/50 0/50 0/50 1/49 3/59 3/60 18/60 19/60 Fibroma or Fibrosarcoma 1/50 1/50 0/50 2/50 3/60 3/60 21/60 22/60 Mammary Gland Fibroadenoma 14/50 17/50 20/50 5/50 23/60 47/60 52/60 56/60 Liver Hepatocellular Adenoma 0/50 0/50 1/50 0/50 1/60 0/59 1/60 6/60 Clitoral Gland Adenoma or Carcinoma 8/50 12/50 20/50 8/49 14/59 13/57 6/54 3/53 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm Male Mice Average Daily Dose (mg/kg) 0 170 345 690 0 165 360 700 Survival 46/50 46/50 45/50 42/50 52/60 46/60 47/60 5/60 Circulatory System Hemangiosarcoma 1/50 1/50 3/50 2/50 4/60 17/60 55/60 60/60 Large Intestine (Cecum) Carcinoma 1/48 0/48 0/48 0/49 0/56 12/49 9/36 0/44 LungAlveolar/bronchiolar Adenoma or Carcinoma 8/50 14/50 12/50 19/50 14/60 7/60 6/60 0/60
78 o-Nitrotoluene, NTP TR 504 TABLE 25 Comparison of Selected Neoplasms in the 2-Year Feed Studies of p-Nitrotoluene and o-Nitrotoluene p-Nitrotoluene o-Nitrotoluene 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm 0 ppm 1,250 ppm 2,500 ppm 5,000 ppm Female Mice Average Daily Dose (mg/kg) 0 155 315 660 0 150 320 710 Survival 46/50 47/50 43/50 49/50 52/60 46/60 47/60 5/60 Circulatory System Hemangiosarcoma 1/50 1/50 0/50 1/50 0/60 2/60 3/60 50/60 Large Intestine (Cecum) Carcinoma 0/49 0/48 0/48 0/50 0/60 1/60 4/60 3/60 Liver Hepatocellular Adenoma 6/49 3/50 2/50 5/50 7/60 5/59 19/59 29/60 Hepatocellular Carcinoma 3/49 4/50 0/50 1/50 2/60 4/59 6/59 16/60 Hepatocellular Adenoma or Carcinoma 8/49 6/50 2/50 6/50 9/60 9/59 24/59 39/60
o-Nitrotoluene, NTP TR 504 79

CONCLUSIONS

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 increased

incidences 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 rats

based 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 of

the 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 leukemia

occurred 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 
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APPENDIX A

SUMMARY OF LESIONS IN MALE RATS

IN THE 2-YEAR FEED STUDY

OF o-NITROTOLUENE

TABLE A1 Summary of the Incidence of Neoplasms in Male Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

TABLE A2 Individual Animal Tumor Pathology of Male Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

TABLE A3a Statistical Analysis of Primary Neoplasms in Male Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

TABLE A3b Statistical Analysis of Primary Neoplasms in Male Rats

in the 2-Year Stop-Exposure Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . 136

TABLE A4a Historical Incidence of Malignant Mesothelioma in Control Male F344/N Rats . . . . . . . . . 141

TABLE A4b Historical Incidence of Subcutaneous Skin Neoplasms 

in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

TABLE A4c Historical Incidence of Mammary Gland Fibroadenoma 

in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

TABLE A4d Historical Incidence of Liver Neoplasms in Control Male F344/N Rats . . . . . . . . . . . . . . . . 144

TABLE A4e Historical Incidence of Alveolar/bronchiolar Neoplasms 

in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

TABLE A4f Historical Incidence of Circulatory System Neoplasms

in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

TABLE A4g Historical Incidence of Mononuclear Cell Leukemia

in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

TABLE A4h Historical Incidence of Interstitial Cell Adenoma of the Testis

in Control Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

TABLE A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
90 o-Nitrotoluene, NTP TR 504 

TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of o-Nitrotoluenea

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%) 
o-Nitrotoluene, NTP TR 504 91

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%) 

TABLE 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)
92 o-Nitrotoluene, NTP TR 504 

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%) 

TABLE 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 93

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%) 

TABLE 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)
94 o-Nitrotoluene, NTP TR 504 

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 organsb (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%) 

TABLE 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 95

TABLE 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 neoplasmsc

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
96 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 97

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

TABLE 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 Total

Carcass ID Number 233355556001112222233344466666 Tissues/

346823690236891678901267823789 Tumors
98 o-Nitrotoluene, NTP TR 504 

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 + +

TABLE 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
o-Nitrotoluene, NTP TR 504 99

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

TABLE 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 Total

Carcass ID Number 233355556001112222233344466666 Tissues/

346823690236891678901267823789 Tumors
100 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 101

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

TABLE 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 Total

Carcass ID Number 233355556001112222233344466666 Tissues/

346823690236891678901267823789 Tumors
102 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 103

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

TABLE 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 Total

Carcass ID Number 028212211028778899127777000111 Tissues/

527440881330781289663456147025 Tumors
104 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 105

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

TABLE 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 Total

Carcass ID Number 028212211028778899127777000111 Tissues/

527440881330781289663456147025 Tumors
106 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 107

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

TABLE 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 Total

Carcass ID Number 028212211028778899127777000111 Tissues/

527440881330781289663456147025 Tumors
108 o-Nitrotoluene, NTP TR 504 

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 + + + +++++ + +++++ ++

TABLE 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
o-Nitrotoluene, NTP TR 504 109

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

TABLE 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 Total

Carcass ID Number 578563536544473734446853447574 Tissues/

062605562458194774603323392707 Tumors
110 o-Nitrotoluene, NTP TR 504 

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 +

TABLE 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
o-Nitrotoluene, NTP TR 504 111

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

TABLE 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 Total

Carcass ID Number 578563536544473734446853447574 Tissues/

062605562458194774603323392707 Tumors
112 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 113

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

TABLE 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 Total

Carcass ID Number 578563536544473734446853447574 Tissues/

062605562458194774603323392707 Tumors
114 o-Nitrotoluene, NTP TR 504 

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 + + + ++++ ++++++++++++++ + +++

TABLE 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
o-Nitrotoluene, NTP TR 504 115

