NATIONAL TOXICOLOGY PROGRAM 

Technical Report Series 

No. 442 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF p-NITROBENZOIC ACID 

(CAS NO. 62-23-7) 

IN F344/N RATS AND B6C3Fl MICE 

(FEED STUDIES) 

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. 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. Selection per se is not an indicator of a 

chemical's carcinogenic potential. 

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

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

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

Management, NIEHS, P.O. Box 12233, MD A0-01, Research Triangle Park, NC 27709 (919-541-1371). 
NTP TECHNICAL REPORT 

ON THE 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF p-NITROBENZOIC ACID 

(CAS NO. 62-23-7) 

IN F344/N RATS AND B6C3F1 MICE 

(FEED STUDIES) 

NATIONAL TOXICOLOGY PROGRAM 

P.O. Box 12233 

Research Triangle Park, NC 27709 

December 1994 

NTP TR 442 

NIH Publication No. 95-3358 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Public Health Service 

National Institutes of Health 
2 p-Nitrobenzoic Acid, NTP TR 442 

CONTRIBUTORS 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

C.J. Alden, Ph.D. 

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

D.A~ Bridge, B.S. 

J.R. Bucher, Ph.D. 

J.K. Dunnick, Ph.D. 

S.L. Eustis, D.V.M., Ph.D. 

T.J. Goehl, Ph.D. 

J.R. Harley, D.V.M. 

J.K. Haseman, Ph.D. 

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

J.H. Roycroft, Ph.D. 

B.A. Schwetz, D.V.M., Ph.D. 

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

D.B. Waiters, Ph.D. 

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

Microbiological Associates, Inc. 

Conducted 14-day and 13-week studies, evaluated pathology 

findings 

L.T. Mulligan, Ph.D. 

B.S. Levine, D.Sc. 

L.H. Brennecke, D.V.M. 

R.M. Kovach, D.V.M. 

Southern Research Institute 

Conducted 2-year studies, evaluated pathology findings 

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

C. Lindamood III, Ph.D. 

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

H.D. Giles, D.V.M., Ph.D. 

R.B. Thompson, D.V.M., Ph.D. 

Experimental Pathology Laboratories, Inc. 

Provided pathology quality assurance 

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

B.F. Hamilton, D.V.M., Ph.D. 

Dynamac Corporation 

Prepared quality assurance audits 

S. Brecher, Ph.D., Principal Investigator 

NTP Pathology Working Group 

Evaluated slides, prepared pathology report on rats 

(02 April 1992) 

M.A. Stedham, D.V.M., M.S., Chair 

Pathology Associates, Inc. 

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

National Toxicology Program 

B.F. Hamilton, D.V.M., Ph.D. 

Experimental Pathology Laboratories, Inc. 

W. Macklin, D.V.M., Ph.D. 

Burroughs Wellcome 

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

National Toxicology Program 

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

National Toxicology Program 

Evaluated slides, prepared pathology report on mice 

(14 May 1992) 

M.A. Stedham, D.V.M., M.S., Chair 

Pathology Associates, Inc. 

S.D. Fitzgerald, D.V.M., Ph.D. 

Purdue University 

J. Fournie, Ph.D. 

Environmental Protection Agency 

J.R. Harley, D.V.M. 

National Toxicology Program 

B.F. Hamilton, D.V.M., Ph.D. 

Experimental Pathology Laboratories, Inc. 

W.E. Hawkins, Ph.D. 

Gulf Coast Research Laboratories 

D. Meuten, D.V.M., Ph.D. 

North Carolina State University 

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

National Toxicology Program 

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

National Toxicology Program 

Biotechnical Services, Inc. 

Prepared Technical Report 

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

P.R. Dennis, M.C.M. 

G. Gordon, M.A. 

L.M. Harper, B.S. 
3 

CONTENTS 

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

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

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

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

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

APPENDIX I Feed and Compound Consumption in the 2-Year Feed Studies 

APPENDIX J Ingredients, Nutrient Composition, and Contaminant Levels 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ............... 10 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE ................................... 11 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS ........... 12 

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

MATERIALS AND METHODS .................................................... 17 

RESULTS .................................................................... 27 

DISCUSSION AND CONCLUSIONS ................................................ 59 

REFERENCES ................................................................ 67 

of p-Nitrobenzoic Acid ............................................... 75 

of p-Nitrobenzoic Acid ............................................... 127 

of p-Nitrobenzoic Acid ........... ..................................... 167 

of p-Nitrobenzoic Acid ............................................... 199 

.APPENDIX E Genetic Toxicology ................................................. 241 

APPENDIX F Organ Weights and Organ-Weight-to-Body-Weight Ratios .................... 251 

APPENDIX G Hematology and Clinical Chemistry Results .............................. 263 

APPENDIX H Chemical Characterization and Dose Formulation Studies ................... 275 

of p-Nitrobenzoic Acid ............................................... 287 

in NIH-07 Rat and Mouse Ration ...................................... 293 

APPENDIX K Sentinel Animal Program ............................................ 299 
4 p-Nitrobenzoic Acid, NTP TR 442 
5 

ABSTRACT 

COOH 

p-NITROBENZOIC ACID 

CAS NO.62-23-7 

ChemicalFormula: C+H,NO, Molecular 167.12 Weight:

Synonyms: 4-Nitrobenzoic acid; nitrodracylic acid; p-nitrobenzenecarboq4ic acid; p-carboxynitrobenzene 

p-Nitrobenzoic acid is produced in large volumes for 

organic synthesis and as an intermediate in the 

manufacture of pesticides, dyes, and industrial solvents. Groups of male and female F344/N rats and 

B6C3Flmice were exposed to p-nitrobenzoic acid 

(>99% pure) in feed for 14 days, 13 weeks, or 

2 years for toxicity and carcinogenicitystudies. 

Genetic toxicology studies were conducted in in vim 

assayswith Salmonella typhimurium and cultured 

Chinese hamster ovary cells, and in in vivo studies of 

erythrocyte micronucleus formation in mice in the 

13-week study. 

14-DAY STUDY IN RATS 

Groups of five male and five female rats were given 

0, 2,500, 5,000, 10,000, 20,000, or 40,000ppm 

p-nitrobenzoic acid in feed for 14 days. All rats 

survived until the end of the study. Male and female 

rats given 20,000 and 40,000 ppm lost weight. The 

final mean body weights of 10,000,20,000, and 40,000 

ppmmales were 82%, 60%, or 52% thatofthe 

controls, and the final mean body weights of 10,000, 

20,000, and 40,000 ppmfemales were 87%, 68%, and 

65% that of the controls. There were no clinical 

findings that were characteristic of organ-specific 

. toxicity. 

Absolute and relative spleen weights were significantlyincreased in rats exposed to 10,000, 20,000, 

and 40,000 ppm. There were decreases in erythrocyte 

count and hemoglobin and hematocrit values and 

increasesinreticulocyte count, nucleated erythrocytes, and methemoglobin concentration that were 

most pronounced in the 20,000 and 40,000 ppm 

groups. Congestion of the spleen occurred in 

10,000ppmmales and in 20,000 and 40,000 ppm 

females. Hypertrophy of the follicular epithelium of 

the thyroid gland was present in male and female rats 

exposed to 10,000, 20,000, or 40,000 ppm p-nitrobenzoic acid, follicular while hyperplasia was

observedin the 40,000 ppmmales and females. 

Atrophy of the testis was observed in 20,000 and 

40,000 ppm males. Other lesions observed in 20,000 

and 40,000 ppm rats included atrophy of the thymus 

in males and atrophy of the ovary, bone marrow, and 

thymus in females. 

14-DAY STUDY IN MICE 

Groups of five male and five female mice were given 

0, 2,500, 5,000, 10,000, 20,000, or 40,000ppm 

p-nitrobenzoic acid in feed for 14 days. Three males 

and two femalesgiven40,000ppm died during the 

study. All other animals survived until the end of the 
6 p-Nitrobenzoic Acid, NTP TR 442 

study. Male mice given 20,000 and 40,000 ppm and 

femalesgiven20,000ppm lost weight. Meanbody 

weight,gains of 20,000 and 40,000 ppmmales and 

10,000, 20,000, and 40,000 ppm females were significantly lower than those of the controls. There were 

no clinical findings related to organ-specific toxicity 

although lethargy and ataxia were observedin 

40,000 ppm mice. 

Relative liver weights were significantly increased in 

20,000 and 40,000 ppm males and females and in 

10,000ppmfemales. Absolute and relative thymus 

weightsof20,000 and 40,000 ppmmales and of 

10,000,20,000, and 40,000 ppm femaleswere reduced. 

No significant differences in hematology parameters 

occurred in exposedmice. Testicular degeneration 

was observed in three 20,000 ppm and two

40,000ppmmales. Bone marrow hemorrhage and 

atrophy occurred in 40,000 ppm females. 

13-w~~~ STUDY IN RATS 

Groups of 10 male and 10 female rats were given 0, 

630,1,250,2,500,5,000, or 10,000 ppmp-nitrobenzoic 

acid in feed for 13 weeks resulting in approximate 

daily doses of 40,70, 160,310, or 660 mg/kg to males 

and 40, 80, 170, 340, or 680 mg/kg to females. All 

rats survived until the end of the study. Mean body 

weightgains and final mean bodyweightswere 

significantly less than those of the controls in 2,500, 

5,000, and 10,000ppmmales and in 5,000 and 

10,000 ppm females. There were no clinical findings 

related to organ-specific toxicity. 

Differences in spleen weights and hematology parameters characteristic of regenerative anemia were 

observed in males and females, primarily in groups 

given 10,000 ppm. The absolute and relative spleen 

weights were significantly increasedin10,000ppm 

males and females and the relative spleen weights 

were significantly increased in 5,000 ppm males and 

females. Methemoglobin, Heinz bodies, and reticulocyte counts were increased and erythrocyte counts, 

hemoglobin, and hematocrit values were decreased in 

10,000 ppm males and females. 

Congestion, pigmentation, and accumulation of 

macrophages in the spleen and pigmentation in the 

kidney occurred in 2,500, 5,000, and 10,000 ppm 

males. Congestion and pigmentation of the spleen 

occurred in 10,000 ppm females. A yellowish brown 

pigment (hemosiderin) in the spleen and kidney was 

associated with hemolytic anemia. Mild cytoplasmic 

hyaline droplet accumulation was present in renal 

tubule epithelial cells in 10,000 ppm males while 

karyomegalywas present in male and female rats 

exposed to 2,500, 5,000, and 10,000 ppm p-nitrobenzoic acid. A chemical-related testicular lesion, 

consisting of atrophy of the seminiferous tubules, 

occurred in 10,000 ppm males. 

U-WEEK STUDY IN MICE 

Groups of 10 male and 10 female mice were given 0, 

1,250, 5,000, 10,000, or 20,000 ppm p-nitrobenzoic 

acidinfeed for 13 weeks resulting in approximate 

dailydoses of 170, 330, 670,1,900, or 4,000 mg/kg 

bodyweight to males and 240, 460,970,2,500, or 

4,900 mgkg to females. All mice survived until the 

end of the study, except one 1,250 ppm female that 

was killed accidentally. Final mean body weights and 

mean body weight gains of all exposed males and of 

5,000, 10,000, and 20,000 ppm females were significantly lower than those of the controls. No clinical 

findings or differences in organ weights or histopathology related to organ-specific toxicity were 

observed in exposed mice. 

%YEAR STUDY IN RATS 

Groups of 60 male and 60 female rats were given 0, 

1,250,2,500, or 5,000 ppmp-nitrobenzoic acid in feed 

for 2 years. Ten males and 10 females from each 

exposure group were evaluated at 15 months. 

Survival, Body Weights, Feed Consumption, 

and Clinical Findings 

Two-year survival rates of 1,250 and 2,500 ppm males 

were similar to that of the controls. Two-year 

survival of 5,000 ppm males was marginally greater 

than that of the controls and was attributed in part to 

a decrease in the severity of nephropathy and a 

decrease in the incidence of mononuclear cell leukemia. Survival of exposed females was similar to that 

of the controls. Mean body weights of 5,000 ppm 

males were 2% to 8% lower than those of the controls through week 80. Final mean body weights of 

exposed males were similar to that of the controls. 

Mean body weights of 5,000ppm females were 2% to 

9% lower than those of the controls during the first 

year of the study and were 10% to 16% lower during 

the second year of the study. Final mean body 

weights of exposed females were 97% (1,250 ppm), 

92% (2,500 ppm), and 84% (5,000 ppm) that of the 
p-Nitrobenzoic Acid, NTP TR 442 7 

controls. Feed consumption by exposed males and 

females was similar to that by the controls. Dietary 

levels of 1,250,2,500, or 5,000 ppm p-nitrobenzoic 

acid delivered approximately 50, 100, or 210 mgkg 

bodyweight per day to males and 60, 125, or 

250 mgkg per day to females. There were no clinical 

findings attributable to organ-specific toxicity. 

Pathology Findings 

There were increases in the incidences of clitoral 

gland adenoma and of clitoral gland adenoma or 

carcinoma (combined) (4/50, 14/49,15/49,15/50)in 

exposedfemales. The incidences of clitoral gland 

adenoma or carcinoma (combined) in the exposed 

groups (29% to 31%) exceeded the historical control 

mean incidence (11%) and range (2% to 21%) in 

female F344/N rats in recent 2-year NTP feed studies. 

The increased incidences of clitoral gland neoplasms 

were considered to be some evidence of carcinogenic 

activity in female rats exposed top-nitrobenzoic acid. 

The incidences of hyperplasia of the clitoral gland in 

exposed females were marginally lower than that of 

the controls (10/50, 6/49, 6/49, 7/50). 

There was a chemical-related decrease in the severity 

of nephropathy in male rats. Male rat kidneys were 

examined using both single and step-section analyses, 

and the incidences of renal tubule neoplasms were 

not statistically greater than those of the controls. 

Mild hyaline droplet accumulation was observed in 

renal tubule epithelial cells in 10,000 ppm males in 

the 13-week study, but this effect was not severe 

enough to lead to a chemical-related neoplastic 

response in the 2-year study as has been observed 

with other chemicals. 

At the 15-month interim evaluation, hematologic 

parameters characteristic of a mild regenerative 

anemia and significant differences in spleen weights 

were noted in 5,000 ppm females. These differences 

included decreases in erythrocyte count, hemoglobin, 

and hematocrit, increases in spleen weights, and 

hemosiderin accumulation in splenic macrophages. 

At 2 years, significant decreases in the incidences of 

mononuclear cell leukemia were in observed 

5,000 ppm males and 2,500 and 5,000 ppm females 

(males:29/50,35/50,26/50,2/50;females:17/50, 

11/50, 3/50, 0/50). While the mechanism for this 

decrease is unknown, decreases in the incidence of 

mononuclear cell leukemia have also been observed 

in 2-year studies with other amine/nitro compounds. 

%YEAR STUDY IN MICE 

Groups of 60 male and 60 female mice were given 0, 

1,250,2,500, or 5,000ppmp-nitrobenzoic acid in feed 

for 2 years. Ten males and 10 females from each 

exposure group were evaluated at 15 months. 

Survival, Body Weights, Feed Consumption, 

and Clinical Findings 

Two-year survival rates of exposed mice were similar 

to those of the controls. Mean body weights of 

5,000 ppm males were 6% to 12% lower than those 

of the controls after week 17, and mean body weights 

of 5,000 ppm females were 12% to 24% lower than 

those of the controls after week 16. The final mean 

body weight of 5,000 ppm females was 19% less than 

that of the controls; final mean body weightsof males 

were similar to that of the controls. Feed consumption by exposed mice was similar to that by the controls. Dietary levels of 1,250,2,500, or 5,000 ppm 

p-nitrobenzoic acid delivered approximately 150,300, 

or 675 mg/kg per day to males and 170, 365, or 

905 mgkg per day to females. There were no clinical 

findings of organ-specific toxicity. No chemicalrelated effects on hematology parameters were noted 

at the 15-month interim evaluation. 

Pathology Findings 

There were no increases or decreases in neoplasms in 

male or femalemice that were considered to be 

related to chemical administration. 

GENETIC TOXICOLOGY 

p-Nitrobenzoic acid was mutagenic in Salmonella 

typhimurium strain TAlOO with and without S9. No 

mutagenicactivity was noted in strains TA98, 

TA1535, or TA1537, with or without S9. p-Nitrobenzoic acid induced sister chromatid exchanges and 

chromosomal aberrations in cultured Chinese hamster ovary cells in the absence of S9; with S9, results 

of both tests were negative. In vivo, no increase in 

micronuclei was observed in peripheral blood erythrocytes of male or female mice administered p-nitrobenzoic acid in dosed feed for 13weeks. 
8 p-Nitrobenzoic Acid, NTP TR 442 

CONCLUSIONS 

Under the conditions of these 2-year feed studies, 

there was no evidence of carcinogenic activity* of 

p-nitrobenzoic acid in male F344/Nrats exposed to 

1,250,2,500, or 5,000 ppm. There was some evidence 

of carcinogenic activity of p-nitrobenzoic acid in 

female F344/N rats based on increases in the incidences of clitoral gland adenoma and of clitoral gland 

adenoma or carcinoma (combined). There was no 

evidence ofcarcinogenic activity ofp-nitrobenzoic acid 

inmale or, female B6C3Fl miceexposed to 1,250, 

2,500, or 5,000 ppm. 

There were chemical-related decreases in the incidences of mononuclear cell leukemia in exposed male 

and female rats. p-Nitrobenzoic acid causedmild 

hematologic toxicity in female rats. 

* Explanation of Levels of Evidence of Carcinogenic Activity ison page 10. A summary of the Technical Reports Review 

Subcommittee comments and the public discussion on this Technical Report appears on page 12. 
p-Nitrobenzoic Acid, NTP TR 442 9 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studiesof p-Nitrobenzoic Acid 

F344/N Rats 

Male Female 

F344/N Rats 

Male 

B6C3Fl Mice 

Female 

B6C3Fl Mice 

Doses 

100, or 210 mgkg/day) 

(approximately 50, 

5,000 ppm in feed 

0, 1,250, 2,500,or 

125, or 250 mgkg/day) 

(approximately60, 

5,000ppm in feed 

0, 1,250, 2,500,or 

675 mg/kg/day) 

150, 300, or 

(approximately 

5,000ppm in feed 

0, 1,250, 2,500,or 

905 mgkglday) 

170, 365,or 

(approximately 

5,000ppm in feed 

0, 1,250, 2,500, or 

Body weights 

to control 

Dosed groups similar 

control 

groups lower than 

High- and mid-dose 

lower than control 

High-dose group 

lower than control 

High-dose group 

rates 

2-Year survival 

13/50, 21/50 

12/50, 13/50, 

2/50, 21/50 

27/50, 23/50, 

39/50, 44/50 

39/50, 36/50, 

33/50, 30/50 

38/50, 36/49, 

effects 

Nonneoplastic 

None 

toxicity 

Mild hematologic None None 

Neoplastic effects None 

14/49, 15/49, 15/50> 

(combined) (4/50, 

or carcinoma 

5/49, 4/50), adenoma 

carcinoma (1/50, 2/49, 

10/49, 12/50), 

adenoma (41.50,12/49, 

Clitoral gland: None None 

incidences 

Decreased 

35/50, 26/50, 2/50) 

leukemia (29/50, 

Mononuclear cell 

11/50, 3/50,0/50) 

leukemia (17/50, 

Mononuclear cell None None 

activity 

of carcinogenic 

Level of evidence 

No evidence Some evidence No evidence No evidence 

< 

Genetic toxicology 

Micronuclei in mouse peripheral blood cells: 

Chromosomal aberrations 

Sister chromatid exchanges 

Salmonella typhimuriurn gene mutation: 

Cultured Chinese hamster ovary cellsin vitro: 

Cultured Chinese hamster ovary cellsin vitro: 

TA1535, and TA1537, with and without S9 

Positive in strain TAlOO with and without S9; negative in strains TA98, 

Positive without S9; negative with S9 

Negative at 13weeks 

Positive without S9; negative with S9 
10 p-Nitrobenzoic Acid, NTP TR 442 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTMTY 

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. 

l 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. 

l 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. 

l JQpivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal 

increase of neoplasms that may be chemical related. 

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

increases in malignant or benign neoplasms. 

l 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. 

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: 

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

l progression (or lack thereot) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; 

l some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, 

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

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

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

organ or tissue; 

l latency in tumorinduction; 

l multiplicity in site-specificneoplasia; 

l metastases; 

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

(same lesion in another sex or species); 

l presence or absence of doserelationships; 

l statistical significance of the observed tumor increase; 

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

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

l structure-activitycorrelations;and 

l in somecases,genetictoxicology. 
p-Nitrobenzoic Acid, NTP TR 442 11 

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 p-nitrobenzoic acid 

on June 22, 1993, 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 NTP studies: 

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

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

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

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

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

Curtis D. Klaassen, Ph.D., Chair 

Department of Pharmacology and Toxicology 

University of Kansas Medical Center 

Kansas City, KS 

Paul T. Bailey, Ph.D. 

Environmental and Health Sciences Laboratory 

Mobil Oil Corporation 

Princeton, NJ 

Louis S. Beliczky, M.S., M.P.H. 

Department of Industrial Hygiene 

United Rubber Workers International Union 

Akron,OH 

Arnold L. Brown, M.D. 

University of Wisconsin Medical School 

Madison, WI 

Kowetha A. Davidson, Ph.D. 

Health and Safety Research Division 

Oak Ridge National Laboratory 

Oak Ridge,TN 

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

Medical Research Division 

American Cyanamid 

Pearl River, NY 

Daniel S. Longnecker, M.D.* 

Department of Pathology 

Dartmouth Medical School 

Lebanon, NH 

* Didnot attend 

Louise Ryan, Ph.D. 

Division of Biostatistics 

Harvard School of Public Health and 

Dana-Farber Cancer Institute 

Boston, MA 

Ellen K. Silbergeld, Ph.D.* 

University of Maryland Medical School 

Baltimore, MD 

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

Department of Pharmacology 

Howard University College of Medicine 

Washington, DC 

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

Department of Safety Assessment 

Merck Research Laboratories 

West Point, PA 

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

National Cancer Institute 

Frederick, MD 

Lauren Zeise, Ph.D. 

Reproductive and Cancer Hazard Assessment Section 

California Environmental Protection Agency 

Berkeley, CA 
12 p-Nitrobenzoic Acid, NTP TR 442 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMIWE COMMENTS 

OnJune 22, 1993, the draft Technical Report onthe 

toxicology and carcinogenesis studies of p-nitrobenzoic acid received public review by the National 

Toxicology Program 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 p-nitrobenzoic acid by 

discussing the uses and rationale for study, describing 

the experimental design, reporting on survival and 

bodyweighteffects, and commenting on chemicalrelated neoplastic lesions in female rats and nonneoplastic lesions in male (nephropathy) and female 

(hematologic toxicity) rats. Additional step-sections 

of the kidney were performed in male rats. The proposed conclusions were no evidence of carcinogenic 

activity in male F344/Nrats, some evidence of carcinogenic activity in female F344/Nrats, and no evidence 

of carcinogenic activity in male or female B6C3Fl 

mice. 

Dr. Brown, a principal reviewer, agreedwith the 

proposed conclusions. He asked for comment on the 

seeminglyparadoxical decrease in the incidence of 

mononuclear cell leukemia in exposed rats and the 

increased weight of the spleen. Dr. Ward noted that 

there was hematopoietic toxicity associated with the 

chemical and speculated that the stem cell in the 

bone marrow or spleen from which the leukemia 

derives may be one of the targets of the chemical 

resulting in an inhibition of leukemogenesis. 

Dr. van Zwieten, the second principal reviewer, 

agreed with the proposed conclusions. He asked for 

substantiation of the conclusion that preputial gland 

and clitoral gland neoplasms were potentially lethal, 

because, in his experience, these neoplasms tend to 

be quite small and well circumscribed. Dr. S.L. 

Eustis, NIEHS, responded that the preputial gland 

neoplasms are not lethal in the sense of causing the 

animal's death, but as they get quite large with some 

becoming ulcerated, the animals are killed. Dr. J.K. 

Haseman, NIEHS, added that if a neoplasm were 

incidental, one would expect it to be more or less 

evenly distributed among the animals that died 

naturally and those that survived. However, in this 

study, the likelihood of observing a preputial gland 

neoplasmin an animal that died early was almost 

three times as high as in a surviving animal. 

Dr. Ryan, the third principal reviewer, deferred her 

opinion of the proposed conclusions pending further 

discussion of exposure-related effects on clitoral 

gland and preputial gland lesions. She said there 

were inconsistencies in how body weight differences 

were discussed. For instance, decreased body weight 

in rats is offered as a possible explanation for the 

exposure-related decrease in leukemia. On the other 

hand, lack of an exposure-response for clitoral gland 

neoplasms was the main reason for some evidence 

rather than clear evidence for female rats, but was 

likely due, in her opinion, also to decreased body 

weight. Dr. Dunnick said the conclusion in female 

rats was based primarily on there being increases in 

neoplasms, mostly adenomas, at all three exposure 

levels. She agreed that body weight can affect the 

incidence of neoplasms, but the decrease in leukemia 

was believed to be more of a chemical effect than a 

bodyweighteffect.Based on preputial gland neoplasms, Dr. Haseman said it was a close call between 

no evidence and equivocal evidence of carcinogenic 

activityin male rats. Dr. Eustis noted that the 

incidence of preputial gland carcinoma at the highest 

exposure level was within the historical control range. 

Dr. Ward askedfor comment on the presence of 

hyaline droplets in the kidneys of rats in subchronic 

studies and whether they were associated with a,-

globulin accumulation. Dr. Eustis said there was no 

evidencefor accumulation of a,-globulin in this 

study. 

Dr. Brown moved that the Technical Report on 

p-nitrobenzoic acid be accepted with the revision 

discussed and with the conclusions as written for 

male rats and male and female mice, no evidence of 

carcinogenic activity, and for female rats, some evidence of carcinogenic activity. Dr. Taylor seconded 

the motion, which was accepted unanimously with ten 

votes. 
13 

INTRODUCTION 

COOH 

NO2 

p-NITROBENZOIC ACID 

CAS NO. 62-23-7 

Chemical Formula: qH,NO, Molecular Weight: 167.12 

Synonyms: 4-Nitrobenzoic acid; nitrodracylic acid;p-nitrobenzenecarboxylicacid; p-carboxynitrobenzene 

CHEMICAL AND PHYSICAL PROPERTIES 

p-Nitrobenzoic acid is a yellow-to-white crystalline 

material with no odor. Ithas a density of 1.61 at 

20"C, and a melting point of 242"C. It is insoluble 

in water and petroleum ether; slightly soluble in acetone, benzene, and carbon disulfide; and soluble in 

methyl alcohol (1 g/110mL) andethanol 

(1 dl10 mL). The pK, ofp-nitrobenzoic acid is 3.4 

(Sax, 1979; Merck Index,1983;Lide, 1992). 

USE AND HUMAN EXPOSURE 

p-Nitrobenzoic acid is used in organic synthesis, in 

the manufacture of intermediates, and as a reagent 

for alkaloids and thorium (Merck Index, 1983). 

Nitrobenzoates are usedin the manufacture of 

pesticides,dyes,explosives, and industrial solvents 

(Groenewegen et aZ., 1992). Exposure to p-nitrobenzoic acid may occur through exposure to other 

chemicals that are metabolized or hydrolyzed to 

p-nitrobenzoic acid, including p-nitrobenzoyl 

chloride (Radding, 1977), p-nitrotoluene (Chism 

et al., 1984), and 5-(4-nitrophenyl)-2,4-pentadienal 

(spy dust) (Burka et aZ., 1987). 

p-Nitrobenzoic acid was not found in a survey of 

717 hazardous waste sites (USEPA, 1987). p-Nitrobenzoic acid is metabolized under aerobic and 

anaerobic conditions by bacteria, as well as when 

mixedwith a representative municipal sewage 

sample, suggesting that under natural conditions 

bacteria would metabolize the chemical (Hallas and 

Alexander, 1983). 

The U.S. International Trade Commission (USITC) 

did not report the production volume for p-nitrobenzoicacid or p-nitrobenzoyl chloride for 1988 

(USITC, 1989). Other sources estimate the production of p-nitrobenzoic acid at 450 to 900 tons per 

year (4 to 8 x lo6 kg/year), the production of 

p-aminobenzoic acid, a major metabolite of p-nitrobenzoic acid, at 250 tonsbear (2 x lo6 kg/year) 

(Kirk-Othmer, 1978),and the production of p-nitrobenzoyl chloride at 3 x lo6 kg/year (NCI, 1980). The 

National Institute for Occupational Safety and 

Health (NIOSH) has estimated thatthereare 

42,700workers potentially exposed top-nitrobenzoic 

acid in 16 different industries (NIOSH, 1993). 

ABSORPTION, DISTRIBUTION,

METABOLISM, AND EXCRETION 

Experimental Animals 

The metabolism of nitroaromatic compounds varies 

with species of animal and with the isomeric configuration of the chemical, as has been noted for some 

representative nitroaromatic chemicals such as 

nitrobenzene and 0-,m-,andp-nitrotoluene (Rickert, 

1987;NTP, 1992). One common pathway for metabolism of nitroaromatic compounds is reduction of the 

nitro groups (Rickert, 1987). 
14 

Nitroreductase activities are found both in mammalian tissues and in gastrointestinal microflora, and 

both can contribute to the eventual metabolism of 

nitroaromatic compounds (Zachariah and Juchau, 

1974). In mammalian species, two nitro-reducing 

systemshave been characterized in the liver, 

including one associated with the endoplasmic 

reticulum of the liver and one with the soluble 

cellular fractions (Carlson and Dubois, 1970; 

Mitchard, 1971). Escherichia coli and other bacteria 

contain various enzymes capable of reducingp-nitrobenzoic acid to p-aminobenzoic acid(Saz and 

Martinez, 1956; Thijssen and Henderson, 1973). 

Intestinal microbial flora appear to be responsible 

for at least some of the in vivo reduction ofp-nitrobenzoic acid because the intestinal contents of rats 

receiving antibiotics indicated diminished reduction 

ofp-nitrobenzoic acid (Zachariah and Juchau, 1974; 

Gardner and Renwick, 1978). Germ-free rats

converted about 1% of p-nitrobenzoic acid to 

p-aminobenzoic acid, while conventional rats 

converted 25% to p-aminobenzoic acid (Wheeler 

et al., 1975). 

In addition to reduction of the nitro group, p-nitrobenzoic acid metabolism may occur by a number of 

pathways involving conjugation of the carboxylic acid 

p-Nitrobenzoic Acid, NTP TR 442 

group with glycine or glucuronic acid and reduction 

to p-aminobenzoic acid, which may then be conjugated at the carboxylic acid group or acetylated at 

the amino substituent (Williams, 1959). 

Nitroaromatic compounds are characteristically toxic 

to the hematopoietic system (NTP, 1992). Aromatic 

and heterocyclic nitro compounds require reduction 

of the nitro group for the expression of these toxicologicactivities(McCalla and Voutsinos, 1974; 

Wheeler et al., 1975; Reddyet al., 1976). A common 

feature of chemicals that cause hematopoietic toxicity 

characterized by methemoglobin formation is a free 

aromatic amine functional group. Hydroxylation of 

this aromatic amine group to a phenylhydroxylamine 

is thought to account for the formation of methemoglobinemia and the subsequent hematologic toxicity 

(Bus and Popp, 1987). This is supported by studies 

that show that hydroxylamino compounds produce 

methemoglobin in vivo and in vitro, and arylamines 

or nitrobenzenes themselves cannot oxidize hemoglobin in vitro (Facchini and Griffiths, 1981). Using 

liver homogenates from Wistar rats, Kat0 etal. 

(1969) identified p-hydroxylamino benzoic acid as a 

metabolite of p-nitrobenzoic acid, and a metabolic 

scheme (Figure 1) for p-nitrobenzoic acid has been 

proposed (Gillette et al., 1968; Mitchard, 1971). 

HOOC *NO, - HOOC eHNOH - HOOC NH, - Conjugation

p-Nitrobenzoic p-Hydroxylamino Acid Acid p-Aminobenzoic

Benzoic Acid 

FIGURE1 

Metabolic Pathwayof p-Nitrobenzoic Acid 

[Proposed by Gillette et al. (1968) and Mitchard (1971)l 
Introduction 

Quantitative information on pharmacokinetics, 

distribution, and elimination of metabolites of 

p-nitrobenzoic acid in vivo is limited. After an oral 

or intraperitoneal dose of 25 mg ['4C]-p-nitrobenzoic 

acid, female Wistar rats excreted 83% to 94% of the 

radiolabel in the urine within 24 hours. Urine 

metabolites were 2% free p-aminobenzoic acid, 

18% conjugated aminobenzoic acid, 42% p-nitrobenzoic acid, and 13% conjugatedp-nitrobenzoic acid 

(Gardner and Renwick, 1978). Studies to quantify 

the urinary metabolites ofp-nitrobenzoic acid in the 

F344 rat and B6C3Fl mouse have not been conducted. 

Pharmacokinetic studies in marmosets receiving oral 

doses of 4-nitro[carbo~y-'~C]benzoic acid 

(0.4 mmolkg) found that peak blood levelswere 

reached in 30 to 40 minutes and the terminal halflife of the chemical in the blood was estimated at 

1hour. Distribution and elimination of metabolites 

could not be determined from these studies (Kuzniar 

and James, 1981). 

Humans 

No information onthe absorption, distribution, 

metabolism, or excretion of p-nitrobenzoic acid in 

humans was found in the literature. 

TOXICITY 

Experimental Animals 

Methemoglobin formation and hematopoietic toxicity 

are found after administration of aromatic nitro and 

amino compounds, and this toxicity is often more 

severe in rats than in mice (Beard and Noe, 1981; 

Rickert, 1987; NTP, 1992). For example, in studies 

of o-,m-,andp-nitrotoluene hematologic toxicity was 

characterized by increased methemoglobin, Heinz 

body formation, and hematopoiesis and by hemosiderin deposition and congestion in the spleen of 

rats. 

p-Nitrobenzoic acid was reported to have LD,, values 

of 1.47,0.88, and 0.77 g/kg in female Swiss mice after 

oral, intraperitoneal, and intravenous administration, 

respectively. Theoral LD,,was1.96g/kg and the 

intravenous LD,, was 1.21 g/kg in female Wistarrats. 

Rats receiving LD,, levels ofp-nitrobenzoic acid had 

liver infiltration with redbloodcells and myeloid 

metaplasia of the red pulp of the spleen (Caujolle 

et al., 1966). 

15 

Humans 

No information on the toxicity ofp-nitrobenzoic acid 

in humans was found in the literature. 

REPRODUCTIW AND DEVELOPMENTAL 

TOXICITY 

Experimental Animals 

Chapin et al. (1987) evaluated the effects ofp-nitrobenzoic acid on sperm morphology and vaginal 

cytologyin rats and mice exposed to 0, 630 (rats), 

1,250,2,500, 5,000, 10,000, or 20,000 (mice) ppm 

p-nitrobenzoic acid in feed for 13weeks. Final body 

weights of 5,000 and 10,000 ppm male rats were 8% 

to 16% lower than that of the controls. The sperm 

count and right caudal, epididymal, and testis weights 

were reduced in 10,000 ppm male rats. Final body 

weights of 10,000 and 20,000 ppm male mice were 

14% to 31% lower than those of the controls. In 

mice 20,000 receiving ppm, the right caudal,

epididymal, and testis weights were reduced, but the 

sperm count was not significantly reduced. At lower 

exposure levels, there were no chemical-related 

effects in male rats and only minor effects in male 

mice.Significantly reduced body weightswere 

observed10,000ppm in female rats and in

20,000 ppm female mice. Some of these females had 

prolonged estrous cycles that were apparently related 

to the reduced body weights (Chapin et al., 1987). 

Continuous breeding studies were conducted in Swiss 

(CD-l@) mice exposed to 7,500 or 15,000 ppm 

p-nitrobenzoic acid ad libitum in feed for a 7-day precohabitation period followed by a 98-day cohabitation period (Hope et al., 1990). Final body weights 

of 15,000 ppm malesand females were 93% and 88% 

that of the corresponding control groups, respectively. Feed consumption was similar between 

exposed and control groups. Pairs of mice exposed 

to 7,500 and 15,000 ppm had fewer litters and fewer 

live pups per litter and their pups weighed less than 

those of pairs receiving control feed. Crossover 

matings of exposed F, females to control males also 

resulted in fewer live pups per litter and lower pup 

weights, indicating that reproductive toxicity was 

primarily due to effects in females. Hope et al. 

(1990)concluded thatthe general toxic effects of 

p-nitrobenzoic acid (as measured by decreases in 

bodyweight of exposed animals) were not severe 

enough to cause impairment of fertility and reproduction, and the studies did not identify the mechanism responsible for this toxicity. 
16 

Humans 

No information on reproductive and developmental 

toxicity of p-nitrobenzoic acid in humans was found 

in the literature. 

CARCINOGENICITY 

Experimental Animals 

No studies describing the carcinogenic potential of 

p-nitrobenzoic acid in experimental animals were 

found in the literature. 

Humans 

No published information on carcinogenic potential 

of p-nitrobenzoic acid in humans is available. 

GENETICTOXICITY 

p-Nitrobenzoic acid was positive, in the absence of 

S9 activation, in the Bacillus subtilis rec assay for 

growth inhibition due to DNA damage (Shimizu and 

Yano, 1986), and it induced gene mutations in 

Salmonella typhimurium, with and without S9 (Chiu 

et al., 1978; Sundvall et al., 1984; Shimizu and Yano, 

1986; Zeiger et al., 1987; Dellarco and Prival, 1989). 

No induction of unscheduled DNA synthesis was 

noted in rat hepatocytes treated in vino with up to 

1,000 nmol p-nitrobenzoic acid/mL (Probst et al., 

1981). Unpublished NTP data show that p-nitrobenzoic acid induces sister chromatid exchanges and 

chromosomal aberrations in cultured Chinese hamster ovary cells.However, no increase in the frequency of micronucleated erythrocytes was observed 

in the peripheral blood of male and female mice 

administeredp-nitrobenzoic acid in feed for 13weeks 

(Appendix E). 

Thestructural analogue, m-nitrobenzoic acid, was 

also positive in the B. subtilis rec assay (Shimizu and 

p-Nitrobenzoic Acid, NTP TR 442 

Yano, 1986) and S. typhimurium gene mutation 

assays (Chiu et al., 1978; Sundvall et al., 1984; 

Shimizu and Yano, 1986; Zeiger et al., 1987). 

Unpublished NTP data show no induction of chromosomal aberrations or sister chromatid exchanges 

in cultured Chinese hamster ovary cells and no 

increase in the frequency of micronucleated erythrocytes in the blood of male or female mice receiving 

m-nitrobenzoic acid in feed for 13weeks. o-Nitrobenzoic acid did not induce chromosomal aberrations 

in cultured Chinese hamster ovary cells, but it did 

induce sister chromatid exchanges (NTP, unpublished 

data) and, like the p- and m-isomers, it was mutagenic in the S. typhimuriumassay (Zeiger et al., 

1987). 

STUDYRATIONALE 

p-Nitrobenzoic acid is a hydrolysis product ofp-nitrobenzoyl chloride. p-Nitrobenzoyl chloride was

originally nominated for testing by the National 

Cancer Institute because it is an acyl chloride and 

aromatic nitro compound with a large import volume 

(10,000kgjyear).However, because p-nitrobenzoyl 

chloride is unstable in feed and undergoes rapid 

hydrolysis top-nitrobenzoic acid,p-nitrobenzoic acid 

was selected for study. p-Nitrobenzoic acid was 

selected because of workplace exposure through its 

use in manufacturing chemical intermediates, because 

it is a metabolite of other chemicals, and because of 

the lack of existing information on its chronic toxic 

and carcinogenic effects. 

Interest in the potential carcinogenicity of p-nitrobenzoic acid is also based on its structure as a single, 

aromatic, nitro compound and the finding that other 

members of this chemical class are carcinogenic in 

rodents (Clayson and Garner, 1976; Ashby and 

Tennant, 1991). 
17 

MATERIALS AND METHODS 

PROCUREMENT AND 

CHARACTERIZATION 

OF ~NITROBENZOIC ACID 

p-Nitrobenzoic acid was obtained from E.I. du Pont 

de Nemours and Company, Inc. (Wilmington, DE), 

in one lot (40). Identity, purity, and stability analyses 

were conducted by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO) 

(Appendix H). Reports on the analyses performed 

in support of the p-nitrobenzoic acid studies are on 

file at he National Institute of Environmental 

Health Sciences. 

The chemical, a light yellow, crystalline solid, was 

identified as p-nitrobenzoic acid by infrared, ultraviolethisible, and nuclear magnetic resonance spectroscopies. Purity was determined by elemental 

analyses,Karl Fischer water analysis, functional 

group titration, thin-layer chromatography, and 

high-performance liquid chromatography. Elemental 

analyses for carbon, hydrogen, and nitrogen were in 

agreement with the theoretical values for p-nitrobenzoic acid. Karl Fischer indicated analysis 

0.08% water. Functional group titration indicated a 

purity of 100.1%. Thin-layer chromatography using 

two systems detected one major spot and one trace 

impurity. No impurities with areas greater than 

0.1% relative to the major peak area were observed 

using high-performance liquid chromatography. The 

overall purity was determined to be greater than 

99%. 

Stability studies performed using high-performance 

liquid chromatography indicated that p-nitrobenzoic 

acid was stable when stored in the dark for 2 weeks 

at temperatures up to 60" C. The study laboratory 

stored the bulk chemical in sealed containers, protected from light, at room temperature. Purity and 

stability were monitored during the 2-year study by 

high-performance liquid chromatography and functional group titration. No degradation of the bulk 

chemical was observed. 

PREPARATIONANDANALYSIS 

OF DOSEFORMULATIONS 

The dose formulations were prepared once in the 

14-day studies, every 2 weeks in the 13-week studies, 

and weekly in the 2-year studies by mixing p-nitrobenzoic acid and feed (Table Hl). Homogeneity and 

stability studies of the 400 ppm concentration were 

performed by Midwest Research Institute using highperformance liquid chromatography. Homogeneity 

was confirmed, and the stability of the dose formulations when stored in the dark at room temperature 

was confirmed for at least 3 weeks. Dose formulations open to air and light were stable for 1week. 

Periodic analyses of the dose formulations ofp-nitrobenzoic acid were conducted at the study laboratory 

and analytical chemistry laboratory using high-performanceliquidchromatography. Dose formulations 

wereanalyzed once during the 14-day studies and 

werewithin 10% of the target concentrations 

(Table H2). Dose formulations for the 13-week 

studies were analyzedpre-study, during week 1, at 

study mid-point, and at the final mix (Table H3). 

During the 2-year studies, the dose formulations 

wereanalyzedapproximatelyevery two months 

(Table H4). All dose formulations were within 10% 

of the target concentrations during the 13-week 

studies; 95% (160/168) of the formulations were 

within 10% of the target concentrations during the 

2-year studies. Results of the periodic referee 

analyses performed by the analytical chemistry 

laboratory were in good agreement with the results 

obtained by the study laboratory (Table H5). 

14-DAY STUDIES 

The 14-day studies were conducted to evaluate the 

cumulativetoxiceffectsof repeated exposure to 

p-nitrobenzoic acid and to determine the appropriate 

doses to be used in the 13-week studies. 

Male and female F344N rats and B6C3Fl mice were 

obtained from Frederick Cancer Research Facility 
18 

(Frederick, MD). At receipt, the animals were 

4 weeks old. The rats were quarantined for 16 days 

before dosing began; the mice were quarantined for 

15 days. Before the beginning of the studies, two 

male and two female rats and mice were randomly 

selected for parasite evaluation and gross observation 

for evidence of disease. 

Groups of five male and five female rats and mice 

received 0, 2,500, 5,000, 10,000, 20,000, or 

40,000 ppm p-nitrobenzoic acid in feed for 14 days. 

Water and feed were available ad libitum. Feed 

consumption was measured twice weekly for rats and 

weekly for mice. Clinical observations were recorded 

twice daily. Animals were weighed at the beginning 

of the studies and weekly thereafter. Rats were 

housed five per cage; mice were housed individually. 

At the end of the studies, blood was collected from 

theorbital sinus for hematology analyses. Automated hematologic determinations, except platelet 

counts, were performed using a Baker Series 7000 

cell counter; platelet counts were determined using 

a Baker Series 810 whole blood platelet analyzer 

(Baker Instruments, Allentown, PA). Reagents were 

obtained from Baker Instruments. The clinical 

pathology parameters measured are listed in Table 1. 

The brain, heart, right kidney, liver,lungs, spleen, 

right testis, and thymus of all surviving animals were 

weighed. A necropsy was performed on all animals. 

Tissues for microscopic examination were fixed and 

preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned 

to a thickness of 6 pm, and stained with hematoxylin 

and eosin. A complete histopathologic examination 

was performed on all controls and all 40,000 ppm 

animals at the end of the studies. Table 1 lists the 

tissues and organs routinely examined. 

ISWEEK STUDIES 

The 13-week studies were conducted to evaluate the 

cumulative toxic effects of repeated exposure to 

p-nitrobenzoic acid and to determine the appropriate 

doses to be used in the 2-year studies. 

Male and female M44/N rats and B6C3Fl mice were 

obtained from Simonsen Laboratories, Inc. 

(Gilroy, CA). At receipt, the animals were 3 to 

p-Nitrobenzoic Acid, NTP TR 442 

4 weeks old. The rats were quarantined for 13 days 

before dosing began; the mice were quarantined for 

11 days. Before the beginning of the studies, five 

male and five female rats and mice were randomly 

selected for parasite evaluation and gross observation 

for evidence of disease. At the end of the studies, 

serologic analyses were performed on five male and 

five female control mice using the protocols of the 

NTP Sentinel Animal Program (Appendix IC). 

Groups of 10 male and 10 female rats received 0, 

630,1,250,2,500, 5,000, or 10,000ppm p-nitrobenzoic acid in feed for 13 weeks. Groups of 

10 male and 10 female mice received0, 1,250,2,500, 

5,000, 10,000, or 20,000 ppm p-nitrobenzoic acid in 

feed for 13 weeks. The brain, heart, right kidney, 

liver,lungs, spleen, right testis, and thymus of all 

surviving animals were weighed. 

Water and feed were available ad libitum. Feed 

consumption was measured weekly. Clinical observations were recorded twice daily. Animals were 

weighed at the beginning of the studies and weekly 

thereafter. Rats were housed five per cage; mice 

were housed individually. 

Special study groups of 10 male and 10 female rats 

received 0, 630, 2,500, or 10,000 ppmp-nitrobenzoic 

acid for 13weeks. On days 7,30, 60, and 90, blood 

sampleswere collected from the orbital sinus for 

hematology and clinical chemistryanalyses. Hematologyanalyses were performed as in the 14-day 

studies. Clinicalchemistry parameters were measured using a Centrifichem-400 chemistry analyzer 

(Baker Instruments). Reagents were obtained from 

Baker Instruments or Sigma Diagnostics (St. Louis, 

MO). The clinical pathology parameters measured 

are listed in Table 1. 

A necropsy was performed on all core study animals. 

Tissues for microscopic examination were fixed and 

preservedin 10%neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned 

to a thickness of 6 pm, and stained with hematoxylin 

and eosin. A complete histopathologic examination 

was performed on all controls, all animals dying 

before the end of the studies, and all 10,000 ppm rats 

and 20,000 ppmmicesurviving to the end of the 

studies. Table 1lists the tissues and organs routinely 

examined. 
Materials and Methods 19 

2-Ym STUDIES 

Study Design 

Groups of 60 male and 60 female rats and mice 

received 0, 1,250,2,500, or 5,000 ppmp-nitrobenzoic 

acid in feed for 103 weeks. Ten male and 10 female 

rats and mice from each group were evaluated at 

15 months. 

Source and Specification of Animals 

Male and female F344/N rats and B6C3Fl mice were 

obtained from Taconic Laboratory Animals and 

Services (Germantown, NY) for use in the 2-year 

studies. The animals were quarantined for 12 days 

before the beginning of the studies. Five male and 

five female rats and mice were selected for parasite 

evaluation and gross observation of disease. 

Serology samples were collected for viral screening. 

Rats and mice were approximately 6 weeks old at the 

beginning of the 2-year studies. The health of the 

animals was monitored during the course of the 

studies according to the protocols of the NTP 

Sentinel Animal Program (Appendix K). 

Animal Maintenance 

Rats were housed five per cage; mice were housed 

individually. Feed and water were available

ad libitum. Feed consumption was measured every 

4 weeks.Cages were rotated twice a week for rats 

and once a week for mice; racks were rotated every 

twoweeks during the studies. Further details of 

animal maintenance are given in Table 1. Information on feed composition and contaminants is provided in Appendix J. 

Clinical Examinations and Pathology 

All animals were observed twice daily.Clinical 

observations and body weights werecorded at study 

initiation, weekly for 13weeks, and monthly thereafter. Blood samples were collected from the retroorbital sinus at the 15-month interim evaluations for 

hematologyanalyses. Automated determinations 

were performed using an Ortho ELT-8 hematology 

analyzer (Ortho Instruments, Westwood, MA). 

Methemoglobin was measured using the Roche 

Cobas Fara (Roche Diagnostic Systems, Inc., 

Montclair, NJ). Reagents were obtained from the 

instrument manufacturer. The clinical pathology 

parameters measured are listed in Table 1. The right 

kidney, liver, and spleen wereweighed at the 

15-month interim evaluations. 

A necropsy was performed on all animals. At 

necropsy, all organs and tissues were examined for 

grosslesions, and all major tissues were fixed and 

preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned, 

and stained with hematoxylin and eosin for microscopic examination. Complete histopathologic 

examinations were performed on all animals. Tissues 

examined are listed in Table 1. 

Microscopic evaluations were completed by the study 

laboratory pathologist, and the pathology data were 

entered intothe Toxicology Data Management 

System. The microscope slides, paraffin blocks, and 

residual wet tissues were sent to the NTP Archives 

for inventory, slideblock match, and wet-tissue audit. 

The slides, individual animal data records, and 

pathology tables were evaluated by an independent 

pathologyqualityassessment laboratory. The individual animal records and tables were compared for 

accuracy, the slide and tissue counts were verified, 

and the histotechnique was evaluated by the quality 

assessment laboratory. The quality assessment 

pathologist microscopically reviewed the clitoral 

gland, kidney, liver, preputial gland, spleen, stomach, 

and uterus of all rats to confirm the incidences of 

neoplasms and nonneoplastic lesions. For mice, the 

qualityassessment pathologist reviewed the forestomach, kidney, liver, lung, and thyroid gland to 

confirm the incidences of neoplasms and nonneoplastic lesions. In addition, each tissue with a 

neoplasmdiagnosisfrom all rats and mice was 

microscopically reviewed. 

The quality assessment report and slides were submitted tothe NTPathology Working Group 

(PWG) chair, who reviewed representative examples 

of potential chemical-related lesions including 

neoplasms of the clitoral gland, kidney, liver, preputial gland, spleen, and thyroid gland from rats; the 

forestomach, kidney, liver,lung, and thyroid gland 

frommice; and any other tissues when there was 

disagreement in diagnosis between the laboratory and 

qualityassessment pathologist. Examples of disagreements in diagnoses between the laboratory and 

quality assessment pathologist or lesions of general 

interest were presented by the chair to the PWG for 

review. The PWG consisted of the quality assessment pathologist and other pathologists experienced 

in rodent toxicologic pathology. This group examined the tissues without knowledge of dose groups or 

previously rendered diagnoses. When the PWG 
20 

consensus differed from the opinion of the laboratory 

pathologist, the diagnosis was changed. Thus, the 

final diagnoses represent a consensus of contractor 

pathologists 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 analysis of pathology data, 

the diagnosed lesions for each tissue type were 

evaluated separately or combined according to the 

guidelines of McConnell et al. (1986). 

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 found to 

be 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 

as presented in Tables Al, A5, B1, B5, C1, C5, Dl, 

and D5 are given as the number of animals bearing 

such lesions at a specific anatomic site and the 

number of animals with that site examined microscopically. For calculation of statistical significance, 

the incidences of most neoplasms (Tables A3, B3, 

0, and D3) and of all nonneoplastic lesions are 

given as the numbers of animals affected at each site 

examinedmicroscopically.However,whenmacroscopic examination was required to detect neoplasms 

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

gland, and mammary gland) 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. 

Analysis of Neoplasm Incidence 

The majority of neoplasms in these studies were 

considered to be incidental to the cause of death or 

not rapidly lethal. Thus, the primary statistical 

method used was logistic regression analysis, which 

assumed that the diagnosed neoplasms were discovered as the result of death from an unrelated 

p-Nitrobenzoic Acid, NTP TR 442 

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

this approach, neoplasm prevalence was modeled as 

a logistic function of chemical exposure and time. 

Both linear and quadratic terms in time were incorporated initially, and the quadratic term was eliminated if the fit of the model was not significantly 

enhanced. The neoplasm incidences of exposed and 

control groups were compared on the basis of the 

likelihood score test for the regression coefficient of 

dose. This method of adjusting for intercurrent 

mortality is the prevalence analysis of Dinse and 

Lagakos (1983), further described and illustrated by 

Dinse and Haseman (1986). When neoplasms are 

incidental, this comparison of the time-specific 

neoplasm prevalences also provides a comparison of 

the time-specific neoplasm incidences (McKnight and 

Crowley,1984). 

In addition to logistic regression, other methods of 

statistical analysis were used, and the results of these 

tests are summarizedin the appendixes. These 

methods include the life table test (Cox,1972; 

Tarone, 1975), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the CochranArmitage trend test (Armitage, 1971; Gart et al., 

1979), procedures based on the overall proportion of 

neoplasm-bearing animals. 

Tests of significance included pairwise comparisons 

of each exposed group with controls, and a test for 

an overall dose-related trend. Continuity-corrected 

tests were used inthe analysis of neoplasm incidence, 

and reported P values are one sided. The procedures 

described in the preceding paragraphs were also used 

to evaluate selected nonneoplastic lesions. For 

further discussion of these statistical methods, refer 

to Haseman (1984). 

Analysis of Nonneoplastic Lesion Incidences 

Because all nonneoplastic lesions in this study were 

considered to be incidental to the cause of death or 

not rapidly lethal, the primary statistical analysis used 

was a logistic regression analysis in which nonneoplastic lesion prevalence was modeled as a logistic 

function of chemical exposure and time. For lesions 

detected at the interim evaluation, the Fisher exact 

test was used, a procedure based on the overall 

proportion of affected animals. 

Analysis of Continuous Variables 

Two approaches were employed to assess the significance of pairwise comparisons between exposed and 
Materials and Methods 21 

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

approximately normal distributions, were analyzed 

using the parametric multiple comparison procedures 

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

Hematology and clinical chemistry data, which have 

typicallyskewed distributions, were analyzed using 

the nonparametric multiple comparison methods of 

Dunn (1964) and Shirley (1977).Jonckheere’s test 

(Jonckheere, 1954) wasused to assess the significance of the dose-related trends and to determine 

whether a trend-sensitive test (Williams’ or Shirley’s 

test) was more appropriate for pairwise comparisons 

than a test that does not assume a monotonic doserelated trend (Dunnett’s or Dunn’s test). Average 

severity values were analyzed for significance using 

the Mann-Whitney U test (Hollander and Wolfe, 

1973). 

Historical Control Data 

Although the concurrent control group is always the 

first and most appropriate control group used for 

evaluation, historical control data can be helpful in 

the overall assessment of lesion incidence in certain 

instances. Consequently, neoplasm incidencesfrom 

the NTP historical control database (Haseman et al., 

1984,1985) are included in the NTP reports for 

neoplasms appearing to show compound-related 

effects. Step-section historical data is taken from 

other NTP technical reports. 

Quality Assurance Methods 

The 13-week and 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 

covering completeness and accuracy of the pathology 

data, pathology specimens, final pathology tables, and 

staff review draft of this NTP Technical Report were 

conducted. Audit procedures and findings are 

presented in the reports and are on file at NIEHS. 

Theaudit findings were reviewed and assessed by 

NTP staff, so all discrepancies had been resolved or 

were otherwise addressed during the preparation of 

this Technical Report. 

GENETIC TOXICOLOGY 

The genetic toxicology of p-nitrobenzoic acid 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, and 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 ofp-nitrobenzoic acid are 

part of a larger effort by the NTP to develop a 

database that would permit the evaluation of carcinogenicity in experimental animals from the structure 

and responses of the chemical in short-term in vitro 

and invivo genetic toxicity tests. These genetic 

toxicity tests were originally developed to study 

mechanisms of chemically induced DNA damage and 

to predict carcinogenicity in animals, based on the 

electrophilic theory of chemical carcinogenesis and 

the somatic mutation theory (Miller and Miller, 

1977; Straw, 1981; Crawford, 1985). 

There is a strong correlation between a chemical’s 

potential electrophilicity (structural alert to DNA 

reactivity), mutagenicity in Salmonella, and carcinogenicityin rodents, The combination of electrophilicity and Salmonella mutagenicityishighly 

correlated with the induction of carcinogenicity in 

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

and Tennant, 1991). Other invitro genetic toxicity 

tests do not correlate well with rodent carcinogenicity (Tennant et al., 1987; Zeiger et al., 1990), 

although these other tests can provide information 

on the types of DNA and chromosome effects that 

can be induced by the chemical being investigated. 

Data from NTP studies show that a positive response 

in Salmonella is currently the most predictive in vitro 

test for rodent carcinogenicity (89% of the 

Salmonella mutagens were rodent carcinogens), and 

that there is no complimentarity among the in vitro 

genetic toxicity tests. That is, no battery of tests that 

included the Salmonella test improved the predictivity of the Salmonella test alone. The predictivity 

forcarcinogenicity of a positive response in bone 

marrow chromosome aberration or micronucleus 

tests is not yet defined. 
22 p-Nitrobenzoic Acid, NTP TR 442 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studiesof p-Nitrobenzoic Acid 

14-Day Studies 

Study Laboratory 

Microbiological Associates, Inc. 

(Bethesda, MD) 

Strain and Species 

Rats: F344/N 

Mice:B6C3Fl 

AnimalSource 

Frederick Cancer Research Facility 

(Frederick, MD) 

Size of Study Groups . 

5 males and 5 females 

Time Held Before Studies 

Rats: 16 days 

Mice:15days 

Average Age When Studies Began 

6 weeks 

Date of First Dose 

Rats: 13 December 1985 

Mice: 12 December 1985 

Duration of Dosing 

14 days 

Date of Last Dose 

Rats:26December1985 

Mice:25December1985 

Method of Sacrifice 

CO, and exsanguination 

13-Week Studies 

Microbiological Associates, Inc. 

(Bethesda, MD) 

Rats: F344/N 

Mice:B6C3Fl 

Simonsen Laboratories, Inc. 

(Gilroy, CA) 

Core study group: 10 male and 

10 female rats and mice 

Special study group: 10 male and 

10 female rats 

Rats: 13 days 

Mice: 11days 

5-6 weeks 

Rats:22May1986 

Mice:20May1986 

13 weeks 

Rats:August1986 

Mice:August1986 

CO, and exsanguination 

2-Year Studies 

Southern Research Institute 

(Birmingham,AL) 

Rats: F344/N 

Mice:B6C3F1 

Taconic Laboratory Animals and 

Services (Germantown,NY) 

60 males and 60 females 

12 days 

44 days 

Rats: 11May 1988 

Mice:25May1988 

103 weeks 

Rats: 1May1990 

Mice: 15 May1990 

CO, and exsanguination 
Materials and Methods 23 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studies of p-Nitrobenzoic Acid(continued) 

14-Day Studies 

Necropsy Dates 

Rats:27December1985 

Mice: 26 December1985 

Average Age at Necropsy 

8 weeks 

Method of Animal Distribution 

Animals were randomized by weight 

with a computer randomization 

program.. 

Animals per Cage 

Rats: 5 

Mice: 1 

Method of Animal Identification 

Ear clip and toe clip 

Diet 

NIH-07 Open Formula Diet 

(powdered) (Zeigler Brothers, Inc., 

Gardners, PA), availablead libitum 

Maximum Storage Time for Feed 

120 days after milling 

Feeders 

Rats:Stainlesssteel(HahnsRoofing 

and Sheet Metal Company, 

Birmingham,AL), changed twice 

weekly 

Mice:Stainlesssteel (Lab Products, 

Inc., Rochelle Park, NJ), changed 

weekly 

Water 

Automaticwateringsystem Same14-day (Edstrom as studies 

Industries, Inc., Waterford, WI), 

available ad libitum 

13-Week Studies 

Rats:August1986 

Mice:August1986 

18-19 weeks 

Same as 14-day studies 

Rats: 5 

Mice: 1 

Ear clip and toe clip 

Same as 14-day studies 

120 days after milling 

Stainless steel (Hahns Roofing and 

Sheet Metal Company, Birmingham, 

AL), changed weekly 

2-Year Studies 

Rats:9-10May1990(males), 

10-11 May 1990 (females) 

Mice:23-24May1990(males) 

24-29 May 1990 (females) 

772-778 days 

Same as 14-day studies 

Rats: 5 

Mice: 1 

Toe clip 

NIH-07 Open Formula Mash (Zeigler 

Brothers, Inc., Gardners, PA), 

available ad libitum 

120 days after milling 

Stainless steel (Lab Products, 

Maywood, NY, Hoeltge, Inc., 

Cincinnati, OH or Automated 

Precision, Madison, AL), changed 

weekly 

Same as 14-day studies 
24 p-Nitrobenzoic Acid, NTP TR 442 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studiesof p-Nitrobenzoic Acid (continued) 

14-Day Studies 13-Week Studies 

Cages 

Polycarbonate (Lab Products, Inc., Polycarbonate (Lab Products, Inc., 

Rochelle Park, NJ), changed weekly Rochelle Park, NJ), changed twice 

for rats and twice weekly for mice weekly for rats and weekly for mice 

Bedding 

BetaChipse (Northeastern Product BetaChipse (Northeastern Product 

Corporation, Warrensburg, NY), Corporation, Warrensburg, NY), 

changed weekly for rats and twice changed twice weekly for rats and 

weekly for mice weekly for mice 

Cage Filters 

Spun-bonded polyester (Snow Same as 14-day studies 

Filtration Company, Cincinnati,OH), 

changed once every 2 weeks 

Racks 

Stainless steel (Lab Products, Inc., Same as 14-day studies 

Rochelle Park, NJ), changed once 

every 2 weeks 

Animal Room Environment 

Average temperature: Averagetemperature:22" C 

22" C (rats), 21" C (mice) Relative humidity: 

Relative humidity: 71% (rats), 72% (mice) 