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

TABLE 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 Total

Carcass ID Number 333914292499012203944094010340 Tissues/

679426976339101280258287580803 Tumors
116 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 117

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

TABLE 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 Total

Carcass ID Number 333914292499012203944094010340 Tissues/

679426976339101280258287580803 Tumors
118 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 119

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

TABLE 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 Total

Carcass ID Number 333914292499012203944094010340 Tissues/

679426976339101280258287580803 Tumors
120 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 121

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

TABLE 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 Total

Carcass ID Number 045103439923951534590244559155 Tissues/

538297021938269599482018793313 Tumors
122 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 123

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

TABLE 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 Total

Carcass ID Number 045103439923951534590244559155 Tissues/

538297021938269599482018793313 Tumors
124 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 125

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

TABLE 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 Total

Carcass ID Number 045103439923951534590244559155 Tissues/

538297021938269599482018793313 Tumors
126 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 127

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

TABLE 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 Total

Carcass ID Number 678897826626892281106290260012 Tissues/

367850404592311356767772880586 Tumors
128 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 129

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

TABLE 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 Total

Carcass ID Number 678897826626892281106290260012 Tissues/

367850404592311356767772880586 Tumors
130 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 131

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

TABLE 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 Total

Carcass ID Number 678897826626892281106290260012 Tissues/

367850404592311356767772880586 Tumors
132 o-Nitrotoluene, NTP TR 504 

TABLE 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 ratea 2/60 (3%) 4/60 (7%) 6/60 (10%) 2/59 (3%)

Adjusted rateb 3.7% 8.9% 16.3% 8.9% 

Terminal ratec 2/39 (5%) 1/18 (6%) 0/3 (0%) 0/0

First incidence (days) 729 (T) 567 531 471

Poly-3 testd 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 
o-Nitrotoluene, NTP TR 504 133

TABLE 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
134 o-Nitrotoluene, NTP TR 504 

TABLE 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
o-Nitrotoluene, NTP TR 504 135

TABLE 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 adrenal gland, liver, 

lung, pituitary gland, preputial gland, testes, 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 that 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.
136 o-Nitrotoluene, NTP TR 504 

TABLE 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 ratea 2/60 (3%) 4/60 (7%) 2/60 (3%) 

Adjusted rateb 3.7% 10.1% 9.9%

Terminal ratec 2/39 (5%) 2/11 (18%) 0/0

First incidence (days) 729 (T) 569 567

Poly-3 testd 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
o-Nitrotoluene, NTP TR 504 137

TABLE 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
138 o-Nitrotoluene, NTP TR 504 

TABLE 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
o-Nitrotoluene, NTP TR 504 139

TABLE 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
140 o-Nitrotoluene, NTP TR 504 

TABLE 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 adrenal gland, liver, 

lung, pancreas, pituitary gland, preputial gland, testes, 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 that 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 504 141

TABLE A4a

Historical Incidence of Malignant Mesothelioma in Control Male F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Citral (feed) 4/100

p,pN-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 Instituteb

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
142 o-Nitrotoluene, NTP TR 504 

TABLE 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 Dieta

Citral (feed) 0/100 2/100 2/100 4/100

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 143

TABLE A4c

Historical Incidence of Mammary Gland Fibroadenoma in Control Male F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Citral (feed) 8/100

p,pN-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 Instituteb

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
144 o-Nitrotoluene, NTP TR 504 

TABLE 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 Dieta

Citral (feed) 0/100 0/100 0/100 0/100 0/100

p,pN-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 Instituteb

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
TABLE 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 Dieta

Citral (feed) 3/100 0/100 3/100

p,pN-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 Instituteb

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

o-Nitrotoluene, NTP TR 504 145
146 o-Nitrotoluene, NTP TR 504 

TABLE 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 Dieta

Citral (feed) 1/100 0/100 1/100

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 147

TABLE A4g

Historical Incidence of Mononuclear Cell Leukemia in Control Male F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Citral (feed) 68/100

p,pN-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 Instituteb

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
148 o-Nitrotoluene, NTP TR 504 

TABLE 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 Dieta

Citral (feed) 96/100

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 149

TABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of o-Nitrotoluenea

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
150 o-Nitrotoluene, NTP TR 504 

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

TABLE 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 504 151

TABLE 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%) 
152 o-Nitrotoluene, NTP TR 504 

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%) 

TABLE 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 504 153

TABLE 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%) 
154 o-Nitrotoluene, NTP TR 504 

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%)

TABLE 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 504 155

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%) 

TABLE 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)
156 o-Nitrotoluene, NTP TR 504 
157

APPENDIX B

SUMMARY OF LESIONS IN FEMALE RATS

IN THE 2-YEAR FEED STUDY

OF o-NITROTOLUENE

TABLE B1 Summary of the Incidence of Neoplasms in Female Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

TABLE B2 Individual Animal Tumor Pathology of Female Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

TABLE B3 Statistical Analysis of Primary Neoplasms in Female Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

TABLE B4a Historical Incidence of Subcutaneous Skin Neoplasms

in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

TABLE B4b Historical Incidence of Mammary Gland Neoplasms 

in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

TABLE B4c Historical Incidence of Hepatocellular Adenoma 

in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

TABLE B4d Historical Incidence of Mononuclear Cell Leukemia 

in Control Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

TABLE B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
158 o-Nitrotoluene, NTP TR 504 

TABLE B1

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of o-Nitrotoluenea

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%) 
o-Nitrotoluene, NTP TR 504 159

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%) 

TABLE 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
160 o-Nitrotoluene, NTP TR 504 

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 organsb (60) (60) (60) (60)

Leukemia mononuclear 21 (35%) 6 (10%) 4 (7%) 5 (8%) 

Lymphoma malignant 1 (2%) 1 (2%) 

TABLE 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 504 161

Neoplasm Summary

Total animals with primary neoplasmsc 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 1

Total 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

TABLE 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
162 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 163

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

TABLE 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 Total

Carcass ID Number 558888800000777855566668899999 Tissues/

351234512345789078912346801234 Tumors
164 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 165

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

TABLE 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 Total

Carcass ID Number 558888800000777855566668899999 Tissues/

351234512345789078912346801234 Tumors
166 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 167

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

TABLE 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 Total

Carcass ID Number 226667111133342223334444445555 Tissues/

450690124568906780252356780689 Tumors
168 o-Nitrotoluene, NTP TR 504 

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 ++++++++++++++++++++++++++++++