59% (rats), 45% (mice) Fluorescentlight: 12 houdday 

Fluorescentlight: 12 houdday Room air changes: minimumof 

Room air changes: minimumof 12 changeshow 

12 changeshour 

Doses 

0,2,500, 5,000, 10,000, 20,000,or Rats: 0, 630, 1,250,2,500, 5,000, or 

40,000 p-nitrobenzoic acid in feed, 10,000 ppm p-nitrobenzoic acidin 

available ad libitum feed, availablead libitum 

Mice: 0, 1,250,2,500,5,000, 10,000, 

or 20,000 ppmp-nitrobenzoic acid in 

feed, available ad libitum 

Type and Frequency of Observation 

Animalswereobservedclinical were and and Animals observedclinical 

observationswererecordedobservationsrecorded twice were twice 

daily;animalswereweighedondaysdaily;animalswereweighedinitially 

1, 8, and15. consumptionand Feed wasweekly thereafter. Feed 

measured weekly twice for rats and was weekly. consumptionmeasured 

once weekly for mice. 

~~~~ ~~ 

2-Year Studies 

Polycarbonate (Lab Products, 

Maywood, NJ), changed twice weekly 

for rats and weekly for mice 

Sani-Chips (P.J. Murphy Forest 

Products Corporation, Montville, NJ), 

changed twice weekly for rats and 

weekly for mice 

Remap spun-bonded polyester 

(Andico, Birmingham,AL), changed 

every 2 weeks 

Stainless steel (Lab Products, Inc., 

Maywood, NJ), changed every 

2 weeks 

Averagetemperature:22" C 

Relativehumidity: 50%-51% 

Fluorescentlight: 12 hours/day 

Room air changes: minimum of 

10 changeshour 

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

p-nitrobenzoic acid in feed, available 

ad libitum 

Animals were observed twice daily. 

Clinical observations and body 

weights were recorded initially, 

weekly during first13 weeks, and 

monthly thereafter. Feed 

consumption was measured every 

4 weeks. 
Materials and Methods 25 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studies of p-Nitrobenzoic Acid(continued) 

14-Day Studies 13-Week Studies 2-Year Studies 

Necropsy 

Necropsywas on performed all Necropsyperformed was on all core was on Necropsy all performed 

animals.Organsweighedbrain, Organs wereanimals. weighed wereanimals. weighedbrain, Organs at 

heart, rightkidney,liver,heart,kidney,lungs, lungs,rightliver, 15 months liver, werekidney, right

spleen,testis right and thymus.thymus. spleen, testis, andright and spleen.

Clinical Pathology 

Blood samples were collected from Blood samples were collected from Blood samples were collected from 

the orbital sinusof all animals at the orbital sinusof special studyrats the retroorbitalsinus of all animals at 

necropsy. on days 7, 30,60, and 90 at exposure the 15-month interim evaluation. 

Hemato@: hematocrit,hemoglobin, levels of 0, 630, 2,500, and HaMtol0gy.- hematocrit,hemoglobin, 

erythrocytes, reticulocytes, mean 10,000 ppm. erythrocytes, mean erythrocyte 

erythrocyte volume, leukocyte count Hemuto@: hematocrit,hemoglobin, volume, mean erythrocyte 

and differential, and methemoglobin erythrocytes, reticulocytes, mean hemoglobin, mean erythrocyte 

erythrocyte volume, mean erythrocyte hemoglobin concentration, platelets, 

hemoglobin, mean erythrocyte reticulocytes, leukocyte count and 

hemoglobin concentration, leukocyte differential, and methemoglobin 

count and differential, Heinz bodies, 

and methemoglobin 

ChidChmimy: alkaline 

phosphatase, alanine 

aminotransferase, and sorbitol 

dehydrogenase 

Histopathology 

Complete histopathologic Except for special study rats, Complete histopathologic 

examinations were performed on all complete histopathologic examinations were performed onall 

controls and all 40,000 ppm animals examinations were performed onall animals. In addition to gross lesions, 

at the end of the studies. In addition controls, all animalsdying before the the tissues examined included: 

to gross lesions, the tissues examined end of the studies, and all adrenal gland, bone and marrow, 

included adrenalgland,boneand 10,000 ppm rats and 20,000 ppm mice brain, clitoral gland, epididymis, 

marrow, brain, epididymis, esophagus, surviving to the end of the studies. In esophagus, gallbladder (mice), heart, 

gallbladder (mice), heart, kidney, addition to gross lesions, the tissues kidney, large intestine (cecum, colon, 

large intestine (cecum, colon, examinedincluded:adrenalgland, rectum), liver, lung, lymph nodes 

rectum), liver, lung, lymph nodes bone and marrow, brain, clitoral (mandibular and mesenteric), 

(mandibular and mesenteric), gland (rats), epididymis, esophagus, mammary gland, nose, ovary, 

mammary gland, nose, ovary, gallbladder (mice), heart, kidney, pancreas, parathyroid gland, pituitary 

pancreas, parathyroid gland, pituitary large intestine (cecum, colon, gland, preputial gland, prostate gland, 

gland, prostate gland, salivary gland, rectum), liver, lung, lymph nodes salivary gland, seminal vesicle, small 

seminal vesicle, small intestine (mandibular and mesenteric), intestine (duodenum, jejunum, ileum), 

(duodenum, jejunum, ileum), spleen, mammary gland, nose, ovary, spleen, stomach (forestomach and 

stomach (forestomach and glandular), pancreas, parathyroid gland, pituitaly glandular), testis, thymus, thyroid 

testis, thymus, thyroid gland, trachea, gland, preputial gland (rats), prostate gland, trachea, urinary bladder, and 

urinary bladder, and uterus. Selected gland, salivary gland, seminal vesicle, uterus. 

organs and gross lesions were small intestine (duodenum, jejunum, 

examined in lower exposure groups ileum), spleen, stomach (forestomach 

until a no-effect level was observed. and glandular), testis, thymus, thyroid 

Selected organs were bone marrow, gland, trachea, urinary bladder,and 

ovary (rats), testis, thymus (rats), uterus. 

thyroid gland (rats), and spleen (rats). 
26 p-Nitrobenzoic Acid, NTP TR 442 
27 

RESULTS 

RATS 

14-DAY STUDY 

All rats survived to the end of the study (Table 2). of the groups that lost weight, there was a reduction 

Mean body weight gains were significantly reduced inin feedconsumption (Table 2). Feed spillagewas not 

males exposedto 10,000 ppm, and males and females measured. Feed consumption by the other exposure 

exposed to 20,000 and 40,000 ppmlost weight. Males groups was similar to that by the controls. Dietary 

and females exposed to 10,000, 20,000, and levels of 2,500,5,000,10,000,20,000,and 40,000 ppm

40,000 ppm had finalmean body weights significantly p-nitrobenzoic acid resulted in average daily doses of 

lower than those of the controls. There were no 240,450,810, 1,170, and 2,260 mgkg body weightto 

clinicalfindings relating to organ-specifictoxicity, males and 230,430,840,930, and 2,840 mgkgto 

although animals in the 40,000ppm groups were females. The estimate for 40,000ppmfemales may 

lethargic during the second week on study. In most exceed the actual value because of scattering of feed. 

TABLE2 

Survival, Mean Body Weights, and Feed Consumption of Rats in the 14-Day Feed Study 

of p-Nitrobenzoic Acid 

Final Weight 

Mean Body Weightb (9) Feed Relative 

Initial SurvivalaDose Final ChangetoControlsConumption‘ 

(PP@ (“/.I Week 1 Week 2 

Male 

0 152f 5 210 f 4 58 f 4 16.3 16.4 

2,500 145 f 5 205 f 6 60 f 2 98 16.2 17.4 

5,000 145 & 3 198 f 3 53 f 2 94 14.6 16.0 

10,000 148 k 4 173 & 4** 25 f 2** 82 12.0 13.9 

20,000 143 4 127 ? 5** -16 f 2** 60 6.8 9.0 

40,000 141 k 5 110 f 6** -31 f 4** 52 3.8 10.5 

Female 

0 117 f 3 140 f 3 23 f 1 10.8 11.6 

2,500 116 f 4 139 f 4 24f1 99 11.1 12.5 

5,000 117 f 2 134 & 3 18 f 1 96 10.1 11.4 

10,000 102 f 4** 123 f 3** 21 f 4 87 8.3 10.5 

20,000 114 f 3 95 f 4** -19 f 2** 68 4.7 5.0 

40.000 116 f 3 91 f 4** -25 f 4** 65 3.4 11.3 

** Significantly different (PSO.01)from the control group by Williams’ or Dunnett’s test 

a Number of animals surviving/number initially in group 

Weights and weight changesare given as mean & standard error. ‘ Feed consumption is expressed as grams of feed consumed per animal per day and was not corrected for feed spillage (scattering). 
28 

The absolute and relative thymus weights of 20,000 

and 40,000 ppm males and females and the absolute 

thymusweights of 10,000 ppmmales and females 

weresignificantlylower than those of the controls 

(Table Fl). The thymus weight effects were considered to be related to lower body weight, lower feed 

intake, and stress. Decreases in absolute weights and 

increases in relative weights of other organs except 

the spleen were attributed to decreased body weights. 

There were statistically significant increasesin the 

absolute and relative spleen weights of 10,000,20,000, 

and 40,000 ppm males and females and differences in 

the hematology parameters characteristic of a regenerative anemia, which wasprobablyhemolyticin 

nature. There were statistically significant reductions 

in erythrocyte count, hemoglobin, and hematocrit 

values and statistically significant increases in reticulocyte count, nucleated erythrocytes, and methemoglobin concentrations (except in 10,000 ppm males) 

in the 10,000,20,000, and 40,000 ppm groups 

(Table Gl). Less pronounced differences in these 

parameters were observed in theother exposure 

groups. A significant leukocytosis with lymphocytes 

was present in 20,000 and 40,000 ppm males. 

There were no gross lesions observed at necropsy that 

were considered to be related to chemical administration. Hypertrophy of the follicular epithelium of the 

thyroid gland was present in all male and female rats 

receiving10,000, 20,000, and 40,000 ppm p-nitrobenzoic acid, while follicular cell hyperplasia was 

observed in four 40,000 ppmmales and two 

p-Nitrobenzoic Acid, NTP TR 442 

40,000 ppmfemales (Table 3). Atrophy (degeneration) of the testis was observedin20,000 and 

40,000 ppmmales. Congestion of the spleen was 

observed in one 5,000 ppm male, in all 10,000 ppm 

males, and in all 20,000 and 40,000 ppm males and 

females. Other microscopic findings observed in the 

20,000 and 40,000 ppm groups, including atrophy in 

the thymus of 20,000 and 40,000 ppm males and 

atrophy in the ovary, bone marrow, and thymus of 

20,000 and 40,000 ppmfemales, were considered 

secondary to stress and inanition. 

Hypertrophy of the follicular epithelium was diffuse 

and ranged from minimal to moderate in severity, in 

a dose-related manner, across exposure groups. 

Thyroidglandsinexposed animals had a predominance of large follicles. The follicular epithelium was 

tall columnar to low cuboidal, the cytoplasm was 

slightly basophilic, nuclei were vesiculate, and the 

colloid was pale eosinophilic. Minimal thyroid 

follicularhyperplasia was characterized by focal 

stacking of epithelial cells that did not maintain 

contact with the basement membranes. Testicular 

atrophy was characterized by reduced seminiferous 

tubule size and fewer spermatogenic cells. Affected 

seminiferous tubules contained multinucleated giant 

cells that represented fused spermatid nuclei. 

Based on the decreased mean bodyweights and 

clinical pathologic and histopathologic findings at 

20,000 and 40,000 ppm, the high dose selected for the 

13-week study was 10,000 ppm. 
Results 29 

TABLE3 

Incidences of Selected Nonneoplastic Lesions in Rats in the14-Day Feed Study of p-Nitrobenzoic Acid 

Dose (PPm) 0 2,500 5,000 10,000 20,000 40,000 

Male 

Thyroid Glanda 5 5 5 5 

Hypertrophy, Follicular Epithelium' 0 5** (l.O)d 5** (1.5) 5** (2.5) 

Hyperplasia, Follicular Epithelium 0 0 0 4* 

Testis 5 5 5 5 

Atrophy, Germinal Epithelium 0 0 1 4+ 

Spleen 5 5 5 5 

Congestion 0 5** 5** 5** 

Thymus 5 5 5 5 

Atrophy, Cortex 0 0 2 2 

Bone Marrow 5 - - 5 

Atrophy 0 - - 0 

Female 

Thyroid Gland 5 5 5 5 

Hypertrophy, Follicular Epithelium 0 5** (1.0) 5** (1.5) 5** (2.5) 

Hyperplasia, Follicular Epithelium 0 0 0 2 

Spleen 5 5 5 5 

Congestion 0 0 5*' 5** 

Thymus 5 5 5 5 

Atrophy, Cortex 0 0 1 4* 

Bone Marrow 5 5 5 5 

Atrophy 0 0 1 5** 

Ovary 5 5 5 5 

Atrophy 0 0 2 4* 

* Significantly different (P50.05) from the control group by the Fisher exact test 

** P50.01 

a Number of animals with organ examined microscopically 

Organ not examined in this exposure group 

Number of animals with lesion 

Average severity of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe 
30 

13-WEEK STUDY 

All rats in the 13-week study survived until the end 

of the study (Table 4). Mean body weight gain and 

final mean body weights were significantly lower than 

those of the controls in 2,500 ppm males and in 5,000 

and 10,000 ppm males and females. There were no 

clinical findings that could be clearly related to 

p-nitrobenzoic acid exposure. Feed consumption by 

males and females was similar to that by the controls. 

Dietary levels of 630,1250, 2,500, 5,000, or 

10,000ppm p-nitrobenzoic acid deliveredaverage 

TABLE4 

p-Nitrobenzoic Acid, NTP TR 442 

daily doses of 40,70,160,210, or 660 mgkg to males 

and 40, 80, 170, 340, or 680 mgkg to females. 

The absolute and relative spleen weights were significantlyincreasedinmales and females exposed to 

10,000ppm, and the relative spleen weights were 

significantly increased in males and females exposed 

to 5,000 ppm (Table E).These differences were 

probably a result of a hemolytic anemia. Differences 

in the absolute and relative weights of other organs 

were considered to be related to decreased body 

weights. 

Survival, Mean Body Weights, and Feed Consumption of Rats in the 13-Week Feed Study 

of p-NitrobenzoicAcid 

Mean Body Weightb (e) Relative 

Final Weight 

Feed 

Dose Survivala Initial Final Change to Controls Consumption' 

(PPm) (W Week 1 Week 13 

Male 

0 lono 354 116 2 238 4 f 6 2 5 15.6 11.8 

630 10/10 119 358 f 239 4 f 5 f 5 101 16.8 12.3 

lono & 4 

lono 114 k 3 

1,250 114 227 

2,500 

15.3341 f10.5 6 96 f 5 

335 f 4* 221 f 4* 95 11.1 16.8 

144 f 14.6 3**

5,000 1ono 107 f 5 322 f 6** 216 f 4** 91 15.8 10.4 

10,000 lono 117 +. 4 261 f 3** 74 10.3 

10.0 9.5 

Female 

0 lono 97 f 2 204 f 4 106 f 4 

630 lono 202 100 f 3 102 2 3 99 9.7 9.7 k 3 

1,250 lono 97 f 2 200 f 3 103 -C 3 98 9.0 10.0 

2,500 lono 98 k 3 98 195 f 3 96 f 2 10.3 9.5 

87 f 3**10.0 8.6 

70 f 2*9.8* 5,000 10/10 95 f 2 182 f 2** 89 

10,000 1ono 1699 f 2 f 2.' 83 8.5 

* Significantly different (PSO.05) from the control group by Williams' test 

** P50.01 

a Number of animals surviving/number initially in group 

Weights and weight changes are given as mean f standard error.

' Feed consumption is expressed as grams of feed consumed per animal per day. 
Results 31 

Differences in hematology and clinical chemistry 

parameters were observed primarily in the

10,000ppm rats (Table G2). These differences 

included increases in methemoglobin and Heinz 

bodies as early as day 7 and increased reticulocyte 

counts, decreased erythrocyte counts, and decreased 

hemoglobin and hematocrit values as early as day 30 

of the study. In addition, slight increases in mean 

erythrocyte hemoglobin at days 30 and 60 inmale 

rats and increases in mean erythrocyte volume in 

male and female rats at days 30 and 60 were supportive of a regenerative anemia. The slight 

increases in leukocyte count in 10,000 ppm femalesat 

days 7 and 30 may have been associated with a 

hemolytic anemia. Slight increases in alanine aminotransferase were present in 10,000ppmmales and 

females at days 7 and 30. 

Chemical-related histopathologic lesions were

observed in the testis, spleen, and kidney. As in the 

14-daystudy, the testicular lesion in 10,000 ppm 

males consisted of atrophy of the seminiferous 

tubules characterized by mild to severe depletion of 

spermatogenic cells and by pyknotic cells and multinucleated cells in the lumen of the seminiferous 

tubules. 

Yellow-brown pigmentation of the red pulp was 

present in the spleen of 2,500,5,000, and 10,000 ppm 

males and females, and congestion was observed in 

the spleen of 2,500,5,000, and 10,000 ppm males and 

10,000 ppm females (Table 5). Splenic sinusoids 

were ectatic, and macrophages contained yellowbrownpigment consistent with hemosiderin.Mild 

pigmentation was also present in the kidney of 5,000 

and 10,000ppmmales. The pigmentation was 

present in the cytoplasm of renal tubule epithelial 

cells. The yellow-brown pigment, consistent with 

hemosiderin, in the spleen and kidney was associated 

with the hemolytic anemia. 

Mild karyomegaly of renal tubule epithelial cells was 

observed in the kidney of 5,000 ppmfemales and 

10,000ppmmales and females. The mild karyomegaly was more prominent in the outer cortex and 

was scattered in renal tubule epithelial cells. Karyomegalywas characterized by nuclei that were 4 to 

6 times larger than normal. Occasional nuclei were 

pleomorphic and contained two prominent nucleoli. 

Male rats exposed to 10,000 ppm had mild cytoplasmichyaline droplet accumulation in renal tubule 

epithelial cells within the outer renal cortex. Multiple hyaline droplets were most commonly present 

within the cytoplasm of cells, but the droplets were 

alsoobserved protruding from the cell, and were 

often observed within renal tubule lumens. Hyaline 

droplets were eosinophilic, crystalline-shaped or 

amorphous to spherical, and variable in size. The 

droplets were more frequent and larger than the 

smaller, more uniform protein "reabsorption droplets" 

typically present in the kidney of male control rats. 

Minimalhyaline droplet accumulation was also 

observed in 630, 1,250, 2,500, and 5,000 ppm males. 

Using the Mallory-Heidenhain stain for proteins, the 

cytoplasmichyaline droplets appeared intensely 

eosinophilic, similar to the staining observed for the 

protein "reabsorption droplets." There was no degeneration or necrosis of the renal tubule epithelial cells 

and no mineralization or granular casts was observed. 

Dose Selection Rationale: Based on lower mean body 

weights and increased severity of splenic lesions, 

dietary levels of p-nitrobenzoic acid selected for the 

2-yearfeedstudyin rats were 0, 1,250,2,500, and 

5,000 ppm. 
32 p-Nitrobenzoic Acid, NTP TR 442 

~ 

0 630 2,500 1,250 5,000 10,000 

Male 

-

b Spleena 10 10 10 10 10 

Congestion' 0 - 0 2 (l.O)d lo** (1.0) 10.. (1.5) 

Pigmentation 0 - 0 6**(1.0) lo** (1.0) lo** (1.6) 

Kidney 10 10 10 10 10 10 

Pigmentation 0 0 0 10.' (1.0) lo** (2.0) lo** (2.0)

Cytoplasmic Hyaline 

Droplet Accumulatione 0 lo** (1.0) lo** (1.0) lo** (1.0) lo** (1.0) lo** (2.0)

Karyomegaly 0 0 0 6**(1.0) lo** (1.0) 10- (2.0) 

Female 

Spleen 10 - 10 10 10 10 

Congestion 0 - 0 0 0 lo** (1.5) 

Pigmentation 0 - 0 10.. (1.0) lo** (1.2) lo** (1.5) 

Kidney 10 10 10 10 10 10 

Karyomegaly 0 0 0 10.. (1.0) lo'* (2.0) 10- (2.0) 

** Significantly different (PSO.01) from the control group by the Fisher exact test 

a Number of animals with organ examined microscopically 

Organ not examined in this exposure group ' Number of animals with lesion 

Average severity of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe 

e Diagnosed as cytoplasmic change by the study pathologist 
Results 33 

2-YEARSTUDY 

Survival 

Estimates of survival probabilities for male and 

female rats are shown in Table 6 and in the KaplanMeier curves in Figure 2. Two-year survival of males 

receiving 1,250 and 2,500 ppm was similar to that of 

the controls at the end of the study. The survival of 

5,000 ppm males was marginally greater than that of 

the controls, which was attributed to a decrease in 

the severity of nephropathy and a decrease in the 

incidence of mononuclear cell leukemia in this 

exposure group. Survival of exposed females was 

similar to that of the controls. 

Body Weights, Feed Consumption,

and Clinical Findings 

The mean body weights of 5,000 ppm males’were 2% 

to 8% lower than those of the controls through 

week 80 (Table 7 and Figure 3). Final mean body 

weights of exposed males were similar to that of the 

controls. The meanbodyweights of 5,000 ppm 

females were 2% to 9% lower than those of the 

controls during the first year of the study and 10% to 

16% lower during the second year of the study 

(Table 8 and Figure 3). Feed consumption by 

exposed groups was similar to that by the controls 

(Tables I1 and 12). Dietary levels of 1,250, 2,500, or 

5,000 ppm p-nitrobenzoic acid delivered approximately 50, 100, or 210 mgkg body weight per day to 

males and 60, 125, or 250 mgkg per day to females. 

There were no clinical findings attributable to organspecific toxicity. 

Hematology 

Marginal differences werenoted between the hematologic profile of 5,000 ppm females and that of the 

controls. These differences did not appear to affect 

the well-being of the animals. Atthe 15-month 

interim evaluation, leukocyte count was significantly 

increasedin 5,000 ppm males and females 

(Table G3). In 5,000 ppm females, erythrocyte count, 

hemoglobin, hematocrit, mean erythrocyte hemoglobin, and mean erythrocyte hemoglobin concentration values were significantly lowerthan those of the 

controls. Nucleated erythrocyte, segmented neutrophil,lymphocyte, and platelet values were significantly greater than those of the controls in 5,000 ppm 

females. 
34 p-Nitrobenzoic Acid, NTP TR 442 

Survival of Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid 

TABLE6 

Dose (PPm) 0 1,250 2,500 5,000 

Male 

Animals initially in study 60 

15-Month interim evaluationa 

60 60 

10 10 10 10 

Moribund 34 

Natural deaths 6 3 3 

34 32 

Animals surviving to study termination 13 12 13 21 

Percent probability of survival at end of studyb 

Mean survival (days)' 

survival analysesd 

Female 

Animals initially in study 

15-Month interim evaluationa 

Moribund 

Natural deaths 

Animals surviving to study termination 

Percent probability of survival at end of study 

Mean survival (days) 

Survival analyses 

a Censoredfromsurvivalanalyses 

25 

587 

26 

607 

P=0.038N P=0.651N 

21 

10 

60 

659 

54 

27 

2 

. 

23 

10 

60 

633 

46 

23 

4 

P=O.250 P=O.400 

26 

618 

P=0.563N 

60 

10 

27 

2 

21 

43 

616 

P=O.231 P=O.137 

42 

618 

P=0.053N 

60 

10 

26 

3 

21 

42 

635 

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

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

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 exposure columns. A negative trend or a lower mortality in an exposure group is indicated 

by N. 
Results 35 

WEEKS ON STUDY 

1.0 

0.9 

0.0 

I

2

3 0.7 

v) 

I& 

0 

OA 

rl 

m

2 0.5 

8 

a. 0.4 

0.1 

0.1 

FIGURE2 

Kaplan-Meier Survival Curvesfor Rats Administered 

p-Nitrobenzoic Acid in Feed for 2 Years 
36 p-Nitrobenzoic Acid, NTP TR 442 

TABLE7 

Mean Body Weights and Survival of Male Rats in the 2-Year Feed Studyof p-NitrobenzoicAcid 

on 

Weeks 

Av. Wt. No. of 

0 ppm 

Av. Wt.Wt. (%of 

1,250 ppm 

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

2,500 ppm 

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

5,000 ppm 

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

1 128 60 126 98 60 124 96 60 126 98 60 

2 169 60 168 100 60 166 98 60 164 97 60 

3 207 60 206 99 60 200 97 60 198 96 60 

4 235 60 231 99 60 227 97 60 221 94 60 

5 262 60 259 99 60 258 99 60 240 92 60 

6 283 60 281 100 60 280 99 60 262 93 60 

7 300 60 299 100 60 298 99 60 278 93 60 

8 313 60 310 99 60 311 99 60 294 94 60 

9 321 60 320 100 60 322 100 60 304 94 60 

10 338 60 337 100 60 337 100 60 315 93 60 

11 347 60 350 101 60 350 101 60 329 95 60 

12 352 60 355 101 60 356 101 60 334 95 60 

13 362 60 360 99 60 359 99 60 338 93 60 

17 387 60 381 99 60 391 101 60 366 95 60 

21 409 60 409 100 60 412 101 60 389 95 60 

25 426 60 421 99 60 427 100 60 400 94 60 

28 435 60 431 99 60 435 100 60 410 94 59 

33 450 60 450 100 60 447 99 60 426 95 59 

37 460 59 461 100 60 464 101 60 442 96 59 

41 469 59 470 100 60 466 99 60 446 95 59 

45 475 58 475 100 60 480 101 60 459 97 59 

49 478 58 485 102 59 484 101 60 464 97 59 

53 480 58 488 102 59 485 101 60 463 97 59 

57 482 56 491 102 58 486 101 60 464 96 59 

61 484 55 484 100 57 480 99 60 461 95 59 

65 483 54 487 101 57 486 101 59 465 96 57 

69a 482 43 490 102 46 485 101 49 466 97 46 

73 474 42 478 101 45 472 99 49 458 97 45 

77 475 40 480 101 41 474 100 43 465 98 41 

80 472 34 471 100 39 469 99 41 458 97 39 

85 463 31 463 100 35 469 101 37 459 99 38 

89 462 30 468 101 33 473 102 34 462 100 36 

93 455 27 458 101 30 470 103 31 457 101 34 

97 442 24 447 101 27 451 102 26 447 101 32 

101 449 17 441 98 20 460 102 19 444 99 26 

Mean for weeks 

1-13 278 277 100 276 99 262 94 

14-52 443 443 100 445 100 422 95 

53-101 469 473 101 474 101 459 98 

a Interim evaluation occurred during week 66. 
Results 37 

TABLE8 

Mean Body Weights and Survival of Female Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid 

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 k!) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors 

1 105 60 106 101 60 103 98 60 103 98 60 

2 125 60 125 100 60 125 100 60 122 98 60 

3 137 60 137 100 60 134 98 60 131 96 60 

4 149 60 149 100 60 146 99 60 143 96 60 

5 156 60 157 100 60 154 99 60 150 96 60 

6 166 60 168 101 60 164 99 60 160 97 60 

7 173 60 174 101 60 170 98 60 167 97 60 

8 175 60 177 101 60 173 99 60 171 98 60 

9 178 60 179 101 60 176 99 60 172 97 60 

10 184 60 185 101 60 183 100 60 179 98 60 

11 190 60 191 101 60 188 99 60 183 96 60 

12 191 60 193 101 60 191 100 60 187 98 60 

13 192 60 192 100 60 188 98 60 184 96 60 

17 203 60 205 101 60 193 95 60 199 98 60 

21 211 60 211 100 60 207 98 60 203 96 60 

25 218 60 218 100 59 212 97 60 206 95 60 

29 217 60 220 102 59 216 100 60 208 96 60 

33 228 60 226 99 59 221 97 60 212 93 60 

37 236 60 235 100 59 227 97 59 219 93 60 

41 242 60 243 101 59 234 97 59 224 93 60 

45 249 60 249 100 59 242 97 59 230 92 60 

50 262 60 261 100 59 254 97 59 237 91 60 

53 271 60 269 99 59 262 97 59 243 90 60 

57 280 60 276 99 59 267 95 57 245 87 60 

61 290 60 282 97 59 273 94 57 253 87 60 

65 298 60 289 97 58 280 94 56 257 86 59 

69a 306 50 297 97 48 286 94 45 267 87 49 

73 308 49 299 97 46 290 94 42 264 86 47 

77 316 49 304 96 44 292 92 41 270 86 44 

81 321 48 308 96 43 298 93 39 274 86 42 

85 324 47 313 97 41 296 92 38 270 84 40 

89 334 46 323 97 41 306 92 37 282 85 38 

93 332 45 324 98 40 305 92 36 283 85 36 

97 337 40 327 97 35 316 94 30 289 86 35 

101 341 31 331 97 25 314 92 24 288 84 28 

Mean for weeks 

1-13 163 164 101 161 99 158 97 

14-52 230 230 100 223 97 215 93 

53-101 312 303 97 291 93 268 86 

a Interim evaluation occurred during week 66. 
38 p-Nitrobenzoic Acid, NTP TR 442 

i I ............................. 

0 

0 ....; ................... 

i :................... :................... :................... :................. 

... 

B i ......................................................... I ................... 1................... i................... I ................ ... 

... ... 

I 

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

I 

I ................... 

I 

15 

$ ................... ;................... ................... 

I I I 

so 45 60 

WEEKS ON STUDY 

I 

75 I 

90 

... 

-

105 

-

105 

FIGURE3 

Growth Curves for Rats Administered p-Nitrobenzoic Acid in Feed for 2 Years 
Results 39 

Pathology and Statistical Evaluation 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences of 

mononuclear cell leukemia and neoplasms or nonneoplastic lesions of the preputiallclitoral gland, 

kidney, spleen, liver, and thyroid gland.Summaries 

of the incidences of neoplasms and nonneoplastic 

lesions, individual animal tumor diagnoses, statistical 

analyses of primary neoplasms that occurred with an 

incidenceof at least 5% in at least one animal group, 

and historical incidences for the biologically significant neoplasms mentioned in this section are presented in Appendix A for male rats and Appendix B 

for female rats. 

PreputiallClitoral gland: The incidence of preputial 

gland carcinoma in 5,000 ppm males was significantly 

greater than that of the controls by the logistic 

regression test but not by the life table test. However, the incidences of preputial gland adenoma and 

of preputial gland adenoma or carcinoma (combined) 

in exposed males were not statistically different from 

those of the controls (Table 9). Twenty-one of the 

25 preputial gland neoplasms occurred in animals 

sacrificed in a moribund condition prior to the end of 

the study, suggesting that these were generally lethal 

neoplasms. Thus, the most appropriate test for these 

neoplasms is life table analysis rather than logistic 

regression. These preputial gland neoplasmswere 

observed on gross pathologyexamination and the 

average diameter of the preputial gland carcinomas 

was 31 mm. No preputial gland hyperplasia was 

observed and few preputial gland adenomas were 

observed at 15 months. 

At the end of the study, all exposed female groups 

hadincidences of clitoral gland adenoma and 

adenoma or carcinoma (combined) that were significantly greater than those of the controls (Tables 10 

and B3). The incidences of clitoral gland adenoma 

and of clitoral gland adenoma or carcinoma (combined) in groups exposed to p-nitrobenzoic acid 

exceeded the historical control ranges in female 

F344lN rats in recent 2-year NTP feed studies 

(Table B4a). Clitoral gland hyperplasia was not 

observed at the 15-month interim evaluation, and the 

incidences of hyperplasia in exposed females were 

marginally lower than that of the controls at 2 years 

(Tables 10 and B5). Because there was no dose 

response for clitoral gland neoplasms and the time to 

neoplasm occurrence was similar (approximately 

700 days) in control and exposed groups, and because 

there was no increase in the incidence of clitoral 

gland hyperplasia, the increased incidences of clitoral 

glandneoplasmswere considered to be only some 

evidence of carcinogenicactivity in female rats 

exposed to p-nitrobenzoic acid. 

Preputial and clitoral gland adenomas were generally 

circumscribed and sometimes caused compression of 

the surrounding tissue. The neoplastic cells formed 

acini and clusters, which were spherical to elongated 

in shape and varied in size. Many of the neoplastic 

cellshad discrete borders and granular cytoplasm. 

Foci of cellular debris, necrosis, and cysts were often 

present. Carcinomas were generally larger masses 

and less circumscribed than adenomas and often 

infiltrated the adjacent normal tissue. 
40 p-Nitrobenzoic Acid, NTP TR 442 

in the 2-Year Feed Study of p-Nitrobenzoic Acid 

Incidences of Neoplasms and Nonneoplastic Lesions of the Preputial Gland in Male Rats 

TABLE9 

Dose (PPm) 0 1,250 2,500 5,000 

15-Month Interim Evaluation 

Adenomab 

Preputial Glanda 

1 

10 

0 

10 

1 

10 

1 

10 

2-Year Study 

Preputial Gland 

Hyperplasia 

50 

4 (2.O)C 0 

50 49 

1(2.0) 3 (1.3) 

50 

Adenoma 

overall rated 

Adjusted ratee 

Terminal ratef 

First incidence (days)

Life table testg 

Logistic regression testg 

3/50 (6%)

21.1% 

2/12 (17%)

689 

P=0.382N 

P=O.522N P=0,611N 

P=0.603N 

588 

0/13 (0%) 

13.6% 

3/50 (6%) 

P=O.560 

P=O.555 

532 

1/13 (8%) 

16.5% 

4/49 (8%) 

10.4% 

3/50 (6%) 

P=0.554N 

P=0.434N 

617 

1/21 (5%) 

Carcinoma 

Overall rate 1/50 (2%) 1/50 (2%) 4/49 (8%) 6/50 (12%) 

Adjusted rate 2.7% 2.9% 15.9% 15.0% 

Terminal rate OD2 (0%) 0/13 (0%) 0/13 (0%) on1 (0%) 

incidenceFirst (days) 548 602 651 518

test table Life P=O.O31 P=0.744N P=O.234 P=O.O94 

Logistictest regression P=O.O02 P=O.743 P=O.192 P=O.O09

Adenoma or Carcinomah 

Overall rate 4/50 (8%) 4/50 (8%) 8/49 (16%) 9/50 (18%) 

Adjusted rate 23.2% 16.1% 29.8% 23.8% 

Terminal rate 2/12 (17%) 0/13 (0%) 1/13 (8%) 1/21 (5%) 

548 588 532 518 First incidence (days)

Life table test P=O.176 P=O.579N P=O.254 P=O.278 ' 

Logistic regression test P=O.O24 P=0.607N P=O.219 P=O.O55 

a Number of animals with preputial gland examined microscopically 

Number of animals with lesion 

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

Number of animals with neoplasm per numberof animals examined microscopically 

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

Observed incidence in animals survivinguntil the end of the study 

g In the control column are the P values associated with the trend test. In the exposure group mlbmns are the P values 

corresponding to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in 

animals dying prior to terminal sacrifice as being (directly or indirectly) the cause of. death. The logistic regression test regards 

these lesions as nonkatal. A negative trend or a lower incidence in an'exposure group is indicated by N. 

Historical incidence for 2-yearNTP feed studies with untreated control groups (mean rt: standard deviation): 

139/1,169 (11.9% k 7.8%), range 2%-30% 
Results 41 

TABLE10 

Incidences of Neoplasms and Nonneoplastic Lesions of the Clitoral Gland in Female Rats 

in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

Dose (PP@ 0 1,250 2,500 5,000 

Clitoral Glanda 50 49 49 50 

Hyperplasiab 10 (2.0)c 6 (1.8) 6 (2.2) 7 (2.3) 

Adenoma 

Overall rated 4/50 (8%) 12/49 (24%) 10/49 (20%) 12/50 (24%) 

Adjusted ratee 11.9% 42.5% 33.7% 42.1% 

Terminal ratef 2/27 (7%) 7/22 (32%) 4/20 (20%) 7/21 (33%) 

First incidence (days) 653 665 496 483 

Life table testg P=O.O34 P=O.O13 P=O.O30 P=O.O13 

Logistic regression testg P=O.O46 P=O.O13 P=O.O50 P=O.O23 

Carcinoma 

Overall rate 1/50 (2%) 2/49 (4%) 5/49 (10%) 4/50 (8%) 

Adjusted rate 3.7% 6.0% 19.3% 11.7% 

Terminal rate 1/27 (4%) on2 (0%) 3/20 (15%) on1 (0%) 

First incidence (days) 730 (TI 694 499 528 

Life table test P=O.O85 P=O.460 P=O.O56 P=O.139 

Logistic regression test P=O.117 P=O.459 P=O.O84 P=O.224 

Adenoma or Carcinomah 

Overall rate 4/50 (8%) 14/49 (29%) 15/49 (31%) 15/50 (30%) 

Adjusted rate 11.9% 45.9% 48.9% 47.7% 

Terminal rate 2/27 (7%) 7/22 (32%) 7/20 (35%) 7/21 (33%) 

First incidence (days) 653 665 496 483 

Life table test P=O.O08 P=O.O05 P=O.OOl P=0.002 

Logistic regression test P=O.O11 P=O.O04 P=O.O03 P=O.O04 

(TITerminal sacrifice 

a Number of animals with clitoral gland examined microscopically 

Number of animals with lesion 

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

Number of animals with neoplasm per number of animals examined microscopically 

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

Observed incidence in animals surviving until the end of the study 

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

corresponding to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in 

animals dying prior to terminal .sacrifice as being (directlyor indirectly) the cause of death. The logistic regression test regards 

these lesions as nonfatal. 

Historical incidence for 2-year NTP feed studies with untreated control groups (mean ? standard deviation): 

120/1,096 (10.9% & 5.3%), range 2%-21% 
42 p-Nitrobenzoic Acid, NTP TR 442 

Kidney: The severity of nephropathy was decreased at plasia of renal tubules was also present in one 

15 months in malesexposed to 2,500 and 5,000 ppm control male, one 2,500ppm male, and five 

p-nitrobenzoic acid. At2 years, the incidences of 5,000 ppmmales. One 1,250 ppm female and five 

nephropathy for all groups of rats were 98%to 100% 5,000 ppm females had oncocytic hyperplasia; how-

(Tables 11, A5, and BS); however, the severityever, there were no renal tubule adenomas or carcidecreased as exposure level increasedinmales and 

females. Nephropathy was less severe in females 

than in males, consistent with differencesnormally 

seen between aging male and female rats. Nephropathy was characterized by glomerulosclerosis, thickening of renal tubule basement membrane, degeneration and atrophy of tubule epithelium, dilatation of 

tubule lumens by pale pink acellular material (hyaline 

casts), interstitial fibrosis, and chronic inflammation. 

Regeneration of the renal tubule epithelium was also 

observed and the extent and severity of this process 

paralleled the overall severity of the degenerative 

changes. Few hyaline droplets were observed in renal 

tubule epithelial cells of male rats at the end of the 

2-yearstudy.Itwasdifficult tofurther evaluate 

karyomegalyof renal tubule cells in males and 

females at 2 years because the karyomegaly could not 

be differentiated from regenerative changes associated 

with nephropathy. 

There was an increase in the severity of pigmentation 

of renal tubule epithelial cells at he 15-month 

interim evaluation and at 2 years (Table 11). The 

pigment was primarily in the cytoplasm of proximal 

convoluted renal tubule epithelial cells and was 

variably brown to light brown or golden. Representative sections of kidney were stained for iron by Perls’ 

method. Positive ferric iron staining was characterized by a distinct medium to dark blue coloration 

primarily in very small granules, most were 1micron 

or less in size. These granules were irregularly 

distributed, primarily in proximal convoluted renal 

tubule epithelial cells in theouter cortex. The 

positive staining indicated the presence of ferric iron, 

a form compatible with that present in hemosiderin. 

The severity of the pigment stained by Perls’ method 

was similar in almost all instances to the severity of 

yellowish brown pigment deposition detected in the 

hematoxylin and eosin slides. 

Proliferative lesions and neoplasms were also present 

in the kidneys of males. In the 2,500 ppm group, one 

male had a renal tubule adenoma and one male had 

a renal tubule carcinoma (Tables 11and Al). Renal 

tubule hyperplasia, a possible precursor of adenomas, 

occurred in three 1,250 ppm males, one 2,500 ppm 

male, and one 5,000 ppmmale.Oncocytichypernomas in females. 

Initially, a single hematoxylin and eosin-stained 

section of each kidneywas prepared. Primarily 

because of the increased incidence of renal tubule 

hyperplasia in exposed males, because of the adenoma and carcinoma observed in 2,500 ppm males, 

and because of the unusual occurrence of oncocytic 

hyperplasiain 5,000 ppmmales, additional step 

sections of kidney were prepared from the remaining 

formalin-fixed tissues. Six to eight additional kidney 

sections taken at 1mm intervals were prepared for 

each male. Additional males with focal hyperplasia 

or adenoma were identified. The incidences of these 

proliferative lesions in the step sections and in the 

single and step sections combined are shown in 

Table 11. There were no significant increases in the 

incidence of renal tubule neoplasms. 

Renal tubule hyperplasia, as defined in this study, was 

distinguishedfrom regenerative epithelial changes 

commonly seen as a part of nephropathy and was 

considered a preneoplastic lesion. Renal tubule 

hyperplasia, adenoma, and carcinoma are part of a 

morphologic continuum. Hyperplasia was generally 

a focal, minimal to mild lesion consisting of tubules 

that were dilated to 1.5 to 2 times normal diameter 

and were lined by increased numbers of tubule 

epithelial cells, which partially or totally filled the 

tubule lumen. Cells within hyperplastic lesions varied 

slightly in size and sometimes stained more basophilic 

than normal cells but otherwise appeared similar to 

normal tubule epithelial cells. Renaltubuleadenomaswere larger discrete lesions, ranging from 

greater than five tubule diameters to 1mm or more 

in size. Cells within adenomas were mildly to moderately pleomorphic, sometimes had vacuolated 

cytoplasm, and tended to form complex patterns, 

particularly microtubular structures. A few adenomas 

contained varying amounts ofhyaline basement 

membrane material that divided the epithelial cells 

into small irregular clusters. 

Oncocytic hyperplasiawas characterized by individual 

tubules or small clusters of tubules, which were 

somewhat dilated and totally filled by large polygonal 
Results 43 

TABLE11 

Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Rats in the 2-Year Feed Study 

of p-Nitrobenzoic Acid 

Dose (PP@ 

Male 

15-Month Interim Evaluation 

Kidn$ 

Pigmentationb 

Nephropathy 

Renal Tubule Hyperplasia 

2-Year Study 

Single Sections (Standard Evaluation) 

Kidney 

Pigmentation 

Nephropathy 

Oncocytic Hyperplasia 

Renal Tubule Hyperplasia 

Renal Tubule Adenoma 

Renal Tubule Carcinoma 

Renal Tubule Adenoma or Carcinomad 

Step Sections (Extended Evaluation) 

Oncocytic Hyperplasia 

Renal Tubule Hyperplasia 

Renal Tubule Adenoma 

Single and Step Sections Combined 

Oncocytic Hyperplasia 

Renal Tubule Hyperplasia 

Renal Tubule Adenoma 

Renal Tubule Carcinoma 

Renal Tubule Adenoma or Carcinomae 

0 

10 

10 (1.O)C 

10 (2.0) 

0 

50 

50 (1.8) 

50 (2.7) 

1(2.0) 

0 

0 

0 

0 

0 

1 

1 

1 

1 

1 

0 

1 

1,250 

10 

10 (1.1) 

10 (2.0) 

0 

50 

50 (2.3)** 

50 (2.5) 

0 

3 (1.7) 

0 

0 

0 

0 

4 

1 

0 

I* 

1 

0 

1 

2,500 

10 

10 (1.9)** 

10 (1.8) 

1 (2.0) 

50 

50 (2.4)** 

50 (1.9)** 

1 (1.0) 

1 (1.0) 

1 

1 

2 

0 

4 

1 

1 

5 

2 

1 

3 

5,000 

10 

10 (2.4)** 

10 (1.7) 

0 

50 

50 (2.6)** 

50 (1.6)** 

5 (1.6) 

1 (2.0) 

0 

0 

0 

5 

4 

3 

10* 

4 

3 

0 

3 
44 p-Nitrobenzoic Acid, NTP TR 442 

TABLE11 

Incidences of Neoplasms and Nonneoplastic Lesionsof the Kidney in Rats in the &Year Feed Study 

of p-Nitrobenzoic Acid (continued) 

~~ 

0 1,250 2,500 5,000 

15-Month Interim Evaluation 

Kidney 10 10 10 10 

Pigmentation 10 (1.1) 10 (2.2)** 10 (2.9)** 10 (2.9):' 

Nephropathy 10 (1.4) 10 (1.0) 9 (1.1) 9 (1.3) 

2-Year Study 

Kidney 50 50 50 50 

Pigmentation 50 (1.8) 50 (2.1) 50 (2.4)** 50 (2.7)** 

Nephropathy 50 (1.9) 49 (1.6) 49 (1.3)** 49 (1.2)** 

Oncocytic Hyperplasia 0 1(1.0) 0 5. (1.2) 

Renal Tubule Hyperplasia 0 0 1 (1.0) 0 

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

** Significantly different (PSO.01) from the control group by the Mann-Whitney U test 

a Number of animals with kidney examined microscopically 

Number of animals with lesion 

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

Historical incidence for 2-yearNTP feed studies with untreated control groups (mean & standard deviation): 

15/1,251 (1.2% & 1.7%), range 0%-6% 

e Historical incidence for 2-year NTP feed, gavage, and inhalation studies with untreated control groups: 22/608(3.6% f 2.7%), 

range 0%-8% 
Results 45 

cells with abundant brightly eosinophilic granular 

cytoplasm and small, often centrally located, basophilic nuclei (oncocytes). These lesions are thought 

to arise from the distal tubule epithelium. 

Spleen: There were significant increases in absolute 

and relative spleen weights of females, but not of 

males, at 15 months (Table F3). Associated with the 

increases in splenic weight were increases in yellowbrown pigment in the red pulp consistent with the 

accumulation of hemosiderin in splenic macrophages 

at 15 months (Tables A5 and B5). The severity of 

pigmentation generally increasedas the exposure 

level increased (severity in males: 1.0,1.0,1.5, and 

1.9;females: 2.0,1.9,2.3, and 3.0). At the end of 

2 years, it was difficult to evaluate the severity of 

splenic pigmentation because rats with mononuclear 

cell leukemia usually hadgreatly distended spleens 

that were packed with leukemia cells. Due to obliteration of the spleen with mononuclear cell leukemia, 

the Pathology Working Group could not confirm a 

chemical-related effect for splenic pigmentation at 

the end of the 2-year study. As with splenic pigmentation, it was difficult to assess extramedullary hematopoiesis because of the mononuclear cell leukemia. 

The incidences of bone marrow hypercellularity were 

not supportive of any chemical-related effect on 

hematopoietic cell proliferation. Furthermore, the 

majority of animals with increased hematopoietic cell 

proliferation had complicating neoplasms or inflammatory lesions, which probably accounted for

increased hematopoietic cell proliferation. 

Mononuclear cell leukemia: There were significant 

dose-related trends in the incidences of mononuclear 

cell leukemia in males and females (Table 12). The 

incidences of mononuclear cell in leukemia 

5,000 ppm males(29/50,35/50,26/50,2/50;Table A3) 

and in 2,500 and 5,000 ppmfemales(17/50,11/50, 

3/50,0/50; Table B3) were significantly lower than 

those of the controls. The incidences in controls 

(males, 58%; females, 34%), while within the range 

of historical controls (males, 32% to 62%, Table A4c; 

females, 14% to 52%, Table B4b), were greater than 

the mean historical rates. The decrease in the 

incidences of total malignant neoplasms in 5,000 ppm 

males and females and increased survival of 

5,000 ppmmales were attributed to the decreased 

incidences of mononuclear cell leukemia in these 

groups. 

Liver: The incidences of fatty cellular change (males: 

15/49, 13/50, 11/50, 7/50; females: 14/50, 13/50, 7/50, 

7/50), multifocalhyperplasia(males:13/49,12/50, 

13/50,4/50;females:9/50, 10/50, 3/50,2/50), and 

centrilobular atrophy (males: 22/49,27/50,23/50, 

5/50; females:14/50,11/50,4/50,2/50) (Tables A5 

and B5) in 5,000 ppm rats were generally lower than 

those of the controls. The fatty change, focal hyperplasia, and centrilobular atrophy were secondary to 

the mononuclear cell leukemia, and the lower incidences of these lesions parallel those of mononuclear 

cell leukemia. 

Thyroid gland: The incidence of thyroid gland C-cell 

adenoma in 5,000 ppm females was marginally lower 

than that of the controls (9/50, 5/49,4/50, 2/50; 

Table B3). No chemical-related differences in the 

incidences of C-cell carcinoma or C-cell hyperplasia 

were observed. C-cell adenomas are common lesions 

of aging F344 rats. C-cell adenomas may be single, 

multiple, or bilateral. Theyoccur as discrete focal 

masses of C-cells but may contain widely separated, 

isolated follicles. The neoplasm isusuallywell 

demarcated and causes some compression of the 

surrounding parenchyma. There were no chemicalrelated decreased incidences of thyroid gland C-cell 

neoplasms in male rats. 
46 p-Nitrobenzoic Acid, NTP TR 442 

Dose(PPm) 0 1,250 2,500 5,000 

Male 

15-Month Interim Evaluation 

Mononuclear Cell Leukemiaa on0 1no on0 on0 

2-Year Study 

Mononuclear Cell Leukemiab 

Logistic regression teste 

Life table teste 

First incidence (days) 

Terminal rated 

Adjusted rate' 

Overall rate 

76.2% 

29/50 (58%) 

P<O.OOlN 

P<O.OOlN 

503 

4/12 (33%) 

P=O.177 

P=O.424 

415 

5/13 (38%) 

79.9% 

35/50 (70%) 

P=0.127N 

P=0.215N 

506 

7/13 (54%) 

76.3% 

26/50(52%) 

4.9% 

2/50 (4%) 

P<0.001N 

P<0.001N 

445 

on1 (0%) 

Female 

15-Month Interim Evaluation 

Mononudear Cell Leukemia on0 1/10 on0 

2-Year Study 

Mononuclear Cell Leukemiaf 

Overall rate 17/50 (34%) 3/50 (22%) 11/50 (6%) 0/50 (0%) 

Adjusted rate 8.5% 38.6%

Terminal rate 3/27 (11%) 4/23 (17%) 

32.5% 0.0% 

0/2l (0%) on1 (0%) 

First incidence (days) 492 490 566 2 

Life table test P<0.001NP=0.008N P=O.272N P<0.001N 

Logistic regression test Pc0.001NP<O.OOlN P=0.159N P<O.OOlN 

a Number of animals with neoplasm per number of animals necropsied 

Historical incidence for 2-yearNTP feed studies with untreated control groups (mean +. standard deviation): 

603/1,253 (48.1% 2 8.7%), range 32%-62% 

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

Observed incidence in animals surviving until the end of the study 

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

corresponding to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in 

animals dying prior to terminal sacrifice as being (directlyor indirectly) the cause of death. The logistic regression test regards 

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

Historicalincidence: 324fl,251 (25.9% f 8.6%),range 14%-52% 

g Not applicable; no neoplasms in animal group 
Results 47 

MICE 

14-DAY STUDY 

Three males and two females receiving 40,000 ppm and ataxia were observed in 40,000 ppm mice. Feed 

died during days 5 through 8 of the study. All other consumption by males and females was similar to that 

animals survived until the end of the study by the controls, although the feed consumption data 

(Table 13). Males exposedto 20,000 and 40,000 ppm varied. Scattering of feed by these mice might have 

p-nitrobenzoic acid and females exposed to contributed to the variability in the data. Dietary

20,000 ppm lost weight. Mean body weight gains of levels of 2,500, 5,000, 10,000, 20,000, or 40,000 ppm 

20,000 and 40,000 ppm males and of 10,000,20,000, p-nitrobenzoic acid delivered average daily doses of 

and 40,000 ppm females were significantly lower than 1,000, 2,000, 3,500, 8,500, or 14,000mg/kgbody

those of the controls. There were no clinical findings weight to males and 1,000, 2,000, 4,000, 9,500, or 

relating to organ-specific toxicity, although lethargy 21,500 mg/kg to females. 

TABLE13 

Survival, Mean Body Weights, and Feed Consumptionof Mice in the 14-Day Feed Study 

of p-Nitrobenzoic Acid 

Final Weight 

Mean Body Weightb (e) Feed Relative 

Dose Survivala Initial Change Final to Consumption' Controls 

(PPm) (W Week 1 Week 2 

Male 

0 18.2 f 1.0 19.5 f 0.9 1.3 f 0.2 5.4 5.7 

2,500 18.3 f 0.6 19.1 f 0.7 0.8 f 0.3 98 8.0 6.8 

5,000 17.8 f 0.5 18.4 f 0.5 0.5 f 0.2 94 6.2 8.3 

10,000 18.4 f 0.4 19.0 f 0.4 0.7 f 0.4 98 5.3 7.9 

20,000 18.4 f 0.3 17.9 f 0.5 -0.5 f 0.4** 92 6.4 8.6 

40.000 18.2 f 0.8 18.0 f 0.4 -0.7 f 0.4** 92 4.0 8.5 

Female 

0 15.5 f 0.7 16.8 f 0.7 1.3 r 0.5 6.3 7.1 

2,500 15.6 f 0.3 16.5 f0.4 0.8 f 0.4 98 6.8 8.3 

5,000 15.7 f 0.6 15.9 f 0.4 0.2 k 0.3 95 5.8 7.6 

10,000 15.4 f 0.2 15.3 f 0.2* -0.1 f 0.1' 91 5.0 8.0 

20,000 15.2 k 0.6 14.6 f 0.3" -0.6 f 0.3* 87 6.9 7.3 

40,000 15.1 f 0.7 15.3 f0.7* 0.8 f 0.9* 91 8.8 7.5 

* Significantly different (PSO.05) from the control group by Williams' test 

** PSO.01 

a Number of animals survivinglnumber initially in group 

Weights and weight changes are given as mean f standard error. Subsequent calculations are based on animals survivingto the 

end of the studies. 

Feed consumption is expressed as gramsof feed consumed per animal per day. 

Dayof death 5, 56 

e Dayof death 7,8 
48 

Absolute and relative thymus weights of 20,000 ppm 

males and 10,000, 20,000, and 40,000 ppmfemales 

were significantly less than those of the controls 

(Table F4). Relative liver weights of 20,000 and 

40,000ppmmales and females and of 10,000 ppm 

females were significantly greater than those of the 

controls (Table F4). Differences in the absolute and 

relative weights of other organs were related to 

decreased body weights. No biologicallysignificant 

differences in hematology parameters occurred in 

exposed males or females (Table G4). 

There were no gross lesions observed at necropsy that 

were considered to be related to chemical administration. Degeneration of the germinal epithelium of 

T~BLE14 

p-NitrobenzoicAcid, NTP TR 442 

the testis was observed in 20,000 and 40,000 ppm 

males (Table 14). Microscopically, testicular degeneration was characterized by multinucleated giant cells, 

pyknosis, and cytoplasmic vacuolization of germinal 

cells. Bone marrow hemorrhage and bone marrow 

atrophy occurred in the 40,000 ppm female mice that 

died early. It was uncertain if these effects were due 

to the reduced body weight or if they were a chemical 

effect. Other lesions, including bone marrow atrophy 

and hemorrhage in males, were considered secondary 

to stress and inanition. 

Based on mortality and reduced mean body weights 

at 40,000 ppm, the high concentration selected for 

the 13-week feed study in mice was 20,000 ppm. 

Incidences of Selected Nonneoplastic Lesions in Mice in the 14-Day Feed Studyof p-Nitrobenzoic Acid ~~ ~ ~~ 

0 2,500 5,000 

~ ~~ 

10,000 

Male 

Hemorrhage 

Atrophy' 

Bone Marrog 

Testis 

Degeneration, Germinal 

Epithelium 0 

5 -

-

-

- 0 

5 

Female 

Hemorrhage 

Atrophy 

Bone Marrow 

' Number of animals with lesion 

a Number of animals with organ examined microscopically 

Organ not examined in this exposure group 

20,000 40,000 

0 

0 

5 

3 

5 

3 

2 

5 

2 

5 

0 

0 

5 

2 

3 

5 
Results 49 

13-WEEK STUDY 

One female exposed to 1,250 ppm was accidentally 

killed during week 3 of the study. All other mice 

survived until the end of the study (Table 15). Final 

mean body weights and mean body weight gains of all 

exposedmales and of females exposed to 5,000, 

10,000, and 20,000 ppm were significantly lower than 

those of controls. No chemical-related clinical 

findings were observed. Feed consumption by 

exposed groups was similar to or greater than that by 

the controls throughout the study. Dietary levels of 

1,250,2,500, 5,000, 10,000, or 20,000 ppm p-nitrobenzoic acid delivered average daily doses of 170,330, 

670, 1,900, or 4,000 mgkg body weight to males and 

240, 460, 970, 2,500, or 4,900 mgkg to females. 

TABLE15 

Differences in absolute and relative organ weights in 

exposed mice were considered to be related to lower 

bodyweights (Table F5). Microscopically,minimal 

degeneration of the germinal epithelium of the 

seminiferous tubules was observed in the testis of six 

20,000ppmmales. Testicular degeneration was 

considered to be related to the reduced body weight 

effect of p-nitrobenzoic acid exposure. 

Dose Selection Rationale: Based on lower final mean 

body weights, the dietary levels ofp-nitrobenzoic acid 

selected for the 2-year feed study in mice were 0, 

1,250, 2,500, and 5,000 ppm. 

Survival, Mean Body Weights, and Feed Consumption of Mice in the 13-Week Feed Study 

of p-Nitrobenzoic Acid 

Final Weight 

Mean Body Weightb (g) Feed Relative 

Initial SurvivalaDose Final Change to ControlsConumption' 

(PPm) (%) Week 1 Week 13 

Male 

0 1ono 33.8 21.5 f 12.4 0.4 f 0.6 3.8 3.5 f 0.6 

1,25010/1021.6 31.5 f 0.6 9.9 f 0.7.. f 0.7. 93 3.5 3.8 

f 0.6** 93 3.4 3.7 

7.6 f 0.7** 88 3.6 3.3 

2,500 1on0 21.9 31.6f 0.4 f 0.7**9.7

5,000 lono 22.09.6f 0.4 f 0.7** 

10,000 lono 25.3 0.3**4.0

21.9 23.42 0.6 2 0.4**1.6 69 4.6 5.0 f 0.9** 

17.5 26.3 f 8.8 0.2 2 0.5 4.2 3.6 ? 0.4 

17.6 25.3f 0.2 7.7 f 4.4 0596 3.9 f 0.5 

21.826.8f 0.6 f 0.3** 4.9 79 

20,000 lono

Female 

0 10110 

1,250 mod 

2,500 lono 17.6 f 0.2 26.0 8.4 f4.4 99 3.7 0.3 f 0.3 

10/1017.6 5,000 24.7 f 0.4 f 0.3** 7.1 4.6 3.6 94 f 0.3** 

10,000 1ono 17.6 f 0.3 22.6 f 0.3** 5.0 f5.8 0.3**4.2

17.5 20.1 f 0.2 2.6 f 0.4** 4.8 76 4.3 f 0.5'. 

86 

20,000 1ono 

* Significantly different (PSO.05) from the control group by Williams' test 

** PL<O.Ol 

a Number of animals surviving/number initially in group 

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

end of the studies. 

Feed consumption is expressed as gramsof feed consumed per animal per day. 

Week of death 3 (accidental) 
50 

2-y~STUDY 

Survival 

Estimates of survival probabilities for male and 

female mice are presented in Table 16 and in the 

Kaplan-Meier curves in Figure 4. Two-year survival 

rates of exposed mice were similar to those of the 

controls. 

Body Weights, Feed Consumption,

and Clinical Findings 

Mean body weights of 5,000 ppm males were 6% to 

12% lower than those of the controls after week 17 

of the study (Table 17 and Figure 5). Mean body 

weights of 5,000 ppmfemales were 12% to 24% 

TABLE16 

p-Nitrobenzoic Acid, NTP TR 442 

lower than those of the controls after week 16 

(Table 18and Figure 5). The final mean body weight 

of 5,000 ppm males was 90% that of the controls, and 

the final mean body weightof 5,000 ppm females was 

81% that of the controls. Mean body weights of the 

other exposure groups were similar to those of the 

controls. 

Feed consumption by exposed groups was similar to 

that by the control groups (Tables I3 and 14). 

Dietary levels of 1,250, 2,500, or 5,000 ppm p-nitrobenzoic acid delivered 150, 300,or 675 mgkg body 

weight per day to males and 170, 365, or 905 mgkg 

per day to females. There were no clinical findings of 

organ-specific toxicity in mice. 

Survival of Mice in the 2-Year Feed Studyof p-NitrobenzoicAcid 

Dose (PPI4 

Male 

Animals initially in study 

15-Month interim evaluationa 

Accidental deathsa 

Moribund 

Natural deaths 

Animals surviving to study termination 

Percent probability of survival at end of studyb 

Mean survival (days)' 

Survival analysesd 

Female 

Animals initially in study 

15-Month interim evaluationa 

Accidental deathsa 

Moribund 

Natural deaths 

Animals surviving to study termination 

Missinga 

Percent probability of survival at end of study 

Mean survival (days) 

Survival analyses 

a Censoredfromsurvivalanalyses 

0 1,250 2,500 5,000 

~ 

60 60 60 60 

10 10 10 10 

1 2 

9 4 10 2 

2 9 1 2 

39 36 39 44 

78 74 78 92 

666 654 664 641 

P=0.077N P=O.764 P=l.OOO P=0.125N 

60 60 60 60 

10 10 10 10 

1 2 

9 8 12 10 

3 5 4 8 

38 36 33 30 

1 

77 74 67 63 

660 658 649 625 

P=O.118 P=O.925 P=O.465 P=O.194 

~ 

Kaplan-Meier determinations basedon the number of animals aliveon first day of terminal sacrifice 

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

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

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

by N. 
51 

1.0-

-I 

4 ........... 0.9 L

2 

3 

in 

L 

0 

g .......... 0.8 

2 

m

< 

m 

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

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

............. ..;............... .;................ .;................ 

B MALE MICE 1 a 0 PPY 

0 1.25OPPY 

A 2.500 PPU 

13 5,000 PPM 

0.7 n ... ... ................. ... 

0.6 

15 

I 

30 

I 

45 

I 

60 

I 

75 

r -

f 105 

WEEKS ON STUDY 

1.0. 

-I 

-l 0.9. $ 

3 

v) 

L

0 

0.8 

2 

m< 

m

0 

KK 

a 0.7 

CI 2.500 PPY 

0.6 1 I I I I I 1 

15 30 45 60 75 90 105 

WEEKS ON STUDY 

FIGURE4 

Kaplan-Meier Survival Curvesfor Mice Administered 

p-Nitrobenzoic Acid in Feedfor 2 Years 
52 p-Nitrobenzoic Acid, NTP TR 442 

TABLE17 

Mean Body Weights and Survival of Male Mice in the 2-Year Feed Study ofp-NitrobenzoicAcid 

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

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

Study (8) Survivors (8) controls) SuMvors (8) controls) SuMvors (8) controls) SuMvors 

1 23.7 60 22.7 96 60 23.0 97 60 22.9 97 60 

2 24.8 60 24.3 98 59 24.4 98 60 23.9 96 58 

3 26.0 60 25.4 98 59 25.7 99 60 25.1 97 58 

4 26.8 60 26.5 99 59 26.4 99 60 25.9 97 58 

5 27.5 60 27.3 99 59 27.4 100 60 26.8 98 58 

6 28.1 60 27.9 99 59 27.9 99 60 27.3 97 58 

7 28.6 60 28.0 98 59 28.4 99 60 27.5 96 58 

8 29.3 60 28.8 98 59 29.3 100 60 28.3 97 58 

9 30.5 60 29.9 98 59 30.2 99 60 29.3 96 58 

10 31.1 60 30.1 97 59 30.8 99 60 29.5 95 58 

11 32.0 60 31.2 98 59 31.7 99 60 30.3 95 58 

12 31.7 60 31.8 100 59 31.5 99 60 30.7 97 58 

13 33.1 60 32.8 99 59 33.1 100 60 31.7 96 58 

17 35.4 60 34.9 99 59 34.9 99 60 33.2 94 58 

21 37.2 60 36.5 98 59 36.4 98 60 34.5 93 58 

25 38.4 60 38.0 99 59 37.8 98 60 35.3 92 58 

29 40.8 60 40.2 99 59 39.8 98 60 37.5 92 57 

33 41.9 60 41.9 100 59 41.6 99 60 38.7 92 57 

37 43.4 60 43.5 100 59 43.3 100 60 40.8 94 57 

41 45.5 60 45.1 99 59 44.8 99 60 41.8 92 57 

45 46.1 60 45.4 99 59 45.4 99 60 41.9 91 57 

49 47.5 60 46.9 99 59 46.7 98 60 43.3 91 57 

53 47.4 60 47.3 100 59 46.7 99 60 43.7 92 57 

57 47.6 60 46.8 98 59 46.3 97 60 42.9 90 56 

61 47.3 59 46.5 98 59 46.0 97 60 42.8 91 56 

65 47.9 59 47.5 99 58 46.2 97 60 43.5 91 56 

69a 47.8 49 48.0 100 48 46.3 97 49 42.8 90 46 

73 49.5 49 48.9 99 48 48.2 97 48 44.4 90 46 

77 49.2 48 48.8 99 48 48.2 98 47 44.7 91 46 

81 49.2 47 48.1 98 47 47.1 96 47 43.5 88 46 

85 48.0 47 48.4 101 46 47.3 99 46 43.6 91 45 

89 48.9 46 48.8 100 45 47.4 97 46 43.3 89 45 

93 48.0 46 49.0 102 45 47.5 99 45 43.1 90 45 

97 46.5 43 47.5 102 44 46.6 100 44 41.8 90 44 

101 45.9 43 47.7 104 39 46.4 101 40 41.4 90 44 

Mean for weeks 

1-13 28.7 28.2 98 28.4 99 27.6 96 

14-52 41.8 41.4 99 41.2 99 38.6 92 

53-101 47.9 47.9 100 46.9 98 43.2 90 

~ 

a Interim evaluation occurred during week 66. 
Results 53 

TABLE18 

Mean Body Weights and Survival of Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 

Weeks 0 ppm 1,250 ppm 2,500 ppm 5.000 ppm 

on Av. Wt. No. of Av. Wt. Wt. (%of No. of Av. W tW t (% of No. of Av. Wt. Wt. (% of No. of 

Study (SI Survivors (9) controls) Survivors (g) controls) Survivors (9) controls) Survivors 

1 17.9 60 18.0 101 60 17.7 99 60 17.8 99 60 

2 20.4 60 20.2 99 60 20.1 99 59 19.8 97 58 

3 22.4 60 22.3 100 60 22.1 99 59 21.5 96 58 

4 23.1 60 23.0 100 60 22.7 98 59 21.9 95 58 

5 24.1 60 24.1 100 60 23.6 98 59 22.7 94 58 

6 24.3 60 24.5 101 60 24.2 100 59 23.3 96 58 

7 25.2 60 25.5 101 60 24.9 99 59 23.9 95 58 

8 25.4 60 25.8 102 60 25.3 100 59 24.5 97 58 

9 25.9 60 25.8 100 60 25.4 98 59 24.5 95 58 

10 27.1 60 27.0 100 60 26.1 96 59 25.3 93 58 

11 28.2 60 28.0 99 60 27.1 96 59 26.0 92 58 

12 28.8 60 28.5 99 60 27.6 96 59 26.4 92 58 

16 31.2 60 30.2 97 60 29.7 95 59 27.4 88 58 

20 33.4 60 32.3 97 60 31.5 94 59 28.9 87 58 

24 34.3 60 33.7 98 60 32.1 94 59 29.7 87 58 

28 36.8 60 35.7 97 60 34.8 95 59 31.1 85 58 

32 39.2 60 38.1 97 60 37.2 95 59 32.9 84 58 

36 40.4 60 39.7 98 60 38.6 96 59 34.1 84 58 

40 42.6 60 42.6 100 60 41.0 96 59 35.6 84 58 

44 44.6 60 44.1 99 60 42.5 95 59 36.4 82 58 

48 46.0 60 45.7 99 60 44.0 96 59 38.1 83 58 

52 47.0 60 46.3 99 60 45.0 96 59 38.5 82 58 

56 46.3 59 45.8 99 60 43.8 95 59 37.0 80 58 

60 47.0 59 46.9 100 59 44.5 95 59 37.2 79 58 

64 48.8 58 48.0 98 59 44.7 92 59 37.6 77 58 

6ga 48.5 48 48.7 100 49 45.9 95 49 38.0 78 46 

72 49.7 48 49.5 100 49 47.2 95 47 39.0 79 45 

76 51.2 48 51.4 100 47 49.0 96 47 40.0 78 44 

80 52.1 46 51.0 98 46 48.2 93 47 39.9 77 44 

84 51.2 46 51.0 100 46 48.2 94 46 40.5 79 41 

88 52.0 44 50.6 97 44 48.1 93 45 39.6 76 41 

93 50.6 43 49.5 98 43 47.4 94 42 38.5 76 37 

96 49.9 43 48.5 97 42 46.5 93 41 39.3 79 35 

100 49.8 41 48.6 98 38 46.2 93 38 38.8 78 33 

104 48.2 38 47.7 99 36 45.2 94 33 38.8 81 30 

Mean for weeks 

1-13 24.4 24.4 100 23.9 98 23.1 95 

38.8 98 37.6 95 33.3 84 

49.0 99 46.5 94 38.8 78 

a Interim evaluation occurred during week 66. 

14-52 39.6 

53-10449.6 
54 p-Nitrobenzoic Acid, NTP TR 442 

0 15 so 45 60 75 90 1 '5 

WEEKS ON STUDY 

55 

i . .; ip 0 00;. 50 _ ...................................... j ................... i.. ............. ................@ ..!...."."....""..,.. : ..."..

i 00 jnAA.~AOO~ 

4; : AA cn 45 _ ................ :.................. :................. ........ i ..................................... 4 

I 

4 

w 

(3 4: ................ j ................... j ...... 8.......... j ................ .i.................. b...B..O..D.'i................... ~ 4o i 0; I- :mn io . io j oo 0

I oQ"" ; UiA 

(3 0 5 s5- ............... i ............... 2.;........... Q..... i ................... i ........................................................... 

3 : ioo ;

> i c3 .a 0 30-................. Q........ ...... j ................... 1.. ................ 1................... i................... 1................... 

i o

h .... i................. i ................... 1...................i........................ FEMALE MICE ..... 

i 0 1.25OPPM 

..: ................... t ................... i ...................................... .i.... A 2.500 F"M ".'. 

l i 0 5.OOOPPU 

15-

0 

I 

15 I 30 

1 

45 I 

60 

I 

75 I 

90 105 

WEEKS O N STUDY 
Results 55 

Hematology 

The results of hematology evaluations are shown in 

Table G5. No chemical-related effects on hematology 

parameters were observed at 15 months. 

Pathology and Statistical Evaluation 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences of 

neoplasms or nonneoplastic lesions of the lung and 

kidney. 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 biologically significant neoplasms mentioned in 

this section are presented in Appendix C for male 

mice and Appendix D for female mice. 

Lung: The incidences of alveolarbronchiolar 

adenoma or carcinoma (combined) in 1,250 and 

5,000 ppm females were significantly greater than that 

of the controls by painvise comparisons but not by 

the trend statistics, and the highest incidence in these 

groups (20%) fell within the historical control range 

of 2% to 26% (Tables 19, D3,and D4). The historical incidence of alveolar/bronchiolar adenoma or 

carcinoma (combined) in control female B6C3Fl mice 

from recent NTP studies is 106/1,371(7.7%). At the 

15-month interim evaluation, no hyperplasia or 

neoplasms were observed in females. At 2 years, the 

incidences of alveolar epithelial hyperplasia in 

exposed females were similar to that of the controls. 

At the 15-month interim evaluation, a few alveolar/ 

bronchiolar adenomas were observed in exposed male 

mice. In addition, alveolar epithelial hyperplasia was 

observed in one 1,250 ppm and one 2,500 ppm male 

butnot in controls. At 2 years,incidences of 

alveolarbronchiolar adenoma or carcinoma (combined) in exposed groups of male mice were not 

significantly different than that of the controls 

(Tables 19 and 0). The incidence of alveolar 

epithelial hyperplasia in 5,000 ppm males was greater 

than that of the controls. 

Alveolar epithelial hyperplasia is considered a precursor lesion of alveolar/bronchiolar adenoma and 

carcinoma. Hyperplasia consists of a focal increase in 

cellularity of the alveolar epithelium with retention of 

the alveolar architecture. In contrast, alveolar/ 

bronchiolar adenomas are discrete expansile masses 

that compress adjacent tissue. Adenomas lack 

normal architecture and consist of somewhat pleomorphic to columnar cells arranged in regular or 

papillary patterns. Alveolarbronchiolar carcinomas 

are similar but consist of heterogenous cell populations with varying degrees of cellular pleomorphism 

and atypia. Adenocarcinomas are larger, highly 

anaplastic neoplasms, often containing areas of 

hemorrhage or necrosis. 

Kidney: The relative kidney weight of females 

exposed to 5,000 ppm was significantly greater than 

that of the controls at 15 months (Table F6); however, this effect 'was considered to be related to the 

lower mean body weight in this exposure group. The 

incidence of mineralization of the kidney was lower 

than that of the controls in 5,000 ppm females(15/50, 

7/49, 7/50, 5/50; Table D5). The mineralization was 

minimal in severity (1.0,1.0,1.0,1.2) and was not 

considered to be chemical related. The incidences of 

mineralization in malesexposed to 2,500 and 

5,000 ppm were also lower than that of the controls 

(41/50,33/49,23/50,31/48; Table C5). At 15 months 

and 2 years, there were marginal decreases in the 

incidence of renal tubule regeneration in male and 

female mice. In addition, at 2 years, there were 

marginal decreases in the incidence of cortical cysts 

in male mice. 
56 p-Nitrobenzoic Acid, NTP TR 442 

TABLE19 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Mice in the 2-Year Feed Study 

of p-Nitrobenzoic Acid 

Dose (PPW 0 1,250 2,500 5,000 

Male 

15-Month Interim Evaluation 

Lunga 10 10 10 10 

Alveolar Epithelial Hyperplasiab 0 1 1 0 

Alveolarbronchiolar Adenoma 0 1 2 1 

2-Year Study 

Lung 50 50 50 50 

Alveolar Epithelial Hyperplasia 2 7 7 8* 

Alveolarbronchiolar Adenoma 

Overall rate' 6/50 (12%) 12/50 (24%) 8/50 (16%) 9/50 (18%) 

Adjusted rated 15.4% 29.2% 20.5% 20.5% 

Life table testf 

First incidence (days) 

Terminal ratee 6/39 (15%) 

P=O.545 

729 (9 

P=O.O77 

537 

8/36 (22%) 

P=O.385 

729 0

8/39 (21%) 

729 (T> 

9/44 (20%) 

P=O.378 

Logistic regression testf P=O.393 P=O.O91 P =0.385 P=O.378 

Alveolarbronchiolar Carcinoma 

Overall rate 1/50 (2%) 3/50 (6%) 2/50 (4%) 5/50 (10%) 

Terminal rate 

Adjusted rate 

1/39 (3%) 

2.6% 

2/36 (6%) 

8.0% 

2/39 (5%) 

5.1% 11.4% 

5/44 (11%) 

Life table test 

First incidence (days) 

P=O.116 

729 (T> 

P=O.279 

725 

P=O.500 

729 (T> 729 (T>

P=O.133 

Logistic regression test P=O.O95 P=O.279 P=O.500 P=O.133 

Alveolarbronchiolar Adenoma or Carcinomag 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

7/39 (18%) 

17.9% 

7/50 (14%) 

P=O.303 

729 (T> P=O.O53 

537 

10/36 (28%) 

34.2% 

14/50 (28%) 

10/39 (26%) 

25.6% 

10/50 (20%) 

P=O.293 

729 (T> 

13/44 (30%) 

29.5% 

13/50 (26%) 

P=O.166 

729 (T> 

Logistic regression test P=O.165 P=O.O64 P=O.293 P=O.166 

(continued) 
Results 57 

TABLE19 

Incidences of Neoplasms and Nonneoplastic Lesionsof the Lung in Mice in the 2-Year Feed Study 

of p-Nitrobenzoic Acid (continued) 

0 1,250 2,500 5,000 

Female 

Lung 50 49 50 50 

Alveolar Hyperplasia Epithelial 3 3 0 1

Alveolarbronchiolar Adenoma 

Overall rate 3/50 (6%) 5/49 (10%) 3/50 (6%) 8/50 (16%) 

Adjusted rate 7.5% 12.9% 8.7% 24.3% 

Terminal rate 2/38 (5%) 3/36 (8%) 2/33 (6%) 6/30 (20%) 

First incidence (days) 689 685 715 570 

Life table test P=O.O35 P=O.324 P=O.599 P=O.O50 

Logistic regression test P=O.O52 P=O.343 P=O.643 P=O.O71 

Alveolarbronchiolar Carcinoma 

Overall rate 0/50 (0%) 5/49 (10%) 1/50 (2%) 1/50 (2%) 

Adjusted rate 0.0% 13.9% 2.9% 3.3% 

Terminal rate 0/38 (0%) 5/36 (14%) 0133 (0%) 1/30 (3%) h First incidence (days) - 720 730 (T) 730 (T> Life table test P=0.572N P=O.O29 P=O.468 P=O.453 

Logistic regression test P=0.568N P=O.O29 P=O.491 P =0.453 

Alveolarbronchiolar Adenoma or Carcinoma' 

Overall rate 3/50 (6%) 10/49 (20%) 4/50 (8%) 9/50 (18%) 

Adjusted rate 7.5% 26.1% 11.3% 27.5% 

Terminal rate 2/38 (5%) 8/36 (22%) 2/33 (6%) 7/30 (23%) 

First incidence (days) 689 685 715 570 

Life table test P=O.O63 P=O.O31 P =0.428 P =0.027 

Logistic regression test P=O.O88 P=O.O31 P=O.475 P=O.O39 

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

(9Terminal sacrifice 

a Number of animals with lung examined microscopically 

Number of animals with lesion 

Number of animals with neoplasm per number of animals examined microscopically 

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

e Observed incidence in animalssurvivinguntil theend ofthestudy 

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

corresponding to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in 

animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these 

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

g Historical incidence for 2-yearNTP feed studies with untreated control groups (mean & standard deviation): 

242/1,369 (17.7% & 7.3%), range 4%-30% 

Not applicable; no neoplasmsin animal group 

Historical incidence: 106/1,371 (7.7% -r- S.O%), range 2%-26% 
58 

GENETICTOXICOLOGY 

p-Nitrobenzoic acid, tested in a preincubation protocol at concentrations of 1 to 3,333 pg/plate, with and 

without induced rat or hamster S9, was mutagenic in 

strain TAlOO (Table El; Zeiger et al., 1987). No 

mutagenicity was detected in strains TA98, TA1535, 

or TA1537, with or without S9. 

In cytogenetic tests with cultured Chinese hamster 

ovarycells, p-nitrobenzoic acid induced significant 

increases in sister chromatid exchanges (Table E2; 

Zeiger et al., 1987) and chromosomal aberrations 

(Table E3; Zeiger et al., 1987) at dose levels which 

induced cell cycle delay in the absence of S9; no 

increases in either endpoint were observed in the 

presence of S9. In the sister chromatid exchange test 

without S9, doses ranging from 498 to 1,000 pg/mL 

p-Nitrobenzoic Acid, NTP TR 442 

produced positive responses, induced cell cycle delay, 

and required use of an extended harvest-protocol to 

allow accumulation of sufficient cells for metaphase 

analysis.Doses producing positive responses in the 

chromosomal aberrations assay without S9 ranged 

from 875 to 1,750 pg/mL p-nitrobenzoic acid. As 

with the sister chromatid exchange test, cell harvest 

was delayed to permit a sufficient number of cells to 

progress to metaphase for analysis. 

Despite the positive results obtained in the in vitro 

studies, results of a single NTP in vivo genotoxicity 

study were negative. In this study, the frequencies of 

micronucleated normochromatic erythrocytes in the 

peripheral blood of male and female mice were 

unaffected by exposure to p-nitrobenzoic acid in feed 

for 13 weeks (Table E4). 
59 

DISCUSSION AND CONCLUSIONS 

p-Nitrobenzoic acid is produced in large volumes for 

organic synthesis and as an intermediate in the 

manufacture of pesticides, dyes, and industrial solvents. p-Nitrobenzoic acid is also a hydrolysis product or metabolite of other high-volume production 

chemicalsincluding p-nitrobenzoyl chloride and 

o-nitrotoluene. Despite the widespread use and 

occurrence of p-nitrobenzoic acid, there is little 

information on the toxic and carcinogenic effects of 

this chemical after long-term exposure. These 

toxicity and carcinogenicity rodent studies were 

conducted to provide this information. 

Many nitroaromatic compounds are toxic to the 

hematopoietic system (Beard and Noe, 1981; Beutler, 

1985;Bus and Popp, 1987). Generalized toxicity to 

the hematopoietic system of the rat was observed in 

studies of the nitroaromatic compounds o-,rn-, and 

p-nitrotoluene (NTP, 1992), p-chloroaniline (NCI, 

1979a), and o-nitroanisole (NTP, 1993). The 

mechanism of the anemia is thought to involve 

oxidative damage to hemoglobin leading to Heinz 

body formation and decreased erythrocyte survival, 

followed by macrophage ingestion of the injured 

erythrocytes and removal of macrophages by the 

spleen resulting in splenic congestion and 

hemosiderin accumulation. The characteristic hematologic toxicity induced by amine/nitroaromatic 

compounds is attributed tothe formation of a 

hydroxylamino compound (Facchini and Griffiths, 

198l), and it has been observed in a variety of 

animals, including rodents, dogs, and cats, exposed to 

nitroaromatic compounds (Kiese, 1966) and in 

humans exposed to aniline and nitroaromatic compounds (Finch, 1948; Smith, 1991). p-Nitrobenzoic 

acid was toxic to the hematopoietic system of rats in 

the current studies. 

In the 14-day rat study, there were decreasesin 

erythrocyte count and hemoglobin and hematocrit 

values, and increases in nucleated erythrocytes, 

reticulocyte counts, and methemoglobin concentration, which were most pronounced in the 20,000 and 

40,000ppm groups. Hypertrophy of the follicular 

epithelium of the thyroid gland was observed in rats 

at doses of 10,000 ppm and greater in the 14-day 

studies, but this change was not seen in the 13-week 

or 2-year studies, probably because lower doses were 

used and rats were able to adapt io this effect. In the 

13-week rat study, a mild hemolytic (regenerative) 

anemia was characterized by decreases in hematocrit 

and hemoglobin values and increases in methemoglobin concentration, reticulocyte counts, and Heinz 

body formation in 10,000ppm rats. Hemosiderin 

accumulation in the spleen was present in 2,500, 

5,000, and 10,000 ppm male and female rats, and 

congestion was observedin2,500 and 5,000 ppm 

males and 10,000 ppmmales and females. There 

were increased incidences of renal tubule pigmentation (hemosiderin) in 5,000 and 10,000 ppmmales. 

Increases in absolute and relative spleen weights were 

observed in 10,000 ppmrats in the 13-week study. At 

the 15-month interim evaluation in the 2-year rat 

study, there were decreases in erythrocyte count, 

hemoglobin, and hematocrit levels, and increases in 

nucleated erythrocytes that were most pronounced in 

5,000 ppmfemales. In exposed males and females, 

increased severity of pigmentation (hemosiderin) of 

renal tubule epithelial cells was also supportive of 

this hemolytic anemia. 

Hematologic toxicity was not 'observed in mice in 

these studies. In studies of other nitroaromatic 

compounds (e.g., nitrotoluenes, p-chloroaniline, and 

p-nitroanisole) the hematologic toxicity was also less 

severe in mice than in rats. Studies with p-chloroaniline hydrochloride (NTP, 1989a) and aniline 

hydrochloride (NCI, 1978) suggest that these chemicals are cleared from blood more quickly in mice 

than in rats (McCarthy et al., 1985). Species differences in clearance of p-nitrobenzoic acid and its 

metabolites from blood may also account for the fact 

that mice are less susceptible to hematologic toxicity 

than rats. Methemoglobin can be reduced to hemoglobin in mammalian species by NADH-dependent 

methemoglobin reductase located in erythrocytes. 

Micehavehigherlevels of this reductase than rats 

(Smith, 1991), and species differences in the ability to 

reduce methemoglobin may be another reason why 

mice are less susceptible than rats to the hematologic 

toxicity of p-nitrobenzoic acid. 
60 

Becauseof the hematologic toxicity in rats and 

decreased bodyweights of 10,000,20,000, and 

40,000 ppm rats and mice in the 14-day studies and 

10,000 ppm rats and mice in the 13-week studies, the 

highest exposure level selected for the 2-year studies 

was 5,000 ppm. While there were no chemicalrelated decreases in survival of exposed groups in the 

2-year studies, the mean body weights of 5,000 ppm 

female rats, 5,000 ppm male mice, and 5,000 ppm 

female mice were consistently lower than those of the 

respective control groups. The 2-year studies were 

considered to be adequate assessments of the carcinogenic potential ofp-nitrobenzoic acid in the F344/N 

rat and B6C3F1 mouse. 

In the 2-year study, the incidences of mononuclear 

cell leukemia were decreased in exposed groups of 

male and female rats. The incidences of mononuclear cell leukemia in 5,000 ppm male rats (4%) 

and 2,500 (6%) and 5,000 ppm (0%) female rats were 

below the historical control ranges from recent NTP 

2-yearfeed studies (males: range 32% to 62%, 

603/1,253, mean 48%; females: range 14% to 52%, 

324/1,251, mean 26%). While the mechanism for this 

decrease is unknown, a decrease in the incidence of 

mononuclear cell leukemia has also been observed 

with other aminehitro compounds including aniline 

hydrochloride (NCI, 1978) andp-chloroaniline (NCI, 

1979a). Injury to splenic cells associated with hematologic toxicity may decrease the chance for the 

development of mononuclear cell leukemia, which 

arises from splenic cells in the Fischer rat (Losco and 

Ward, 1984; Stromberg, 1985). The splenic toxicity 

appears to be less severe with p-nitrobenzoic acid 

than with aniline hydrochloride or p-chloroaniline 

(Stefanski et al., 1990). In studies of aniline hydrochloride (NCI, 1978), p-chloroaniline (NCI, 1979a), 

o-toluidine (NCI, 1979b), and D&C Red No. 9 (NTP, 

1982a) splenic damage was more extensive and led to 

fibrosis and the development of sarcomas of the 

spleen (Goodman et al., 1984;Weinberger et aL, 

1985). 

While decreased incidences of mononuclear cell 

leukemia have been observed in rats treated with 

aminehitro compounds (Table 17), not all chemicals 

of this class produced this effect [e.g., o-nitroanisole 

(NTP, 1993)]. Decreased incidences of mononuclear 

cell leukemia have also been observed with aromatic 

compounds that donot contain the aminehitro 

substitution [e.g., 4-hexylresorcinol (NTP, 1988a) and 

p-Nitrobenzoic Acid, NTP TR 442 

a-methylbenzyl alcohol (NTP, 199O)J Some of the 

chemicals that caused decreased incidences of mononuclear cell leukemia [nitrobenzoic acid (King and 

Henschel, 1941; Rosenthal and Bauer, 1941) and 

4-hexylresorcinol (NTP, 1988a; Burnens and 

Vurma-Pupp, 1989; Collins and Levett, 1989)] share 

an antibacteriostatic activity, but studies to determine 

if this activity is related to inhibition of nucleic acid 

synthesis or cell proliferation/growth have not been 

reported. It has previously been reported (Raoet al., 

1987) that decreased incidencesof naturally occurring 

neoplasms (e.g., neoplasms of the liver or mammary 

gland) may occur when a chemical causes a decrease 

inbodyweight.In the present 2-year studies of 

p-nitrobenzoic acid, there were decreases in body 

weights in exposed groups of rats where there were 

also decreases in the incidences of mononuclear cell 

leukemia. Further studies are needed to explain the 

relationship between bodyweight and neoplasm 

occurrence in rodents and to determine if body 

weight was a factor in the decreased incidences of 

mononuclear cell leukemia in rats exposed to 

p-nitrobenzoic acid. 

In the 13-week rat study, chemical-related effects on 

the kidney included hyaline droplet accumulation in 

males and karyomegaly in males and females. In the 

2-year rat study, chemical-related effects included 

tubule epithelial cell hyperplasia in 1,250 ppm males 

and oncocytic hyperplasiain 5,000 ppm males and 

females. Also at 2 years, nephropathy severity 

decreasedwith increasing dose in both males and 

females.However, the association between hyaline 

droplet accumulation (aw-globulin-associated nephrotoxicity) and kidney neoplasms was not observed in 

the 2-year study.Chemicals that cause protein 

droplet accumulation have been found to bind to 

aw-globulin (Dietrich and Swenberg, 1991). Evidence 

suggests that the chemical binding is responsible for 

the accumulation of this protein. Chemical-mediated 

accumulation of crw-globulin is thought to be responsible for cell death, which in turn stimulates cell 

division as the kidney attempts to repair itself. With 

prolongedchemical exposure, repeated cycles of 

cytotoxicity and reparative replication are proposed 

to be responsible for the observed tumorigenic 

response (USEPA, 1991). In the present studies, the 

typicalcytotoxicity associated with hyaline droplet 

(aw-globulin-associated) nephrotoxicity (such as 

single cell necrosis of the P2 segment epithelium, 

accumulation of granular casts, linear mineralization 
Discussion and Conclusions 61 

of Mononuclear Cell Leukemia in Male and Female Fisher Rats 

Results of Carcinogenicity Tests of Selected Chemicals Causing Decreased Incidences 

TABLE17 

Chemical 

Incidences of Leukemiaa 

Male Rats Female Rats 

Rat 

d 

Rat Mouse Mouse 

0 d 0 

Carcinogenicity 

Result 

Test 

Salmonella 

NTP TR 233 

Hydrochloride 

2-Biphenylamine 

15/50, 1/50, 4/50 5/50, 1/49, 3/50 

NH, l HCI 

NTP TR 351 

Hydrochloride 

p-Chloroaniline 

I 

NH, 0 HCI 3 

CI 

21/49, 3/50, 2/50, 3/50 10/50,2/50,1/50, 1/50 

S 

+ -

L,He 

+ - + 

NTP TR 211 

C.I. Acid Orange 10 

22/90, 4/50, 3/50 16/88, 2/50, 0/50 

NTPTR 222 

C.I. Disperse Yellow 3 

13/50,2/50, 1/50 8/50, 2/50, 1/50 

L 

+ - -

L 

+ + 

NTP TR 226 

C.I. Solvent Yellow 14 

11/50, 2/49, 0/49 

L 

+ 

L 

+ - - + 
62 p-Nitrobenzoic Acid, NTP TR 442 

TABLE17 

Results of Carcinogenicity Testsof Selected Chemicals Causing Decreased Incidences 

of Mononuclear Cell Leukemia in Male and Female Fisher Rats(continued) 

Incidences of Leukemia Carcinogenicity SahmUu 

Male Rats Female Rats d 0 d 0 Test 

Chemical Rat Rat Mouse Mouse Result 

D&CRed9 

NTP TR 225 

12/50,4/50,3/5011/50,5/50,3/50 + 

Ba 

S,L 

Nfl-Dimethylaniline 

NTP TR 360 

H,C\/CH3 

13/50,4/50,3/5011/50,7/50, 0/50 + 

S 

4-Hexylresorcinol 

NTP TR 330 

OH 

HO Q 12/49,1/50,1/5016/50,3/50,2/50 - - - - -

CHz(CHz)&W 

a-Methylbenzyl Alcohol 

NTP TR 369 

I 

OH 

I 

HC -CH, 15/50,2/50,0/5012/50,2/50,2/50 

K 

+ 

Monuron 

NTP TR 266 

0 

Cl 
- - 

Discussion and Conclusions 63 

TABLE17 

Results of Carcinogenicity Testsof Selected Chemicals Causing Decreased Incidences 

of Mononuclear Cell Leukemia in Male and Female Fisher Rats (continued) 

Incidences of Leukemia Carcinogenicity SalmoneUa 

Male Rats Female Rats d 0 d 0 Test 

Chemical Rat Rat Mouse Mouse Result 

Nalidivic Acid 

NTP 368 

CH,CH7 

p-Nitmbenzoic Acid 

NTP TR 442 

COOH 29/50,35/50,26/50, 2/50 17/50, 11/50, 3/50,0/50 - + t 

C 

4,4' -0xydianiline 

NCI TR 205 

27/50,4/50, 4/50, 3/50 3/50, 1/50, 2/50,0150 + + + + t 

L,T L,T,H L,T L,T,H

a Incidences given in increasing order of dose level beginning with control. 

Levels of carcinogenic evidence: -+ = some or clear evidence, -

H = harderian gland, He = hemangiosarcoma,K = kidney, L = 

of tubules within the renal papilla, hyperplasia of the 

renal pelvis, or significant increases in the incidences 

of kidney neoplasms) was not observed. In addition, 

compared to cuw-globulin-associated nephrotoxicity 

where there is an exacerbation of spontaneous 

chronic progressive nephropathy, there was a 

decrease in the severity of nephropathy in rats 

exposed to p-nitrobenzoic acid. The lack of cytotoxicity in male rats with hyaline droplet accumulation would suggest that p-nitrobenzoic acid might 

not bind strongly to cuw-globulin, thus cuw-globulin 

accumulation might be below the concentration that 

elicits a cytotoxic response and subsequently a carcinogenic effect. 

= no or equivocal evidence; C = clitoral gland, 

liver, P = preputial gland, S = spleen, T = thyroid gland. 

In NTP studies, oncocytic hyperplasia has not 

commonly been associated with the spectrum of renal 

proliferative lesions thought to be important in the 

development of renal tubule adenomas and 

carcinomas. Renal tubule neoplasms are thought to 

originate from proximal tubules, while oncocytic 

proliferative lesions are thought to originate from 

distal tubules (Bannasch et al., 1986). In addition, 

oncocytic proliferative lesions are different from renal 

tubule proliferative lesions in that cells in oncocytic 

proliferative lesions are usually much larger and have 

a more densely packed granular eosinophilic

cytoplasm; the characteristic granular appearance of 

the cytoplasm iscaused by populations of atypical 
64 

mitochondria. In the present studies, oncocytic 

hyperplastic lesions did not progress to oncocytomas. 

Rat renal oncocytomas appear to be benign end stage 

lesions that do not progress to malignant neoplasms 

(Bannasch et al., 1986). In the NTP database, kidney 

proliferative lesions and neoplasms include 

documented oncocytic hyperplasias and oncocytomas, 

but no malignant oncocytic neoplasmshave been 

observed. 

Thestep sections revealed additional renal tubule 

neoplasms and hyperplasia in control and exposed 

male rats, but, in exposed males, incidences of renal 

tubule adenoma or carcinoma (combined) from both 

the single- and step-section evaluations were similar 

to that of the controls (1/50, 1/50,3/50,3/50). The 

incidences of renal tubule hyperplasia in 1,250 ppm 

males and of oncocytic hyperplasiain 5,000 ppm 

males were significantly greater than those of the 

controls. An increase in nonneoplastic lesions alone 

is not considered to be evidence of a carcinogenic 

effect. Further,the incidences of renal tubule 

adenoma (1/50,1/50,1/50,3/50) from the step-section 

evaluation fell within the historical range for renal 

tubule adenoma from step-section evaluations in 

male control rats from other NTP studies (range 0% 

to 8%, 18/608, mean 3%; Table A4b). Thus,p-nitrobenzoic acid did not cause chemical-related increases 

in the incidences of kidney neoplasms either by the 

initial single-section evaluation or by the step-section 

evaluation. There were no renal tubule neoplasms in 

female rats. 

In the 2-year rat study, the incidences of clitoral 

gland adenoma in1,250,2,500, and 5,000 ppm 

females, the incidence of clitoral gland carcinoma in 

2,500ppmfemales, and the incidences of clitoral 

gland adenoma or carcinoma (combined) in all 

exposed groups of females (0 ppm, 4/50; 1,250 ppm, 

14/49;2,500 ppm, 15/49; 5,000 ppm,15/50)were 

significantly greater than those in the controls by 

both the life table and logistic regression tests. The 

incidences of clitoral gland adenoma or carcinoma 

(combined) in each exposed group (29% to 31%) 

were greater than that in historical controls (mean 

incidence, 11%; range, 2% to 21%). Based on these 

clitoral gland neoplasms, there was some evidence of 

a carcinogenic effect of p-nitrobenzoic acid in the 

female rat. The neoplasm incidences were not 

considered to represent a clear carcinogenic response 

because the incidences of clitoral gland neoplasms 

were approximately the same in each exposure group, 

p-Nitrobenzoic Acid, NTP TR 442 

despite a fourfold increase in dose from the lowest 

exposure level to the highest. In addition, there was 

no notable decrease in the time to occurrence of 

neoplasm (mean time to diagnosis: 0 ppm, 699 days; 

1,250 ppm, 712days;2,500 ppm, 672days; or 

5,000 ppm,683days) nor was there an increased 

incidence in clitoral gland hyperplasia. 

A chemical-related increase in the incidence of 

clitoral gland neoplasms is often accompanied by an 

increase in the incidence of preputial gland neoplasms (male counterparts of clitoral gland neoplasms). In this study, a slight increase in the incidence of preputial gland neoplasms was observed in 

animalsreceiving 5,000 ppm p-nitrobenzoic acid. 

However, because of improved survival in this group, 

some increase in neoplasm incidence would be 

expected to occur by chance, and the life table test 

indicated that the slight increase was not statistically 

significant. The life table test was givenprimary 

emphasis because 21 of the 25 neoplasms occurred in 

animals that died early,suggesting thatthese neoplasms may have contributed to their deaths. Moreover, the neoplasm incidence in 5,000 ppmmales 

(18%) fell well within the historical control range 

(2%-30%), and there was no chemical-related 

increase in preputial gland hyperplasia. Thus, the 

slight increase in preputial gland neoplasms was not 

considered to be chemical related. 

The mechanism for the formation of clitoral gland 

neoplasms following exposure to p-nitrobenzoic acid 

could not be fullyexplained by the results of the 

present studies. Clitoral gland neoplasms have 

usually been observed with those chemicals that are 

genotoxic(Ashby and Tennant, 1991). Chemicals 

shown to induce clitoral gland neoplasms generally 

are strong mutagens in a variety of Salmonella test 

strains, and also induce neoplasms of the Zymbal's 

gland, skin, mammary gland, or a combination of 

these sites (Copeland-Haines and Eustis, 1990). 

p-Nitrobenzoic acid was positive in only one of the 

Salmonella test strains'used and was negative in the 

in vivo mouse micronucleus test. 

The incidences of thyroid gland C-celladenoma (9/50, 

5/49,4/50,2/50) and adenoma or carcinoma (combined) (10/50, 5/49,6/50,2/50) were significantly 

decreasedin 5,000 ppm female. rats. This response 

could not be conclusively related to p-nitrobenzoic 

acid exposure because the incidence ofC-cell 

adenoma or carcinoma (combined) is highly variable 
Discussion and Conclusions 65 

in historical controls. There were no chemicalrelated decreased incidences of thyroid gland C-cell 

neoplasms in male rats. 

At the end of the 2-year mouse study, the incidences 

of alveolarbronchiolar adenoma or carcinoma (combined) in 1,250 and 5,000 ppm females were significantly greater than that of the controls by pairwise 

comparison (3/50, 10/49,4/50,9/50).However, the 

occurrence of lung neoplasms was not considered to 

be related to chemical administration because these 

neoplasms were not increased by the trend statistic; 

the incidences of alveolar/bronchiolar adenoma or 

carcinoma (combined) were within the historical 

range for control female B6C3Fl mice in recent NTP 

feed studies (range 2% to 26%, 106/1,371, mean8%); 

and there was no increase in the incidence of alveolar 

epithelial hyperplasia, a preneoplastic lesion. In 

other NTP studies where the lung was a target site 

for chemical-induced neoplasms in mice, the chemical 

is usualIy genotoxic, there are preneoplastic lesions, 

and males and females are both affected. 

Nitroaromatic compounds are an important class of 

chemicals and it is estimated that 10% of chemicals 

used in various chemical industries are nitroaromatic 

chemicals. In a review of 301 chemicals studied by 

NTP, there were 84 aromatic amino/nitro-type 

chemicals: 59 were carcinogenic (93% of these 

chemicals were positive in the S. typhimurium assay); 

8 gaveonly equivocal evidence of a carcinogenic 

response (63% were positive in the S. typhimurium 

assay); and 17 gave no evidence of a carcinogenic 

response (71% were positive in the S. typhimurium 

assay)(Ashby and Tennant, 1991). p-Nitrobenzoic 

acid fell into the class of aromatic aminohitro-type 

chemicals that gave only some evidence for carcinogenic activity in the rodent. The benzoic acid moiety 

on the aromaticring allows forp-nitrobenzoic acid to 

be conjugated with glucuronic acid, and may allow for 

more rapid excretion of the chemical in the urine 

than the other aromatic/amino chemicals that have 

been shown to cause some or clear evidence of 

carcinogenic activity in the rodent. 

The regional position of substitutions onthe 

aromatic ring plays an important role in the eventual 

metabolism and carcinogenic activity of the chemical 

(Jakoby et al., 1982; Rickert, 1987). In several series 

of aromatic isomers tested for carcinogenic activity, 

the ortho-substituted chemical was more carcinogenic 

than the meta- or para-substituted chemicals. For 

example, in a 13-week study of 0-,m-, and p-nitrotoluene, o-nitrotoluene caused mesothelioma and 

mesothelial cell hyperplasia in male rats, but no 

preneoplastic lesions or neoplasms were observed 

with m- or p-nitrotoluene (NTP, 1992). Weisburger 

et al. (1978) reported that ortho-substituted aromatic 

compounds are more potent carcinogens than corresponding isomers with meta- or para-substitutions. 

This was observed witho-,m-, andp-toluidine, where 

o-toluidine was carcinogenic in rats, while carcinogenicactivitywas not reported in rats treated with 

m- or p-toluidine. Information on the carcinogenic 

potential of m- and o-nitrobenzoic acid is not available, and the results on the carcinogenic activity of 

p-nitrobenzoic acid reported here may not be predictive of the carcinogenic activity of the other isomers. 

CONCLUSIONS 

Under the conditions of these 2-year feed studies, 

there was no evidence of carcinogenicactivity*of 

p-nitrobenzoic acid in male F344/N rats exposed to 

1,250,2,500, or 5,000 ppm. There was some evidence 

of carcinogenicactivityof p-nitrobenzoic acidin 

femaleF344/N rats based on increases in the incidences of clitoral gland adenoma and of clitoral gland 

adenoma or carcinoma (combined). There was no 

evidence of carcinogenic activity ofp-nitrobenzoic acid 

in male or female B6C3Flmiceexposed to 1,250, 

2,500, or 5,000 ppm. 

There were chemical-related decreases in the incidences of mononuclear cell leukemia in exposed male 

and female rats. p-Nitrobenzoic acid causedmild 

hematologic toxicity in female rats. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 10. A summary of the Technical Reports Review 

Subcommittee comments and the public discussion on this Technical Report appears on page 12. 
66 p-Nitrobenzoic Acid, NTP TR 442 

\ 
67 

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(1990). Spleen, lymph nodes, and thymus. In

Pathology of the Fischer Rat: Reference and Atlas 

(G.A. Boorman, S.L. Eustis, M.R. Elwell,C.A. 

Montgomery, Jr., and W.F. MacKenzie,Eds.), 

pp. 369-403. Academic Press, Inc., San Diego, CA. 

Straus, D.S. (1981). Somatic mutation, cellular 

differentiation, and cancer causation. JNCI 67, 

233-241. 

p-Nitrobenzoic Acid, NTP TR 442 

Stromberg, P.C. (1985). Animal model of human 

disease: Large granular lymphocyte leukemia in 

F344rats: Model for human Ty lymphoma, 

malignant histiocytosis, and chronic T-cell 

lymphocytic leukemia. Am. J. Pathol. 119,517-519. 

Sundvall, A., Marklund, H., and Rannug, U. (1984). 

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generated in the production of nitrobenzoic acids 

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Tennant, R.W., Margolin, B.H., M.D., Shelby,

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74 p-Nitrobenzoic Acid, NTP TR 442 
75 

APPENDIX A 

SUMMARY OF LESIONS IN MALE RATS 

IN THE 2-YEAR FEED STUDY 

OF p-NITROBENZOIC ACID 

TABLEA1 Summary of the Incidence of Neoplasms in Male Rats 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 77 

TABLEA2 Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 82 

TABLEA3 Statistical Analysis of Primary Neoplasms in Male Rats 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 106 

TABLE A4a Historical Incidence of Renal Tubule Neoplasms 

in Untreated Male F344/N Rats .......................................... 112 

TABLE A4b Historical Incidence of Renal Tubule Lesions 

from Single and Step Sections in Male F344/N Rats .......................... 113 

TABLE A4c Historical Incidence of Preputial Gland Neoplasms 

in Untreated Male F344/N Rats .......................................... 115 

TABLEA4d Historical Incidence of Mononuclear Cell Leukemia 

in Untreated Male F344/N Rats .......................................... 116 

TABLEA5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in the 2-Year Feed Study ofp-Nitrobenzoic Acid ............................. 117 
76 p-Nitrobenzoic Acid, NTP TR 442 
Lesions in Male Rats n 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acida 

Disposition Summary 

Animals initially in study 

lS-bfonthintaim evahiim 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

Animals examined microscopically 

Endocrine System 

Adrenal medulla 

Pheochromocytoma benign 

Pituitary gland 

Pars distalis, adenoma 

Genital System 

Preputial gland 

Adenoma 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Histiocytic sarcoma 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammary gland 

Fibroadenoma 

Respiratory System 

Lung 

Alveolarlbronchiolar adenoma 

~ ~~ ~ 

0 PPm 

60 

10 

32 

6 

12 

60 

(10) 

1 (10%) 

(10) 

4 (40%) 

5(50%) 

(10) 

(10) 

(10) 

(10) 

(9) 

(10) 