TABLE 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
o-Nitrotoluene, NTP TR 504 169

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

TABLE 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 Total

Carcass ID Number 226667111133342223334444445555 Tissues/

450690124568906780252356780689 Tumors
170 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 171

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

TABLE 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 Total

Carcass ID Number 226667111133342223334444445555 Tissues/

450690124568906780252356780689 Tumors
172 o-Nitrotoluene, NTP TR 504 

TABLE 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
o-Nitrotoluene, NTP TR 504 173

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

TABLE 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 Total

Carcass ID Number 112888999902277777788889900000 Tissues/

680678078908945678903454512345 Tumors
174 o-Nitrotoluene, NTP TR 504 

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 + +

TABLE 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
o-Nitrotoluene, NTP TR 504 175

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

TABLE 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 Total

Carcass ID Number 112888999902277777788889900000 Tissues/

680678078908945678903454512345 Tumors
176 o-Nitrotoluene, NTP TR 504 

Urinary System

Kidney ++++++++++++++++++++++++++++++

Urinary bladder ++++++++++++++++++++++++++++++

Papilloma

Systemic Lesions

Multiple organs ++++++++++++++++++++++++++++++

Leukemia mononuclear X X X

Lymphoma malignant

TABLE 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
o-Nitrotoluene, NTP TR 504 177

Urinary System

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Papilloma X 1

Systemic Lesions

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Leukemia mononuclear X 4

Lymphoma malignant X 1

TABLE 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 Total

Carcass ID Number 112888999902277777788889900000 Tissues/

680678078908945678903454512345 Tumors
178 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 179

TABLE 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 Total

Carcass ID Number 898883347734444555556666677778 Tissues/

903458901421689123680134507890 Tumors

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
180 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 181

TABLE 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 Total

Carcass ID Number 898883347734444555556666677778 Tissues/

903458901421689123680134507890 Tumors

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
182 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 183

TABLE 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 Total

Carcass ID Number 898883347734444555556666677778 Tissues/

903458901421689123680134507890 Tumors

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
184 o-Nitrotoluene, NTP TR 504 

TABLE 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 ratea 12/59 (20%) 9/57 (16%) 3/54 (6%) 3/53 (6%)

Adjusted rateb 21.5% 17.0% 6.2% 6.3%

Terminal ratec 11/46 (24%) 9/45 (20%) 3/36 (8%) 3/32 (9%)

First incidence (days) 726 729 (T) 729 (T) 729 (T)

Poly-3 testd 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
o-Nitrotoluene, NTP TR 504 185

TABLE 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
186 o-Nitrotoluene, NTP TR 504 

TABLE 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 clitoral 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 that 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.
o-Nitrotoluene, NTP TR 504 187

TABLE 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 Dieta

Citral (feed) 1/100 0/100 1/100

p,pN-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 Instituteb

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
188 o-Nitrotoluene, NTP TR 504 

TABLE 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 Dieta

Citral (feed) 53/100 0/100 53/100

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 189

TABLE B4c

Historical Incidence of Hepatocellular Adenoma in Control Female F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Citral (feed) 0/100

p,pN-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 Instituteb

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
190 o-Nitrotoluene, NTP TR 504 

TABLE B4d

Historical Incidence of Mononuclear Cell Leukemia in Control Female F344/N Rats

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Citral (feed) 24/100

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 191

TABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of o-Nitrotoluenea

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
192 o-Nitrotoluene, NTP TR 504 

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%) 

TABLE 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 504 193

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%) 

TABLE 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
194 o-Nitrotoluene, NTP TR 504 

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%) 

TABLE 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

TABLE C1 Summary of the Incidence of Neoplasms in Male Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

TABLE C2 Individual Animal Tumor Pathology of Male Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

TABLE C3 Statistical Analysis of Primary Neoplasms in Male Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

TABLE C4a Historical Incidence of Hemangiosarcoma (All Sites)

in Control Male B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

TABLE C4b Historical Incidence of Large Intestine (Cecum) Carcinoma

in Control Male B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

TABLE C4c Historical Incidence of Hepatocellular Neoplasms in Control Male B6C3F1 Mice . . . . . . . . 230

TABLE C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
196 o-Nitrotoluene, NTP TR 504 

TABLE C1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of o-Nitrotoluenea

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%) 
o-Nitrotoluene, NTP TR 504 197

TABLE 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)
198 o-Nitrotoluene, NTP TR 504 

TABLE 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%) 
o-Nitrotoluene, NTP TR 504 199

TABLE 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 organsb (60) (60) (60) (60)

Histiocytic sarcoma 1 (2%) 1 (2%) 

Lymphoma malignant 3 (5%) 1 (2%) 

Neoplasm Summary

Total animals with primary neoplasmsc 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
200 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 201

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

TABLE 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 Total

Carcass ID Number 001111112222333334444555603555 Tissues/

670123470568356891278568020019 Tumors
202 o-Nitrotoluene, NTP TR 504 

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 ++++++++++++++++++++++++++++++

TABLE 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
o-Nitrotoluene, NTP TR 504 203

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

TABLE 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 Total

Carcass ID Number 001111112222333334444555603555 Tissues/

670123470568356891278568020019 Tumors
204 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 205

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

TABLE 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 Total

Carcass ID Number 001111112222333334444555603555 Tissues/

670123470568356891278568020019 Tumors
206 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 207

TABLE 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 Total

Carcass ID Number 890000111166677789999011789001 Tissues/

542789237812558922359456486061 Tumors

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
208 o-Nitrotoluene, NTP TR 504 

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 ++++++++++++++++++++++++++++++

TABLE 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
o-Nitrotoluene, NTP TR 504 209

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

TABLE 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 Total

Carcass ID Number 890000111166677789999011789001 Tissues/

542789237812558922359456486061 Tumors
210 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 211

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

TABLE 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 Total

Carcass ID Number 890000111166677789999011789001 Tissues/

542789237812558922359456486061 Tumors
212 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 213

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

TABLE 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 Total

Carcass ID Number 555763223356234736627453727764 Tissues/

986081585705907832733629219462 Tumors
214 o-Nitrotoluene, NTP TR 504 

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 + +

TABLE 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
o-Nitrotoluene, NTP TR 504 215