~~~~~~ 

1,250 ppm 

60 

10 

34 

3 

13 

60 

(?I 

(10)

7 (70%) 

1 (10%) 

(10) 

2,500 ppm 

60 

10 

34 

3 

13 

60 

(10)

1 (10%) 

(10)

7 (70%) 

2(20%) 

5,000 ppm 

60 

10 

25 

4 

21 

60 

(10)

1 (10%) 

(10) 

1 (10%) 
78 p-Nitrobenzoic Acid, NTP TR 442 

TABLEAI 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

15-Month Interim Evaluation (continued) 

Systemic Lesions 

Multiple organsb (10)

Histiocytic sarcoma 

Leukemia mononuclear 

Systems Examined WUh No Neoplasms Observed 

Cardiovascular System 

General Body System 

Musculoskeletal System 

Nervous System 

Special Senses System 

Urinary System 

2-Year Study 

Alimentary System 

Intestine large, colon 

Polyp adenomatous 

Intestine large, rectum 

Intestine large, cecum 

Hemangiosarcoma 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Osteosarcoma, metastatic, bone 

Mesentery 

Carcinoma, metastatic, kidney 

Schwannoma malignant, metastatic, 

peripheral nerve 

Pancreas 

Schwannoma malignant, metastatic, 

peripheral nerve 

Acinar cell, adenoma 

Pharynx 

Palate, squamouscell papilloma 

Salivary glands 

Schwannoma malignant 

Stomach, forestomach 

Stomach, glandular 

Leiomyoma 

Tongue 

Squamous cell papilloma 

(49) 

(49) 

(49)

1 (2%) 

(48) 

(49)

2 (4%) 

(48) (49) 

(49)

3 (6%) 

(50) 

2 (4%) 2 (4%) 1 (2%) 

2 (4%) 

1 (2%) 

(13)

1 (8%) 

1 (8%) 

(49) (50) (49) 

1 (2%) 

2 (4%) 3 (6%) 

1 (100%)

(1) 

1 (2%)

(49) 

(50) 

(50) 
Lesions in Male Rats 79 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid (continued) 

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

&Year s&dy (continued) 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Bilateral, carcinoma 

Duct, squamous cell papilloma 

Prostate 

Seminal vesicle 

Schwannoma malignant, metastatic, 

peripheral nerve 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, kidney 

Pancreatic, carcinoma, metastatic, kidney 

Renal, carcinoma, metastatic, kidney 

(50) 

(50) 

(50)

2 (4%) 

8 (16%) 

2 (4%) 

(49)

2 (4%) 

1 (2%) 

(48) 

(49)

19 (39%) 

(49)

4 (8%) 

(50)

34 (68%) 

10 (20%) 

(50) 

(24) 

(50) 

7 (14%) 

4 (8%) 

(50)

1 (2%) 

(49) 

(50)

12 (24%) 

1 (2%) 

(49)

2 (4%) 

1 (2%) 

(50) 

(50) 

2 (4%) 

1 (2%) 

1 (2%) 

(50) 

(26) 

(50)

1 (2%) 

(50)

1 (2%) 

5 (10%) 

2 (4%) 

(49)

2 (4%) 

(47) 

(49)

16(33%) 

(49)

2 (4%) 

3 (6%) 

(49) 

(49) 

4 (8%) 

3(6%) 

1 (2%) 

1 (2%) 

(50) 

(49) 

(49) 

35 (71%) 

9 (18%) 

(49) 

(49)

12 (24%) 

(50)

3 (6%) 

1 (2%) 

1 (2%) 

(50)

21 (42%) 

15 (30%) 
80 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid (continued) 

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

2-Yef.Wstudy (continued) 

Hematopoietic System (continued) 

Lymph node, mandibular 

Lymph node, mesenteric 

Carcinoma, metastatic, kidney 

Spleen 

Fibrosarcoma 

Histiocytic sarcoma 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

.!Keratoacanthoma 

Squamous cell papilloma 

Trichoepithelioma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, sarcoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Schwannoma malignant, metastatic, 

peripheral nerve 

Nervous System 

Brain 

Glioma malignant 

Peripheral nerve 

Schwannoma malignant 

Spinal cord 

Respiratory System 

Lung 

Alveolaribronchiolar adenoma 

Alveolaribronchiolar carcinoma 

Carcinoma, multiple, metastatic, kidney 

Osteosarcoma, multiple, metastatic, bone 

Squamous cell carcinoma 

Nose 

Nasolacrimal duct, squamous cellcarcinoma 

Trachea 

(49) 

(49) 

(50) 

(48)

1 (2%) 

(49)

2 (4%) 

(50) 

3 (6%) 

4 (8%) 

1 (2%) 

1 (2%) 

(50) 

(1) 

1 (100%) 

(50) 

(50)

1 (2%) 

(49) 

(49) 

(50) 

(50) 

(50)

1 (2%) 

(49)

1 (2%) 

(50) 

3 (6%) 

6 (12%) 

1 (2%) 

(50)

2 (4%) 

(49)

4 (8%) 

(50)

1 (2%) 

3 (6%) 

2 (4%) 

4 (8%) 

(50)

1 (2%) 

(1) 

(46)

2 (4%) 

(50)

1 (2%) 

2 (4%) 

1 (2%) 

7 (14%) 

4 (8%) 

1 (2%) 

(50)

1 (2%) 

(1) 
Lesions in Male Rats 81 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid(continued) 

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

2-Yef.W shdy (continued) 

Special Senses System 

Ear 

Pinna, fibrosarcoma 

Zymbal's gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Pelvis, transitional epithelium, papilloma 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Urinary bladder 

Leiomyosarcoma 

Schwannoma malignant, metastatic, 

peripheral nerve 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

Mesothelioma benign 

Mesothelioma malignant 

NeoplasmSummary 

Total animals with primary neoplasms' 

15-Month interim evaluation 

2-Year study 

Total primary neoplasms 

15-Month interim evaluation 

2-Year study 

Total animals with benign neoplasms 

15-Month interim evaluation 

2-Year study 

Total benign neoplasms 

15-Month interim evaluation 

2-Year study 

Total animals with malignant neoplasms 

15-Month interim evaluation 

2-Year study 

Total malignant neoplasms 

15-Month interim evaluation 

2-Year study 

Total animals with metastatic neoplasms 

2-Year study 

Total metastatic neoplasms 

2-Year study 

(2)

1 (50%) 

1 (50%) 

(50)

1 (2%) 

(50) 

1 (2%) 

(50) 

29(58%) 

9 

48 

12 

146 

9 

47 

12 

99 

39 

47 

1 

1 

(1)

1 (100%) 

(501 

(50) 

1 (2%) 

(50) 

35(70%) 

2 (4%) 

9 

50 

11 

138 

9 

48 

10 

94 

1 

38 

1 

44 

.2 

5 

(1) 

(50) 

1 (2%) 

1 (2%) 

(49) 

(50) 

26 (52%) 

1 (2%) 

1 (2%) 

10 

49 

16 

139 

10 

48 

16 

101 

34 

38 

2 

8 

(50) 

(50) 

(50)

1 (2%) 

2 (4%) 

1 (2%) 

4 

46 

5 

108 

4 

41 

4 

85 

1 

19 

1 

23 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Number of animals with any tissue examined microscopically ' Primaryneoplasms:allneoplasmsexceptmetastaticneoplasms 
82 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Hemangiosarcoma 

Intestine small, duddenum 

Intestine small, jejunum 

Carcinoma \ 

Intestine small, ileum 

Liver !

Hepatocellular carcinoma 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Acinar cell, adenoma 

Pharynx 

Palate, squamous cell papilloma 

Salivary glands 

Schwannoma malignant 

Stomach, forestomach 

Stomach, glandular 

~ ~~ 

Cardiovascular System 

Blood vessel 

,IHeart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets,pancreatic . 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Cell, adenoma 

General Body System 

Tissue NOS 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

2233444555555555555666666 

4989135001344455688011344 

6912981357517866148277155 

0000000000000000000000000 

5111023144013402114203423 

6290841346378975641964560 

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

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

++++++++++A++++++++++++++ 

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

X 

++++++++++AM+++++++++++++ 

++++++++++A++++++++++++++ 

++++++++++A+++++M++++++++ 

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

X X 

X 

++ + +++ + + + 

++++++++++A++++++++++++++ 

++++++++++M++++++++++++++ 

X 

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

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

++++I++++++++++++++++++++ 

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

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

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

xx 

X 

++++++++++A++++++++++++++ 

++++++++++M++++++++++++++ 

+++++++M+++++++++++++++++ 

x x xxx 

++++++++++M++++++++++++++ 

X 

+ 

M Missing tissue X Lesion present 

I Insufficient tissue Blank: Not examined 
Lesions in Male Rats 83 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Hemangiosarcoma 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Acinar cell, adenoma 

Pharynx 

Palate, squamous cell papilloma 

Salivary glands 

Schwannoma malignant 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell,adenoma 

General Body System 

Tissue NOS 

6666666677777777777777777 

6777’888801122222333333333 

8334009920424999000000000 

0000000000000000000000000 Total 

2041240320234234001123334 Tissues/ 

12780353398522281415767’90 Tumors 

......................... 50 

......................... 49 

......................... 49 

......................... 49 

1 

......................... 48 

......................... 49 

X X 2 

......................... 48 

......................... 49 

X 3 

X 2 

+ + ++ + + + + 17 

......................... 49 

xx 2 

+ 1 

X 1 

......................... 49 

1 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

xx 2 

xx X X X X 8 

X 2 

......................... 49 

X X 2 

X 1 

++M++++++++++++++++++++++ 48 

......................... 49 

X x x xx x x xx x xxxx 19 

......................... 49 

X X X 4 

1 
84 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Schwannoma malignant, metastatic, 

peripheral nerve 

Nervous System 

Brain 

Glioma malignant 

Peripheral nerve 

Schwannoma malignant 

Spinal cord 

2233444555555555555666666 

4989135001344455688011344 

6912981357517866148277155 

0000000000000000000000000 

5111023144013402114203423 

6290841346378975641964560 

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

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

X 

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

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

......................... xxxxx x xx xx x x x

xx xx X X X 

......................... + +++ ++ + + + +++ 

++++++++++M++++++++++++++ 

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

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

++++++++++++++++++++M++++ 

~~ ~ 

++++++++M++++++++++++++++ 

......................... X 

X 

X 

X 

......................... + 

X 

......................... X 

+ 

X 

+ 
Lesions in Male Rats 85 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Studyofp-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Schwannoma malignant, metastatic, 

peripheral newe 

Nervous System 

Brain 

Glioma malignant 

Peripheral nerve 

Schwannoma malignant 

Spinal cord 

6666666677777777777777777 

6777888801122222333333333 

8334009920424999000000000 

0000000000000000000000000 Total 

2041240320234234001123334 Tissues/ 

1278035339852228141576790 Tumors 

......................... 50 

......................... 50 

X xx 3 

1 

......................... 50 

......................... 50 

......................... 50 

xxx x xxxx xxxxxxxxxxxxx 34 

X X X 10 

......................... 50 

+ +++++ +++++ + 24 

......................... 49 

......................... 49 

......................... 50 

++M++++++++++++++++++++++ 48 

X 1 

......................... 49 

X X 2 

......................... 50 

X X 3 

xx X 4 

1 

1 

......................... 50 

1 

1 

......................... 50 

1 

+ 2 

1 

+ 2 
86 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Nose 

Nasolacrimal duct, squamous cell 

carcinoma 

Trachea 

Special Senses System 

Zymbal’s gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Pelvis, transitional epithelium, 

papilloma 

Urinary bladder 

Leiomyosarcoma 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

2233444555555555555666666 

4989135001344455688011344 

6912981357517866148277155 

0000000000000000000000000 

5111023144013402114203423 

6290841346378975641964560 

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

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

++++++++++M++++++++++++++ 

+ + 

X 

X 

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

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

......................... xxxxxx x x x x xx x x 
Lesions in Male Rats 87 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Nose 

Nasolacrimal duct, squamous cell 

carcinoma 

Trachea 

Special Senses System 

Zymbal’s gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Pelvis, transitional epithelium, 

papilloma 

Urinary bladder 

Leiomyosarcoma 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

4444444477777777777777777 

4’777888801122222333333333 

8334009920424999000000000 

0000000000000000000000000 Total 

2041240320234234001123334 Tissues/ 

1278035339852228141576790 Tumors 

......................... 50 

......................... 50 

X 1 

......................... 49 

2 

1 

1 

......................... 50 

X 1 

......................... 50 

X 1 

......................... 50 

xxxxxxx x x x x xx x x 29 
88 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 1,250 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Schwannoma malignant, metastatic, 

peripheral nerve 

Pancreas 

Schwannoma malignant, metastatic, 

peripheral nerve 

Acinar cell, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

Tissue NOS 

3344455555555555666666666 

1915902333368889023345677 

0254357228914781241772533 

1000000001000111000010000 

0688878660997000679708879 

9772821234035821561860914 

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

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

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

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

A+M++++++++++++++++++++++ 

++++++++++A++++++++++++++ 

++A++++++++++++++++++++++ 

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

X 

+ + + + + + 

X 

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

X 

++M++++++++++++++++++++++ 

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

+++ ..................... 

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

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

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

......................... X 

x x ......................... X 

++M++++++++++++++++++++++ 

......................... xxx X X X X 

++M++++++++++++++++++++++ 

X 
Lesions in Male Rats 89 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Schwannoma malignant, metastatic, 

peripheral nerve 

Pancreas 

Schwannoma malignant, metastatic, 

peripheral nerve 

Acinar cell, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

Tissue NOS 

6667777777777777777777777 

7770000122222222222222222 

3440022022889999999999999 

0000000101000000000000111 Total 

9667779080786667889999001 Tissues/ 

7190946733364681452589050 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 48 

......................... 49 

.......................... 49 

......................... 50 

X X 2 

X 2 

+ + + +++ + 13 

1 

......................... 50 

1 

X X X 3 

......................... 49 

......................... 50 

......................... -49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

x x xx X X 7 

X X 4 

......................... 50 

1 

......................... 49 

......................... 50 

X X X xx 12 

X 1 

......................... 49 

X 2 

X 1 

+ + 2 
90 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of p-NitrobenzoicAcid 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Prostate 

Seminal vesicle 

Schwannoma malignant, metastatic, 

peripheral nerve 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Schwannoma malignant 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolarcarcinoma 

Osteosarcoma, multiple, metastatic, 

bone 

Nose 

Trachea 

3344455555555555666666666 

1915902333368889023345677 

0254357228914781241772533 

1000000001000111000010000 

0688878660997000679708879 

9772821234035821561860914 

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

......................... X 

X 

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

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

X 

......................... x xx xx xx x x xx 

x xx x X X xx X 

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

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

++M++++++++++++++++++++++ 

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

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

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

++M++++++++++++++++++++++ 

......................... xx 

X X 

......................... X X 

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

+ ++ ++ 

X 

++ ++ 

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

X 

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

++M++++++++++++++++++++++ 
Lesions in Male Rats 91 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Prostate 

Seminal vesicle 

Schwannoma malignant, metastatic, 

peripheral nerve 

Testes 

Bilateral, interstitial cell, adenoma

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

~ ~~ 

Nervous System 

Brain 

Peripheral nerve 

Schwannoma malignant 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma 

Osteosarcoma, multiple, metastatic, 

bone 

Nose 

Trachea 

6667777777777777777777777 

7770000122222222222222222 

3440022022889999999999999 

0000000101000000000000111 Total 

9667779080786667889999001 Tissues/ 

7’190946733364687452589050 Tumors 

......................... 50 

......................... 50 

X 2 

1 

X 1 

......................... 50 

......................... 50 

1 

......................... 50 

xxxxxxxxxxxxxxxxxxxxxxxxx 36 

9 

......................... 50 

++++ + + + 26 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 49 

X 1 

.......................... 50 

X 3 

X X X X 6 

X 1 

......................... 50 

2 

~ ~ ~~ ~~ ~ 

......................... 50 

5 

1 

4 

......................... 50 

X 1 

1 

......................... 50 

......................... 49 
92 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Pinna, fibrosarcoma 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Schwannoma malignant, metastatic, 

peripheral nerve 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

3344455555555555666666666 

1915902333368889023345677 

0254357228914781241772533 

1000000001000111000010000 

0688878660997000679708879 

9772821234035821561860914 

+ 

X 

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

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

X 

......................... xxx xxxxxx xx xxxxxxxxx 
Lesions in Male Rats 93 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Pinna, fibrosarcoma 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Schwannoma malignant, metastatic, 

peripheral nerve 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

6667777777777777777777777 

7770000122222222222222222 

3440022022889999999999999 

0000000101000000000000111 Total 

9667779080786667889999001 Tissues/ 

7190946733364687452589050 Tumors 

1 

1 

+ 1 

......................... 50 

......................... 50 

1 

......................... 50 

xx xxxxxx xxx X x xx 35 

X X 2 
94 

TABLEA2 

Individual Animal Tumor Pathology of 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Polyp adenomatous 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Osteosarcoma, metastatic, bone 

Mesentery 

Carcinoma, metastatic, kidney 

Pancreas 

Pharynx 

Palate, squamous cell papilloma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Leiomyoma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Ccell, adenoma 

C-cell, carcinoma 

General Body System 

None 

p-Nitrobenzoic Acid, NTP TR 442 

Male Rats in the 2-Year Feed Study of p-NitrobenzoicAcid 2,500 ppm 

4555555555555666666666666 

2000013456778000334556777 

6.566672168038449123178234 

1111111111111111111111111 

5345623265735332643563245 

9186780293080793675743694 

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

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

+++++++++++A+++++++++++++ 

+++++++++++A+++++++++++++ 

+++++++++++A+++++++++++++ 

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

+++++++++++A+++++++++++++ 

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

X 

+ ++ + + 

X 

+++++++++++A+++++++++++++ 

+ 

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

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

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

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

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

......................... A

.I 

......................... X 

X X 

+++++++++++A+++++++++++++ 

+++++++++++A++M++++++++++ 

+++I+++++++++++++++++++++ 

X xx xx xx 

+++++++++++A+++++++++++++ 

X 
Lesions in Male Rats 95 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 2,500ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Polyp adenomatous 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Osteosarcoma, metastatic, bone 

Mesentery 

Carcinoma, metastatic, kidney 

Pancreas 

Pharynx 

Palate, squamous cell papilloma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Leiomyoma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

6666677777777777777777777 

7778900002222222222222222 

4997602292889999999999999 

1111111111111111111111111 Total 

5466562642452223334444566 Tissues/ 

SO20114545221792461356838 Tumors 

......................... 50 

......................... 49 

X 1 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 50 

X 1 

1 

+ + + + + + + + 13 

1 

......................... 49 

++ 3 

xx 2 

......................... 50 

......................... 50 

++ ...................... 49 

X 1 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

1 

X X X 5 

X X 2 

......................... 49 

X X 2 

+M+++++++++++++++++++++++ 47 

......................... 49 

xx X xx X x xx 16 

......................... 49 

X X 2 

X X 3 
96 p-NitrobenzoicAcid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, carcinoma 

Duct, squamous cell papilloma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, 

kidney 

Pancreatic, carcinoma, metastatic, 

kidney 

Renal, carcinoma, metastatic, kidney 

Lymph node, mandibular 

Lymph node, mesenteric 

Carcinoma, metastatic, kidney 

Spleen 

Fibrosarcoma 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammaly gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneoustissue, fibroma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Nervous System 

Brain 

Peripheral newe 

Spinal cord 

4555555555555666666666666 

2000013456778000334556777 

6566672168038449123178234 

1111111111111111111111111 

5345623265735332643563245 

9186780293080193675143694 

+++++++++++M+++++++++++++ 

+++++++++++M+++++++++++++ 

X X 

xx 

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

+++++++++++M+++++++++++++ 

+++++++++++M+++++++++++++ 

xxxxxxx x x x xx xx x 

xx X X 

......................... + ++ + + ++ + + + ++++ + 

X 

X 

X 

+++++++++++A+++++++++++++ 

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

X ......................... 

+++++++++++++++++++++++M+ 

X 

++++M++++++++++++++++++++ 

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

X X 

......................... X 

+ 

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

+ 
Lesions in Male Rats 97 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, carcinoma 

Duct, squamous cell papilloma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitialcell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, 

kidney 

Pancreatic, carcinoma, metastatic, 

kidney 

Renal, carcinoma, metastatic, kidney 

Lymph node, mandibular 

Lymph node, mesenteric 

Carcinoma, metastatic, kidney 

Spleen 

Fibrosarcoma 

Thymus 

Epithelial cell, thymoma benign 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

6666611111171111111111171 

1118900002222222222222222 

4991602292889999999999999 

1111111111111111111111111 Total 

5466562642452223334444566 Tissues/ 

502011454522119246135~6838 Tumors 

......................... 49 

......................... 49 

X X 4 

X 3 

X 1 

X 1 

......................... 50 

......................... 49 

......................... 49 

x x x x x x x x x x x x x x x x x 35 

X x xx x 9 

......................... 50 

+ ++++ + ++ + + + + + 30 

1 

1 

1 

......................... 49 

......................... 49 

1 

......................... 50 

X 1 

+++++++++M+++++++++++++++ 48 

1 

......................... 49 

xxx X 4 

......................... 50 

X 1 

X X X 3 

X X 2 

xx 4 

......................... 50 

1 

1 

......................... 50 

+ 1 

+ 2 
98 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Carcinoma, multiple, metastatic, 

kidney 

Osteosarcoma, multiple, metastatic, 

bone 

Nose 

Trachea 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma benign 

Mesothelioma malignant 

4555555555555666666666666 

2000013456778000334556777 

6566672168038449123178234 

1111111111111111111111111 

5345623265735332643563245 

9186780293080793675743694 

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

X 

X 

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

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

+ 

+ 

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

X 

+++++++++++M++++++++++++t 

......................... x xx x x xx x x x x x x x
Lesions in Male Rats 99 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System ~~~~ 

Lung 

Alveolaribronchiolar adenoma 

Carcinoma, multiple, metastatic, 

kidney 

Osteosarcoma, multiple, metastatic, 

bone 

Nose 

Trachea 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma benign 

Mesothelioma malignant 

6666677777777777771777177 

7778900002222222222222222 

4997602292889999999999999 

1111111111111111111111111 Total 

5466562642452223334444566 Tissues/ 

5020114545221792461356838 Tumors 

......................... 50 

X 1 

1 

1 

......................... 50 

......................... 50 

1 

t 2 

......................... 50 

X 1 

1 

......................... 49 

......................... 50 

x xx x x x xx xx x 26 

X 1 

X 1 
100 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 5,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Salivary glands 

Schwannoma malignant 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS 

1444455555556666666666677 

9345800134460111458899900 

1458366822878578741945603 

1221112121222121222222221 

9229890808012809022110129 

2253880165567745938431947 

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

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

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

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

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

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

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

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

X 

+++ +++ ++ + + + ......................... 

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

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

.......................... + 

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

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

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

......................... X 

X xx X 

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

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

......................... x x ......................... X 

X 

+ 
Lesions in Male Rats 101 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Salivary glands 

Schwannoma malignant 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS 

7777777777777777777777777 

1112222222222222222222222 

0090999999999999999999999 

1122111111112222222222222 Total 

8912888899990000111112223 Tissues/ 

6650234901492378012781690 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X 2 

X 2 

+ + + +++ 17 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

+ 2 

X 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 2 

xx xx x xx 11 

X 1 

......................... 50 

+++++M+++++++++++++++++f+ 49 

++I++++++++++++++++++++++ 49 

X xxx xx x xx x 12 

......................... 50 

X X 3 

1 

X 1 

+ 2 
102 p-NitrobenzoicAcid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Studyof p-NitrobenzoicAcid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Histiocytic sarcoma 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Trichoepithelioma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Squamous cell carcinoma 

Nose 

Trachea 

1444455555556666666666677 

9345800134460111458899900 

1458366822878578741945603 

1221112121222121222222221 

9229890808012809022110129 

2253880165567745938431947 

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

......................... X X 

xx xxx 

X 

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

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

......................... X X x x x x xx x 

X xx X x x X 

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

+ + +++ +++ ++ + ......................... 

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

......................... X 

M+M++M++++++M++++++++++++ 

+M++++++++++++++M++++++++ 

......................... X 

X X 

X 

X X xx X 

X X 

X 

......................... X 

+ 

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

......................... X 

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

......................... 
Lesions in Male Rats 103 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Histiocytic sarcoma 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Trichoepithelioma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

~~~ 

Lung 

Alveolar/bronchiolar adenoma 

Squamous cell carcinoma 

Nose 

Trachea 

7777777777777777777777777 

1112222222222222222222222 

0090999999999999999999999 

1122111111112222222222222 Total 

8912888899990000111112223 Tissues/ 

6650234901492378012781690 Tumors 

......................... 50 

......................... 50 

A 3 

5 

1 

......................... 50 

......................... 50 

......................... 50 

X xxx x x x xxx xx 21

X X xxx xx X 15 

......................... 50 

+ + + 14 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 46 

+++++++++++++M++++++M++++ 46 

X X 2 

......................... 50 

1 

2 

1 

X X 7 

X X 4 

1 

......................... 50 

1 

1 

......................... 50 

......................... 50 

X 2 

X 1 

......................... 50 

......................... 50 
104 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

Mesothelioma malignant 

1444455555556666666666677 

9345800134460111458899900 

1458366822878578741945603 

1221112121222121222222221 

9229890808012809022110129 

2253880165567745938431947 

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

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

......................... X 

X X 

X 
Lesions in Male Rats 105 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

Mesothelioma malignant 

7777777777777777777777777 

1112222222222222222222222 

0090999999999999999999999 

1122111111112222222222222 Total 

8912888899990000111112223 Tissues1 

6650234901492378012781690 Tumors 

+ 1 

.......................... 5 0

......................... 50 

......................... 50 

1 

2 

1 
106 pNitrobenzoic Acid, NTP TR 442 

TABLEA3 

Statistical Analysisof Primary Neoplasms in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

Adrenal Medulla: Benign Pheochromocytoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd I Fisher exact test 

Adrenal Medulla: Benign or Malignant Pheochromocytoma 

11/50 (22%) 

57.2% 

6/13 (46%) 

591 

P=0.580N 

P=0.587N 

P=O.500 

11/50 (22%) 

57.2% 

6/13 (46%) 

591 

P=0.483N 

P=0.475N 

.P=0.595N 

4/50 (8%) 

23.0% 

2/13 (15%) 

538 

P=O.386 

P=O.385 

P=O.349 

0/50(0%) 

0.0% 

0/13 (0%) -e 

P=0.104N 

P=0.109N 

P=0.117N 

4/50 (8%) 

23.0% 

2/13 (15%) 

538 

P=0.581N 

P=O.592N 

P=0.631N 

7/50 (14%) 

33.4% 

3/13 (23%) 

541 

P=0.228N 

P=0.206N 

P=0.298N 

8/50 (16%) 

35.7% 

3/13 (23%) 

541 

P=0.229N 

P=0.202N 

P=0.306N 

1/50 (2%) 

7.7% 

1/13(8%) 

729 (T)

P=0.465N 

P=0.448N 

P=O.492N 

0/50 (0%) 

0.0% 

0/13 (0%) -

P=0.104N 

P=0.107N 

P=0.117N 

1/50 (2%) 

7.7% 

1/13(8%) 

729 (T)

P=0.154N 

P=0.140N 

P=0.175N 

12/50 (24%) 

42.6% 

6/21 (29%) 

608 

P=0.339N 

P=O.500 

P=O.405 

13/50 (26%) 

44.8% 

6/21 (29%) 

608 

P =0.303N 

P=O.531 

P=O.408 

2/50 (4%) 

7.9% 

1/21 (5%) 

694 

P=0.513N 

P=0.605N 

P=0.684N 

2/50 (4%) 

9.5% 

2/21 (10%) 

729 (TI

P=0.324N 

P=O.422N 

P=0.490N 

4/50 (8%) 

17.1% 

3/21 (14%) 

694 

P=0.381N 

P=0.516N 

P=0.631N 

10/50 (20%) 

44.4% 

3/12 (25%) 

631 

P=0.253N 

P=0.476N 

P=O.406 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Gxhran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Gxhran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

11/50 (22%) 

50.6% 

4/12 (33%) 

631 

P=0.259N 

P=0.494N 

P=O.374 

2/49 (4%) 

11.7% 

1/12 (8%) 

645 

P=0.242N 

P=0.336N 

P=0.424N 

3/49 (6%) 

11.8% 

0/12 (0%) 

617 

P=0.396N 

P=0.476N 

P=0.525N 

4/49 (8%) 

19.2% 

1/12 (8%) 

617 

P=0.277N 

P=O.402N 

P=0.511N 
Lesions in Male Rats 107 

TABLEA3 

Statistical Analysisof Primary Neoplasms in Male Ratsin the 2-Year Feed Study of p-Nitrobenzoic Acid(continued) 

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

Mammary Gland: Fibroadenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreas: Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreatic Islets: Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Preputial Gland: Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

2/50 (4%) 

16.7% 

2/12 (17%) 

729 (T>

P=0.470N 

P=O.552N 

P=O.477 

2/49 (4%) 

16.7% 

2/12 (17%) 

729 (T) 

P=0.032N 

P=0.040N 

P=O.O72N 

3/49 (6%) 

22.2% 

2/12 (17%) 

714 

P=O.O52N 

P=0.065N 

P=0.102N 

19/49 (39%) 

79.7% 

8/12 (67%) 

547 

P=0.013N 

P=0.072N 

P=O.l45N 

3/50 (6%) 

21.1% 

2/12 (17%) 

689 

P=0.382N 

P=0.522N 

P=O.556 

1/50 (2%) 

5.6% 

0/13 (0%) 

710 

P=0.468N 

P=0.442N 

P=0.500N 

3/50 (6%) 

18.9% 

2/13 (15%) 

674 

P=O.538 

P=O.571 

P=O.510 

1/50 (2%) 

3.0% 

0/13 (0%) 

624 

P=0.268N 

P=0.259N 

P=0.301N 

12/50 (24%) 

45.5% 

4/13 (31%) 

505 

P=0.070N 

P =0.054N 

P=0.085N 

3/50 (6%) 

13.6% 

0/13 (0%) 

588 

P=0.603N 

P=0.611N 

P=0.661N 

4/50 (8%) 

24.0% 

1/13(8%) 

709 

P=O.389 

P=O.396 

P=O.339 

0/49 (0%) 

0.0% 

O/l3 (0%) 

-

P=0.217N 

P=0.217N 

P=0.247N 

2/49 (4%) 

13.1% 

1/13 (8%) 

709 

P=0.467N 

P=0.434N 

P=0.500N 

16/49 (33%) 

57.0% 

4/13 (31%) 

506 

P=0.224N 

P=0.188N 

P=0.337N 

4/49 (8%) 

16.5% 

1/13(8%) 

532 

P=O.555 

P=O.560 

P=O.489 

2/50 (4%) 

8.6% 

1/21(5%) 

710 

P=0.492N 

P=0.526N 

P=0.691N 

0/50 (0%) 

0.0% 

on1 (0%) 

-

P-O.124N 

P=O.l24N 

P=O.242N 

0/50 (0%) 

0.0% 

on1 (0%) -

P=0.048N 

P=O.O52N 

P=0.117N 

12/49 (24%) 

48.1% 

9/21 (43%) 

608 

P=0.003N 

P=0.020N 

P=0.096N 

3/50 (6%) 

10.4% 

1/21 (5%) 

617 

P=0.434N 

P=0.554N 

P=0.661N 
10s p-NitrobenzoicAcid, NTP TR 442 

0 PPm 1,2502,500 ppmppm5,000 ppm

Preputial Gland Carcinoma 

Overall rate 1/50 (2%) 1/50 (2%) 4/49 6/50 (12%) (8%) Adjusted rate 2.7% 2.9% 15.9% 15.0% 

Terminal rate on2 (0%) 0/13 (0%) 0/13 (0%) on1 (0%)

First incidence 651 (days) 602 548 518 

Life table test P=O.234 P=0.744N P=O.O31 P=O.O94 

Logistic regressiontest P=O.192 P=O.743 P=O.O02 P=O.O09 

Cochran-Armitage test P=O.O13 

Fisher exact test P=O.175 P=0.753N P=O.O56

Preputial Gland Adenoma or Carcinoma 

Overall rate 4/50 (8%) 4/50 (8%) 8/49 (16%) 9/50 (18%) 

Adjusted rate 23.2% 16.1% 29.8% 23.8% 

Terminal rate 2/12 (17%) 0/13 (0%) 1/13 (8%) 1/21 (5%) 

First incidence (days) 532 

Life table test P=O.254 P=0.579N P=O.176 P=O.278 

Logistic regressiontest P=O.219 P=0.607N P=O.O24 P=O.O55 

Cochran-Armitagetest P=O.O52 

Fisher exact test P=O.168 P=0.643N P=O.117

Skin: Keratoacanthoma 

Overall rate 3/50 (6%) 3/50 (6%) 3/50 (6%) 2/50 (4%) 

Adjusted rate 10.5% 10.5% 19.4% 6.9% 

Terminal rate 0/12 (0%) 0/13 (0%) (15%) 2/13 on1 (0%) 

588 548 518 

First incidence 696 (days)

Life table test P=0.600N P=O.242N P=0.595N P=0.350N

Logistic regressiontest P=0.618NP=0.655NP=0.345N P=0.479N 

Cochran-Armitage test P=0.397N 

Fisher exact test P=0.661N P=0.661N P=O.SOON

Skin: Squamous Cell Papilloma, Keratoacanthoma, Trichoepithelioma, or Basal Cell Adenoma 

Overall rate 3/50 3/50 (6%) (6%) 3/50 (6%) 5/50 (10%)

Adjusted rate 10.5% 10.5% 30.1% 10.1% 

Terminal rate Oh2 (0%) 0/13 (0%) 3/13 (23%) on1 (0%) 

631 505 694 

First incidence (days) 687 505 631 694 

Life table test P=O.433P=0.600N P=O.386N P=O.482N 

Logistic regressiontest P=O.421P=0.655N P=0.535N P=0.633N 

Cochran-Annitagetest P=O.537 

Fisher exact test P=O.357 P=0.661N P=0.661N 

Skin (Subcutaneous Tissue): Fibroma 

Overall rate 4/50 (8%) (12%) 6/504/50 (8%) 7/50 (14%) 

Adjusted rate 20.6% 28.5% 21.7% 21.2% 

Terminal rate 1/12 (8%) (15%) 2/132/13 (15%) 1/21 (5%) 

First incidence 609 (days)

Life table test P=0.583N P=O.441 P=O.509 P=O.496 

Logistic regressiontest P=O.572N P=O.414 P=O.298 P=O.303 

Cochran-Annitagetest P=O.254 

Fisher exact test P=0.643N P=O.370 P=O.262 

392 602 483 
Lesions in Male Rats 109 

TABLEA3 

Statistical Analysisof Primary Neoplasms in Male Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid (continued) 

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

Skin (Subcutaneous Tissue): Fibrosarcoma 

Overall rate 1/50 (2%) Ob0 (0%) Ob0 (0%) 4/50 (8%) 

Adjusted rate 3.7% 0.0% 0.0% 14.1% 

Terminal rate 0/12 (0%) 0/13 (0%) 0/13 (0%) 1/21 (5%) 

First incidence (days) 645 - - 615 

Life table test P=O.O65 P=0.479N P=0.472N P=O.319 

Logistic regression test P=O.O36 P=0.493N P=0.492N P=O.223 

Cochran-Armitage test P=O.O28 

Fisher exact test P=0.500N P=0.500N P=O.181 

Skin (Subcutaneous Tissue): Fibrosarcoma or Sarcoma 

Overall rate 2/50 (4%) 0/50 (0%) 0/50 (0%) 4/50 (8%) 

Terminal rate 

Adjusted rate 5.9% 

0/12 (0%) 0/13 (0%) 

0.0% 

0/13 (0%) 

0.0% 14.1% 

1/21(5%) 

First incidence (days) 451 - - 615 

Life table test P=O.181 P=0.221N P=0.213N P=O.501 

Logistic regression test P=O.104 P=0.270N P=0.307N P=O.346 

Cochran-Armitage test P=O.104 

Fisher exact test P=0.247N P=0.247N P=O.339 

Skin (Subcutaneous Tissue): Fibroma, Fibrosarcoma, or Sarcoma 

Overall rate (12%) 6/50 6/50 (12%) 4/50 (8%) 11/50 (22%) 

Adjusted rate 

Terminal rate 

First 

Life 

ssion Logistic 

Cochran-Armitage 

Fisher exact test 

Testes: Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Thyroid Gland (C-cell): Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

26.3% 

1/12 (8%) 

451 

P=O.294 

P=O.106 

P=O.O86 

44/50 (88%) 

100.0% 

12/12 (100%) 

381 

P<0.001N 

P<0.001N 

P=O.OlON 

4/49 (8%) 

19.0% 

1/12 (8%) 

547 

P=0.290N 

P=0.414N 

P=0.469N 

28.5% 

2/13 (15%) 

392 

P=0.544N 

P=0.595N 

P=0.620N 

45/50 (90%) 

100.0% 

13/13 (100%) 

392 

P=0.374N 

P=0.580N 

P=O.500 

2/49 (4%) 

9.9% 

1/13 (8%) 

538 

P=0.303N 

P=0.315N 

P=0.339N 

20.6% 

2/13 (15%) 

609 

P=0.311N 

P=0.322N 

P=0.370N 

44/49 (90%) 

100.0% 

13/13 (100%) 

505 

P=0.293N 

P=0.437N 

P=O.514 

2/49 (4%) 

15.4% 

2/13 (15%) 

729 (T>

P=0.297N 

P=0.280N 

P=0.339N 

32.5% 

2/21 (10%) 

483 

P=O.399 

P=O.170 

P=O.143 

36/50 (72%) 

91.9% 

18/21(86%) 

483 

P=O.OOlN 

P=0.007N 

P=0.039N 

3/50 (6%) 

11.0% 

1/21 (5%) 

608 

P=0.308N 

P=0.431N 

P =0.489N 
110 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid (continued) 

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

Thyroid Gland (C-cell): Carcinoma 

Overall rate 0/49 (0%) 1/49 (2%) 3/49 (6%) 1/50(2%) 

Adjusted rate 0.0% 7.7% 11.2% 2.9% 

Life table test 

First incidence (days) 

Terminal rate 0/12 (0%) -

P=O.478 

1/13(8%) 

P=O.516 

729 (9 

P=O.159 

604 

0/13 (0%) on1 (0%) 

P=O.545 

618 

Logistic regression test P=O.382 P=O.516 P=O.133 P=0.497 

Cochran-Armitage test P=O.358 

Fisher exact test P=O.500 P=O.121 P=O.505 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Overall rate 4/49 (8%) 3/49 (6%) 5/49 (10%) 4/50 (8%) 

Adjusted rate 19.0% 17.4% 24.9% 13.5% 

Terminal rate 1/12 (8%) 2/13 (15%) 2/13 (15%) 1/21 (5%) 

First incidence (days) 547 538 604 608 

Life table test P=0.396N P=0.456N P =OS75 P=O.442N 

Logistic regression test P=0.567N P=0.464N P=O.569 P=0.587N 

Cochran-Armitage test P=O.513 

Fisher exact test P=0.500N P=O.500 P=0.631N 

All Organs: Mononuclear Cell Leukemia 

Overall rate 29/50 (58%) 35/50 (70%) 26/50 (52%) 2/50(4%) 

Adjusted rate 76.2% 79.9% 76.3% 4.9% 

Terminal rate 4/12 (33%) 5/13 (38%) 7/13 (54%) OD1 (0%) 

First incidence (days) 503 415 506 445 

Life table test P<0.001N P=O.42.4 P=0.215N P<O.OOlN 

Logistic regression test P<0.001N P=O.177 P=0.127N P<O.OOlN 

Fisher exact test 

Cochran-Armitage test P<O.OOlN 

P=O.149 P=0.344N P<0.001N 

All Organs: Benign Neoplasms 

Overall rate 48/50 (96%) 48/50 (96%) 48/50 (96%) 42/50 (84%) 

Adjusted rate 100.0% 100.0% 100.0% 97.7% 

Terminal rate 12/12 (100%) 13/13 (100%) 13/13(100%) 20/21 (95%) 

Life table test 

First incidence (days) 

P=O.O02N 

2.46 

P=O.322N 

392 

P -0.283N 

505 

P=0.003N 

483 

Logistic regression test P<O.OOlN P=0.457N P=0.374N P=0.005N 

Fisher exact test 

Cochran-Armitage test P=O.OlON 

P=0.691N P=0.691N P=0.046N 

Overall rate 

All Organs: Malignant Neoplasms 

39/50 (78%) 38/50 (76%) 35/50 (70%) 19/50 (38%) 

Terminal rate 

Adjusted rate 

5/12 (42%) 

84.6% 

6/13 (46%) 

83.3% 

8/13 (62%) 

86.0% 

5/21 (2.4%) 

50.3% 

Life table test 

First incidence (days) 

P<O.OOlN 

381 

P=0.317N 

310 

P=O.l72N 

506 

P<O.OOlN 

445 

Logistic regression test P<0.001N P=0.478N P=O.O92N P<O.OOlN 

Fisher exact test 

Cochran-Armitage test PK0.001N 

P=0.500N P=0.2.47N P<0.001N 
Lesions in Male Rats 111 

TABLEA3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid (continued) 

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

All Organs: Benign or Malignant Neoplasms 

Overall rate 49/50 (98%) 50/50(100%) 49/50 (98%) 46/50 (92%)

Adjusted rate 100.0% 100.0% 100.0% 100.0% 

Terminal rate 12/12 (100%) 13/13 13/13 21/21 (100%)(100%)(100%) 

First incidence (days) 310 246 505 445 

Life table test P=0.009N P=0.366N P=0.279N P=0.005N

Logistic regression test P=0.026N P=0.297N PP=O.773 =0.002N

Cochran-Armitage test P=0.034N 

Fisher exact test P=0.181N P=0.753N P=O.500

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, 

pancreas, pancreatic islets, pituitarygland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals 

necropsied. 

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

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposure group incidence are the P values corresponding 

to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in animals dying prior to terminal kill 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher 

exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

e Not applicable; no neoplasms in animal group 
112 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA4a 

Historical Incidence of Renal Tubule Neoplasms in Untreated Male F344/N Ratsa 

Incidence in Controls 

Study Adenoma CarcinomaAdenoma or Carcinoma 

Historical Incidence at Southern Research Institute 

C.I. Pigment Red 23 

Benzyl Acetate 

0/50 

0/50 

0150 

0/50 

0/50 

0/50 

Nitrofurantoin 

C.I. Pigment Red 3 

0/50 

0/50 

0/50 

1/50 

0/50 

1/50 

o-Nitroanisole 0/49 0/49 0/49 

Polysorbate 80 0/50 1/50 1/50 

Rhodamine 6G 0150 0/50 0/50 

Roxarsone 1/50 1/50 2/50 

Total 1B99 (0.3%) 3B99 (0.8%) 4B99 (1.0%) 

Standard deviation 0.7% 1.0% 1.5% 

Range 0%-2% 0%-2% 0%-4% 

Overall Historical Incidence 

Total 9/1,251 (0.7%) 6/1,251 (0.5%) 15/1,251 (1.2%) 

Standard deviation 1.5% 1.1% 1.7% 

Range 0%-6% 0%-4% 0%-6% 

a Data as of 20 August 1992 
Lesions in Male Rats 113 

TABLEA4b 

Historical Incidence of Renal Tubule Lesions from Single and Step Sections in Male F344/N Rats 

Incidence in Controls 

Study Hyperplasia AdenomaCarcinomaAdenoma or Carcinoma 

Single Sections (Standard Evaluation) 

Nitrofurantoina 2/50 0/50 0150 0150 

Furosemidea 4/50 1/50 0/50 1/50 

Phenylbutazoneb 3/50 0150 0/50 0150 

a-Methylbenzyl Alcoholb 0150 0150 0150 0150 

Toluene' 4/60 0160 0160 0160 

2,4-Diaminophenol Dihydrochlorideb 0150 0150 1/50 1/50 

Quercetina 

Mercuric Chlorideb 

1/50 

1/50 

0150 

0150 

0150 

0150 

0150 

0/50 

Coumarinb 1/49 1/49 0149 1/49 

C.I. Pigment Red 23a 

o-Benzylp-Chlorophenol 

3,4-Dihydrocoumarinb b 

3/50 

0/50 

0/50 

0150 

1/50 

0/50 

0/50 

0150 

0150 

0150 

1/50 

0/50 

Overall Historical Incidence 

Standard deviation 

Total 191609 (3.1%)

3.0% 

31609 (0.5%)

0.9% 

11609 (0.2%)

0.6% 1.0% 

41609 (0.7%) 

Range 0%-8% 0%-2% 0%-2% 0%-2% 

Step Sections (Exknded Evaluations) 

Nitrofurantoin 9/50 3/50 0150 3/50 

Toluene 

a-Methylbenzyl Alcohol 

Phenylbutazone 

Furosemide 

0160 

1/49 

2/50 

2/50 

5/60 

1/49 

0150 

2/50 

0160 

0/49 

0/50 

0/50 

5/60 

1/49 

0150 

2/50 

Mercuric Chloride 

2,4-Diaminophenol Dihydrochloride 

2/50 

3/50 

4/50 

0/50 

0150 

0150 

4/50 

0150 

Coumarin 

Quercetin 

2/49 

2/50 

0149 

1/50 

0/49 

0150 

0149 

1/50 

C.I. Pigment Red 23 

o-Benzylp-Chlorophenol 

3,4-Dihydrocoumarin 

3/50 

3/50 

0/50 

1/50 

0/50 

1/50 

0150 

0150 

0/50 

1/50 

0150 

1/50 

Overall Historical Incidence 

Standard deviation 

Total 

4.6% 

291608 (4.8%) 

3.0% 

18/608 (3.0%) 01608 (0%) 

3.0% 

18/608 (3.0%) 

Range 0%-18% 0%-8% 0%-8% 
114 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA4b 

Historical Incidence of Renal Tubule Lesions from Single and Step Sections in Male F344/N Rats (continued) 

Study 

Single and Step Sections Combined 

Nitrofurantoin 

Furosemide 

Phenylbutazone 

a-Methylbenzyle Alcohol 

Toluene 

2,4-Diaminophenol Dihydrochloride 

Mercuric Chloride 

Quercetin 

Coumarin 

3,4-Dihydrocoumarin 

o-Benzyl-p-Chlorophenol 

C.I. Pigment Red 23 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

a Feedstudy 

Gavage study 

Inhalation study 

Incidence in Controls 

HyperplasiaAdenoma Adenoma Carcinoma 

10/50 3/50 0/50 

6/50 3/50 0/50 

5/50 0/50 0/50 

1/49 1/49 0/49 

4/60 5/60 0/60 

3/50 0/50 1/50 

3/50 4/50 0/50 

3/50 1/50 0/50 

3/49 1/49 0/49 

0/50 1/50 0/50 

3/50 1/50 0/50 

6/50 1/50 0/50 

471608 (7.7%) 21/608 (3.5%) 1/608 (0.2%) 

5.2% 2.9% 0.8% 

0%-20% 0%-8% 0%-2% 

or Carcinoma 

3/50 

3/50 

0/50 

1/49 

5/60 

1/50 

4/50 

1/50 

1/49 

1/50 

1/50 

1/50 

22/608 (3.6%) 

2.7% 

0%-8% 
Lesions in Male Rats 115 

TABLEA4c 

Historical Incidence of Preputial Gland Neoplasms in Untreated Male F344DJ Ratsa 

Incidence in Controls 

Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at Southern Research Institute 

Benzyl Acetate 2/50 0/50 2/50 

C.I. Pigment Red 23 3/49 2/49 5/49 

C.I. Pigment Red 3 6/49 1/49 7/49 

Nitrofurantoin 6/48 6/48 12/48 

o-Nitroanisole 4/50 7/50 11/50 

Polysorbate 80 5/48 5/48 10/48 

Rhodamine 6G 2/49 2/49 4/49 

Roxarsone 7/49 0149 7/49 

Total 351392 (8.9%) 231392 (5.9%) 58/392 (14.8%) 

Standard deviation 4.0% 5.7% 7.4% 

Range 4%-14% 0%-14% 4%-25% 

Overall Historical Incidence 

Totalb 94/1,169 (8.0%) 46/1,169 (3.9%) 139/1,169 (11.9%) 

Standard deviation 5.6% 4.0% 7.8% 

Range 2%-24% 0%-14% 2%-30% 

a Data as of 20 August 1992 

Data from Quercetin, TR 409, censored due to low denominator (adenoma,2/13; carcinoma, l/l$ adenoma or carcinoma, 3/13) 
116 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA4d 

Historical Incidence of Mononuclear Cell Leukemia in Untreated Male F344DJ Ratsa 

Incidence in Controls 

Study Mononuclear Cell Leukemiab 

Historical Incidence at Southern Research Institute 

Benzyl Acetate 16/50 

C.I. Pigment Red 23 28/50 

C.I. Pigment Red 3 22/50 

Nitrofurantoin 23/50 

o-Nitroanisole 26/50 

Polysorbate 80 23/50 

Rhodamine 6G 27/50 

Roxarsone 27/50 

Total 192/400 (48.0%) 

Standard deviation 7.9% 

Range 32%-56% 

Overall Historical Incidence 

Total 603/1,255 (48.1%) 

Standard deviation 8.1% 

Range 32%-62% 

a Data as of 20 August 1992 

Includes incidencesof lymphocytic, monocytic, or undifferentiated leukemia. 
Lesions in Male Rats 117 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyofp-Nitrobenzoic Acida 

Disposition Summary 

Animals initially in study 

1SMd intsrinevddon 

Early deaths 

Moribund 

Natural deaths 

SuMvors 

Terminal sacrifice 

Animals examined microscopically 

15-Month Interim Evaluation 

Alimentary System 

Intestine large, colon 

Parasite metazoan 

Intestine large, rectum 

Parasite metazoan 

Liver 

Basophilic focus 

Clear cell focus 

Degeneration, cystic 

Fatty change 

Hepatodiaphragmatic nodule 

Inflammation, focal 

Mixed cell focus 

Bile duct, hyperplasia 

Mesentery 

Accessory spleen 

Fat, necrosis 

Pancreas 

Accessory spleen 

Atrophy, focal 

Stomach, forestomach 

Mineralization, focal 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Focal cellular change 

Pituitary gland 

Angiectasis 

Cyst

Pars distalis, focal cellular change 

Thyroid gland 

Degeneration, cystic 

Ultimobranchial cyst 

C-cell, hyperplasia 

Follicular cell, hyperplasia 

0 PPm 

60 

10 

32 

6 

12 

60 

(10) 

(10)

2 (20%) 

(10)

5(50%) 

2 (20%) 

10 (100%) 

8 (80%) 

10(100%) 

(1)

1 (100%) 

(10) 

7 (70%) 

(10)

1 (10%) 

1,250ppm 

60 

10 

34 

3 

13 

60 

(10) 

(10)

1 (10%) 

(10)

1 (10%) 

1 (10%) 

8 (80%) 

6 (60%) 

1 (10%) 

10(100%) 

(1) 

2,500 ppm 

60 

10 

34 

3 

13 

60 

(10)

5 . (50%) 

1 (10%) 

10 (100%) 

5 (50%) 

7 (70%) 

10 (100%) 

(2)

1 (50%) 

1 (50%) 

(10) 

4(40%) 

(10) 

5,000 ppm 

60 

10 

25 

4 

21 

60 

9 (90%) 

6 (60%) 

10 (100%) 

(3) 

3 (100%) 

(10)

1 (10%) 

6 (60%) 

(10) 

(10)

1 (10%) 

1 (10%) 

(10)

1 (10%) 

1 (10%) 

3(30%) 

(10) 

1 (10%) 

1 (10%) 

a Number o f animals examined microscopically at site and number of animals with lesion 
118 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid 

(continued) 

1IMonth Interim Evaluation (continued) 

Genital System 

Epididymis 

Inflammation, chronic 

Preputial gland 

Degeneration, cystic 

Inflammation, chronic 

Prostate 

Inflammation, suppurative 

Testes 

Bilateral, interstitial cell, hyperplasia 

Germinal epithelium, degeneration 

Interstitial cell, hyperplasia 

Hematopoietic System 

Bone marrow 

Myelofibrosis 

Lymph node 

Mediastinal, congestion 

Mediastinal, hyperplasia 

Lymph node, mandibular 

Congestion 

Hyperplasia 

Lymph node, mesenteric 

Edema 

Hyperplasia, lymphoid 

Spleen 

Pigmentation 

Thymus 

Cyst 

Integumentary System 

Mammary gland 

Dilatation 

Skin 

Exudate 

Hemorrhage, focal 

Inflammation, chronic, focal 

Ulcer 

Nervous System 

Brain 

Compression 

(10) 

(10)

10 (100%) 

1 (10%) 

(10) 

8 (80%) 

(10)

1 (10%) 

1 (10%) 

5 (50%) 

(10) 

(2)

2 (100%) 

(10) 

(10) 

(10) 

10 (100%) 

(9) 

(10)

6 (60%) 

(10) 

1 (10%) 

2 (20%) 

(10)

1 (10%) 

(10) 

1 (10%) 

(10)

1 (10%) 

1 (10%) 

(10) 

10 (100%) 

(9) 

2 (20%) 

(10) 

(2)

2 (100%) 

1 (50%) 

(10)

1 (10%) 

1 (10%) 

(10) 

(10) 

10 (100%) 

(10) 

3 (30%) 

(10) 

(1)

1 (100%) 

(10) 

(10) 

1 (10%) 

(10) 

10 (100%) 

(10)

1 (10%) 
Lesions in Male Rats 119 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

0 PPm 

15-Month Interim Evaluation (continued) 

Respiratory System 

Lung (10)

Alveolar epithelium, hyperplasia 

Nose (10)

Fungus 

Inflammation, suppurative 

RespiratoIy epithelium, hyperplasia,focal 

Urinary System 

Kidney (10)

Nephropathy 10 (100%) 

Pelvis, dilatation 

Renal tubule, hyperplasia 

Renal tubule, pigmentation 10 (100%) 

Systems Examined WuhNo Leswm Observed 

Cardiovascular System 

General Body System 

Musculoskeletal System 

Special Senses System 

2-Year Study 

Alimentary System 

Intestine large, colon (49)

Parasite metazoan 2 (4%) 

Intestine large, rectum (49)

Parasite metazoan 1 (2%) 

Intestine large, cecum (49)

Inflammation, chronic 

Parasite metazoan 

Intestine small, duodenum 

Mucosa, hyperplasia 

Intestine small, jejunum 

Necrosis 

Ulcer 

Intestine small, ileum 

Diverticulum 

Fibrosis 

Inflammation, chronic, focal 

Ulcer 

1,250 ppm 

(10) 

(10)

1 (10%) 

1 (10%) 

(10)

10 (100%) 

1 (10%) 

10 (100%) 

(50)

3 (6%) 

(50)

1 (2%) 

1 (2%) 

(48) 

(49)

1 (2%) 

1 (2%) 

(49)

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2,500 ppm 

(10)

1 (10%) 

(10) 

1 (10%) 

(10) 

10 (100%) 

1 (10%) 

10 (100%) 

5,000 ppm 

(10)

1 (10%) 

(10)

1 (10%) 

1 (10%) 

1 (10%) 

(10) 

10 (100%) 

10 (100%) 
120 p-Nitrobenzoic Acid, NTP TR 442 

shdy (continued) 

Alimentary System (continued) 

Liver 

Angiectasis 

Atrophy, focal 

Autolysis 

Basophilic focus 

Clear cell focus 

Congestion, focal 

Degeneration, cystic 

Developmental malformation 

Eosinophilic focus 

Fatty change 

Fibrosis, focal 

Focal cellular change 

Hematopoietic cell proliferation 

Hemorrhage, focal 

Hepatodiaphragmatic nodule 

Hepatodiaphragmatic nodule, multiple 

Hyperplasia, histiocytic 

Hyperplasia, lymphoid 

Hyperplasia, multifocal 

Infiltration cellular, mixed cell 

Inflammation, focal 

Mixed cell focus 

Necrosis, focal 

Pigmentation 

Thrombosis 

Bile duct, hyperplasia 

Centrilobular, atrophy 

Centrilobular, congestion 

Centrilobular, necrosis 

Mesentery 

Accessory spleen 

Angiectasis 

Fibrosis 

Hemorrhage 

Inflammation, chronic 

Fat, necrosis 

Pancreas 

Accessory spleen. Atrophy, diffuse 

Atrophy, focal 

Edema 

Inflammation, chronic 

Necrosis 

Acinar cell, focal cellular change 

Acinar cell, hyperplasia 

Artery, inflammation, chronic 

Duct, dilatation 

Salivary glands 

Atrophy, focal 

0 PPm 

(49)

4 (8%) 

1 (2%) 

13 (27%) 

6 (12%) 

3 (6%) 

7 (14%) 

5 (10%) 

15 (31%) 

7 (14%) 

2 (4%) 

1 (2%) 

1 (2%) 

13 (27%) 

7 (14%) 

10 (20%) 

2(4%) 

1 (2%) 

48(98%) 

22(45%) 

1 (2%) 

2(4%) 

(17) 

1 (6%) 

1 (6%) 

12 (71%) 

(49)

1 (2%) 

23(47%) 

1 (2%) 

1 (2%) 

(49) 

1,250 ppm 

(50)

6(12%) 

18 (36%) 

8 (16%) 

1 (2%) 

10 (20%) 

5(10%) 

13 (26%) 

1 (2%) 

2(4%) 

1 (2%) 

12 (24%) 

5 (10%) 

10 (20%) 

2(4%) 

6 (12%) 

48(96%) 

27(54%) 

(13)

2(15%) 

1 (8%) 

4 (31%) 

(50) 

1 (2%) 

28 (56%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

~~ ~ 

2,500 ppm 

(50)

6(12%) 

1 (2%) 

25(50%) 

9 (18%) 

10(20%) 

7(14%) 

11 (22%) 

1 (2%) 

1 (2%) 

1 (2%) 

7(14%) 

1 (2%) 

13 (26%) 

4 (8%) 

15(30%) 

1 (2%) 

3(6%) 

2(4%) 

49(98%) 

23 (46%) 

1 (2%) 

(13) 

1 (8%) 

1 (8%) 

3 (23%) 

(49) 

25(51%) 

1 (2%) 

2 (4%) 

1 (2%) 

2(4%) 

(50) 

~~ 

5,000 ppm 

(50)

1 (2%) 

33 (66%) 

12 (24%) 

12 (24%) 

1 (2%) 

5 (10%) 

7 (14%) 

2 (4%) 

1 (2%) 

9(18%) 

4 (8%) 

3 (6%) 

26 (52%) 

2(4%) 

1 (2%) 

48(96%) 

5 (10%) 

1 (2%) 

(17)

1 (6%) 

1 (6%) 

1 (6%) 

13 (76%) 

(50) 

21 (42%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 
Lesions in Male Rats 121 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

study (continued) 

Alimentary System (continued) 

Stomach, forestomach 

Edema 

Erosion 

Inflammation, chronic 

Ulcer 

Mucosa, hyperplasia 

Stomach, glandular 

Erosion 

Hyperplasia, focal, lymphoid 

Pigmentation, focal 

Mucosa, hyperplasia 

Tongue 

Hyperplasia, squamous 

Cardiovascular System 

Blood vessel 

Mesenteric artery, inflammation, chronic 

Heart 

Inflammation, chronic, focal 

Thrombosis 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Angiectasis 

Atrophy 

Congestion 

Focal cellular change 

Hematopoietic cell proliferation 

Hyperplasia, focal 

Infiltration cellular, lymphocyte 

Vacuolization cytoplasmic 

Adrenal medulla 

Angiectasis 

Hyperplasia 

Pituitary gland 

Angiectasis 

Cyst

Hemorrhage 

Pars distalis, focal cellular change 

Pars distalis, hyperplasia, focal 

Pars nervosa, focal cellular change 

Thyroid gland 

Degeneration, cystic 

Ultimobranchial cyst 

C-cell, hyperplasia 

Follicle, dilatation 

Follicular cell, hyperplasia 

0 PPm 

(50)

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

4 (8%) 

(50) 

4 (8%) 

2 (4%) 

(49)

1 (2%) 

(50)

1 (2%) 

(50)

2 (4%) 

2 (4%) 

3 (6%) 

1 (2%) 

1 (2%) 

11 (22%) 

(49)

1 (2%) 

3 (6%) 

1 (2%) 

5 (10%) 

3 (6%) 

(49)

1 (2%) 

5 (10%) 

1 (2%) 

2 (4%) 

1,250 ppm 

(50)

1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 

3 (6%) 

(49)

2 (4%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 

(50)

1 (2%) 

(50)

1 (2%) 

7 (14%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 

8 (16%) 

(50)

3 (6%) 

3 (6%) 

2 (4%) 

7 (14%) 

(49)

1 (2%) 

10 (20%) 

1 (2%) 

1 (2%) 

2,500 ppm 

(50)

1 (2%) 

3 (6%) 

4 (8%) 

5 (10%) 

(49)

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

(50) 

2 (4%) 

(50)

2 (4%) 

1 (2%) 

1 (2%) 

9 (18%) 

(50) 

15 (30%) 

(49)

2 (4%) 

3 (6%) 

1 (2%) 

6 (12%) 

4 (8%) 

1 (2%) 

(49)

1 (2%) 

2 (4%) 

5 (10%) 

1 (2%) 

5,000 ppm 

(50) 

(50) 

(2)

1 (50%) 

(50)

2 (4%) 

(50)

1 (2%) 

1 (2%) 

(50)

2 (4%) 

1 (2%) 

8(16%) 

1 (2%) 

(50) 

9 (18%) 

(49)

3 (6%) 

3 (6%) 

9 (18%) 

1 (2%) 

(50)

2 (4%) 

1 (2%) 

9 (18%) 
122 p-Nitrobenzoic Acid, NTP TR 442 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

%Year Study (continued) 

General Body System 

Tissue NOS 

Anterior, fibrosis 

Anterior, inflammation, chronic 

Oral, inflammation, chronic 

Genital System 

Epididymis 

Inflammation, chronic 

Epithelium, degeneration 

Preputial gland 

Degeneration, cystic 

Hyperplasia 

Inflammation, chronic 

.Prostate 

Hemorrhage 

Inflammation, suppurative 

Epithelium, hyperplasia, focal 

Seminal vesicle 

Dilatation 

Inflammation, chronic 

Testes 

Congestion 

Mineralization, focal 

Artery, inflammation, chronic 

Bilateral, interstitialcell, hyperplasia 

Germinal epithelium, degeneration 

Interstitialcell, hyperplasia 

Hematopoietic System 

Bone marrow 

Hemorrhage 

Hypercellularity 

Hyperplasia, focal, histiocytic 

Metaplasia, osseous 

Myelofibrosis 

Lymph node 

Inguinal, hyperplasia 

Lumbar, hyperplasia 

Mediastinal, angiectasis 

Mediastinal, congestion 

Mediastinal, hyperplasia 

Mediastinal, pigmentation 

Pancreatic, angiectasis 

Pancreatic, edema 

Pancreatic, hyperplasia 

Pancreatic, hyperplasia, lymphoid 

Pancreatic, hyperplasia, macrophage 

0 PPm 

(1)

1 (100%) 

1 (100%) 

(50)

47(94%) 

4 (8%) 

1 (2%) 

(50) 

37(74%) 

1 (2%) 

(50)

2(4%) 

(50) 

1 (2%) 

6(12%) 

10(20%) 

5 (10%) 

1 (2%) 

1 (4%) 

1 (4%) 

1 (4%) 

2 (8%) 

2 (8%) 

1,250 ppm 

(2) 

1 (2%) 

(50)

47(94%) 

(50)

1 (2%) 

31(62%) 

7(14%) 

(50)

1 (2%) 

1 (2%) 

(50) 

11 (22%) 

13 (26%) 

(50) 

3(6%) 

1 (2%) 

1 (2%) 

1 (2%) 

(26) 

3 (12%) 

2 (8%) 

1 (4%) 

2,500 ppm 

(49) 

(49)

48(98%) 

1 (2%) 

2(4%) 

(50) 

36(72%) 

2(4%) 

(49) 

(49) 

2 (4%) 

13 (27%) 

7(14%) 

6 (12%) 

1 (2%) 

(30)

1 (3%) 

4(13%) 

3(10%) 

1 (3%) 

1 (3%) 

5,000 ppm 

(2) 

1 (50%) 

(50)

2(4%) 

(50)

48(96%) 

3 (6%) 

(50) 

39(78%) 

(50) 

1 (2%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

7(14%) 

14 (28%) 

14 (28%) 

(50)

2(4%) 

5 (10%) 

3 (6%) 

1 (2%) 

(14)

4(29%) 

1 (7%) 

1 (7%) 

4(29%) 

4 (29%) 

1 (7%) 

1 (7%) 

1 (7%) 

1 (7%) 

1 (7%) 
Lesions in Male Rats 123 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

2-YeW sf" (continued) 

Hematopoietic System (continued) 

Lymph node, mandibular 

Angiectasis 

Congestion 

Ectasia 

Edema 

Hemorrhage 

Hyperplasia 

Lymph node, mesenteric 

Congestion 

Edema 

Hyperplasia 

Hyperplasia, lymphoid 

Spleen 

Angiectasis 

Congestion 

cLst 

Degeneration, fatty 

Fibrosis 

Hematopoietic cell proliferation 

Necrosis, focal 

Pigmentation 

Red pulp, hyperplasia, focal, histiocytic 

Thymus 

Congestion 

Cyst

Hemorrhage 

Epithelial cell, hyperplasia 

Integumentary System 

Mammary gland 

Dilatation 

Hemorrhage 

Hyperplasia 

Inflammation, chronic 

Skin 

Cyst epithelial inclusion 

Hemorrhage, focal 

Hyperkeratosis, focal 

Hyperplasia, focal 

Inflammation, chronic, focal 

Ulcer 

Epidermis, hyperplasia, focal 

Subcutaneous tissue, angiectasis 

Subcutaneous tissue, congestion 

Subcutaneous tissue, inflammation, chronic, 

focal 

-

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

(49) (49) (49)

1 (2%) 

(50) 

1 (2%) 1 (2%) 1 (2%) 2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

7(14%) 7(14%) 6 (12%) 16 (32%) 

(49) (50) (49) (50)

1 (2%) 

5 (10%) 

1 (2%) 

3 (6%) 1 (2%) 

2 (4%) 1 (2%)

7 (14%) 

1 (2%) 2 (4%) 

1 (2%)

(50) (50) (50) 

1 (2%) 

1 (2%) 

1 (2%) 

12 (24%) 14 (28%) 12 (24%) 7 (14%) 

1 (2%) 2 (4%) 7 (14%) 9 (18%) 

1 (2%) 

50 (100%) 50 (100%) 

4 (8%) 1 (2%) 

50 (100%) 50 (100%) 

1 (2%) 

(48)

1 (2%) 

(50) (48) (46) 

1 (2%) 

1 (2%) 

1 (2%) 

(49)

18 (37%) 

(49)

11 (22%) 

(49)

18 (37%) 

(46)

9 (20%) 

6 (12%) 

2 (4%) 

3 (6%) 2 (4%) 2 (4%) 

1 (2%) 1 (2%) 1 (2%) 

1 (2%)

(50) (50) (50) (50) 

1 (2%) 

2 (4%) 

2 (4%) 

4 (8%) 2 (4%) 

1 (2%) 

1 (2%) 3 (6%) 1 (2%) 

1 (2%) 

2 (4%) 4 (8%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 
124 p-Nitrobenzoic Acid, NTPTR 442 

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

study (continued) 

Bone 

Musculoskeletal System 

Trabecula, proliferation 

Hyperostosis 

Fracture 

Inflammation, chronic 

Skeletal muscle 

(50) 

1 (2%) 

1 (2%) 

(1) 

(50) 

1 (2%) 

2(4%) 

(1)

1 (100%) 

Nervous System 

Compression 

Brain (50)

8 (16%) 

(50) 

4 (8%) 

(50)

5 (10%) 

(50)

2 (4%) 

Hemorrhage 2 (4%) 4 (8%) 

Meninges, fibrosis, focal 

Spinal cord 

Demyelination 

Hemorrhage, focal 

Respiratory System 

Lung 

Congestion 

Edema 

Fibrosis, focal 

Hemorrhage, focal 

Hyperplasia, diffuse, macrophage 

Hyperplasia, focal, macrophage 

Infiltration cellular, mixed cell 

Necrosis, focal 

Alveolar epithelium, hyperplasia 

Nose 

Fungus 

Inflammation, suppurative 

Respiratory epithelium, ulcer 

Special Senses System 

Eye

Atrophy 

Cataract 

(2) 

1 (50%) 

(50)

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

2(4%) 

1 (2%) 

6(12%) 

(50)

6(12%) 

8 (16%) 

1 (2%) 

(4)

1 (25%) 

1 (25%) 

3 (6%) 

1 (2%) 

2 (4%) 

6(12%) 

(50)

8 (16%) 

10 (20%) 

1 (2%) 

(1) 

1 (100%) 

(2) 

(50)

2(4%) 

1 (2%) 

3 (6%) 

2 (4%) 

1 (2%) 

6(12%) 

(50)

8 (16%) 

11 (22%) 

(2)

1 (50%) 

2 (100%) 

(50)

2(4%) 

2 (4%) 

3 (6%) 

(50)

7 (14%) 

8 (16%) 

(1)

1 (100%) 

1 (100%) 

Retina, degeneration 1 (100%) 2 (100%) 1 (100%) 
Lesions in Male Rats 125 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

Urinary System 

Kidney 

Autolysis 

Congestion 

cLst 

Infarct 

Nephropathy 

Thrombosis 

Pelvis, dilatation 

Pelvis, transitional epithelium, hyperplasia 

Renal tubule, hyperplasia 

Renal tubule, hyperplasia, oncocytic 

Renal tubule, pigmentation 

Renal tubule, vacuolization cytoplasmic 

Urinary bladder 

Dilatation 

Hemorrhage 

Inflammation, chronic 

Transitional epithelium, hyperplasia 

0 PPm 

50 (100%) 

1 (2%) 

1 (2%) 

1 (2%) 

50 (100%) 

(50)

1 (2%) 

2 (4%) 

1 (2%) 

1,250 ppm 

2 (4%) 

3 (6%) 

50(100%) 

(50) 

1 (2%) 

2,500 ppm 

(50) 

1 (2%) 

1 (2%) 

50 (100%) 

1 (2%) 

1 (2%) 

1 (2%) 

50 (100%) 

(49) 

5,000 ppm 

1 (2%) 

50(100%) 

1 (2%) 

5 (10%) 

50 (100%) 

1 (2%) 

(50) 

1 (2%) 
126 p-Nitrobenzoic Acid, NTPTR 442 
127 

APPENDIX B 

SUMMARY OF LESIONS IN FEMALE RATS 

IN THE 2-YEAR FEED STUDY 

OF p-NITROBENZOIC ACID 

TABLEB1 Summary of the Incidence of Neoplasms in Female Rats 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 129 

TABLEB2 Individual Animal Tumor Pathology of Female Rats 

in the 2-Year Feed Study ofp-Nitrobenzoic Acid ............................. 134 

TABLE B3 Statistical Analysis of Primary Neoplasms in Female Rats 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 152 

TABLEB4a Historical Incidence of Clitoral Gland Neoplasms 

in Untreated Female F344/N Rats ........................................ 157 

TABLEB4b Historical Incidence of Mononuclear Cell Leukemia 

in Untreated Female F344/N Rats ........................................ 158 

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

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 159 
128 p-Nitrobenzoic Acid, NTP TR 442 
Lesions in Female Rats 129 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of p-Nitrobenzoic Acida 

Early deaths 

1SMd inprim evnhotion 

Animals initiallyin study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

survivors 

10 

60 

2 

21 

27 

10 

60 

4 

23 

23 

10 

60 

2 

27 

21 

10 

60 

3 

26 

21 

Animals examined microscopically 60 60 60 60 

Thyroid gland 

Pituitary gland 

Endocrine System 

C-cell, adenoma 

Pars distalis, adenoma 

Uterus 

Genital System 

Endometrium, sarcoma stromal 

Endometrium, polyp stromal 

(10) (10)

3 (30%) 

(10)

3 (30%) 

1 (10%) 

(10)

1 (10%) 

Spleen 

Lymph node, mesenteric 

Lymph node 

Hematopoietic System 

Fibroadenoma 

Mammary gland 

Integumentary System 

Multiple organsb 

Systemic Lesions 

Leukemia mononuclear 
130 p-Nitrobenzoic Acid, NTPTR442 

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

15-Month Interim Evaluation (continued) 

Systems Examined W~hNo Neoplasms Observed 

Cardiovascular System 

General Body System 

Musculoskeletal System 

Nervous System 

Respiratory System 

Special Senses System 

Urinary System 

2-Year Study 

Alimentary System 

Esophagus 

Carcinoma, metastatic, thyroid gland 

Intestine large, colon 

Leiomyosarcoma, metastatic, uterus 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Sarcoma, metastatic, mesentery 

Mesentery 

Sarcoma 

Schwannoma malignant, metastatic, uterus 

Pancreas 

Acinar cell, adenoma 

Pharynx 

Palate, squamous cell papilloma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Osteosarcoma, metastatic, bone (49) (50)

1 (2%) 

(50) (50) 

Adrenal medulla 

Osteosarcoma, metastatic, bone (49) (50) 

1 (2%) 

(50) (50) 

Pheochromocytoma benign 2 (4%) 1 (2%) 3 (6%)' 

Adenoma 

Islets, pancreatic 

1 (2%)

(50) (50) (49) (49) 
Lesions in Female Rats 131 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid (continued) 

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

%Year StUdy (continued) 

Endocrine System (continued) 

Pituitary gland 

Osteosarcoma, metastatic, bone 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Pars nelvosa, ganglioneuroma 

Thyroid gland 

C-cell, adenoma 

C-cell, adenoma, multiple 

C-cell, carcinoma 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Bilateral, carcinoma 

@sly

Granulosa cell tumor benign 

Neoplasm NOS 

Uterus 

Histiocytic sarcoma 

Leiomyosarcoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Endometrium, sarcoma stromal 

Endometrium, schwannoma malignant 

Hematopoietic System 

Bone marrow 

Lymph node 

Renal, sarcoma, metastatic, mesentely 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Sarcoma, metastatic, mesentery 

Thymus 

18 (36%) 

1 (2%) 

(50)

9 (18%) 

1 (2%) 

(50)

4 (8%) 

1 (2%) 

5 (10%) 

1 (2%) 

(50) 

(13) 

(50) 

(49) 

(50) 

(49) 

27 <54%) 

(49)

4 (8%) 

1 (2%) 

1 (2%) 

(49)

12 (24%) 

2 (4%) 

1 (2%) 

(50) 

1 (2%) 

10 (20%) 

1 (2%) 

1 (2%) 

(50) 

(10) 

(50) 

(50) 

(50) 

(48)-

25 (50%) 

(50)

4 (8%) 

2 (4%) 

(49)

9 (18%) 

4 (8%) 

1 (2%) 

1 (2%) 

(50) 

(50)

1 (2%) 

11 (22%) 

1 (2%) 

1 (2%) 

2 (4%) 

(49) 

23 (47%) 

1 (2%) 

(50)

2 (4%) 

1 (2%) 

(50)

11 (22%) 

3 (6%) 

1 (2%) 

1 (2%) 

(49) 

(49) 

5 (10%) 
132 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

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

&Year Study (continued) 

Integumentary System 

Mammary gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basal cell carcinoma 

Basosquamous tumor malignant 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, schwannoma malignant 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Rhabdomyosarcoma 

Sarcoma 

Nervous System 

Brain 

Astrocytoma NOS 

Glioma malignant 

Peripheral neme 

Schwannoma malignant 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Ahreolar/bronchiolar carcinoma 

Carcinoma, metastatic, thyroid gland 

Osteosarcoma, multiple, metastatic, bone 

Squamous cell carcinoma 

Nose 

Trachea 

Carcinoma, metastatic, thyroid gland 

Special Senses System 

Zymbal’s gland 

Carcinoma 

(50)

1 (2%) 

2 (4%) 

17 (34%) 

5 (10%) 

(50) 

1 (2%) 

2 (4%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

(50) 

(50) 

2 (4%) 

15 (30%) 

7 (14%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

(50) 

(50) 

3 (6%) 

19 (38%) 

7(14%) 

(50) 

(50)

1 (2%) 

(50) 

(50) 

(1)

1 (100%) 

(50) 

2 (4%) 

19 (38%) 

5 (10%) 

(50)

1 (2%) 

1 (2%) 

3 (6%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

(50)

1 (2%) 
Lesions in Female Rats 133 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

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

&Year Study (continued) 

Urinary System 

Kidney 

Pelvis, transitional epithelium, mesenchymal 

(50) (50) 

tumor 

Urinary bladder (50) (50) 

Systemic Lesions 

Multiple organs (50) (50)

Histiocytic sarcoma 

Leukemia mononuclear 17 (34%) 11 (22%) 

Mesothelioma malignant 

Neoplasm Summary 

Total animals with primary neoplasms’ 

15-Month interim evaluation 2 5 

2-Year study 44 48 

Total primary neoplasms 

15-Month interim evaluation 2 7 

2-Year study 95 102 

Total animals with benign neoplasms 

15-Month interim evaluation 2 5 

2-Year study 40 41 

Total benign neoplasms 

15-Month interim evaluation 2 6 

2-Year study 68 79 

Total animals with malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 21 19 

Total malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 27 21 

Total animals with metastatic neoplasms 

2-Year study 2 

Total metastatic neoplasms 

2-Year study 5 

Total animals with uncertain neoplasms 

benign or malignant 

2-Year study 2 

Total uncertain neoplasms 

2-Year study 2 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 

(50)

1 (2%) 

3 (6%) 

5 

50 

6 

107 

4 

44 

5 

84 

1 

21 

1 

22 

2 

4 

1 

1 

(50) 

1 (2%) 

4 

45 

4 

83 

4 

41 

4 

72 

10 

11 

1 

3 
134 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed study Ofp-Nitrobenzoic Acid 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

ovary

Granulosa cell tumor benign 

Uterus 

Endometrium, polyp stromal 

Endometrium, schwannoma malignant 

Vagina 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

4556666666666667771777’777 

9560345566778890000012233 

0665173747990740022942411 

2222222222222222222222222 

8856446587886556646477444 

2094343889.579622577268516 

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

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

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

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

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

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

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

......................... X 

+ ++ + ++ ++ ......................... 

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

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

......................... + 

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

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

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

......................... X X 

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

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

......................... xxxx xxxx x xxx 

X 

......................... X X 

X 

.......................... X X 

......................... X 

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

X X 

X 

+ 

M: Missing tissue X Lesion present 

I: Insufficient tissue Blank: Not examined 
Lesions in Female Rats 135 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

Granulosa cell tumor benign 

Uterus 

Endometrium, polyp stromal 

Endometrium, schwannoma malignant 

Vagina 

7777777777777777777777777 

3333333333333333333333333 

1111111111111111111111111 

2222222222222222222222222 Total 

4455555566667777777888889 Tissues/ 

8901345701680123457134690 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

.......................... 50 

......................... 50 

......................... 50 

X 2 

+ + 10 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

+++++++++++++++++++++M+++ 49 

+++++++++++++++++++++M+++ 49 

2 

......................... 50 

X 1 

+++++++++++++++++++M+M+++ 48 

......................... 50 

X xxx x X 18 

1 

......................... 50 

X xx X X x x 9 

1 

......................... 50 

X X 4 

X 1 

......................... 50 

1 

......................... 50 

xx X 5 

1 

+ + 3 
136 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid:0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basosquamous tumor malignant 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Nervous System 

Brain 

Glioma malignant 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

4556666666666667777.777777 

9560345566778890000012233 

0665173747990740022942411 

2222222222222222222222222 

8856446587886556646477444 

2094343889579622577268516 

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

+ +++ ++ ++ + + ++ ......................... 

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

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

++++++M++++++++++++++++++ 

......................... X 

X 

X xx X X X 

X xx xx .......................... 

X 

X 

......................... X 

......................... X 

+ + 

+ 

.......................... X 

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

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

+ 

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

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

......................... Leukemia mononuclear x xxxx xx xxx X x x x
Lesions in Female Rats 137 

TABLEB2 

Individual Animal Tumor Pathologyof Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone mam 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

MammaIy gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basosquamous tumor malignant 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Nervous System 

Brain 

Glioma malignant 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

.Systemic Lesions 

Multiple organs 

7777777777777777777777777 

3333333333333333333333333 

1111111111111111111111111 

2222222222222222222222222 Total 

4455555566667777777888889 Tissues/ 

8901345701680123457134690 Tumors 

......................... 50 

+ 13 

......................... 50 

++++++M++++++++++++++++++ 49 

......................... 50 

......................... 49 

......................... 50 

1 

X 2 

xxx X xx x xxxx 17 

5 

......................... 50 

X 1 

X 2 

1 

......................... 50 

1 

......................... 50 

1 

2 

1 

......................... 50 

1 

......................... 50 

......................... 50 

+ 2 

......................... 50 

......................... 50 

......................... 50 

Leukemia mononuclear X X X 17 
138 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 1,250 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Leiomyosarcoma, metastatic, uterus 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Osteosarcoma, metastatic, bone 

Adrenal medulla 

Osteosarcoma, metastatic, bone 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Osteosarcoma, metastatic, bone 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, adenoma, multiple 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary

Neoplasm NOS 

Uterus 

Leiomyosarcoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Endometrium, sarcoma stromal 

Vagina 

1444555556666666666666777 

5489124673561771999999000 

0136018602154446456666022 

3333333333333333333333333 

3022031214420231523444312 

5347239671401180031518222 

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

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

X 

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

A+++A++++++++++++A+++++++ 

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

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

A+++A++++++++++++A+++++++ 

......................... + + + ......................... 

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

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

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

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

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

......................... X 

......................... X 

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

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

......................... X 

xx xx xxxxx xx X

+++++++++++++++A+++++++++ 

X X 

.......................... X X xxx 

X X 

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

.......................... X 

xx 

X 
Lesions in Female Rats 139 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acd 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Leiomyosarcoma, metastatic, uterus 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Osteosarcoma, metastatic, bone 

Adrenal medulla 

Osteosarcoma, metastatic, bone 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Osteosarcoma, metastatic, bone 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, adenoma, multiple 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

ovary

Neoplasm NOS 

Uterus 

Leiomyosarcoma 

Endometrium, polyp stromal 

Endometrium,stromal, polyp multiple 

Endometrium, sarcoma stromal 

Vagina 

7777777777777777777777777 

0233333333333333333333333 

8811111111111111111111111 

3333333333333333333333333 Total 

1100000011112223333344444 Tissues/ 

1345678945685890467902369 Tumors 

......................... 50 

......................... 48 

1 

......................... 48 

......................... 47 

......................... 50 

......................... 48 

......................... 47 

......................... 50 

+ + + + + 8 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

1 

X 1 

......................... 50 

......................... 50 

......................... 50 

1 

x xx x x x x x xx x x xxx 27 

......................... 49 

X X 4 

X 1 

X 1 

++++++++++++++++++M++++++ 49 

X x xx x x X 12 

2 

......................... 50 

X 1 

......................... 50 

1 

X xx x xx x x 10 

X 1 

1 

+ 1 
140 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB2 

Individual AnimalTumor Pathologyof Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma,'multiple 

Skin 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, schwannoma 

malignant 

Musculoskeletal System 

Bone 

Osteosarcoma 

Nervous System 

Brain 

Astrocytoma NOS 

Peripheral nerve 

Schwannoma malignant 

Spinal cord 

Respiratory System 

Lung 

Osteosarcoma, multiple, metastatic, 

bone 

Nose 

Trachea 

Special Senses System 

Eye

Lacrimal gland 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

1444555556666666666666777 

5489124673567777999999000 

0136018602154446456666022 

3333333333333333333333333 

3022031214420231523444312 

5347239671401180031518222 

......................... + +++ + + + ......................... 

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

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

+M++++++++++++++M++++++++ 

......................... -X 

X xx xx X X X 

xxx X X 

......................... X 

X 

......................... X 

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

+ + + 

X 

+ + 

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

X 

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

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

+ 

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

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

......................... Leukemia mononuclear x xx x xx 
Lesions in Female Rats 141 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, schwannoma 

malignant 

~~ ~ ~~ 

Musculoskeletal System 

Bone 

Osteosarcoma 

Nervous System 

Brain 

Astrocytoma NOS 

Peripheral nerve 

Schwannoma malignant 

Spinal cord 

Respiratory System 

Lung 

Osteosarcoma, multiple, metastatic, 

bone 

Nose 

Trachea 

Special Senses System 

Eye

Lacrimal gland 

Urinary System 

Kidney 

Urinaly bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

7777771777777777777777777 

0233333333333333333333333 

8 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i l l l l l l l l 

3333333333333333333333333 Total 

1100000011112223333344444 Tissues/ 

1345618945685890467902369 Tumors 

......................... 50 

+ + + 10 

......................... 50 

......................... 50 

......................... 50 

......................... 48 

......................... 50 

X 2 

x xx x X X X 15 

X X 7 

......................... 50 

1 

1 