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

TABLE 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 Total

Carcass ID Number 555763223356234736627453727764 Tissues/

986081585705907832733629219462 Tumors
216 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 217

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

TABLE 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 Total

Carcass ID Number 555763223356234736627453727764 Tissues/

986081585705907832733629219462 Tumors
218 o-Nitrotoluene, NTP TR 504 

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 ++++++++++++++++++++++++++++++

TABLE 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
o-Nitrotoluene, NTP TR 504 219

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

TABLE 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 Total

Carcass ID Number 108081813028229133891290430913 Tissues/

441025420869597949756819066208 Tumors
220 o-Nitrotoluene, NTP TR 504 

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 +

TABLE 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
o-Nitrotoluene, NTP TR 504 221

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

TABLE 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 Total

Carcass ID Number 108081813028229133891290430913 Tissues/

441025420869597949756819066208 Tumors
222 o-Nitrotoluene, NTP TR 504 

Urinary System

Kidney ++++++++++++++++++++++++++++++

Capsule, hemangiosarcoma X

Urethra +

Urinary bladder ++++++++++++++++++++++++++++++

Systemic Lesions

Multiple organs ++++++++++++++++++++++++++++++

TABLE 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
o-Nitrotoluene, NTP TR 504 223

Urinary System

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Capsule, hemangiosarcoma 1

Urethra + 2

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Systemic Lesions

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

TABLE 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 Total

Carcass ID Number 108081813028229133891290430913 Tissues/

441025420869597949756819066208 Tumors
224 o-Nitrotoluene, NTP TR 504 

TABLE 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 ratea 4/60 (7%) 1/60 (2%) 1/60 (2%) 0/60 (0%)

Adjusted rateb 7.0% 2.0% 4.2% 0.0% 

Terminal ratec 4/52 (8%) 1/34 (3%) 0/0 0/0 

First incidence (days) 729 (T) 729 (T) 645 —e

Poly-3 testd 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 
o-Nitrotoluene, NTP TR 504 225

TABLE 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
226 o-Nitrotoluene, NTP TR 504 

TABLE 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
o-Nitrotoluene, NTP TR 504 227

TABLE 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 liver, 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 that 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.
228 o-Nitrotoluene, NTP TR 504 

TABLE C4a

Historical Incidence of Hemangiosarcoma (All Sites) in Control Male B6C3F1 Mice

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Acrylonitrile (gavage) 3/50

Citral (feed) 3/100

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 229

TABLE C4b

Historical Incidence of Large Intestine (Cecum) Carcinoma in Control Male B6C3F1 Mice

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Acrylonitrile (gavage) 0/50

Citral (feed) 0/100

p,pN-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 Instituteb

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
230 o-Nitrotoluene, NTP TR 504 

TABLE C4c

Historical Incidence of Hepatocellular Neoplasms in Control Male B6C3F1 Mice

Incidence in Controls

Study Adenoma Carcinoma Adenoma 

or Carcinoma

Historical Incidence in Controls Given NTP-2000 Dieta

Acrylonitrile (gavage) 23/50 14/50 32/50

Citral (feed) 20/100 13/100 28/100

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 231

TABLE C5

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of o-Nitrotoluenea

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
232 o-Nitrotoluene, NTP TR 504 

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%) 

TABLE 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 504 233

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%) 

TABLE 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
234 o-Nitrotoluene, NTP TR 504 

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%)

TABLE 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 504 235

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%) 

TABLE 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
236 o-Nitrotoluene, NTP TR 504 
237

APPENDIX D

SUMMARY OF LESIONS IN FEMALE MICE

IN THE 2-YEAR FEED STUDY

OF o-NITROTOLUENE

TABLE D1 Summary of the Incidence of Neoplasms in Female Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

TABLE D2 Individual Animal Tumor Pathology of Female Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

TABLE D3 Statistical Analysis of Primary Neoplasms in Female Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

TABLE D4a Historical Incidence of Hemangiosarcoma (All Sites)

in Control Female B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

TABLE D4b Historical Incidence of Hepatocellular Neoplasms 

in Control Female B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

TABLE D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
238 o-Nitrotoluene, NTP TR 504 

TABLE D1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of o-Nitrotoluenea

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%) 
o-Nitrotoluene, NTP TR 504 239

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%) 

TABLE 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
240 o-Nitrotoluene, NTP TR 504 

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%) 

TABLE 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 504 241

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 organsb (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 neoplasmsc 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

TABLE 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
242 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 243

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

TABLE 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 Total

Carcass ID Number 888866667777779990777788899999 Tissues/

234567890123456780678907901234 Tumors
244 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 245

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

TABLE 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 Total

Carcass ID Number 888866667777779990777788899999 Tissues/

234567890123456780678907901234 Tumors
246 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 247

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

TABLE 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 Total

Carcass ID Number 111222231111222223333334455556 Tissues/

789067801345123451345786706780 Tumors
248 o-Nitrotoluene, NTP TR 504 

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 + +

TABLE 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
o-Nitrotoluene, NTP TR 504 249

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

TABLE 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 Total

Carcass ID Number 111222231111222223333334455556 Tissues/

789067801345123451345786706780 Tumors
250 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 251

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

TABLE 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 Total

Carcass ID Number 111222231111222223333334455556 Tissues/

789067801345123451345786706780 Tumors
252 o-Nitrotoluene, NTP TR 504 

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+ + + + + + + + + + + + + + + + + + +

TABLE 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
o-Nitrotoluene, NTP TR 504 253

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

TABLE 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 Total

Carcass ID Number 778000011118888888999990666111 Tissues/

890134512451346789067890125678 Tumors
254 o-Nitrotoluene, NTP TR 504 

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 + +

TABLE 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
o-Nitrotoluene, NTP TR 504 255

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

TABLE 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 Total

Carcass ID Number 778000011118888888999990666111 Tissues/

890134512451346789067890125678 Tumors
256 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 257

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

TABLE 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 Total

Carcass ID Number 778000011118888888999990666111 Tissues/

890134512451346789067890125678 Tumors
258 o-Nitrotoluene, NTP TR 504 

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+ + + + + + + +

TABLE 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
o-Nitrotoluene, NTP TR 504 259