~~~~~~~~~~~~ ~ ~ ~ ~ 

......................... 50 

1 

......................... 50 

X 1 

+ 4 

1 

2 

......................... 50 

1 

......................... 50 

......................... 50 

+ 2 

+ 1 

......................... 50 

......................... 50 

......................... 50 

X X X xx 11 
142 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid: 2,500 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Sarcoma, metastatic, mesentery 

Mesentery 

Sarcoma 

Schwannoma malignant, metastatic, 

uteNS 

Pancreas 

Acinar cell, adenoma 

Phalynx 

Palate, squamous cell papilloma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Blood vessel 

Heart 

~~~~~~~~~ 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

.Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

Cell, carcinoma 

~~ ~~~~~~ 

General Body System 

None 

2334444455556666666666667 

4773699913580444556689990 

1994126988384377034854552 

3334333344333334434333333 

6690997800876880081698686 

9564149737902581500802764 

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

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

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

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

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

+++M+++++++++++++++++++++ 

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

......................... X 

+ + ++++ + 

X 

X 

++++++++++++++++++A++++++ 

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

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

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

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

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

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

......................... X 

++++++++++++++++++A++++++ 

+++++++++++++M+++++++++++ 

......................... xx xx xxx xx X 

......................... X X X 
Lesions in Female Rats 143 

TABLEB2 

Individual Animal Tumor Pathologyof Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Sarcoma, metastatic, mesentery 

Mesentery 

Sarcoma 

Schwannoma malignant, metastatic, 

uterus 

Pancreas 

Acinar cell, adenoma 

Pharynx 

Palate, squamous cell papilloma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

1111177771171711111111171 

0001333333333333333333333 

2894000000000000000000000 

3343333333333333333334444 Total 

9601661171177888999990000 Tissues/ 

3305161234678134125892689 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

......................... 50 

1 

+ + 9 

1 

1 

......................... 49 

X 1 

+ 1 

X 1 

......................... 50 

......................... 49 

......................... 49 

+ 1 

X 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X 3 

......................... 49 

++++++++++M++++++M+++++++ 41 

......................... 50 

X x x xx xxxxx x xxx X 25 

......................... 50 

X 4 

X X 2 
144 p-NitrobenzoicAcid, NTP TR 442 

TABLEB2 

Individual Animal Tumor Pathologyof Female Rats in the 2-Year Feed Studyofp-Nitrobenzoic Acid 2,500ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Bilateral, carcinoma 

ovary

Uterus 

Histiocytic sarcoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Endometrium, sarcoma stromal 

Endometrium, schwannoma malignant 

Vagina 

Hematopoietic System 

Bone marrow 

Lymph node 

Renal, sarcoma, metastatic, mesentery 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Sarcoma, metastatic, mesentery 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Rhabdomyosarcoma 

Nervous System 

Brain 

Peripheral nexve 

Spinal cord 

2334444455556666666666667 

4773699913580444556689990 

1994126988384377034854552 

3334333344333334434333333 

6690997800876880081698686 

9564749737902581500802764 

......................... X X x xx x 

X X 

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

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

X x x X X 

X 

x x 

+ + 

......................... + + + +++ 

X 

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

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

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

X 

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

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

X 

x xx x X x x xx 

xx X 

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

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

+ 

X 

......................... + + 

+ + 
Lesions in Female Rats 145 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats inthe 2-Year Feed Study ofp-Nitrobenzoic Acid: 2,500 ppm (continued)

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Bilateral, carcinoma 

ovary

Uterus 

Histiocytic sarcoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Endometrium, sarcoma stromal 

Endometrium, schwannoma malignant 

Vagina 

Hematopoietic System 

Bone marrow 

Lymph node 

Renal, sarcoma, metastatic, mesentery 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Sarcoma, metastatic, mesentely 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Rhabdomyosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

7777777777777777777777777 

0001333333333333333333333 

2894000000000000000000000 

3343333333333333333334444 Total 

9607667771777888999990000 Tissues/ 

3305161234618134125892689 Tumors 

++++++++++M++++++++++++++ 49 

X X X 9 

X X 4 

X 1 

X 1 

......................... 50 

......................... 50 

X 1 

x xx X X X 11 

X 1 

1 

2 

+ 3 

.......................... 50 

++ + 9 

1 

......................... 50 

......................... 50 

......................... 50 

X 1 

1 

......................... 50 

......................... 50 

X X 3 

X xx X x xx xx X 19 

X X xx 7 

......................... 50 

......................... 50 

X 1 

1 

1 

......................... 50 

2 

2 
146 pNitrobenzoic Acid, NTP TR 442 

TABLEB2 

Individual AnimalTumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid 2,500 ppm (continued) 

2334444455556666666666667 

Number of Days on Study 4773699913580444556689990 

1994126988384377034854552 

3334333344333334434333333 

Carcass ID Number 6690997800876880081698686 

9564749737902581500802764 

Respiratory System 

Lung ......................... Alveolar/bronchiolar adenoma 

Nose ......................... Trachea ......................... 

Special Senses System 

Zymbal’s gland 

Harderian gland

Eye + 

+ 

+ 

Carcinoma X ~ ~~~~~~~~~~~~~~~~~~~~~ 

Urinary System 

Kidney ......................... Pelvis, transitional epithelium, 

mesenchymal tumor 

Urinary bladder ......................... 

Systemic Lesions 

Multiple organs ......................... Histiocytic sarcoma 

Leukemia mononuclear X xx 
Lesions in Female Rats 147 

TABLEB2 

Individual AnimalTumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolarlbronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

Eye

Harderian gland 

Zymbal’s gland 

Carcinoma 

Urinary System 

Kidney 

Pelvis, transitional epithelium, 

mesenchymal tumor 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

7777777777777777777777777 

0001333333333333333333333 

2894000000000000000000000 

3343333333333333333334444 Total 

9607667777777888999990000 Tissues/ 

3305161234618134125892689 Tumors 

......................... 50 

X 1 

......................... 50 

......................... 50 

2 

+ 1 

1 

1 

......................... 50 

X 1 

......................... 50 

......................... 50 

X 1 

3 
148 p-Nitrobenzoic Acid, NTP TR 442 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Carcinoma, metastatic, thyroid gland 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars nervosa, ganglioneuroma 

Thyroid gland 

C-cell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Bilateral, carcinoma 

Ovary

Uterus 

Endometrium, polyp stromal 

4445555555666666666777777 

4891234567011278899000000 

1338821654218247955022788 

4444444444444444444444444 

3452562342545526656426623 

9669168084018357942763813 

......................... X 

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

M++++++++++++++++++++++++ 

+++M+++++++++++++++++++++ 

+++M+++++++++++++++++++++ 

+++M+++++++++++++++++++++ 

M++M++++++++f++++++++++++ 

......................... + + + 

+++M+++++++++++++++++++++ 

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

+++M+++++++++++++++++++++ 

+++M+++++++++++++++++++++ 

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

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

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

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

+++M+++++++++++++++++++++ 

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

......................... xxxxx x x xx xxx xx 

......................... X 

X 

......................... X X X X X 

X X 

X 

+++M+++++++++++++++++++++ 

+++M+++++++++++++++++++++ 

X xx x X 
Lesions in Female Rats 149 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acd 5,000 ppm (continued)

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Carcinoma, metastatic, thyroid gland 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

~ ~ ~ ~-~__ 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars nervosa, ganglioneuroma 

Thyroid gland 

Ccell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Bilateral, carcinoma 

ovary

Uterus 

Endometrium, polyp stromal 

7777777777777777777777777 

0011333333333333333333333 

9944000000000000000000000 

4444444444444444444444444 Total 

4556222333333344444556667 Tissues/ 

3590237124567802459271450 Tumors 

......................... 50 

1 

......................... 50 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 48 

......................... 50 

+ + + 6 

......................... 49 

......................... 50 

......................... 49 

......................... 49 

+ 1 

......................... 50 

. ......................... 50 

......................... 50 

......................... 50 

......................... 49 

+++++++M+++++++++++M+++++ 48 

++++++M++++++++++++++++++ 49 

xx x xx X X xx 23 

X 1 

......................... 50 

X 2 

1 

......................... 50 

xx X xx X 11 

X 3 

X 1 

1 

......................... 49 

......................... 49 

5 
150 p-Nitrobenzoic Acid, NTP TR 442 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 5,000ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basal cell carcinoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Carcinoma, metastatic, thyroid gland 

Squamous cell carcinoma 

Nose 

Trachea 

Carcinoma, metastatic, thyroid gland 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Mesothelioma malignant 

4445555555666666666777777 

4891234567011278899000000 

1338821654218247955022788 

4444444444444444444444444 

3452562342545526656426623 

9669168084018357942763813 

......................... + + + + ++ + ......................... 

+++M+++++++++++++++++++++ 

+++M+++++++++++++++++++++ 

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

......................... X 

xx X X xxxxx x xx 

x x ......................... 

X 

X X 

......................... + 

X 

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

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

X 

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

......................... X 

t 

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

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

......................... 
Lesions in Female Rats 151 

TABLEB2 

Individual AnimalTumor Pathology of Female Rats in the 2-Year Feed Study ofp-Nitrobenzoic Acd 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

~ ~~~~~ 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basal cell carcinoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Carcinoma, metastatic, thyroid gland 

Squamous cell carcinoma 

Nose 

Trachea 

Carcinoma, metastatic, thyroid gland 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Mesothelioma malignant 

7777777777777777777777777 

0011333333333333333333333 

9944000000000000000000000 

4444444444444444444444444 Total 

4556222333333344444556667 Tissues/ 

3590237124567802459271450 Tumors 

......................... 50 

+ 8 

......................... 50 

......................... 49 

......................... 49 

......................... 50 

......................... 50 

X 2 

X x x xx X X 19 

xx X 5 

......................... 50 

1 

X 1 

X 3 

......................... 50 

1 

1 

......................... 50 

......................... 50 

X 1 

1 

X 1 

++++++M++++++++++++++++++ 49 

......................... 50 

1 

1 

......................... 50 

......................... 50 

......................... 50 

X 1 
152 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB3 

Statistical Analysisof Primary Neoplasms in Female Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid 

1,250 ppmppm 2,500 5,000 ppm

Adrenal Medulla: Benign Pheochromocytoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd ! Fisher exact test 

Clitoral Gland Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Clitoral Gland Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Clitoral Gland Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochrari-Armitage test 

Fisher exact test 

Mammary Gland Fibroadenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

2/49 (4%) 

5.4% 

om (0%) 

647 

P=0.298N 

P=O.242N 

P=0.237N 

4/50 (8%) 

11.9% 

2/27 (7%) 

653 

P=O.O34 

P=O.O46 

P=O.O66 

1/50 (2%) 

3.7% 

1/27 (4%) 

730 (TI

P=O.O85 

P=O.117 

P=O.116 

4/50 (8%) 

11.9% 

2/27 (7%) 

653 

P=O.O08 

P=O.O11 

P=O.O18 

22/50 (44%) 

59.6% 

13/27 (48%) 

566 

P=O.161 

P=O.280 

P=O.333 

1/50 (2%) 

4.3% 

1/23 (4%) 

730 (TI

P=0.557N 

P=0.510N 

P=O.492N 

12/49 (24%) 

42.5% 

7/22 (32%) 

665 

P=O.O13 

P=O.O13 

P=O.O24 

2/49 (4%) 

6.0% 

on2 (0%) 

694 

P=O.460 

P=O.459 

P =0.492 

14/49 (29%) 

45.9% 

7/22 (32%) 

665 

P=O.O05 

P=O.O04 

P=O.O08 

22/50 (44%) 

57.1% 

8/23(35%) 

483 

P=O.363 

P=O.502 

P=0.580N 

3/50 (6%) 

11.7% 

2/21 (10%) 

518 

P=O.402 

P=O.526 

P=O.510 

10/49 (20%) 

33.7% 

4/20 (20%) 

496 

P=O.O30 

P =0.050 

P=O.O68 

5/49 (10%) 

19.3% 

3/20 (15%) 

499 

P=O.O56 

P=O.O84 

P=O.O98 

15/49 (31%) 

48.9% 

7/20 (35%) 

496 

P=O.OOl 

P=O.O03 

P=O.O04 

26/50 (52%) 

70.4% 

11/21 (52%) 

379 

P=O.O73 

P=O.187 

P=O.274 

0/50 (0%) 

0.0% 

om (0%) -e

P=0.289N 

P=0.22QN 

P=O.242N 

12/50 (24%) 

42.1% 

7/21 (33%) 

483 

P=O.O13 

P=O.O23 

P=O.O27 

4/50 (8%) 

11.7% 

om (0%) 

528 

P=O.139 

P=O.224 

P=O.181 

15/50 (30%) 

47.7% 

7/21 (33%) 

483 

P=O.O02 

P=O.O04 

P=O.O05 

24/50 (48%) 

61.4% 

7/21 (33%) 

483 

P=O.198 

P=O.327 

P=O.421 
Lesions in Female Rats 153 

TABLEB3 

Statistical Analysisof Primary Neoplasms in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

0 PPm 1,2502,500 ppmppm 5,000 ppm 

Mammary Gland: Carcinoma 

Overall rate 2/50 (4%) 2/50 (4%) 3/50 (6%) 2/50 (4%) 

Adjusted rate 7.0% 6.6% 11.3% 7.0% 

Terminal rate 1/27 (4%) 1/23 (4%) 2/21 (10%) 1/21(5%) 

First incidence (days) 722 570 241 556 

Life table test P=O.481 P=O.636 P=O.404 P=O.605 

Logistic regression test P=0.583N P=O.691 P=O.623 P=0.699N 

Cochran-Armitage test P=O.569 

Fisher exact test P=0.691N P=O.500 P-0.691N 

Mammary Gland Adenoma or Carcinoma 

Overall rate 3/50 (6%) 2/50 (4%) 3/50 (6%) 2/50 (4%) 

Terminal rate 

Adjusted rate 

1/27 (4%) 

9.3% 

1/23 (4%) 

6.6% 11.3% 

2/21 (10%) 

7.0% 

1/21(5%) 

First incidence (days) 667 570 241 556 

Life table test P=0.546N P=O.562N P=O.554 P=0.596N 

Logistic regression test P=O.422N P=0.500N P=0.564N P=0.490N 

Cochran-Amitage test P=0.456N 

Fisher exact test P=OSOON P=0.661N P=0.500N 

Mammary Gland Adenoma or Fibroadenoma 

Overall rate 23/50 (46%) 22/50 (44%) 26/50 (52%) 24/50 (48%) 

Adjusted rate 60.6% 57.1% 70.4% 61.4% 

Terminal rate 13/27 (48%) 8123 (35%) 11/21(52%) 7/21(33%) 

Life table test 

First incidence (days) 

P=O.196 

566 

P=O.427 

483 

P=O.O98 

379 

P=O.247 

483 

Logistic regression test P=O.340 P=0.576N P=O.249 P=O.408 

Cochran-Armitage test P=O.396 

Fisher exact test P=0.500N P=O.345 P=O.500 

Mammary Gland Adenoma, Fibroadenoma, or Carcinoma 

Overall rate 25/50 (50%) 24/50 (48%) 28/50(56%) 26/50 (52%) 

Adjusted rate 64.7% 60.9% 73.9% 65.0% 

Terminal rate 14/27 (52%) 9/23 (39%) 12/21 (57%) 8/21 (38%) 

First incidence (days) 566 483 241 483 

Life table test P=O.186 P=O.409 P=O.O93 P=O.229 

Logistic regression test P=O.362 P,=0.579N P=O.304 P=O.408 

Fisher exact test 

Cochran-Amitage test P=O.396 

P=0.500N P=O.344 P=O.500 

Overall rate 

Pituitary Gland (Pars Distalis): Adenoma 

18/50 (36%) 27/50 (54%) 25/50 (50%) 23/49 (47%) 

Adjusted rate 46.6% 74.8% 76.4% 59.8% 

First incidence (days) 

Terminal rate 

657 

8/27 (30%) 

510 

15/23 (65%) 

538 

14/21 (67%) 

518 

7/20 (35%) 

Life table test P=O.112 P=O.O30 P=O.O23 P=O.O96 

Logistic regression test P=O.200 P=O.O34 P=O.O25 P=O.184 

Fisher exact test 

Cochran-Armitage test P=O.272 

P=O.O54 P=O.113 P=O.184 
154 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of p-Nitrobenzoic Acid (continued) 

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

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma 

Overall rate 19/50 (38%) 27/50 (54%) 25/50 (50%) 23/49 (47%) 

Adjusted rate 48.4% 74.8% 76.4% 59.8% 

Terminal rate 8/27 (30%) 15/23 (65%) 14/21 (67%) 7/20 (35%) 

First incidence (days) 657 510 538 518 

Life table test P=O.139 P=O.O45 P=O.O35 P=O.125 

Logistic regression test P=O.248 P=O.O51 P=O.O38 P=O.237 

Cochran-Armitage test P=O.330 

Fisher exact test P=O.O80 P=O.157 P=O.243 

Skin (Subcutaneous Tissue): Fibroma 

Overall rate 2/50 (4%) 1/50 (2%) 0/50 (0%) 3/50 (6%) 

Adjusted rate 6.6% 3.1% 0.0% 9.9% 

Terminal rate 1/27 (4%) 0/23 (0%) om (0%) 1/21 (5%) 

First incidence (days) 702 696 - 611

Life table test P=O.294 P=O.552N P=0.295N P =0.427 

Logistic regression test P=O.315 P=0.535N P =0.290N P=O.480 

Cochran-Armitage test P=O.337 

Fisher exact test P=0.500N P=0.247N P=O.500 

Thyroid Gland (C-cell): Adenoma 

Overall rate 9/50 (18%) 5/49 (10%) 4/50 (8%) 2/50 (4%) 

Adjusted rate 29.3% 17.4% 13.6% 6.7% 

Terminal rate 7/27 (26%) 3/23 (13%) 1/21 (5%) 1/21 (5%) 

First incidence (days) 556 632 588 483 

Life table test P=0.045N P=0.284N P=0.241N P=0.059N 

Logistic regression test P=0.023N P=0.249N P=0.165N P=O.OWN 

Cochran-Armitage test P=0.019N 

Fisher exact test P=0.205N P=0.117N P=O.OXN 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Overall rate 10/50 (20%) 5/49 (10%) 6/50 (12%) 2/50 (4%) 

Adjusted rate 31.4% 17.4% 22.2% 6.7% 

Terminal rate 7/27 (26%) 3/23 (13%) 3/21 (14%) 1/21 (5%) 

First incidence (days) 556 632 588 483 

Life table test P=0.041N P=0.213N P=0.391N P=0.038N 

Logistic regression test P=0.021N P=0.176N P=0.308N P=0.016N 

Cochran-Armitage test P=0.016N 

Fisher exact test P=0.140N P=0.207N P=0.014N 

Uterus: Stromal Polyp 

Overall rate 5/50(10%) 11/50 (22%) 12/50 (24%) 5/50(10%) 

Adjusted rate 15.9% 38.9% 37.9% 13.2% 

Terminal rate 3/27 (11%) 7/23 (30%) 4/21 (19%) on1 (0%) 

First incidence (days) 490 521 434 565 

Life table test P=O.523 P=O.O50 P=O.O23 P=O.513 

Logistic regression test P=0.406N P=O.O75 P=O.O63 P=0.534N 

Cochran-Armitage test P=0.411N 

Fisher exact test P=O.O86 P=O.O54 P=0.630N 
Lesions in Female Rats 155 

TABLEB3 

Statistical Analysisof Primary Neoplasms in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

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

Uterus: Stromal Polyp or Stromal Sarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Mononuclear Cell Leukemia 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Malignant Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Benign or Malignant Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

5/50(10%) 

15.9% 

3/27 (11%) 

490 

P=O.540 

P=0.379N 

P=0.389N 

17/50 (34%) 

38.6% 

3/27 (11%) 

490 

P<0.001N 

Pc0.001N 

Pc0.001N 

40/50 (80%) 

84.9% 

2007 (74%) 

490 

P=O.128 

P=O.311 

P=O.424 

22/50 (44%) 

49.0% 

6/27 (22%) 

490 

P=0.113N 

P=0.007N 

P=0.017N 

44/50 (88%) 

88.0% 

21/27 (78%) 

490 

P=O.143 

P=O.559 

P=O.519 

12/50 (24%) 

40.7% 

7/23 (30%) 

521 

P=O.O32 

P=O.O47 

P=O.O54 

11/50 (22%) 

32.5% 

4/23 (17%) 

566 

P=0.272N 

P=0.159N 

P=0.133N 

41/50 (82%) 

93.0% 

20/23 (87%) 

483 

P=O.206 

P=O.339 

P=O.500 

19/50 (38%) 

48.3% 

6/23 (26%) 

150 

P=0.541N 

P=0.188N 

P=0.342N 

48/50 (96%) 

98.0% 

22/23 (96%) 

150 

P=O.103 

P=O.156 

P=O.134 

13/50 (26%) 

39.4% 

4/21 (19%) 

434 

P=O.O14 

P=O.O44 

P=O.O33 

3/50 (6%) 

8.5% 

on1 (0%) 

492 

P=0.008N 

Pc0.001N 

Pc0.001N 

44/50 (88%) 

95.6% 

19/21 (90%) 

379 

P=O.O35 

P=O.O72 

P=O.207 

22/50 (44%) 

62.3% 

lOnl (48%) 

241 

P=O.259 

P=0.383N 

P=0.580N 

50/50 (100%) 

100.0% 

21/21 (100%) 

241 

P=O.O17 

P=O.O32 

P=O.O13 

5/50 (10%) 

13.2% 

on1 (0%) 

565 

P=O.513 

P=0.534N 

P=0.630N 

0/50 (0%) 

0.0% 

on1 (0%) -

PC0.001N 

P<0.001N 

Pc0.001N 

41/50 (82%) 

88.8% 

16/21 (76%) 

483 

P=O.150 

P=O.473 

P=O.500 

11/50 (22%) 

31.4% 

2/21 (10%) 

441 

P=0.104N 

P=0.004N 

P=0.016N 

45/50 (90%) 

91.8% 

17/21 (81%) 

441 

P=O.138 

P=O.564 

P=O.500 
156 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB3 

Statistical Analysisof Primary Neoplasms in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

QTerminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for clitoral gland, 

pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

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

Observed incidence at terminalkill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposure group incidence are the P values corresponding 

to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in animals dying prior to terminal kill 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Annitage and Fisher 

exact tests compare directly the overall incidence rates.For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

e Not applicable; noneoplasms in animal group 
Lesions in Female Rats 157 

TABLEB4a 

Historical Incidence of Clitoral Gland Neoplasms in Untreated Female F344/N Ratsa 

Incidence in Controls 

Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at Southern Research Institute 

Benzyl Acetate 0150 1/50 1/50 

C.I. Pigment Red 23 5/47 3/47 7/47 

C.I. Pigment Red 3 9/47 0/47 9/47 

Nitrofurantoin 1/44 4/44 5/44 

o-Nitroanisole 3/45 4/45 7/45 

Polysorbate 80 3/48 7/48 10/48 

Rhodamine 6G 5/42 1/42 6/42 

Roxarsone 1/44 1/44 2/44 

Total 27/367 (7.4%) 21/367 (5.7%) 47t367 (12.8%) 

Standard deviation 6.4% 5.1% 6.6% 

Range 0%-19% 0%-15% 2%-21% 

Overall Historical Incidence 

Totalb 90/1,096 (8.2%) 31/1,096 (2.8%) 120/1,096 (10.9%) 

Standard deviation 4.6% 4.0% 5.3% 

Range 0%-19% 0%-15% 2%-21% 

a Dataasof 20 August 1992 

Data from Quercetin, TR 409, censored due to low denominator (adenoma, 4/14; carcinoma, 1/14; adenoma or carcinoma, 5/14) 
158 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB4b 

Historical Incidence of Mononuclear Cell Leukemia in Untreated Female F344/N Ratsa 

Incidence in Controls 

Study Mononuclear Cell Leukemiab 

Historical Incidence at Southern Research Institute 

Benzyl Acetate 9/50 

C.I. Pigment Red 23 14/50 

C.I. Pigment Red 3 10/50 

Nitrofurantoin 13/50 

o-Nitroanisole 14/50 

Polysorbate 80 26/50 

- Rhodamine 6G 11/50 

Roxarsone 14/50 

Total 111/400 (27.8%) 

Standard deviation 10.6% 

Range 18%-52% 

Overall Historical Incidence 

Total 324/1,251 (25.9%) 

Standard deviation 8.6% 

Range 14%-52% 

a Data as of 20 August 1992 

Includes incidences of lymphocytic, monocytic, or undifferentiated leukemia. 
Lesions in Female Rats 159 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-NitrobenzoicAcida 

Disposition Summary 

Animals initially in study 

IS-Month intsim evalknri0;l 

Early deaths 

Moribund 

Natural deaths 

SuMvors 

Terminal sacrifice 

Animals examined microscopically 

IS-Month Interim Evaluation 

Alimentary System 

Intestine large, colon 

Parasite metazoan 

Intestine large, rectum 

Parasite metazoan 

Liver 

Basophilic focus 

Clear cell focus 

Fatty change 

Hepatodiaphragmatic nodule 

Inflammation, focal 

Mixed cell focus 

Bile duct, hyperplasia 

Mesentery 

Fat, necrosis 

Pancreas 

Accessory spleen 

Atrophy, focal 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Focal cellular change 

Pituitary gland 

Angiectasis 

Cyst

Pars distalis, focal cellular change 

Pars distalis, hyperplasia, focal 

Thyroid gland 

Ultimobranchial cyst 

C-cell, hyperplasia 

0 PPm 

60 

10 

21 

2 

27 

60 

3 (30%) 

7 (70%) 

1 (10%) 

4 (40%) 

(1)

1 (100%) 

(10)

1 (10%) 

5 (50%) 

(10)

7 (70%) 

1 (10%) 

5 (50%) 

(10) 

1,250 ppm 

60 

10 

23 

4 

23 

60 

(10)

1 (10%) 

(10)

5 (50%) 

1 (10%) 

1 (10%) 

1 (10%) 

9 (90%) 

6 (60%) 

(1)

1 (100%) 

(10)

1 (10%) 

4 (40%) 

(10)

2 (20%) 

2 (20%) 

2 (20%) 

(10)

2 (20%) 

2,500 ppm 

60 

10 

27 

2 

21 

60 

1 (10%) 

4 (40%) 

1 (10%) 

6 (60%) 

(1)

1 (100%) 

(10) 

2 (20%) 

(10) 

2 (20%) 

(9)

5 (56%) 

5 (56%) 

(10)

1 (10%) 

5,000 ppm 

60 

10 

26 

3 

21 

60 

(9)

1 (11%) 

(10)

8 (80%) 

4 (40%) 

4 (40%) 

6 (60%) 

(10) 

(10)

3 (30%) 

2(20%) 

1 (10%) 

1 (10%) 

(10) 

3 (30%) 

a Number of animals examined microscopically at site and number of animals with lesion 
160 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

0 PPm 

IS-Month Interim Evaluation (continued) 

Genital System 

Clitoral gland 

Degeneration, cystic 

ovary 

Cyst

Uterus 

Hydrometra 

Endometrium, hyperplasia, cystic 

Endometrium, infarct 

Vagina 

Cyst 

Hematopoietic System 

Bone marrow 

Hyperplasia, focal, histiocytic 

Lymph node 

Mediastinal, congestion 

Mediastinal, pigmentation 

Pancreatic, congestion 

Pancreatic, pigmentation 

Lymph node, mandibular 

Congestion 

Hyperplasia, lymphoid 

Spleen 

Cyst

Pigmentation 

Integumentary System 

Mammaly gland 

Dilatation 

Hyperplasia 

Musculoskeletal System 

Bone 

Hyperostosis 

Nervous System 

Brain 

Compression 

Respiratory System 

Lung 

Alveolar epithelium, hyperplasia 

(10)

8 (80%) 

(10) 

(10) 

1 (10%) 

(10) 

1 (10%) 

(1) 

1 (100%) 

10 (100%) 

(10)

1 (10%) 

(10) 

1,250 ppm 2,500 ppm 

(1)

1 (100%) 

(10) 

2 (20%) 

(4) (2) 

2 (50%) 2 (100%) 

1 (25%) 

(10) 

10 (100%) 10 (100%) 

(10) (10)

2 (20%) 3 (30%) 

1 (10%) 3 (30%) 

(10) 

5,000 ppm 

(10)

4 (40%) 

(10)

1 (10%) 

(10)

1 (10%) 

2 (20%) 

(2) 

1 (50%) 

(10)

1 (10%) 

10 (100%) 

(10)

2 (20%) 

1 (10%) 
Lesions in Female Rats 161 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

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

15-Month Interim Evaluation (continued) 

Special Senses System 

Eye (2)

Cataract 1 (50%) 

Fibrosis 1 (50%) 

Retina, degeneration 1 (50%) 

Urinary System 

Kidney (10) 

(100%) 10 9 (90%) 

10 (100%) (100%) 10

(10) (10) (10)

Nephropathy 9 (90%) 

Renal tubule, pigmentation 

(100%) 10 

10 (100%) 10 (100%) 

Systems Examined WUh No Lesions Observed 

Cardiovascular System 

General Body System 

2-Year Study 

Alimentary System 

Parasite metazoan 

Intestine large, rectum (50) (48)

6 (13%) 

(50)

2 (4%) 

(49)

3 (6%) 

Parasite metazoan 

Intestine large, cecum (50) (47)

1 (2%) 

(50) (49) 

Angiectasis 

Liver (50)

1 (2%) 

(50)

2 (4%) 

(50) (50)

2 (4%) 

Basophilic focus 32(64%) 38(76%) 44 (88%) 47 (94%) 

Clear cell focus 4 (8%) 6 (12%) 7 (14%) 12 (24%) 

Eosinophilic focus 4 (8%) 4 (8%) 1 (2%) 

Fatty change 14 (28%) 13 (26%) 7 (14%) 7 (14%) 

Fibrosis, focal 1 (2%) 2 (4%) 

Focal cellular change 1 (2%) 1 (2%) 2 (4%) 

Hematopoietic cell proliferation 2 (4%) 2 (4%) 1 (2%) 

Hepatodiaphragmatic nodule 5 (10%) 5 (10%) 11 (22%) 12 (24%) 

Hepatodiaphragmatic nodule, multiple 2 (4%) 2 (4%) 3 (6%) 

Hyperplasia, histiocytic 1 (2%) 

Hyperplasia, lymphoid 1 (2%) 

Hyperplasia, multifocal 9 (18%) 10(20%) 3 (6%) 2 (4%) 

Infiltration cellular, mixed cell 1 (2%) 4 (8%) 3 (6%) 1 (2%) 

Inflammation, focal 26 (52%) 24 (48%) 37(74%) 41 (82%) 

Mixed cell focus 4 (8%) 8 (16%) 3 (6%) 7 (14%) 

Necrosis, focal 2 (4%) 1 (2%) 1 (2%) 

Pigmentation 1 (2%) 

Thrombosis 1 (2%) 

Bile duct, hyperplasia 

Bile duct, dilatation 

23(46%) 

1 (2%) 

19 (38%) 18 (36%) 27(54%) 

Centrilobular, atrophy 14 (28%) 11 (22%) 4 (8%) 2 (4%) 

Centrilobular, congestion 1 (2%) 

Centrilobular, hemorrhage 1 (2%) 

Centrilobular, necrosis 1 (2%) 1 (2%) 
162 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-NitrobenzoicAcid 

(continued) 

&Year Study (continued) 

Alimentary System (continued) 

Mesentery 

Accessory spleen 

Cyst

Inflammation, chronic 

Fat, necrosis 

Pancreas 

Atrophy, diffuse 

Atrophy, focal 

Cyst

Inflammation, chronic 

Acinar cell, depletion secretory 

Duct, dilatation 

Stomach, forestomach 

Edema 

Hemorrhage, focal 

Inflammation, chronic 

Ulcer 

Mucosa, hyperplasia 

Stomach, glandular 

Bacterium 

Erosion 

Inflammation, chronic 

Ulcer 

Tongue 

Hypertrophy, squamous 

Tooth 

Gingiva, hyperplasia 

Cardiovascular System 

Blood vessel 

Mesenteric artery, inflammation, chronic 

Mesenteric artery, thrombosis 

Heart 

Bacterium 

Embolus 

Inflammation, chronic, focal 

Artery, inflammation, chronic 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Angiectasis 

Congestion 

Degeneration, cystic 

Depletion cellular 

Focal cellular change 

Hyperplasia, focal 

Infiltration cellular, lymphocyte 

Vacuolization cytoplasmic 

Capsule, extra adrenal tissue, fibrosis 

0 PPm 

(10) 

6 (60%) 

(50) 

2 (4%) 

16 (32%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

(50)

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

(1)

1 (100%) 

(50)

1 (2%) 

1 (2%) 

(50)

1 (2%) 

1 (2%) 

2 (4%) 

(49)

1 (2%) 

1 (2%) 

4 (8%) 

1 (2%) 

13 (27%) 

1 (2%) 

1 (2%) 

1,250ppm 

(8)

1 (13%) 

1 (13%) 

3 (38%) 

(50)

1 (2%) 

14 (28%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

(50) 

(50) 

2 (4%) 

1 (2%) 

9 (18%) 

1 (2%) 

2,500ppm 

(9) 

1 (11%) 

1 (11%) 

6 (67%) 

(49)

1 (2%) 

19 (39%) 

1 (2%) 

1 (2%) 

(49) 

2 (4%) 

2 (4%) 

1 (2%) 

(49) 

(1) 

2 (4%) 

(50)

1 (2%) 

2 (4%) 

8 (16%) 

5,000 ppm 

(6)

1 (17%) 

1 (17%) 

4 (67%) 

(49)

1 (2%) 

11 (22%) 

1 (2%) 

(49) 

1 (2%) 

2 (4%) 

(49) 

(1)

1 (100%) 

(50)

2 (4%) 

1 (2%) 

(50)

3 (6%) 

2 (4%) 

13 (26%) 

1 (2%) 
Lesions in Female Rats 163 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

shcdy (continued) 

Endocrine System (continued) 

Adrenal medulla 

Hyperplasia 

Pituitaly gland 

Angiectasis 

Cyst

Granuloma 

Hemorrhage 

Pars distalis, cyst 

Pars distalis, focal cellular change 

Pars distalis, hyperplasia, focal 

Rathke’s cleft, hyperplasia, cystic 

Thyroid gland 

Degeneration, cystic 

Inflammation, focal 

Ultimobranchial cyst 

C-cell, hyperplasia 

Follicle, dilatation 

Follicular cell, hyperplasia 

General Body System 

None 

Genital System 

Clitoral gland 

Atrophy 

Cyst

Degeneration, cystic 

Hyperplasia 

Inflammation, chronic 

Duct, hyperplasia, squamous 

ovary

Angiectasis 

Atrophy 

Cyst

Hemorrhage 

Bilateral, cyst 

Corpus luteum, hyperplasia, lymphoid 

Corpus luteum, thecal cell, hyperplasia 

Uterus 

Hydrometra 

Endometrium, cyst 

Endometrium, hyperplasia, cystic 

Endometrium, infarct 

Vagina 

Cyst

Cyst, multiple 

Inflammation, chronic 

Mucosa, hyperplasia 

0 PPm 

(49)

2 (4%) 

(50)

12 (24%) 

3 (6%) 

1 (2%) 

1 (2%) 

6(12%) 

11 (22%) 

(50)

1 (2%) 

10 (20%) 

1 (2%) 

34 (68%) 

10 (20%) 

5 (10%) 

1 (2%) 

2 (4%) 

2 (4%) 

(50)

1 (2%) 

3 (6%) 

1 (2%) 

(3)

2 (67%) 

1 (33%) 

1,250 ppm 

(50)

1 (2%) 

(50)

11 (22%) 

4 (8%) 

1 (2%) 

4 (8%) 

9 (18%) 

1 (2%) 

(49) 

1 (2%) 

8 (16%) 

1 (2%) 

2 (4%) 

(49)

1 (2%) 

1 (2%) 

38 (78%) 

6 (12%) 

1 (2%) 

1 (2%) 

(50) 

6 (12%) 

1 (2%) 

1 (2%) 

(501

4 (8%) 

1 (2%) 

9 (18%) 

(1)

1 (100%) 

2,500 ppm 

(50)

2 (4%) 

(50)

7 (14%) 

2 (4%) 

2 (4%) 

9 (18%) 

2 (4%) 

11 (22%) 

(49) 

1 (2%) 

39 (80%) 

6 (12%) 

1 (2%) 

(50)

1 (2%) 

1 (2%) 

6 (12%) 

1 (2%) 

(50)

3 (6%) 

1 (2%) 

4 (8%) 

(3) 

1 (33%) 

1 (33%) 

1 (33%) 

5,000 ppm 

(50)

2 (4%) 

(49)

8 (16%) 

2 (4%) 

1 (2%) 

2 (4%) 

8 (16%) 

11 (22%) 

2 (4%) 

35 (70%) 

7 (14%) 

(49)

1 (2%) 

8 (16%) 

(49)

2 (4%) 

1 (2%) 

4 (8%) 
164 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

%Year shdy (continued) 

Hematopoietic System 

Bone marrow 

Hypercellularity 

Hyperplasia, focal, histiocytic 

Metaplasia, osseous 

Myelofibrosis 

Lymph node 

Deep ceMcal, angiectasis 

Inguinal, depletion lymphoid 

Inguinal, hyperplasia 

Mediastinal, angiectasis 

Mediastinal, congestion 

Mediastinal, depletion lymphoid 

Mediastinal, hyperplasia 

Mediastinal, hyperplasia, lymphoid 

Mediastinal, hyperplasia, macrophage 

Mediastinal, pigmentation 

Pancreatic, congestion 

Pancreatic, depletion lymphoid 

Pancreatic, edema 

Pancreatic, hyperplasia, lymphoid 

Pancreatic, inflammation, chronic 

Renal, hyperplasia, macrophage 

Renal, pigmentation 

Lymph node, mandibular 

Congestion 

Depletion lymphoid 

Hyperplasia 

Pigmentation 

Lymph node, mesenteric 

Congestion 

Depletion lymphoid 

Hyperplasia 

Hyperplasia, lymphoid 

Spleen 

Fibrosis 

Hematopoietic cell proliferation 

Necrosis, focal 

Pigmentation 

Thymus 

Congestion 

Crst 

Fibrosis 

Hyperplasia, lymphoid 

0 PPm 

(50)

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

(13) 

(50)

1 (2%) 

(49) 

1 (2%) 

(50)

1 (2%) 

2 (4%) 

2 (4%) 

50 (100%) 

(49) 

1 (2%) 

1 (2%) 

1,250 ppm 

(50)

4 (8%) 

2 (4%) 

(10) 

1(10%) 

1 (10%) 

1(10%) 

1(10%) 

1 (10%) 

1(10%) 

(50) 

1 (2%) 

2 (4%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

(50)

6 (12%) 

14 (28%) 

50 (100%) 

(48) 

1 (2%) 

2,500 ppm 

(50)

2 (4%) 

4 (8%) 

(9)

1(11%) 

1(11%) 

5 (56%) 

1(11%) 

2(22%) 

(50)

2 (4%) 

2 (4%) 

(50) 

1 (2%) 

(50) 

9 (18%) 

1 (2%) 

50 (100%) 

(50) 

1 (2%) 

5,000 ppm 

(50)

3 (6%) 

4 (8%) 

(8) 

2(25%) 

1(13%) 

3 (38%) 

1(13%) 

1(13%) 

1(13%) 

1(13%) 

1(13%) 

1(13%) 

(50)

4 (8%) 

1 (2%) 

2 (4%) 

(49) 

(49)

1 (2%) 

8 (16%) 

49 (100%) 

(50)

1 (2%) 
Lesions in Female Rats 165 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-NitrobenzoicAcid 

(continued) 

shdy (continued) 

Integumentary System 

Mammary gland 

Dilatation 

Hyperplasia 

Skin 

Cyst epithelial inclusion 

Hemorrhage, focal 

Inflammation, chronic, focal 

Ulcer 

Subcutaneous tissue, inflammation, 

chronic, focal 

Musculoskeletal System 

Bone 

Fracture healed 

Hyperostosis 

Inflammation, chronic, focal 

Nenous System 

Brain 

Compression 

Demyelination, focal 

Hemorrhage 

Spinal cord 

Hemorrhage, focal 

Respiratory System 

Lung 

Bacterium 

Congestion 

Fibrosis, focal 

Hemorrhage, focal 

Hyperplasia, focal, macrophage 

Infiltration cellular, mixed cell 

Metaplasia, focal, osseous 

Thrombosis 

Alveolar epithelium, hyperplasia 

Mediastinum, infiltration cellular, 

Wphocyte

Nose 

Fungus 

Inflammation, suppurative 

Nasolacrimal duct, cyst 

0 PPm 

(50)

43(86%) 

9(18%) 

(50) 

(50) 

17 (34%) 

1 (2%) 

(50)

8 (16%) 

4 (8%) 

(1) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

3(6%) 

2(4%) 

1,250 ppm 

(50)

38(76%) 

14 (28%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

9(18%) 

(50)

16(32%) 

1 (2%) 

(2) 

(50) 

1 (2%) 

1 (2%) 

3(6%) 

1 (2%) 

(50) 

4 (8%) 

1 (2%) 

2,500 ppm 

(50)

39(78%) 

10(20%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 

9(18%) 

(50)

10 (20%) 

1 (2%) 

1 (2%) 

(2)

1 (50%) 

2(4%) 

2 (4%) 

1 (2%) 

1 (2%) 

4 (8%) 

(50) 

5 (10%) 

5,000 ppm 

(50)

35(70%) 

15 (30%) 

(50) 

(50) 

9(18%) 

(50)

14 (28%) 

1 (2%) 

1 (2%) 

2 (4%) 

3(6%) 

(49)

1 (2%) 

2 (4%) 
166 p-Nitrobenzoic Acid, NTP TR 442 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

2-YkZt study (continued) 

Special Senses System 

Eye

Atrophy 

Cataract 

Hemorrhage 

Inflammation, chronic 

Necrosis, focal 

Retina, degeneration 

Urinary System 

Kidney 

Atrophy, focal 

Bacterium 

Congestion 

Cyst

Fibrosis, focal 

Nephropathy 

Cortex,renal tubule, autolysis 

Glomerulus, autolysis 

Interstitium, infarct 

Papilla, epithelium, hyperplasia, focal 

Renal tubule, hyperplasia 

Renal tubule, hyperplasia, oncocytic 

Renal tubule, pigmentation 

Urinary bladder 

Transitional epithelium, hyperplasia 

0 PPm 

(2) 

2 (100%) 

1 (50%) 

1 (50%) 

2 (100%) 

(50) 

1 (2%) 

50 (100%) 

1 (2%) 

1 (2%) 

50 (100%) 

(50) 

1,250 ppm 

(2) 

1 (50%) 

1 (50%) 

1 (50%) 

1 (50%) 

1 (50%) 

(50) 

1 (2%) 

49 (98%) 

1 (2%) 

50 (100%) 

(50) 

2,500 ppm 

(2)

1 (50%) 

2 (100%) 

1 (50%) 

1 (50%) 

2 (100%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

49(98%) 

1 (2%) 

50 (100%) 

(50)

1 (2%) 

5,000 ppm 

(1) 

1 (100%) 

1 (100%) 

(50) 

1 (2%) 

49(98%) 

1 (2%) 

1 (2%) 

5 (10%) 

50 (100%) 

(50) 
167 

APPENDIX C 

SUMMARY OF LESIONS IN MALE MICE 

IN THE 2-YEAR FEED STUDY 

OF p-NITROBENZOIC ACID 

TABLE C1 Summary of the Incidence of Neoplasms in Male Mice 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 168 

TABLE C2 Individual Animal Tumor Pathology of Male Mice 

in the 2-Year Feed Study ofp-Nitrobenzoic Acid ............................. 172 

TABLE C3 Statistical Analysis of Primary Neoplasms in Male Mice 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 188 

TABLE C4 Historical Incidence of Lung Neoplasms in Untreated Male B6C3Fl Mice .......... 192 

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

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 193 
168 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acida ~ ~~ ~ 

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

Early deaths 

ls-Mmth inraim c&”m 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental deaths 

Terminal sacrifice 

SuMvors 

10 

60 

2 

9 

39 

10 

60 

9 

4 

1 

36 

10 

60 

1 

10 

39 

10 

60 

2 

2 

2 

44 

Animals examined microscopically 60 60 60 60 

Liver 

Alimentary System 

15-Month Interim Evaluation 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

(10) 

1 (10%) 

1 (10%) 

(10) 

1 (10%) 

1 (10%) 

2 (20%) 

(10) 

1 (10%) 

1 (10%) 

Thyroid gland 

Endocrine System 

Follicular cell, adenoma 

Skin 

Integumentary System 

Subcutaneous tissue, lipoma 

Lung 

Respiratory System 

Alveolarbronchiolar adenoma 

(10)

2 (20%) 

Schwannoma benign 

Ear 

Special Senses System 

(1)

1 (100%) 

Urinary System 

Nervous System 

Musculoskeletal System 

Hematopoietic System 

Genital System 

General Body System 

Cardiovascular System 

Systems Examined WdhNo Neoplasms Observed 
Lesions in Male Mice 169 

TABLEC1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid (continued) 

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

2-Year Study 

Alimentary System 

Gallbladder 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Hepatocholangiocarcinoma 

Mesentery 

Sarcoma 

Pancreas 

Sarcoma, metastatic, mesentery 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Carcinoid tumor NOS 

Sarcoma, metastatic, mesentery 

Cardiovascular System 

None 

Endocrine System 

Adrenal cortex 

Sarcoma, metastatic, mesentery 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Epididymis 

Prostate 

Seminal vesicle 

Testes 

Interstitial cell, adenoma 

1 (2%) 

(50)

1 (2%) 

(50) 

(50) 

(46) 

(50)

1 (2%) 

1 (2%) 

(45) 

(43) 

1 (2%) 

(42) 

(50)

1 (2%) 

9(18%) 

4 (8%) 

10 (20%) 

7(14%) 

(1) 

(48) 

(47) 

(47)

1 (2%) 

(49) 

(49) 

(49)

1 (2%) 

(46) 

1 (2%) 

(49) 

1 (2%) 

(49) 

(50) 

12 (24%) 

4(8%) 

5 (10%) 

7(14%) 

1 (2%) 

(2) 

(50) 

(50) 

(50)

1 (2%) 

(50) 

(50) 

(50) 

(46) 

(48) 

4(8%) 

4 (8%) 

11 (23%) 

3 (6%) 

(2) 

(47) 

(47) 

(47) 

(49) 

(49) 

(48) 

(46)

1 (2%) 

(49) 
170 p-Nitrobenzoic Acid, NTP TR 442 

2-YeUr study (continued) 

Hematopoietic System 

Bone marrow 

Hemangioma 

Lymph node 

Lymph node, mandibular 

Mast cell tumor NOS 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Integumentary System 

Skin 

Hemangioma 

Hemangiosarcoma 

Subcutaneous tissue, mast cell tumorNOS 

Musculoskeletal System 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

None 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar multiple adenoma, 

Alveolaribronchiolar carcinoma 

Hepatocellularcarcinoma,metastatic,liver 

Nose 

Nasolacrimal duct, mast cell tumor NOS 

Special Senses System 

Harderian gland 

Adenoma 

Adenoma, multiple 

Urinary System 

Kidney 

Transitional epithelium, carcinoma 

0 PPm 

(50) 

1 (2%) 

(50)

3 (6%) 

3 (6%) 

1 (2%) 

4 (8%) 

(50) 

(1)

1 (100%) 

1,250 ppm 

(49)

1 (2%) 

(49)

1 (2%) 

(50)

10 (20%) 

2 (4%) 

3 (6%) 

2 (4%) 

(49) 

(3)

3 (100%) 

(49)

1 (2%) 

2,500 ppm 

(50) 

1 (2%) 

(1)

1 (100%) 

(50)

6 (12%) 

2 (4%) 

2 (4%) 

6 (12%) 

(49)

1 (2%) 

(3)

2 (67%) 

1 (33%) 

5,000 ppm 

(50)

6 (12%) 

3 (6%) 

5 (10%) 

4 (8%) 

(50) 

(1)

1 (100%) 
Lesions in Male Mice 171 

%Year Study (continued) 

Systemic Lesions 

Multiple organsb 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Neoplasm Summary 

Total animals with primary neoplasms' 

15-Month interim evaluation 

2-Year study 

Total primary neoplasms 

15-Month interim evaluation 

2-Year study 

Total animals with benign neoplasms 

15-Month interim evaluation 

2-Year study 

Total benign neoplasms 

15-Month interim evaluation 

2-Year study 

Total animals with malignant neoplasms 

15-Month interim evaluation 

2-Year study 

Total malignant neoplasms 

15-Month interim evaluation 

2-Year study 

Total animals with metastatic neoplasms 

2-Year study 

Total metastatic neoplasms 

2-Year study 

Total animals with uncertain neoplasms 

benign or malignant 

2-Year study 

Total uncertain neoplasms 

2-Year study 

0 PPm 

(50)

2 (4%) 

2 

33 

3 

48 

2 

20 

3 

26 

18 

22 

5 

7 

1,250 ppm 

(50)

1 (2%) 

5 

36 

5 

59 

3 

29 

3 

37 

2 

19 

2 

21 

2 

2 

1 

1 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Number of animals with any tissue examined microscopically

' Primaryneoplasms:allneoplasmsexceptmetastaticneoplasms 

2,500 ppm 

~~ 

5,000 ppm 

(50) 

1 (2%) 

2 (4%) 

(50)

2 (4%) 

35 

6 

20 

6 

52 

6 

25 

6 

30 

1 

41 

1 

22 

1 

25 

1 

21 14 

6 

24 

6 

16 

4 

4 

3 

1 
172 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC2 

Individual Animal Tumor Pathologyof Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Sarcoma 

Pancreas 

Sarcoma, metastatic, mesentery 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Sarcoma, metastatic, mesentery 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Sarcoma, metastatic, mesentery 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

C-cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Coagulating gland 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

4555666111111111111111111 

1339661222233333333333333 

9217065008800000000000000 

0000000000000000000000000 

2135014002400000011111111 

6830198691412345801234561 

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

++M++++++++++++++++++++++ 

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

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

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

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

......................... X 

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

......................... X X xx X 

X 

xxx 

+ 

A 

+ 

x 

X 

X X xx 

......................... X 

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

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

......................... X 

+ 

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

......................... X 

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

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

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

++++++M++++M+++++++++++++ 

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

X 

X 

+ ......................... 

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

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

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

M: Missing tissue X Lesion present 

I: Insufficient tissue Blank Not examined 
Lesions in Male Mice 173 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Sarcoma 

Pancreas 

Sarcoma, metastatic, mesentery 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Sarcoma, metastatic, mesentery 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Sarcoma, metastatic, mesentery 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

C-cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Coagulating gland 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000000000 

0000000000000000000000000 Total 

2222222233333333344444444 Tissues/ 

0123458901245678901235679 Tumors 

......................... 50 

++++++++M++++++++++++++++ 48 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X 3 

......................... 50 

........................... 50 

X 6 

xx 2 

xx X xx X 14 

X 3 

2 

1 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

1 

+ 2 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

++++++++++++++++++++++M+M 46 

......................... 50 

1 

1 

~ 

1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 
174 p-Nitrobenzoic Acid, NTP TR 442 

Number of Days on Study 

Carcass ID Number 

Genital System (continued) 

Testes 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph 'node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

integumentary System 

MammaIy gland 

Skin 

Hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Eye

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

4555666777777777777777777 

1339661222233333333333333 

9277065008800000000000000 

0000000000000000000000000 

2135014002400000011111111 

6830798697412345801234567 

......................... X 

......................... + + 

+++M+++++++++++++++++++++ 

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

......................... X 

++++++M++++++++++++++M+++ 

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

......................... X 

......................... + 

X 

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

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

X 

X 

X X X 

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

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

+ 

+ 

X 

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

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

......................... 
Lesions in Male Mice 175 

TABLEC2 

Individual AnimalTumor Pathology of Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System (continued) 

Testes 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Eye

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

~~ ~ ~~ ~ 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

1111111111111111111111111 

3333333333333333333333333 

0000000000000000000000000 

0000000000000000000000000 Total 

2222222233333333344444444 Tissues1 

0123458901245618901235619 Tumors 

......................... 50 

1 

......................... 50 

+ + 4 

......................... 49 

......................... 50 

......................... 50 

X xx 4 

++MM++++++++++++++++++M++ 45 

......................... 50 

......................... 50 

1 

......................... 50 

1 

1 

......................... 50 

......................... 50 

X X X 3 

xx 3 

1 

X 4 

......................... 50 

......................... 50 

t 2 

1 

1 

......................... 50 

......................... 50 

......................... 50 

X X 2 
176 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC2 

Individual Animal Tumor Pathologyof Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 1,250 ppm 

0455666667777777777777777 

Number of Days on Study 043’7067890022222233333333 

7275155150105899900000000 

1111011001000000000000000 

Carcass ID Number 1100600870896666666677777 

0846801868903124567901234 

Alimentary System 

Esophagus ......................... Gallbladder AM+A+++++A++A++++++++++++ 

Intestine large, colon AAAAAA+++A++A++++++++++++ 

Intestine large, rectum AAAAA++++A++A++++++++++++ 

Intestine large, cecum AAAAAA+++A++A++++++++++++

Intestine small, duodenum AAAAA++++A++A++++++++++++

Intestine small, jejunum AAAAA++++A++A++++++++++++ 

Adenocarcinoma 

Intestine small, ileum AAAAAA+++A++A++++++++++++ 

Liver ......................... Hemangiosarcoma 

Hepatocellular carcinoma xx x xx xx 

Hepatocellular carcinoma, multiple xxx 

Hepatocellular adenoma xx x X X X 

Hepatocellular adenoma, multiple X X 

Mesentery 

Pancreas ......................... Salivary glands +A+++++++++++++++++++++++ 

Stomach, forestomach +AA++++++A+++++++++++++++ 

Stomach, glandular +AA++++++A+++++++++++++++ 

Carcinoid tumor NOS X 

Cardiovascular System 

Heart +A+++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex ......................... 

Adrenal medulla ......................... Islets, pancreatic +A+++++++++++++++++++++++ 

Adenoma X 

Parathyroid gland +M+++++++++++++++M+++++++ 

Pituitary gland IM+M+++++++++++++++M+++++ 

Pars intermedia,adenoma 

Thyroid gland ......................... Follicular cell, adenoma X

General Body System 

Tissue NOS 

Genital System 

Coagulating gland + + 

Epididymis .......................... Preputial gland ......................... Prostate ......................... Seminal vesicle ......................... Testes +A+++++++++++++++++++++++ 
Lesions in Male Mice 177 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Carcinoid tumor NOS 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

Tissue NOS 

Genital System 

Coagulating gland 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000000000 

0000000000000000000001111 Total 

7777888888889999999990000 Tissues/ 

5789012345671234567892357 Tumors 

......................... 50 

......................... 45 

......................... 42 

......................... 43 

......................... 42 

......................... 43 

......................... 43 

X 1 

......................... 42 

......................... 50 

X 1 

X X 9 

X 4 

X X xx 10 

X xxx X 7 

+ 1 

......................... 48 

......................... 49 

......................... 47 

......................... 47 

1 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

1 

++I++++M+++++++++++++++++ 46 

.......................... 46 

X 1 

......................... 49 

1 

+ 1 

2 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 49 
178 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Hemangioma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangioma 

Musculoskeletal System 

Bone 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Transitional epithelium, carcinoma 

Urethra 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

0455666667777777777771777 

0431067890022222233333333 

7275155150105899900000000 

1111011001000000000000000 

1100600870896666666671777 

0846801868903124567901234 

......................... X 

+A++M++M+++++++++++++++++ 

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

+M+++++++++++++++++++++++ 

X 

++++++M++++M+++++++++++++ 

M++++++++++++++++++++++++ 

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

X 

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

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

......................... xx X xx X X 

X xx 

X 

+A+++++++++++++++++++++++ 

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

+ + 

X X 

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

+ ......................... 

......................... 
Lesions in Male Mice 179 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 1,250ppm (continued) 

Number of Days on Study 

CarcassIDNumber 

Hematopoietic System 

Bone marrow 

Hemangioma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangioma 

Musculoskeletal System 

Bone 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, 

multiple 

Alveolar/bronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Transitional epithelium, carcinoma 

Urethra 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000000000 

0000000000000000000001111 Total 

7’777888888889999999990000 Tissues/ 

5789012345671234567892357 Tumors 

......................... 49 

1 

+ 1 

......................... 47 

......................... 49 

......................... 49 

1 

M++++++++++++++++++++++++ 47 

......................... 49 

......................... 49 

1 

......................... 50 

......................... 50 

......................... 50 

X X X 10 

X X 2 

3 

X 2 

......................... 49 

......................... 50 

+ 3 

X 3 

......................... 49 

X 1 

1 

......................... 50 

......................... 50 

X 1 
180 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC2 

Individual Animal Tumor Pathologyof Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 2,500 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Hepatocholangiocarcinoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

~~~~~~ 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

4455666666777117777777171 

5928378899022222222222222 

1628851135399999999999999 

1111111111111111111111111 

7325632666222222233333333 

6989383786214567901234567 

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

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

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

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

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

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

++A++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

......................... xxx X X X 

X X 

xx 

xx X 

X 

+ .......................... 

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

........................... X 

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

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

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

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

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

+++++++++++++M+++++++++++ 

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

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

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

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

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

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

......................... 
Lesions in Male Mice 181 

TABLEC2 

Individual Animal Tumor Pathologyof Male Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Hepatocholangiocarcinoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

1111111111111111111111111 

2222222222222222222222222 

9999999999999999999999999 

1111111111111111111111111 Total 

4444444444555555555666666 Tissues/ 

0123456189012345618012459 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

.......................... 50 

......................... 50 

......................... 49 

......................... 49 

......................... 50 

xx x xx X 12 

X X 4 

xx X 5 

xx X X 1 

1 

+ 2 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

+ + 2 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 
182 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 2,500 ppm(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Mast cell tumor NOS 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, mastcell tumor 

NOS 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolar/bronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Nasolacrimal duct, mast cell tumor 

NOS 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Adenoma, multiple 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

4455666666777777777777777 

5928378899022222222222222 

1628851135399999999999999 

1111111111111111111111111 

7325632666222222233333333 

6989383786214567901234567 

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

+ + ......................... X 

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

......................... X 

+++++++++++++M+++++++++++ 

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

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

X 

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

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

......................... x x xx 

X 

X X X 

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

X 

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

+ ++ 

xx 

X 

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

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

......................... X 

X 
Lesions in Male Mice 183 

TABLEC2 

Individual Animal Tumor Pathologyof Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Mast cell tumor NOS 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammaly gland 

Skin 

Subcutaneous tissue, mast cell tumor 

NOS 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Nasolacrimal duct, mast cell tumor 

NOS 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Adenoma, multiple 

Urinary System 

Kidney 

Urinaly bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

1111111111111111111111111 

2222222222222222222222222 

9999999999999999999999999 

1111111111111111111111111 Total 

4444444444555555555666666 Tissues/ 

0123456189012345618012459 Tumors 

......................... 50 

+ + 4 

......................... 50 

1 

......................... 50 

......................... 50 

1 

++++M+++M++++++++++++++++ 41 

......................... 50 

......................... 50 

1 

......................... 50 

+ 1 

X 1 

......................... 50 

......................... 50 

X X 6 

X X 2 

X 2 

xx X 6 

+++++++++++M+++++++++++++ 49 

1 

......................... 50 

3 

2 

1 

......................... 50 

......................... 50 

......................... 50 

X 2 

1 
184 

TABLEC2 

Individual Animal Tumor Pathology of 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

p-Nitrobenzoic Acid, NTP TR 442 

Male Mice in the 2-Year Feed Study ofp-NitrobenzoicAcid 5,000 ppm 

0013567777777777777777717 

0087862222222222222222222 

I788279999999999999999999 

1222211111111111111111122 

8130188888888999999999900 

8094761234579012345678901 

+A+++++++++++++++++++++++ 

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

AAA+A++++++++++++++++++++ 

AAA+A++++++++++++++++++++ 

AAA+A++++++++++++++++++++ 

AAA+A++++++++++++++++++++ 

AAA+A++++++++++++++++++++ 

AAA+A++++++++++++++++++++ 

+AA++++++++++++++++++++++ 

X X 

X 

xx X xx X 

X 

+ 

AAA++++++++++++++++++++++ 

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

AAA++++++++++++++++++++++ 

AAA++++++++++++++++++++++ 

+ 

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

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

AA+++++++++++++++++++++++ 

MA+++++++++++++++++++M++M 

MMM+++++M++++++++++++++++ 

X 

+A+++++++++++++++++++++++ 

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

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

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

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

......................... 
Lesions in Male Mice 185 

TABLEC2 

Individual Animal Tumor Pathologyof Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

7777777777777777777777777 

2222222222222222222222222 

9999999999999999999999999 

2222222222222222222222222 Total 

0000000111111112222222222 Tissues/ 

2356789123456890123456789 Tumors 

......................... 49 

......................... 48 

......................... 46 

......................... 46 

......................... 46 

......................... 46 

......................... 46 

......................... 46 

......................... 48 

X X 4 

X xx 4 

X X X X X 11 

X X 3 

+ 2 

......................... 47 

......................... 50 

......................... 47 

......................... 47 

+ 2 

......................... 50 

......................... 49 

......................... 49 

......................... 48 

......................... 46 

......................... 46 

1 

.......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 
186 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC2 

Individual Animal Tumor Pathologyof Male Mice in the 2-Year Feed Studyofp-Nitrobenzoic Acid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

0013567777771177777771777 

0087862222222222222222222 

I788279999999999999999999 

1222211111111111111111122 

8130188888888999999999900 

8094761234579012345678901 

......................... + + ......................... 

MAA++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

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

M+M++++++++++++++++++++++ 

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

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

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

......................... X 

X X 

xx 

X X 

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

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

+ 

X 

+AA++++++++++++++++++++++ 

A+A++++++++++++++++++++++ 

......................... 
Lesions in Male Mice 187 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary Bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

1111111111111111111111111 

2222222222222222222222222 

9999999999999999999999999 

2222222222222222222222222 Total 

0000000111111112222222222 Tissues/ 

2356189123456890123456189 Tumors 

......................... 50 

+ + 4 

......................... 50 

......................... 41 

.......................... 49 

X 1 

++M++++++++++++++++++++++ 41 

......................... 48 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

xxx X X 6 

X 3 

X X X 5 

x x 4 

......................... 50 

......................... 50 

1 

1 

......................... 48 

......................... 48 

......................... 50 

X X 2 
18% p-Nitrobenzoic Acid, NTP TR 442 

TABLEC3 

Statistical Analysisof Primary Neoplasms in Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 

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

Harderian Gland: Adenoma 

Overall ratea 1/50 (2%) 3/50 (6%) 3/50 (6%) 1/50 (2%) 

Adjusted rateb 7.6% 2.3% 6.6% 2.3% 

Terminal rate' OB9 (0%) (3%) 1/36 OB9 (0%) 1/44 (2%) 

First incidence (days) 681 720 638 729 (T) 

Life table testd P=O.284 P=0.473N P=O.303 P=0.744N 

Logistic regressiontestd P=O.298 P=O.538 P=O.761 P=O.394 

Cochran-Armita e testd P=0.500N I Fisher exact test P=O.309 P=0.753NP=O.309 

Liver: Hepatocellular Adenoma 

Overall rate 17/5017/50 (34%)(34%) 12/50 (24%) 14/48 (29%)

Adjusted rate 41.4% 36.4% 29.1% 31.1%

Terminal rate 10/39 (26%) 13/36 (6%) 

First incidence (days) 442 532 

10/39 (26%) 13/44 (30%)

681 667 

Fisher exact test 

Cochran-Armitage test 

Logistic regressiontest 

Life table test 

E=0.276N 

P=0.557NP=0.277N 

P=O.496 P=0.145N 

P=0.583N 

P=0.215N 

P=0.187N 

P=0.250N 

P=0.384N 

' Liver:HepatocellularCarcinoma 

Overall rate 8/50(16%)13/50 (26%) 16/50(32%)8/48(17%) 

Adjusted 17.4%rate 19.1% 29.6% 35.7% 

Terminal rate 6/39 (15%) (17%) 11/3966 (28%) 6/44 (14%)

First incidence 496 (days) 575 597 

Life table test P=O.145 P=0.366N P=O.O60 P=0.531N 

Logistic regressiontest P=O.203 P=O.388 P=O.O52 P=O.421 

Cochran-Armitagetest P=0.513N 

Fisher exact test P=O.163 P=O.572P=Q.OSO 

' Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 22/50(44%) 26/50 (52%) 23/50 (46%) 19/48(40%)

Adjusted 41.3%rate 47.4% 56.0% 50.6% 

Terminal rate 15/3916/36(44%)17/39 (38%) (44%)(39%) 17/44 

First incidence 496 (days). 

Life table test P=O.491 P=0.121N P=O.223 P=0.234N . ..

Logistic regressiontest P=O.548 P=O.566P=O.295P=0.453N . . 

Cochran-Armitagetest P=0.265N 

Fisher exact test P=0.406N P=O.500 P=O.274 

Lung: Alveolarbronchiolar Adenoma 

Overall rate 6/50 (12%) 12/50 (24%) 8/50 (16%) 9/50 (18%) 

Adjusted rate 15.4% 29.2% 20.5% 20.5% 

Terminal rate 8/36 (22%) 

442 532 

6/39 (15%) 8/39 (21%) 9/44 (20%)

537 (T) 

Logistic regressiontest P=O.378 P=O.385 P=O.O91 P=O.393 

Cochran-Armitagetest P=O.413 

Fisher exact test P=O.O96 P=O.288 P=O.387 

First incidence (days) 729 729 (T)

P=O.545 

729 (T)

Life table test P=O.378 P=O.385 P=O.O77
Lesions in Male Mice 189 

TABLEC3 

Statistical Analysisof Primary Neoplasms in Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

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

Lung: Alveolar/bronchiolar Carcinoma 

Adjusted rate 

Overall rate 

2.6% 

1/50 (2%) 

8.0% 

3/50 (6%) 

5.1% 

2/50 (4%) 

11.4% 

5/50(10%) 

Terminal rate 1/39 (3%) 2/36 (6%) 2/39 (5%) 5/44 (1 1%) 

First incidence (days) 729 (T) 725 729 (T) 729 (T) 

Life table test P=O.116 P=O.279 P=O.500 P=O.133 

Logistic regression test P=O.O95 P=O.279 P=O.500 P=O.133 

Cochran-Armitage test P=O.O78 

Fisher exact test P=O.309 P=O.500 P=O.102 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 7/50 (14%) 14/50 (28%) 10/50 (20%) 13/50 (26%) 

Adjusted rate 17.9% 34.2% 25.6% 29.5% 

Terminal rate 7/39 (18%) 10/36 (28%) 10/39 (26%) 13/44 (30%) 

Life table test 

First incidence (days) 

P=O.303 

729 (T> 

P=O.O53 

537 

P=O.293 

729 (T> 

P=O.166 

729 (T> 

Logistic regression test P=O.165 P=O.O64 P=O.293 P=O.166 

Cochran-Armitage test P=O.178 

Fisher exact test P=O.O70 P=O.298 P=O.105 

Small Intestine (Jejunum): Carcinoma 

Adjusted rate 

Overall rate 

7.4% 

3/50 (6%) 

2.8% 

1/50 (2%) 

0.0% 

Ob0 (0%) 

0.0% 

0/50 (0%) 

Life table test 

First incidence (days) 

Terminal rate 

P=0.039N 

728 

2/39 (5%) 1/36 (3%) 

P=0.338N 

729 (T> 

OB9 (0%) 

P=0.126N 

-e 

0/44 (0%) 

P=O.lOSN 

-

Logistic regression test P=O.O42N P=0.336N P=0.131N P=0.113N 

Cochran-Armitage test P=0.044N 

Fisher exact test P=0.309N P=0.121N P=0.121N 

Spleen: Hemangiosarcoma 

Adjusted rate 

Overall rate 

10.3% 

4/50 (8%) 

2.8% 

1/49 (2%) 

2.4% 

1/50 (2%) 

2.3% 

1/49 (2%) 

Life table test 

First incidence (days) 

Terminal rate 

729 (T> 

4/39 (10%) 

P=0.113N 

1/36 (3%) 

P=0.204N 

729 (T> 

P=O.l85N 

695 

0/39 (0%) 1/44 (2%) 

P=0.145N 

729 (9 

Logistic regression test P=0.134N P=0.204N P=0.185N P=0.145N 

Cochran-Armitage test P=0.137N 

Fisher exact test P=0.187N P=0.181N P=0.187N 

All Organs: Hemangiosarcoma 

Adjusted rate 

Overall rate 

14.9% 

6/50 (12%) 

5.6% 

2/50 (4%) 

4.9% 

2/50 (4%) 

2.3% 

1/50 (2%) 

Life table test 

First incidence (days) 

Terminal rate 

P=0.032N 

728 

5/39 (13%) 2/36 (6%) 

P=0.164N 

729 tT> 

P=0.142N 

695 

1/39 (3%) 1/44 (2%) 

P=0.044N 

729 (T> 

Logistic regression test P=0.040N P=O.l62N P=0.143N P=0.049N 

Cochran-Armitage test P=O.O42N 

Fisher exact test P=0.134N P=0.134N P=0.056N 
190 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC3 

Statistical Analysisof Primary Neoplasms in Male Mice in the 2-Year Feed Studyo f p-Nitrobenzoic Acid(continued) 

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

All Organs: Hemangioma or Hemangiosarcoma 

Adjusted rate 

Overall rate 

14.9% 

6/50 (12%) 

7.8% 

3/50 (6%) 

4.9% 

2/50 (4%) 

2.3% 

1/50 (2%) 

Life table test 

First incidence (days) 

Terminal rate 

P=0.028N 

728 

5/39 (13%) 

P=0.284N 

695 

2/36 (6%) 

P=0.142N 

695 

1/39 (3%) 

729 (T> 

1/44 (2%) 

P=0.044N 

Logistic regression test P=0.035N P=0.261N P=0.143N P=0.049N 

Cochran-Armitage test P=0.035N 

Fisher exact test P=0.243N P=0.134N P=0.056N 

All Organs: Malignant Lymphoma (Lymphocytic or Mixed) 

Overall rate 2/50 (4%) 1/50 (2%) 3/50 (6%) 2/50 (4%) 

Adjusted rate 5.1% 2.8% 7.0% 4.5% 

Terminal rate 2/39 (5%) 1/36 (3%) 2/39 (5%) 2/44 (5%) 

Life table test 

First incidence (days) 

P=O.554 

729 (T> 

P=0.528N 

729 (T> 

P=O.502 

451 

P=0.651N 

729 (T> 

Logistic regression test P=O.502 P=0.528N P=O.508 P=0.651N 

Cochran-Armitage test P=O.500 

Fisher exact test P=0.500N P=O.500 P=0.691N 

All Organs: Benign Neoplasms 

Overall rate 20/50 (40%) 29/50 (58%) 22/50 (44%) 23/50 (46%) 

Adjusted rate 43.0% 62.8% 49.6% 50.0% 

Terminal rate 13/39 (33%) 19/36 (53%) 17/39 (44%) 21/44 (48%) 

First incidence (days) 532 442 451 188 

Life table test P=0.351N P=O.O54 P=O.421 P=O.500 

Logistic regression test P=O.496 P=O.O56 P=O.427 P=0.322 

Cochran-Armitage test P=O.519 

Fisher exact test P=O.OS P=O.420 P=O.343 

All Organs: Malignant Neoplasms 

Overall rate 18/50 (36%) 20/50 (40%) 22/50 (44%) 14/50 (28%) 

Adjusted rate 40.6% 44.0% 48.2% 30.4% 

Terminal rate 13/39 (33%) 11/36 (31%) 16/39 (41%) 12/44 (27%) 

First incidence (days) 419 442 451 582 

Life table test P=0.126N P=O.340 P=O.286 P=0.181N 

Logistic regression test P=0.201N P=O.429 P=O.284 P=0.286N 

Fisher exact test 

Cochran-Armitage test P=0.200N 

P=O.418 P=O.270 P =0.260N 

All Organs: Benign or Malignant Neoplasms 

Overall rate 33/50 (66%) 36/50 (72%) 36/50 (72%) 31/50 (62%) 

Terminal rate 

Adjusted rate 

24/39 (62%) 

68.6% 

23/36 (64%) 

73.5% 

27/39 (69%) 

74.8% 

28/44 (64%) 

65.9% 

Life table test 

First incidence (days) 

P=0.137N 

419 

P=O.248 

442 

P=O.361 

451 

P=0.235N 

188 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

P=0.304N 

P=0.496N P=O.337 

P=O.333 

P=O.364 

P=O.333 

P=0.464N 

P=0.418N 
Lesions in Male Mice 191 

TABLEC3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid (continued) 

OTerminal sacrifice 

a Number of neoplasm-bearing animalshumber of animals examined. Denominator is number of animals examined microscopically for liver, lung, 

and spleen; for other tissues, denominator is number of animals necropsied. 