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

TABLE 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 Total

Carcass ID Number 735245353465477467644566347777 Tissues/

886288512784902539602902763567 Tumors
260 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 261

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

TABLE 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 Total

Carcass ID Number 735245353465477467644566347777 Tissues/

886288512784902539602902763567 Tumors
262 o-Nitrotoluene, NTP TR 504 

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

TABLE 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
o-Nitrotoluene, NTP TR 504 263

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

TABLE 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 Total

Carcass ID Number 735245353465477467644566347777 Tissues/

886288512784902539602902763567 Tumors
264 o-Nitrotoluene, NTP TR 504 

TABLE 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 ratea 4/60 (7%) 0/60 (0%) 1/60 (2%) 2/60 (3%) 

Adjusted rateb 7.1% 0.0% 1.8% 5.0%

Terminal ratec 4/52 (8%) 0/46 (0%) 1/47 (2%) 1/5 (20%) 

First incidence (days) 729 (T) —e 729 (T) 630 

Poly-3 testd 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
o-Nitrotoluene, NTP TR 504 265

TABLE 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
266 o-Nitrotoluene, NTP TR 504 

TABLE 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 
o-Nitrotoluene, NTP TR 504 267

TABLE 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 liver, 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 that 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.
268 o-Nitrotoluene, NTP TR 504 

TABLE D4a

Historical Incidence of Hemangiosarcoma (All Sites) in Control Female B6C3F1 Mice

Study Incidence in Controls

Historical Incidence in Controls Given NTP-2000 Dieta

Acrylonitrile (gavage) 4/50

Citral (feed) 0/99

p,pN-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 Instituteb

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
o-Nitrotoluene, NTP TR 504 269

TABLE D4b

Historical Incidence of Hepatocellular Neoplasms in Control Female B6C3F1 Mice

Incidence in Controls

Study Adenoma Carcinoma Adenoma 

or Carcinoma

Historical Incidence in Controls Given NTP-2000 Dieta

Acrylonitrile (gavage) 14/50 7/50 20/50

Citral (feed) 8/99 4/99 12/99

p,pN-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 Instituteb

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
270 o-Nitrotoluene, NTP TR 504 

TABLE D5

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of o-Nitrotoluenea

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
o-Nitrotoluene, NTP TR 504 271

TABLE 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%) 
272 o-Nitrotoluene, NTP TR 504 

TABLE 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%) 
o-Nitrotoluene, NTP TR 504 273

TABLE 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%) 
274 o-Nitrotoluene, NTP TR 504 

TABLE 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

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

RAT AND MOUSE BONE MARROW MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

EVALUATION PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

TABLE E1 Mutagenicity of o-Nitrotoluene in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . 279

TABLE E2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells

by o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

TABLE E3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells

by o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

TABLE E4 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats

Treated with o-Nitrotoluene by Intraperitoneal Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

TABLE E5 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Mice

Treated with o-Nitrotoluene by Intraperitoneal Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

TABLE 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

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL

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.

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS

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 medium

plus 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-division

metaphase 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.

RAT AND MOUSE BONE MARROW MICRONUCLEUS TEST PROTOCOL

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 B6C3F1 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.

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL

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.

EVALUATION PROTOCOL

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

TABLE E1

Mutagenicity of o-Nitrotoluene in Salmonella typhimuriuma

Revertants/Plateb

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 ± 5.9c 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 controld 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 ± 1.2c 0 ± 0.0c 7 ± 0.9 8 ± 0.7 11 ± 1.9 11 ± 1.5

666.0 Toxic 9 ± 3.0

1,000.0 3 ± 1.8c 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 ± 0.0c Toxic 8 ± 2.6 6 ± 1.5 6 ± 1.5 6 ± 1.0

666.0 Toxic 6 ± 1.3

1,000.0 Toxic 0 ± 0.0c

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 ± 0.0c 27 ± 0.7 28 ± 2.6 21 ± 3.7 31 ± 3.8

666.0 0 ± 0.0c 32 ± 0.5

1,000.0 10 ± 5.4c 0 ± 0.0c

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 

TABLE 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 ± 1.9c 132 ± 9.2c 90 ± 7.3c 148 ± 3.3 111 ± 7.2c 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 ± 2.9c 23 ± 2.8c 11 ± 2.0c 16 ± 2.5 12 ± 3.8c 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 ± 1.7c 6 ± 0.3c 10 ± 2.7c 11 ± 0.9 5 ± 0.9c 7 ± 0.9

666.0 Toxic 6 ± 2.1c 6 ± 0.3c

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 ± 0.6c 12 ± 1.8c 27 ± 2.3 36 ± 3.5 27 ± 3.5c 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), and 

4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
o-Nitrotoluene, NTP TR 504 281

TABLE E2

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by o-Nitrotoluenea

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 Chromosomeb

(%)

–S9

Summary: Equivocal

Dimethylsulfoxidec 50 1,035 485 0.46 9.7 25.5

o-Nitrotoluene 117.000 50 1,039 569 0.54 11.4 34.0d 16.87

176.000 50 1,021 557 0.54 11.1 34.0d 16.42

218.000 50 1,035 576 0.55 11.5 34.0d 18.76

282.000 0

P=0.005e

Mitomycin-Cf 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

Cyclophosphamidef 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 

TABLE E3

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by o-Nitrotoluenea

Dose Total Cells Number Aberrations/ Cells with

Compound (μg/mL) Scored of Abberations Cell Aberrations (%)

–S9

Harvest time: 10.5 hours

Summary: Negative

Dimethylsulfoxideb 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=0.873c

Mitomycin-Cd 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

Cyclophosphamided 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 oild 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=0.609e

Cyclophosphamidef 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

TABLE E4

Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats 

Treated with o-Nitrotoluene by Intraperitoneal Injectiona

Number of Rats

Compound Dose with Erythrocytes Micronucleated PCEs/ P Valuec

(mg/kg) Scored 1,000 PCEsb
284 o-Nitrotoluene, NTP TR 504 

TABLE E5

Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Mice 

Treated with o-Nitrotoluene by Intraperitoneal Injectiona

Number of Mice

Compound Dose with Erythrocytes Micronucleated PCEs/ P Valuec

(mg/kg) Scored 1,000 PCEsb

Corn oild 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=0.055e

Cyclophosphamidef 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

TABLE E6

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Administration of o-Nitrotoluene in Feed for 13 Weeksa

Number of Mice 

Concentration with Erythrocytes Micronucleated NCEs/ P Valuec PCEs (%)

(ppm) Scored 1,000 NCEsb

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=0.003d

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

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

TABLE F1 Urinary Biomarker Data for Rats in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . 290

TABLE 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

INTRODUCTION

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. 

MATERIALS AND METHODS

Urinary metabolites were quantitated by high-performance liquid chromatography using a C18 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 DISCUSSION

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 

TABLE F1

Urinary Biomarker Data for Rats in the 2-Year Feed Study of o-Nitrotoluenea

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

TABLE 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. 
TABLE F2

Urinary Biomarker Data for Mice in the 2-Year Feed Study of o-Nitrotoluenea

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 0b

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 ± 0.1c 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 ± 2.6c 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

TABLE 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 complete 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

TABLE 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 

TABLE 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-Nitrotoluenea

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

PROCUREMENT AND CHARACTERIZATION OF O-NITROTOLUENE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

FIGURE H1 Infrared Absorption Spectrum of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

FIGURE H2 Nuclear Magnetic Resonance Spectrum of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

TABLE H1 Gas Chromatography Systems Used in the 2-Year Feed Studies of o-Nitrotoluene . . . . . . . 302

TABLE H2 Preparation and Storage of Dose Formulations in the 2-Year Feed Studies 

of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

TABLE 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

PROCUREMENT AND CHARACTERIZATION OF O-NITROTOLUENE

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.

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS

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 

FIGURE H1

Infrared Absorption Spectrum of o-Nitrotoluene 
o-Nitrotoluene, NTP TR 504 301

FIGURE 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

TABLE 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 headspace

Study Laboratory

Southern Research Institute (Birmingham, AL)

302

TABLE H1

Gas Chromatography Systems Used in the 2-Year Feed Studies of o-Nitrotoluenea

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

TABLE 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 Concentrationa 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, 1996b 625 585 –6

1,250 1,030 –18

2,000 1,500 –25

5,000 3,970 –21
304 o-Nitrotoluene, NTP TR 504 

TABLE 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 1,780c –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 1,860d –7
o-Nitrotoluene, NTP TR 504 305

TABLE 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 518c –17

625 565 –10

625 546c –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 604d –3

625 616d –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, 1996b 625 533 –15

1,250 976 –22

2,000 1,510 –24
306 o-Nitrotoluene, NTP TR 504 

TABLE 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

TABLE 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 558c –11

625 546c –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 709d +13

625 716d +15

June 23-24, 1997b 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 

TABLE 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 2,750c +38

2,000 2,330c +17

2,000 2,240 +12

2,000 2,220 +11

November 7, 1997 November 7, 1997 2,000 2,080d +4

2,000 2,240d +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 623e –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, 1998b 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, 1996b 1,250 730 –42

2,500 1,330 –47

5,000 2,910 –42
o-Nitrotoluene, NTP TR 504 309

TABLE 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, 1996b 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 

TABLE 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, 1997b 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, 1998b 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

TABLE I1 Feed and Compound Consumption by Male Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

TABLE I2 Feed and Compound Consumption by Female Rats

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

TABLE I3 Feed and Compound Consumption by Male Mice 

in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

TABLE 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

TABLE 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

TABLE 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

TABLE 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

TABLE 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

TABLE 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

TABLE J1 Ingredients of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

TABLE J2 Vitamins and Minerals in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . 318

TABLE J3 Nutrient Composition of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . 319

TABLE J4 Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
318 o-Nitrotoluene, NTP TR 504 

TABLE 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 premixa 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

TABLE J2

Vitamins and Minerals in NTP-2000 Rat and Mouse Rationa

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

B12 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

TABLE 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) 1,000a

"-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 B12 (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 

TABLE J4

Contaminant Levels in NTP-2000 Rat and Mouse Rationa

Mean ± Standard

Deviationb 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

TABLE 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

METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
324

SENTINEL ANIMAL PROGRAM

METHODS

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

RATS

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

MICE

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

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

TABLE L1 Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F1 Mice 

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 334

TABLE L2 Cumulative Urinary Excretion of Radioactivity by F344/N Rats 

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 334

FIGURE L1 HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 335

FIGURE L2 HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene

and Incubated with $-Glucuronidase/Sulfatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

FIGURE L3 HPLC Radiochromatograms of Urine Collected from B6C3F1 Mice 0 to 24 Hours 

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 337

TABLE L3 Urinary Metabolite Profile for F344/N Rats and Male B6C3F1 Mice

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 338

TABLE L4 Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F1 Mice 

after a Single Gavage Dose of 2 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . 339

TABLE L5 Urinary Metabolite Profile for F344/N Rats and Male B6C3F1 Mice

after a Single Gavage Dose of 2 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . 340

TABLE L6 Cumulative Excretion of Radioactivity by Male F344/N Rats 

Administered 200 mg/kg [14C]-o-Nitrotoluene in the 14-Day Gavage Study . . . . . . . . . . . . . 341

TABLE L7 Urinary Metabolite Profile for Male F344/N Rats 

Administered 200 mg/kg [14C]-o-Nitrotoluene in the 14-Day Gavage Study . . . . . . . . . . . . . 342

TABLE L8 Urinary Metabolite Profile for Male F344/N Rats 

Pretreated with Buthionine Sulfoxamine after a Single Gavage Dose 

of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

TABLE L9 Urinary Metabolite Profile for Male F344/N Rats Pretreated with Pentachlorophenol 

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . 343

TABLE L10 Plasma Concentration of o-Nitrotoluene in Male F344/N Rats 

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 344

FIGURE 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 [14C]-o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . . 345
328

COMPARATIVE METABOLISM STUDIES 

OF o-NITROTOLUENE 

INTRODUCTION

Studies were conducted in male and female F344/N rats and male B6C3F1 mice to determine and compare the

metabolism and excretion of [14C]-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).

MATERIALS AND METHODS

Nonradiolabeled o-nitrotoluene was obtained from Aldrich Chemical Company (St. Louis, MO) in two lots

(08506MV and 04328DG). Radiolabeled [14C]-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 [14C]-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 B6C3F1 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 CO2. 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

[14C]-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 [14C]-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 [14C]-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 [14C]-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

[14C]-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 [14C]-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 C18 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 [14C]-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 [14C]-o-nitrotoluene. Alternatively, three male rats were injected intraperitoneally with

pentachlorophenol 45 minutes before gavage administration of 200 mg/kg [14C]-o-nitrotoluene. In a third study,

the effect of repeat gavage dosing on the metabolism and excretion of [14C]-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 3N-phosphoadenosine5N-phosphosulfate (PAPS)-generating system. The incubation mixture contained 1 mL rat liver cytosol, 10 mM

[3H]-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 DISCUSSION

Excretion and Urinary Metabolites of [14C]-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

B6C3F1 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 analysis

of 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 B6C3F1

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 [14C]ortho-Nitrotoluene

(ONT) and [14C]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

TABLE L2

Cumulative Urinary Excretion of Radioactivity by F344/N Rats after a Single Gavage Dose

of 200 mg/kg [14C]-o-Nitrotoluenea

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

TABLE L1

Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F1 Mice 

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluenea

Time (hours

after dosing) Urine Feces Total
o-Nitrotoluene, NTP TR 504 335

FIGURE L1

HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours

after a Single Gavage Dose of 200 mg/kg [14C]-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 

FIGURE L2

HPLC Radiochromatograms of Urine Collected from F344/N Rats 0 to 24 Hours 

after a Single Gavage Dose of 200 mg/kg [14C]-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

FIGURE L3

HPLC Radiochromatograms of Urine Collected from B6C3F1 Mice 0 to 24 Hours 

after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene

Metabolites: A=2-nitrobenzoic acid; B=2-nitrobenzyl glucuronide; G=2-nitrobenzyl alcohol
338 o-Nitrotoluene, NTP TR 504 TABLE L3 Urinary Metabolite Profile for F344/N Rats and Male B6C3F1 Mice after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluene Time (hours 2-Nitrobenzoic 2-Nitrobenzyl 2-Aminobenzyl S-(2-Nitrobenzyl)- 2-Nitrobenzyl after dosing) acid glucuronide Ca alcohol Ea N-acetylcysteine alcohol o-Toluidine Ratsb Male 24 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 ± 0.71 48 1.90 ± 1.20 1.29 ± 0.47 0.19d 1.16 ± 0.42 0.30 ± 0.07e 0.51 ± 0.12 0.23d Totalc 20.97 ± 2.03 16.64 ± 2.16 0.19d 18.17 ± 1.89 4.18 ± 0.48 10.38 ± 1.32 1.77 ± 0.75 1.30 ± 0.79 Female24 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 ± 0.67 48 2.06 ± 1.63 1.65 ± 0.94 0.39d 0.65 ± 0.45 0.22 ± 0.12 0.34 ± 0.21 0.14 ± 0.08 0.06 ± 0.05 Total 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 ± 0.62 Micef Male 24 38.2 23.9 —g —— —— — a Metabolite not identified b n=3 at each time point; data are presented as percentage of dose recovered (mean ± standard deviation). c Mean of individual animal cumulative urinary excretion from 0 to 48 hours. d n=1; standard deviation not calculated because of an insufficient number of samples e n=2 f Urine was pooled from four animals; data are presented as percentage of dose recovered. g Metabolite not detected
o-Nitrotoluene, NTP TR 504 339

TABLE L4

Cumulative Excretion of Radioactivity by F344/N Rats and Male B6C3F1 Mice after a Single Gavage Dose 

of 2 mg/kg [14C]-o-Nitrotoluenea

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 72b 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 72b 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 72b 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 504 TABLE L5 Urinary Metabolite Profile for F344/N Rats and Male B6C3F1 Mice after a Single Gavage Dose of 2 mg/kg [14C]-o-Nitrotoluene Time (hours 2-Nitrobenzoic 2-Nitrobenzyl 2-Aminobenzyl S-(2-Nitrobenzyl)- 2-Nitrobenzyl after dosing) acid glucuronide Ca alcohol Ea N-acetylcysteine alcohol o-Toluidine Ratsb Male 24 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 —c Female24 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 — Miced Male 24 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 — a Metabolite not identified b n=3 at each time point; data are presented as percentage of dose recovered (mean ± standard deviation). c Not detected d Urine was pooled from three mice at each time point; data are presented as percentage of dose recovered.
o-Nitrotoluene, NTP TR 504 341

TABLE L6

Cumulative Excretion of Radioactivity by Male F344/N Rats Administered 200 mg/kg [14C]-o-Nitrotoluene

in the 14-Day Gavage Studya

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 72b 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 dose 

recovered (mean ± standard deviation). b Cage rinse included for this collection period
342 o-Nitrotoluene, NTP TR 504 TABLE L7 Urinary Metabolite Profile for Male F344/N Rats Administered 200 mg/kg [14C]-o-Nitrotoluene in the 14-Day Gavage Studya Time (hours after dosing 2-Nitrobenzoic 2-Nitrobenzyl 2-Aminobenzyl S-(2-Nitrobenzyl)- 2-Nitrobenzyl with radiolabel) acid glucuronide Cb alcohol Eb N-acetylcysteine alcohol o-Toluidine 24 19.7 23.1 0.0 12.0 3.1 4.8 1.3 0.8 48 2.3 1.9 0.0 1.0 0.4 0.5 0.1 0.0 Total 22.1 25.0 0.0 12.9 3.6 5.3 1.4 0.8 a Radiolabeled dose was administered on day 12. Urine was pooled from three rats at each time point; data are presented as percentage of dose recovered. b Metabolite not identified TABLE L8 Urinary Metabolite Profile for Male F344/N Rats Pretreated with Buthionine Sulfoxamine after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluenea 2-Nitro- 2-Nitro- 2-Amino- S-(2-Nitro- 2-Nitro- Time (hours benzoic benzyl Cb benzyl Eb benzyl)-N- benzyl o-Toluidine % Excretedc after dosing) acid glucuronide alcohol acetylcysteine alcohol 0 to 24 13.0 12.9 0.0 10.0 3.8 4.7 5.8 0.6 57.2 ± 6.3 24 to 48 2.6 2.5 0.0 1.4 0.5 0.7 0.3 0.0 65.7 ± 4.4 48 to 72 0.4 0.4 0.0 0.2 0.1 0.1 0.0 0.0 67.2 ± 5.0 Total 16.0 15.8 0.0 11.5 4.4 5.5 6.2 0.6 a Urine was pooled from three rats for each collection period; data are presented as percentage of dose recovered. b Metabolite not identified c Cumulative percentage of the dose recovered as all metabolites excreted in the indicated time period (mean ± standard deviation)
o-Nitrotoluene, NTP TR 504 343 TABLE L9 Urinary Metabolite Profile for Male F344/N Rats Pretreated with Pentachlorophenol after a Single Gavage Dose of 200 mg/kg [14C]-o-Nitrotoluenea 2-Nitro- 2-Nitro- 2-Amino- S-(2-Nitro- 2-Nitro- Time (hours benzoic benzyl Cb benzyl Eb benzyl)-N- benzyl o-Toluidine % Excretedc after dosing) acid glucuronide alcohol acetylcysteine alcohol 0 to 4 1.25 1.34 0.00 0.18 0.11 0.19 0.91 0.00 4.2 ± 2.0 4 to 8 4.45 2.73 0.00 1.07 0.60 0.40 0.51 0.21 13.0 ± 1.7 0 to 24 21.52 7.79 2.06 4.08 1.96 1.50 2.43 0.83 51.6 ± 8.5 a Urine was pooled from three rats for each collection period; data are presented as percentage of dose recovered. b Metabolite not identified c Cumulative percentage of the dose recovered as all metabolites excreted in the indicated time period (mean ± standard deviation)
344 o-Nitrotoluene, NTP TR 504 

TABLE L10

Plasma Concentration of o-Nitrotoluene in Male F344/N Rats after a Single Gavage Dose 

of 200 mg/kg [14C]-o-Nitrotoluenea

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 <LDb <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

FIGURE 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 [14C]-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 B6C3F1 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

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351

TABLE M1 Summary of Immunohistochemistry for p53 and $-Catenin Protein Accumulation 

in Spontaneous Hemangiosarcomas in Control B6C3F1 Mice in Selected NTP Studies . . . 355

TABLE M2 Summary of Immunohistochemistry for p53 and $-Catenin Protein Accumulation 

in Hemangiosarcomas in B6C3F1 Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . 355

TABLE M3 Summary of ras, p53, and $-Catenin Mutations in Hemangiosarcomas

from B6C3F1 Mice in the 2-Year Feed Study of o-Nitrotoluene . . . . . . . . . . . . . . . . . . . . . . . 356
348

GENETIC ALTERATIONS IN ras, p53, 

AND $-CATENIN GENES IN HEMANGIOSARCOMAS

OF B6C3F1 MICE FOLLOWING EXPOSURE 

TO o-NITROTOLUENE FOR 2 YEARS

INTRODUCTION

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 B6C3F1 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. 

MATERIALS AND METHODS

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 B6C3F1 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, 5N-TACAGGTAGCATTTTCAGTTCAC-3N and BCAT-2R, 5N-TAGCTTCCAAACACAAATGC-3N

(De La Coste et al., 1998). Inner primers were BCAT-7F, 5N-TAACATACTCTGTTTTTACAGCTG-3N, and

BCAT-8R, 5N-ACATCTTCTTCCTCAGGGTTG-3N. 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. [33P]-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 ["-33P]-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 5N-TCGTACTCATCCACAAAGTG-3N and

5N-ACCTGTCTCTTGGAT-3N for exons 1 and 2 respectively; for H-ras exon 2, the sequencing primer was

5N-TGTGCGCATGTACTG-3N. 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 [33P]-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 B6C3F1 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).

DISCUSSION

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 B6C3F1 mice. Similar to the current study, low frequencies of ras mutations were detected

in spontaneous or thiazolidinedione-induced hemangiosarcomas in the B6C3F1 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

N6-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 N2, 3-ethenoguanine and 3,N4-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.

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o-Nitrotoluene, NTP TR 504 355

TABLE M1

Summary of Immunohistochemistry for p53 and $-Catenin Protein Accumulation

in Spontaneous Hemangiosarcomas in Control B6C3F1 Mice in Selected NTP Studies

Studya 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 hemangiosarcoma. b No positive immunohistochemical response was observed

TABLE M2

Summary of Immunohistochemistry for p53 and $-Catenin Protein Accumulation

in Hemangiosarcomas in B6C3F1 Mice in the 2-Year Feed Study of o-Nitrotoluene

Exposure Concentrationa 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 504 TABLE M3 Summary of ras, p53, and $-Catenin Mutations in Hemangiosarcomas from B6C3F1 Mice in the 2-Year Feed Study of o-Nitrotoluenea Exposure Concentration Tissue K-ras H-ras p53 $-Catenin (ppm) Codon 12 Codon 13 Codon 61 Codon 61 Exon 5 Exon 6 Exon 7 Exon 8 1,250 Skeletal Muscle —b — — — — Codon 190 — — — CAT to CGT (His to Arg) 1,250 Mesentery — — — — — — — — Splicing Intron 2,500 Skeletal Muscle — — — — — Codon 195 — — — GAA to GGA (Glu to Gly) 2,500 Skeletal Muscle — — — — — — — — — 5,000 Skeletal Muscle — — — — — — Codon 243 — Splicing Intron ATG to ATA (Met to Ile) 5,000 Skeletal Muscle — — — — — Codon 220 — — Deletions 25-36 CCC to TCC (Pro to Ser) 5,000 Skeletal Muscle — — — — — Codon 200 Codon 241 — Deletions 5-6 CCC 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 Intron TGT 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 504 357 TABLE M3 Summary of ras, p53, and $-Catenin Mutations in Hemangiosarcomas from B6C3F1 Mice in the 2-Year Feed Study of o-Nitrotoluene Exposure Concentration Tissue K-ras H-ras p53 $-Catenin (ppm) Codon 12 Codon 13 Codon 61 Codon 61 Exon 5 Exon 6 Exon 7 Exon 8 5,000 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 Intron CGG to CAG (Arg to Glu) 5,000 Subcutaneous Tissues — — — — — — — — Splicing Intron Total combined mutations 1/15 (7%) 0/15 (0%) 11/15 (73%) 7/15 (47%) Hemangiomas containing at least one mutation 13/15 (87%) a His=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=glutamine b No mutation identified
358 o-Nitrotoluene, NTP TR 504 
Acetaminophen 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: C23, 43% Chlorine 305

Chlorinated Paraffins: C12, 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 format. 

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 B1 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-Delta9-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.