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

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposure group incidence are the P values corresponding 

to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in animals dying prior to terminal kill 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher 

exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

e Notapplicable;noneoplasmsinanimalgroup 
192 p-Nitrobenzoic Acid, NTP TR 442 

Incidence in Controls 

Study Alveolar/bronchiolar Alveolar/bronchiolarAlveolar/bronchiolar 

AdenomaAdenoma Carcinoma or Carcinoma

Historical Incidence at Southern Research Institute 

Benzyl Acetate 9/50 5/50 14/50 

C.I. Pigment Red 23 4/49 2/49 5/49 

C.I. Pigment Red 3 2/50 0/50 2/50 

Ethylene Glycol 7/54 1/54 7/54 

Nitrofurantoin 5/50 1/50 6/50 

o-Nitroanisole 5/50 1/50 6/50 

Polysorbate 80 5/49 1/49 6/49 

Rhodamine 6G 6/50 3/50 9/50 

Roxarsone 5/50 6/50 11/50 

Total 481452 (10.6%) 20/452 (4.4%) 661452 (14.6%) 

Standard deviation 3.8% 4.1% 7.1% 

Range 4%-18% 2%-12% 4%-28% 

Overall Historical Incidence 

Total 181/1,369 (13.2%) 68/1,369(5.0%) 242/1,369 (17.7%) 

Standard deviation 5.8% 4.0%. 7.3% 

Range 4%-26% 0%-14% 4%-30% 

a Data as of 20 August 1992 
Lesions in Male Mice 193 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acida 

Disposition Summary 

Animals initiallyin study 

IJM& inrabnevrrlkation 

Early deaths 

Accidental deaths 

Moribund 

Natural deaths 

SuMvors 

Terminal sacrifice 

Animals examined microscopically 

15-Month Interim Evaluation 

Alimentary System 

Liver 

Basophilic focus 

Inflammation, subacute 

Karyomegaly 

Mixed cell focus 

Necrosis 

Vacuolization cytoplasmic 

Mesentery 

Fat, necrosis 

Pancreas 

Acinus, atrophy 

Stomach, glandular 

Mineralization 

Endocrine System 

Adrenal cortex 

Hypertrophy 

Pituitary gland 

Pars distalis, hyperplasia 

Thyroid gland 

Follicle, degeneration 

Genital System 

Preputial gland 

Atrophy 

Inflammation, subacute 

Duct, cyst 

0 PPm 

60 

10 

9 

2 

39 

60 

1 (10%) 

2 (20%) 

(1)

1 (100%) 

(10) 

(10) 

(10) 

2 (20%) 

1,250 ppm 

60 

10 

1 

4 

9 

36 

60 

1 (10%) 

2 (20%) 

(10)

1 (10%) 

(10) 

(10)

1 (10%) 

1 (10%) 

2,500 ppm 

60 

10 

10 

1 

39 

60 

(10) 

1 (10%) 

(10) 

3(30%) 

5,000 ppm 

60 

10 

2 

2 

2 

44 

60 

(10)

1 (10%) 

1 (10%) 

1 (10%) 

(10) 

1 (10%) 

3 (30%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
194 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

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

15-Month Interim Evaluation (continued) 

Hematopoietic System 

Lymph node, mesenteric (10) (10) Angiectasis 1 (10%) 1 (10%) 

Spleen (10) (10) Hematopoietic cell proliferation 1 (10%) 1 (10%) 

Hyperplasia, lymphoid 2 (20%) 

Thymus (10) (9) 

Cyst 

Integumentary System 

Skin 

Pinna, inflammation, subacute 

Subcutaneous tissue, hemorrhage 

Respiratory System 

Lung 

Alveolar epithelium, hyperplasia 

Nose 

Lumen, hemorrhage 

Special Senses System 

Eye (1) (2) (1)

Retrobulbar, hemorrhage 1 (100%) 2 (100%) 1 (100%) 

Urinary System 

Kidney 

Fibrosis 

(10)

1 (10%) 

Hydronephrosis 1 (10%) 

Inflammation, subacute 1 (10%) 

Mineralization 9 (90%) 4 (40%) 5 (50%) 4 (40%) 

Renal tubule, casts 1 (10%) 

Renal tubule, regeneration 

Renal tubule, pigmentation 

7 (70%) 6 (60%) 

1 (10%) 

7 (70%) 3 (30%) 

Systems Examined WUh No Lesions Observed 

Cardiovascular System 

General Body System 

Musculoskeletal System 

Nervous System 
Lesions in Male Mice 195 

TABLEC5 

Summary of the ‘Incidenceof Nonneoplastic Lesions in Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

2-Year Study 

Alimentary System 

Gallbladder 

Epithelium, hyperplasia 

Intestine small, jejunum 

Peyer’s patch, hyperplasia, lymphoid 

Liver 

Basophilic focus 

Clear cell focus 

Cytologic alterations 

Eosinophilic focus 

Hematopoietic cell proliferation 

Inflammation, subacute 

Mixed cell focus 

Necrosis 

Regeneration 

Vacuolization cytoplasmic 

Hepatocyte, hypertrophy, focal 

Mesentery 

Fat, inflammation, granulomatous 

Fat, necrosis 

Pancreas 

Edema 

Inflammation, chronic 

Acinus, atrophy 

Acinus, depletion secretory 

Duct, cyst 

Salivary glands 

Acinus, atrophy 

Duct, cyst 

Stomach, forestomach 

Cyst

Hyperplasia 

Inflammation, subacute 

Ulcer 

Stomach, glandular 

Cyst

Ulcer 

Tooth 

Dysplasia 

Inflammation, chronic 

Inflammation, subacute 

Cardiovascular System 

Heart 

Cardiomyopathy 

Fibrosis 

Inflammation, chronic 

Mineralization 

Artery, inflammation, subacute 

0 PPm 

(48) 

(50) 

(50)

1 (2%) 

4 (8%) 

4 (8%) 

2 (4%) 

1 (2%) 

6 (12%) 

4 (8%) 

1 (2%) 

2 (4%) 

(2) 

1 (50%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

(50) 

6 (12%) 

1 (2%) 

1 (2%) 

(50) 

2 (4%) 

(2) 

2 (100%) 

1,250 ppm 

(45) 

(43)

1 (2%) 

(50)

3 (6%) 

6 (12%) 

17 (34%) 

5 (10%) 

1 (2%) 

(1) 

1 (100%) 

(48) 

1 (2%) 

1 (2%) 

(49)

1 (2%) 

1 (2%) 

(47) 

4 (9%) 

3 (6%) 

(47)

1 (2%) 

(49)

1 (2%) 

1 (2%) 

2,500 ppm 

(50) 

(49) 

(50) 

5 (10%) 

1 (2%) 

5 (10%) 

1 (2%) 

5 (10%) 

1 (2%) 

4 (8%) 

(2)

1 (50%) 

1 (50%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

(50) 

(50)

2 (4%) 

(2)

1 (50%) 

1 (50%) 

(50) 

1 (2%) 

1 (2%) 

5,000 ppm 

(48)

1 (2%) 

(46) 

(48)

4 (8%) 

2 (4%) 

4 (8%) 

2 (4%) 

3 (6%) 

1 (2%) 

(2) 

2 (100%) 

(47) 

(50) 

(47)

1 (2%) 

(47)

1 (2%) 

(2)

1 (50%) 

1 (50%) 

1 (50%) 

(50) 

1 (2%) 

1 (2%) 
196 p-Nitrobenzoic Acid, NTP TR 442 

2-h.WhkUfJ (continued) 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Hyperplasia 

Hypertrophy 

Spindle cell, hyperplasia 

Islets, pancreatic 

Hyperplasia 

Pituitary gland 

Pars distalis, cyst 

Thyroid gland 

Follicle, cyst 

Follicular cell, hyperplasia 

General Body System 

None 

Genital System 

Coagulating gland 

Dilatation 

Epididymis 

Cyst, multiple 

Inflammation, granulomatous 

Preputial gland 

Abscess 

Atrophy 

Cyst

Infiltration cellular, subacute 

Inflammation, chronic 

Inflammation, subacute 

Duct, cyst 

Prostate 

Atrophy 

Inflammation, subacute 

Polyarteritis 

Seminal vesicle 

Atrophy 

Dilatation 

Inflammation, chronic 

Inflammation, subacute 

(50)

1 (2%) 

14 (28%) 

(50)

1 (2%) 

(46)

2 (4%) 

(50)

2 (4%) 

15 (30%) 

11 (22%) 

31(62%) 

(50)

1 (2%) 

(50)

1 (2%) 

1 (2%) 

(49)

2 (4%) 

1 (2%) 

17 (35%) 

(49)

3(6%) 

(46)

1 (2%) 

(49)

1 (2%) 

12 (24%) 

2 (4%) 

36(73%) 

(49) 

(49)

1 (2%) 

6 (12%) 

(50)

1 (2%) 

16 (32%) 

1 (2%) 

(50) 

(50)

1 (2%) 

(50) 

8 (16%) 

(50)

1 (2%) 

1 (2%) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

10(20%) 

36 (72%) 

(50) 

1 (2%) 

(50)

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

1 (2%) 

15 (31%) 

1 (2%) 

(48) 

(46)

2 (4%) 

(49)

3 (6%) 

9 (18%) 

(50) 

1 (2%) 

(50)

2 (4%) 

1 (2%) 

1 (2%) 

4 (8%) 

27 (54%) 

(50) 
Lesions in Male Mice 197 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

2-YeUr study (continued) 

Hematopoietic System 

Bone marrow 

Congestion 

Hypercellularity 

Lymph node 

Axillary, hyperplasia, lymphoid 

Bronchial, hyperplasia, lymphoid 

Inguinal, angiectasis 

Inguinal, hyperplasia, lymphoid 

Mediastinal, hyperplasia, lymphoid 

Pancreatic, mineralization 

Renal, hyperplasia, lymphoid 

Lymph node, mandibular 

Hyperplasia, lymphoid 

Infiltration cellular, mast cell 

Lymph node, mesenteric 

Angiectasis 

Atrophy 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Spleen 

Atrophy 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Necrosis 

Thymus 

Atrophy 

Cyst

Necrosis 

Integumentary System 

Skin 

Ulcer 

Dermis, inflammation, chronic 

Dermis, inflammation, subacute 

Subcutaneous tissue, abscess 

Subcutaneous tissue, inflammation, chronic 

Musculoskeletal System 

Bone 

Cranium, hyperostosis 

Femur, fibrous osteodystrophy 

Nervous System 

None 

0 PPm 

1 (25%) 

1 (25%) 

1 (25%) 

(49)

1 (2%) 

(50)

17 (34%) 

1 (2%) 

2 (4%) 

(50)

1 (2%) 

11 (22%) 

1 (2%) 

(45) 

4 (9%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

1,250 ppm 

1 (100%) 

(47) 

(49)

20 (41%) 

1 (2%) 

2 (4%) 

(49)

2 (4%) 

8 (16%) 

1 (2%) 

(47)

1 (2%) 

4 (9%) 

(49)

1 (2%) 

1 (2%) 

1 (2%) 

2,500 ppm 

(50) 

2 (4%) 

(4)

1 (25%) 

1 (25%) 

1 (25%) 

(50)

1 (2%) 

(50)

20 (40%) 

2 (4%) 

2 (4%) 

(50)

3 (6%) 

10(20%) 

1 (2%) 

(47)

2 (4%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

5,000 ppm 

(50)

1 (2%) 

3 (6%) 

(4)

1 (25%) 

2(50%) 

1 (25%) 

(50)

3 (6%) 

1 (2%) 

(47)

17 (36%) 

1 (2%) 

(49)

5 (10%) 

6 (12%) 

2 (4%) 

(47)

1 (2%) 

(50)

1 (2%) 

1 (2%) 

(50) 

1 (2%) 
198 p-Nitrobenzoic Acid, NTP TR 442 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Studyofp-NitrobenzoicAcid 

(continued) 

2-YefW study (continued) 

Respiratory System 

Lung 

Congestion 

Hemorrhage 

Infiltration cellular, histiocyte 

Inflammation, granulomatous 

Inflammation, subacute 

Thrombosis 

Alveolar epithelium, hyperplasia 

Bronchiole, hyperplasia 

Nose 

Congestion 

Glands, inflammation, subacute 

Nasolacrimal duct, ectasia 

Nasolacrimal duct, inflammation, subacute 

Special Senses System 

Eye

Cataract 

Urinary System 

Kidney 

Fibrosis 

Glomerulosclerosis 

Hemorrhage 

Infarct 

Infiltration cellular, lymphocyte 

Inflammation, subacute 

Metaplasia, osseous 

Mineralization 

Cortex, cyst 

Renal tubule, casts 

Renal tubule, regeneration 

Urethra 

Bulbourethral gland, hemorrhage 

Urinary bladder 

Dilatation 

0 PPm 

(50) 

1 (2%) 

2 (4%) 

(50) 

(2)

1 (50%) 

(50) 

4 (8%) 

2 (4%) 

7 (14%) 

1 (2%) 

1 (2%) 

41(82%) 

11 (22%) 

7 (14%) 

43(86%) 

1,250 ppm 

(50) 

1 (2%) 

7 (14%) 

(49)

1 (2%) 

(49)

2 (4%) 

5 (10%) 

4 (8%) 

1 (2%) 

33(67%) 

6 (12%) 

7 (14%) 

2,500 ppm 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

7 (14%) 

1 (2%) 

(49) 

1 (2%) 

1 (2%) 

(50)

2 (4%) 

6 (12%) 

5 (10%) 

1 (2%) 

1 (2%) 

23(46%) 

5 (10%) 

7 (14%) 

35 (70%) 

(50)

1 (2%) 

5,000 ppm 

(50)

1 (2%) 

3 (6%) 

3 (6%) 

8 (16%) 

(50) 

1 (2%) 

(48)

2 (4%) 

2 (4%) 

1 (2%) 

3 (6%) 

31(65%) 

3 (6%) 

3 (6%) 

32 (67%) 
199 

APPENDIX D 

SUMMARY OF LESIONS IN FEMALE MICE 

IN THE 2-YEAR FEED STUDY 

OF p-NITROBENZOIC ACID 

TABLE Dl Summary of the Incidence of Neoplasms in Female Mice 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 201 

TABLE D2 Individual Animal Tumor Pathology of Female Mice 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 206 

TABLE D3 Statistical Analysis of Primary Neoplasms in Female Mice 

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 228 

TABLE D4 Historical Incidence of Lung Neoplasms in Untreated Female B6C3Fl Mice ......... 232 

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

in the 2-Year Feed Study of p-Nitrobenzoic Acid ............................. 233 
200 p-Nitrobenzoic Acid, NTP TR 442 
Lesions in Female Mice 201 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acida 

0 PPm 

~~~ ~ ~ 

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

Early deaths 

ISMd inraimotalkadon 

Animals initiallyin study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental deaths 

Terminal sacrifice 

Missing 

SuMvors 

10 

60 

3 

9 

38 

10 

60 

5 

8 

1 

36 

10 

60 

4 

12 

1 

33 

10 

60 

8 

10 

2 

30 

Animals examined microscopically 60 59 60 60 

15-Month Interim Evaluation 

Liver 

Alimentary System 

Hepatocellular adenoma 

Hepatocellular carcinoma 

(10)

1 (10%) 

(10) 

2 (20%) 

(10) 

4(40%) 

(10) 

1 (10%) 

Lymph node 

Bone marrow 

Hematopoietic System 

Renal, hemangiosarcoma 

Hemangiosarcoma 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Systems ExaminedWUh No Neoplasms Observed 

Gallbladder 

Alimentary System 

2-Year Study 

Polyp adenomatous 

Intestine small, duodenum 

Histiocytic sarcoma 

Sarcoma, metastatic, mesentexy 

Intestine small, ileum 

Adenocarcinoma 

Intestine small, jejunum 

(49) 

(50) 

(49) 

(49) 
202 p-Nitrobenzoic Acid, NTP TR 442 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid (continued) 

%Year shdy (continued) 

Alimentary System (continued) 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Sarcoma 

Pancreas 

Histiocytic sarcoma 

Sarcoma, metastatic, mesentery 

Salivary glands 

Stomach, forestomach 

Hepatocellular carcinoma, metastatic, liver 

Squamous cell papilloma 

Stomach, glandular 

Histiocytic sarcoma 

Tooth 

Histiocytic sarcoma 

Cardiovascular System 

Heart 

Hepatocellular carcinoma, metastatic,liver 

Endocrine System 

Adrenal cortex 

Histiocytic sarcoma 

Sarcoma, metastatic, mesentery 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Carcinoma 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

0 PPm 

(50)

1 (2%) 

4 (8%) 

8 (16%) 

3 (6%) 

1 (2%) 

(4) 

(50) 

(50) 

(50) 

1 (2%) 

(49) 

(50) 

(49) 

(48)

7 (15%) 

1 (2%) 

(50) 

1 (2%) 

1,250 ppm 

(49) 

5 (10%) 

9 (18%) 

4 (8%) 

2 (4%) 

(8)

1 (13%) 

(47)

1 (2%) 

(49) 

(49)

1 (2%) 

2 (4%) 

(49)

1 (2%) 

(49)

1 (2%) 

(48)

1 (2%) 

(48) 

(49) 

(48)

6 (13%) 

1 (2%) 

(49)

1 (2%) 

~~ ~~ 

2,500 ppm 

(50) 

4 (8%) 

1 (2%) 

11 (22%) 

2 (4%) 

3 (6%) 

(5) 

(49)

1 (2%) 

(50) 

(49) 

2 (4%) 

(49) 

(1)

1 (100%) 

(50) 

(50) 

(49)

1 (2%) 

(49)

9 (18%) 

(50)

2 (4%) 

5,000 ppm 

(50) 

6 (12%) 

1 (2%) 

4 (8%) 

1 (2%) 

(6) 

1 (17%) 

1 (17%) 

(49) 

1 (2%) 

(50) 

(49) 

1 (2%) 

(49) 

1 (2%) 
Lesions in Female Mice 203 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

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

%Year &ddy (continued) 

Genital System 

Ovary 

Cystadenoma 

Histiocytic sarcoma 

Luteoma 

Sarcoma, metastatic, mesentery 

Uterus 

Hemangiosarcoma 

Histiocytic sarcoma 

Leiomyoma 

Leiomyosarcoma 

Polyp stromal 

Sarcoma, metastatic, mesentery 

Sarcoma stromal 

Vagina 

Squamous cell papilloma 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Axillary, fibrosarcoma, metastatic, skin 

Iliac, histiocytic sarcoma 

Inguinal, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Pancreatic, sarcoma, metastatic, mesentery 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Sarcoma, metastatic, mesentery 

Thymus 

Fibrosarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, liver 

Histiocytic sarcoma 

(50)

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(1)

1 (100%) 

(48)

2 (4%) 

2 (4%) 

1 (2%) 

(49) 

2 (4%) 

1 (2%) 

(49)

1 (2%) 

1 (2%) 

(10) 

1 (10%) 

1 (10%) 

1 (10%) 

1 (10%) 

(49)

1 (2%) 

(49)

1 (2%) 

(49)

1 (2%) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

(49)

1 (2%) 

1 (2%) 

(50)

1 (2%) 

1 (2%) 

2 (4%) 

(1) 

(48)

1 (2%) 

1 (2%) 

(13)

1 (8%) 

1 (8%) 

1 (8%) 

1 (8%) 

1 ' (8%)

(50)

1 (2%) 

(48)

1 (2%) 

(49)

1 (2%) 

1 (2%) 

(48)

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 
204 p-Nitrobenzoic Acid, NTP TR 442 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

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

ZYear Study (continued) 

Integumentary System 

Mammary gland 

Adenoma 

Skin 

Schwannoma NOS 

Subcutaneous tissue, basal cell carcinoma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, histiocytic sarcoma 

Subcutaneous tissue, sarcoma 

Subcutaneous tissue, schwannomamalignant 

Subcutaneous tissue, thymoma malignant, 

metastatic, thymus 

Musculoskeletal System 

Bone 

Femur, osteosarcoma 

Skeletal muscle 

Fibrosarcoma 

Hemangiosarcoma 

Hepatocellular carcinoma, metastatic, liver 

Sarcoma, metastatic, mesentery 

Nervous System 

Brain 

Carcinoma, metastatic, pituitary gland 

Glioma NOS 

Meninges, histiocytic sarcoma 

Olfactory lobe, histiocytic sarcoma 

Respiratory System 

Lung 

Alveolaribronchiolar adenoma 

Alveolaribronchiolar carcinoma 

Alveolar/bronchiolar carcinoma, multiple 

Fibrosarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, liver 

Osteosarcoma, metastatic, bone 

Sarcoma, metastatic, mesentery 

Mediastinum, hemangiosarcoma, metastatic, 

spleen 

Nose 

Glands, histiocytic sarcoma 

Special Senses System 

Harderian gland 

Adenoma 

(50)

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

(1) 

1 (100%) 

(49)

1 (2%) 

(50)

3 (6%) 

1 (2%) 

1 (2%) 

(50) 

(3)

3 (100%) 

(49) 

(49) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

(1) 

1 (100%) 

(49) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

5 (10%) 

4 (8%) 

1 (2%) 

4 (8%) 

(49)

1 (2%) 

(2)

1 (50%) 

(50) 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

(2)

2 (100%) 

(50) 

1 (2%) 

(50)

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

(48) 

(49)

1 (2%) 

(50) 

1 (2%) 

(50)

1 (2%) 

(2) 

1 (50%) 

(50) 

(50)

8 (16%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 
Lesions in Female Mice 205 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid(continued) 

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

2-Year Study (continued) 

Urinary System 

Kidney (50)

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Urinary bladder (50) 

Systemic Lesions 

Multiple organsb (50) (49)

Histiocytic sarcoma 1 (2%) 2 (4%) 

Lymphoma malignant lymphocytic 6 (12%) 8 (16%) 

Lymphoma malignant mixed 3 (6%) 3 (6%) 

Neoplasm Summary 

Total animals with primary neoplasms' 

15-Month interim evaluation 1 2 

2-Year study 36 38 

Total primary neoplasms 

15-Month interim evaluation 1 2 

2-Year study 53 61 

Total animals with benign neoplasms 

15-Month interim evaluation 2 

2-Year study 22 22 

Total benign neoplasms 

15-Month interim evaluation 2 

2-Year study 29 33 

Total animals with malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 23 23 

Total malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 24 27 

Total animals with metastatic neoplasms 

2-Year study 3 4 

Total metastatic neoplasms 

2-Year study 3 9 

Total animals with uncertain neoplasms 

benign or malignant 

2-Year study 1 

Total uncertain neoplasms 

2-Year study 1 

a Number of animals examined microscopically at site and number of animals with lesion 

Number of animals with any tissue examined microscopically ' Primaryneoplasms:allneoplasmsexceptmetastaticneoplasms 

(50)

3 (6%) 

7 (14%) 

2 (4%) 

4 

35 

6 

61 

4 

23 

4 

32 

1 

23 

2 

28 

2 

4 

1 

1 

(50) 

5 (10%) 

2 (4%) 

1 

32 

1 

42 

1 

20 

1 

23 

17 

19 

2 

12 
206 p-Nitrobenzoic Acid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

Follicular cell, carcinoma 

General Body System 

None 

+: Tissue examined microscopically 

A Autolysis precludes examination 

3455566667777777777777777 

6233813892223333333333333 

4333969933885555555555555 

2222222222222222222222222 

5859547568474444444555555 

5610398097132345678246789 

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

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

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

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

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

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

X X 

X X xx 

X X 

+ + + .......................... 

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

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

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

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

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

......................... X 

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

++++++M+++M+++M+++++++MMM 

+M+++++++++++++++++++++++ 

X X xx 

X 

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

M Missing tissue X Lesion present 

I: Insufficient tissue Blank Not examined 
Lesions in Female Mice 207 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

Follicular cell, carcinoma 

General Body System 

None 

7777777777777777777777777 

3333333333333333333333333 

5555555555555555555555555 

2222222222222222222222222 Total 

6666666667777777788888888 Tissues1 

0123456780124567901234589 Tumors 

......................... 50 

......................... 50 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 50 

X 1 

X X 4 

xx X X 8 

X 3 

X 1 

+ 4 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 49 

......................... 50 

++++++++ ++++++++++++++++ 49 

++++++++ ++++++++++++++++ 49 

1 

......................... 50 

+++++M+++++MM+++++M++++.++ 40 

++++++M++++++++++++++++++ 48 

X X X 7 

1 

......................... 50 

X 1 
208 p-Nitrobenzoic Acid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid:0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Uterus 

Histiocytic sarcoma 

Leiomyosarcoma 

Sarcoma stromal 

Vagina 

Squamous cell papilloma 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Skin 

Subcutaneous tissue, sarcoma 

Subcutaneous tissue, schwannoma 

malignant 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Carcinoma, metastatic, pituitary 

gland 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Hepatocellular carcinoma, metastatic, 

liver 

Mediastinum, hemangiosarcoma, 

metastatic, spleen 

Nose 

Trachea 

3455566667777777777717777 

6233813892223333333333333 

4333969933885555555555555 

2222222222222222222222222 

5859547568474444444555.555 

5610398097132345678246789 

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

......................... X 

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

+ 

X 

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

+ ++ +++ + ......................... 

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

......................... X 

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

......................... X 

......................... X 

X 

......................... + 

X 

++++++++++++++++++++M++++ 

X 

......................... X 

X 

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

......................... 
Lesions in Female Mice 209 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Uterus 

Histiocytic sarcoma 

Leiomyosarcoma 

Sarcoma stromal 

Vagina 

Squamous cell papilloma 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Skin 

Subcutaneous tissue, sarcoma 

Subcutaneous tissue, schwannoma 

malignant 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Carcinoma, metastatic, pituitaq 

gland 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Hepatocellular carcinoma, metastatic, 

liver 

Mediastinum, hemangiosarcoma, 

metastatic, spleen 

Nose 

Trachea 

1111111111111111111111111 

3333333333333333333333333 

5555555555555555555555555 

2222222222222222222222222 Total 

6666666661111111188888888 Tissues/ 

0123456180124561901234589 Tumors 

......................... 50 

......................... 50 

1 

......................... 50 

X 1 

X 1 

X 

......................... 50 

X 1 

+ 8 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

1 

......................... 50 

1 

1 

......................... SO 

1 

1 

......................... 49 

1 

......................... 50 

X X 3 

X 1 

1 

......................... 50 

......................... 50 
210 p-Nitrobenzoic Acid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the %Year Feed Study Ofp-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Eye

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

3455566667777777777777777 

6233813892223333333333333 

4333969933885555555555555 

2222222222222222222222222 

5859547568474444444555555 

5610398097132345678246789 

+ + 

+ ++ 

X xx 

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

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

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

X xx X 

X X 
Lesions in Female Mice 211 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Eye

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

1111111111111111111111111 

3333333333333333333333333 

5555555555555555555555555 

2222222222222222222222222 Total 

6666666661111111188888888 Tissues/ 

0123456180124561901234589 Tumors 

2 

3 

3 

......................... 50 

......................... 50 

......................... 50 

X 1 

X X 6 

X 3 
212 p-Nitrobenzoic Acid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the %Year Feed Study Ofp-Nitrobenzoic Acid: 1,250 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Hepatocellular carcinoma, metastatic, 

liver 

Squamous cell papilloma 

Stomach, glandular 

Histiocytic sarcoma 

Cardiovascular System 

Heart 

Hepatocellular carcinoma, metastatic, 

liver 

Endocrine System 

Adrenal cortex 

Histiocytic sarcoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

455556666667777777777777 

025992518880033333333333 

060480781551111111111111 

333333333333333333333333 

201030533232400000111111 

067872023553313459012345 

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

++++++A+++++++++++++++++ 

X 

A+A++A++++++++++++++++++ 

A+A++A++++++++++++++++++ 

A+A++A++++++++++++++++++ 

A+A++A++++++++++++++++++ 

A+A++A++++++++++++++++++ 

A+A++A++++++++++++++++++ 

........................ X X 

X X X X 

xx 

xx 

+ + ++ 

X 

++A+++A+++++++++++++++++ 

X 

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

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

X X 

........................ X 

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

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

X 

++++++++++ +++++++++++++ ........................ 

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

+++++++M++++++++++++++++ 

X 

........................ 
Lesions in Female Mice 213 

TABLE D2 

Individual Animal Tumor Pathologyof Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acd: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Hepatocellular carcinoma, metastatic, 

liver 

Squamous cell papilloma 

Stomach, glandular 

Histiocytic sarcoma 

Cardiovascular System 

Heart 

Hepatocellular carcinoma, metastatic, 

liver 

Endocrine System 

Adrenal cortex 

Histiocytic sarcoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

1111111111111111111111111 

3333333333333333333333333 

1111111111111111555555555 

3333333333333333333333333 Total 

1112222333333444222444444 Tissues/ 

6891249014689012618456189 Tumors 

......................... 49 

......................... 48 

1 

......................... 46 

......................... 46 

......................... 46 

......................... 46 

......................... 46 

......................... 46 

......................... 49 

xx X 5 

x xx X X 9 

X X 4 

2 

+ + + + 8 

1 

......................... 47 

1 

......................... 49 

......................... 49 

X 1 

2 

......................... 49 

1 

......................... 49 

X 1 

......................... 48 

1 

......................... 48 

......................... 49 

M+++++++++++++M++++++++++ 41 

......................... 48 

X X X x x 6 

X 1 

......................... 49 

X 1 
214 p-Nitrobenzoic Acid, NTP TR 442 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Histiocytic sarcoma 

Luteoma 

Uterus 

Histiocytic sarcoma 

Polyp stromal 

Hematopoietic System 

Blood 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Iliac, histiocytic sarcoma 

Inguinal, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, basal cell 

carcinoma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, thymoma 

malignant, metastatic, thymus 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hepatocellular carcinoma, metastatic, 

liver 

455556666667777777777777 

025992578880033333333333 

060480781551111111111111 

333333333333333333333333 

201030533232400000111111 

067872023553313459012345 

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

+++++++++++++++M++++++++ 

X 

xx 

........................ xx 

+ ........................ X 

X 

++ + + ++ 

X 

X 

X 

X 

........................ X 

........................ X 

........................ X 

X 

M+++++++++++++++++++++++ 

X 

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

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

X 

........................ 
Lesions in Female Mice 215 

TABLED2 

Individual Animal Tumor Pathology of Female Micein the 2-Year Feed Study ofp-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Histiocytic sarcoma 

Luteoma 

Uterus 

Histiocytic sarcoma 

Polyp stromal 

Hematopoietic System 

Blood 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Iliac, histiocytic sarcoma 

Inguinal, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

~~ ~~ ~~~~~ ~~~~ 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, basal cell 

carcinoma 

Subcutaneous fibrosarcoma tissue,

Subcutaneous tissue, thymoma 

malignant, metastatic, thymus 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hepatocellular carcinoma, metastatic, 

liver 

1111111111111171111111111 

3333333333333333333333333 

1111111111111111555555555 

3333333333333333333333333 Total 

1112222333333444222444444 Tissues/ 

6891249014689012618456789 Tumors 

......................... 49 

......................... 48 

X 2 

2 

X 1 

......................... 49 

2 

X 1 

1 

......................... 49 

1 

1 

+ + ++ 10 

1 

1 

1 

1 

......................... 49 

1 

......................... 49 

1 

......................... 49 

1 

1 

......................... 48 

X 1 

1 

......................... 49 

......................... 49 

1 

X 1 

X 1 

......................... 49 

+ 1 

X 1 
216 p-Nitrobenzoic Acid, NTP TR 442 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Nervous System 

Brain 

Glioma NOS 

Meninges, histiocytic sarcoma 

Olfactory lobe, histiocytic sarcoma 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar carcinoma, 

multiple 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Glands, histiocytic sarcoma 

Trachea 

Special Senses System 

Ear 

Eye

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

455556666667777777177777 

025992578880033333333333 

060480781551111111111111 

333333333333333333333333 

201030533232400000111111 

067872023553313459012345 

........................ X 

X 

X 

........................ xx X 

X X 

X 

X 

........................ X 

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

+ 

+ 

........................ xx ........................ 

........................ xx 

X xx 
Lesions in Female Mice 217 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid 1,250 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Nervous System 

Brain 

Glioma NOS 

Meninges, histiocytic sarcoma 

Olfactory lobe, histiocytic sarcoma 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar carcinoma, 

multiple 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Glands, histiocytic Sarcoma 

Trachea 

Special Senses System 

Ear 

Eye

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic Sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

11171111111111111111171~7 

3333333333333333333333333 

1111111111111111555555555 

3333333333333333333333333 Total 

1112222333333444222444444 Tissues/ 

6891249014689012618456189 Tumors 

......................... 49 

1 

1 

1 

......................... 49 

X X 5 

X X 4 

1 

xx X 4 

......................... 49 

1 

.......................... 49 

+ 

+ 

X 

......................... 49 

2 

......................... 49 

......................... 49 

2 

X X x xx 8 

xx X 3 
218 

TABLED2 

Individual Animal Tumor Pathology of 

Number of Days on Study 

CarcassID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Histiocytic sarcoma 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

p-Nitrobenzoic Acid, NTP TR 442 

Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid: 2,500 ppm 

0445566666667777771777777 

0896822357880012233333333 

7009949818097750011111111 

4343333333343333333333333 

0607697879906987966666677 

4270528224524345813678913 

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

AA+A+++++++++++++++++++++ 

A++A+++++++++++++++++++++ 

A++A+++++++++++++++++++++ 

AA+A+++++++++++++++++++++ 

AA+A+++++++++++++++++++++ 

AA+A+++++++++++++++++++++ 

AA+A+++++++++++++++++++++ 

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

X X X X X 

X 

X X 

+ + 

A++++++++++++++++++++++++ 

X ......................... 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

+ 

X 

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

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

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

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

X 

M++++++++++++++++++++++++ 

M++++++++++++++++++++++++ 

X xx X X 

......................... X X 
Lesions in Female Mice 219 

TABLED2 

Individual Animal Tumor Pathologyof Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid:2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocelluiar adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Histiocytic sarcoma 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

7777777777777777777777777 

3333333333333333333333333 

1111111111111111111111111 

3333333333333333344444444 Total 

7777888888889999900000001 Tissues1 

4679013567890167901356890 Tumors 

......................... 50 

......................... 47 

......................... 48 

......................... 48 

......................... 47 

......................... 47 

......................... 47 

X 1 

......................... 47 

......................... 50 

xx X X 4 

X 1 

X xx x X X 11 

X 2 

X 3 

+ + + 5 

......................... 49 

1 

......................... 50 

......................... 49 

xx 2 

......................... 49 

1 

1 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

1 

++++++++++++++++++M++++.++ 48 

......................... 49 

xx X X 9 

......................... 50 

L 
220 p-NitrobenzoicAcid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Histiocytic sarcoma 

Oviduct 

Uterus 

Hemangiosarcoma 

Histiocytic sarcoma 

Polyp stromal 

Vagina 

Hematopoietic System 

Blood 

Bone marrow ~ 

Hemangiosarcae.; 

Histiocytic sardma 

Lymphnode ~ 

Axillary, fibrosarcoma, metastatic, 

skin 

Iliac, histiocytic sarcoma 

Inguinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangosarcoma 

Histiocytic sarcoma 

Thymus 

(Fibrosardma, metastatic, skin 

Integumentary System 

Mammary gland 

Skin 

Schwannoma NOS 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, histiocytic 

sarcoma 

Subcutaneous tissue, sarcoma 

0445566666667777777777777 

0896822357880012233333333 

7009949818097750011111111 

4343333333343333333333333 

0607697879906987966666677 

4270528224524345813678913 

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

A++++++++++++++++++++++++ 

X 

X 

......................... X 

+++++++++MI++++++++++++++ 

x 

X 

++++++ + + 

X 

X 

X 

X 

X 

......................... X 

A+++++++++I++++++++++++++ 

X 

A++++++++++++++++++++++++ 

X 

X 

A++++++++M+++++++.++++++++ 

X 

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

......................... X 

X 

X 

X 

X 
Lesions in Female Mice 221 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital 

~~ 

System 

Clitoral gland 

ovary

Cystadenoma 

Histiocytic sarcoma 

Oviduct 

Uterus 

Hemangiosarcoma 

Histiocytic Sarcoma 

Polyp stromal 

Vagina 

Hematopoietic System 

Blood 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Axillary, fibrosarcoma, metastatic, 

skin 

Iliac, histiocytic sarcoma 

Inguinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic Sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Fibrosarcoma, metastatic, skin 

Integumentary System 

Mammary gland 

Skin 

Schwannoma NOS 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, histiocytic 

sarcoma 

Subcutaneous tissue, sarcoma 

1111111111111111111111111 

3333333333333333333333333 

1111111111111111111111111 

3333333333333333344444444 Total 

1111888888889999900000001 Tissues/ 

4619013561890161901356890 Tumors 

......................... 50 

......................... 49 

1 

1 

+ 1 

......................... 50 

1 

X 1 

X X 2 

+ 1 

+ 1 

......................... 48 

1 

1 

+ ++ + + 13 

1 

1 

1 

1 

1 

......................... 50 

1 

......................... 48 

1 

......................... 49 

1 

1 

......................... 48 

1 

......................... 50 

......................... 50 

1 

1 

1 

1 

1 
222 p-Nitrobenzoic Acid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Musculoskeletal System 

Bone 

Skeletal muscle 

Fibrosarcoma 

Nervous System 

Brain 

Meninges, histiocytic sarcoma 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

k+hec$pmcbiplar prcinoma 

Fibrosarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Ear 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinaly bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

0445566666661111111111171 

0896822357880012233333333 

1009949818091750011111111 

4343333333343333333333333 

0601697819906987966666677 

4210528224524345813618913 

++++++++++I++++++++++++++ 

+ + 

X X 

......................... X 

......................... X X 

X 

X 

A+++++++++I++++++++++++++ 

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

......................... X 

A++++++++++++++++++++++++ 

......................... X X 

X 

X X 
Lesions in Female Mice 223 

TABLED2 

Individual Animal Tumor Pathology of Female Micen the 2-Year Feed Study ofp-Nitrobenzoic Acid 2,500 ppm (continued)

Number of Days on Study 

Carcass ID Number 

Musculoskeletal System 

Bone 

Skeletal muscle 

Fibrosarcoma 

Nervous System 

Brain 

Meninges, histiocyticsarcoma 

Respiratory System 

Lung 

Alveolarbronchiolaradenoma 

Alveolarbronchiolarcarcinoma 

Fibrosarcoma, metastatic, skin 

Hepatocellularcarcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Ear 

Urinary System 

Kidney 

Histiocyticsarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

1111111111111111111111111 

3333333333333333333333333 

7777777777777777777777777 

3333333333333333344444444 

7777888888889999900000001 

4619013567890167901356890 

Total 

Tissues/ 

Tumors 

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

2 

2 

49 

......................... 1 50 

......................... X 

1 

1 

3 

50 

X 

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

......................... 50 48 

1 

+ 1 

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

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

49 

1 

50 

......................... X 

x xx x x X 

2 

7 

3 

50 
p-Nitrobenzoic Acid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathologyof Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid: 5,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Polyp adenomatous 

Sarcoma, metastatic, mesentery 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Osteosarcoma, metastatic, bone 

Sarcoma 

Pancreas 

Sarcoma, metastatic, mesentery 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular ~. System 

Heart 

Endocrine System 

Adrenal cortex 

Sarcoma, metastatic, mesentery 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, carcinoma 

General Body System 

None 

~ ______ ~~~~ ~~~~ 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Sarcoma, metastatic, mesentery 

Uterus 

Leiomyoma 

Sarcoma, metastatic, mesentery 

0044455556666666777777777 

0057916781333467001133333 

7771095046067658092500000 

4444444444444444444444444 

3446424536633366526222222 

0685462381296787153812347 

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

M+++++A++++++++++++++++++ 

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

A+++++A+++++A++++++++++++ 

A+++++A+A+++A++++++++++++ 

A+++++A+A++++++A+++++++++ 

X 

X 

A+++++A+A++++++A+++++++++ 

A+++++A+A+++A++A+++++++++ 

......................... X 

X 

X 

+ + + + + 

X 

X 

++++++A++++++++++++++++++ 

X 

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

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

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

......................... X 

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

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

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

+++++++++++++++++++++M+++ 

......................... X 

+M+++++++++++++++++++++++ 

+++++++++++++++++++++M+++ 

X 

X 

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

X 
Lesions in Female Mice 225 

TABLED2 

Individual Animal Tumor Pathology of Female Micen the 2-Year Feed Study ofp-Nitrobenzoic Acid 5,000 ppm (continued)

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Polyp adenomatous 

Sarcoma, metastatic, mesentery 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Osteosarcoma, metastatic, bone 

Sarcoma 

Pancreas 

Sarcoma, metastatic, mesentery 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal Artex 

Sarcoma, metastatic, mesentery 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary

Cystadenoma 

Sarcoma, metastatic, mesentery 

Uterus 

Leiomyoma 

Sarcoma, metastatic, mesentery 

1117111111111111111111111 

3333333333333333333333333 

0000000011111111111111111 

4444444444444444444444444 Total 

2333334444445555555566661 Tissues/ 

9123450135190245618904690 Tumors 

......................... 50 

......................... 48 

......................... 48 

......................... 41 

......................... 46 

......................... 46 

1 

1 

......................... 46 

......................... 45 

......................... 50 

X x xx x 6 

1 

X X X 4 

X 1 

+ 6 

1 

1 

......................... 49 

1 

......................... 50 

......................... 49 

X 1 

......................... 49 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

+++++++++++++++I+++++++++ 49 

++++++++++++++++M++++++++ 48 

xx X X X 5 

......................... 50 

1 

......................... 49 

+++++++++++++++++M+++++++ 48 

1 

1 

......................... 50 

X 1 

1 
226 p-Nitrobenzoic Acid, NTP TR 442 

TABLED2 

Individual Animal Tumor Pathology of Female Micen the 2-Year Feed Study ofp-Nitrobenzoic Acid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, sarcoma, metastatic, 

mesentery 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Sarcoma, metastatic, mesentev 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Skin 

Subcutaneous tissue, sarcoma 

Musculoskeletal System 

Bone 

Femur, osteosarcoma 

Skeletal muscle 

Sarcoma, metastatic, mesentery 

Nervous System 

Brain 

Respiratory System 

Lung' 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma, 

multiple 

Osteosarcoma, metastatic, bone 

Sarcoma, metastatic, mesentery 

Nose 

Trachea 

Special Senses System 

None 

Urinary System 

Kidney 

Osteosarcoma, metastatic, bone 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

0044455556666666777777777 

0057916781333467001133333 

7771095046067658092500000 

4444444444444444444444444 

3446424536633366526222222 

0685462381296787153812347 

......................... ++ ++++ ++++ 

X 

M++++++++++++++++++++++++ 

AM+++++++++++++++++++++++ 

......................... X 

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

M++++++++++++++++++++++++ 

......................... X 

......................... X 

+ + 

X 

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

++++-+++++++,+++,+,++.++,+ 

X X X 

X 

X 

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

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

......................... X 

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

......................... xx 

xx 
Lesions in Female Mice 227 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study ofp-Nitrobenzoic Acid: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, sarcoma, metastatic, 

mesentery 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Sarcoma, metastatic, mesentery 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Skin 

Subcutaneous tissue, sarcoma 

Musculoskeletal System 

Bone 

Femur, osteosarcoma 

Skeletal muscle 

Sarcoma, metastatic, mesentery 

Nervous System 

Brain 

Respiratory System 

Ling.-'.

Alveolaribronchiolar adenoma 

Alveolaribronchiolar carcinoma, 

multiple 

Osteosarcoma, metastatic, bone 

Sarcoma, metastatic, mesentery 

Nose 

Trachea 

Special Senses System 

None 

Urinary System 

Kidney 

Osteosarcoma, metastatic, bone 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

7777777777777777777777777 

3333333333333333333333333 

0000000011111111111111111 

4444444444444444444444444 Total 

2333334444445555555566667 Tissues/ 

9123450135790245678904690 Tumors 

......................... 50 

++ ++ + 15 

1 

......................... 49 

......................... 48 

......................... 50 

1 

++++++++++M+++MM+++++++M+ 46 

......................... 49 

X 1 

......................... 50 

1 

......................... 50 

1 

2 

1 

......................... 50 

......................... 50 

X X xx X 8 

X 1 

1 

1 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

X X X 5 

2 
228 p-Nitrobenzoic Acid, NTP TR 442 

Harderian Gland: Adenoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd I Fisher exact test 

Liver: Hepatocellular Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

Liver: Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 

Adjusted rate 

Tenninal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

3/50 (6%) 

7.2% 

OB8 (0%) 

639 

P=0.061N 

P=0.047N 

P=0.044N 

11/50 (22%) 

27.2% 

9/38 (24%) 

589 

P=O.l62N 

P=0.080N 

P=O.OSN 

4/50 (8%) 

9.8% 

3/38 (8%) 

533 

P=O.112 

P=O.164 

P=O.210 

15/50 (30%) 

36.1% 

12/38 (32%) 

533 

P=O.418N 

P=0.240N 

P=0.171N 

3/50 (6%) 

7.5% 

2/38 (5%) 

689 

P=O.O35 

P=O.O52 

P=O.O79 

1/49 (2%) 

2.8% 

1/36 (3%) 

730 (T>

P=0.334N 

P=0.312N 

P=0.316N 

13/49 (27%) 

31.9% 

9/36 (25%) 

620 

P=O.359 

P=O.382 

P=O.385 

5/49 (10%) 

13.0% 

3/36 (8%) 

685 

P=O.466 

P=O.487 

P=O.487 

16/49 (33%) 

38.6% 

11/36 (31%) 

620 

P=O.436 

P=O.473 

P=O.473 

5/49 (10%) 

12.9% 

3/36 (8%) 

685 

P=O.324 

P=O.343 

P=O.346 

0/50 (0%) 

0.0% 

OB3 (0%) -e

P=0.149N 

P=0.121N 

P=0.121N 

13/50 (26%) 

34.0% 

9/33 (27%) 

480 

P=O.290 

P =0.390 

P=O.408 

5/50 (10%) 

15.2% 

5/33 (15%) 

730 (T> P=O.420 

P=O.489 

P-0.500 

15/50 (30%) 

39.5% 

11/33 (33%) 

480 

P-0.433 

P=O.570 

P-0.586N 

3/50 (6%) 

8.7% 

2/33 (6%) 

715 

P=O.599 

P=O.643 

P=0.661N 

0/50 (0%) 

0.0% 

OB0 (0%) -

P=0.171N 

P=0.128N 

P=0.121N 

5/50 (10%) 

15.6% 

4/30 (13%) 

637 

P='O.l92N 

P=0.131N 

P=0.086N 

7/50 (14%) 

21.0% 

5/30 (17%) 

616 

P=O.163 

P=O.232 

P=O.262 

11/50 (22%) 

32.3% 

8/30 (27%) 

616 

P=0.483N 

P=0.326N 

P=0.247N 

8/50 (16%) 

24.3% 

6/30 (20%) 

570 

P=O.O50 

P=O.O71 

P=O.lOO 
Lesions in Female Mice 229 

TABLED3 

Statistical Analysisof Primary Neoplasms in Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid (continued) 

0 PPm 1,2502,0 ppmppm 5,000 ppm 

Lung: Alveolar/bronchiolar Carcinoma 

Overall rate 0/50 (0%) 5/49 (10%) 1/50 (2%) 1/50 (2%) 

Adjusted rate 0.0% 13.9% 2.9% 3.3% 

Terminal rate OB8 (0%) 5/36 (14%) OB3 (0%) 1/30 (3%) 

Life table test 

First incidence (days) 

P=O.572N 

-

P=O.O29 

730 (.r) 

P =0.468 

720 730 (r)

P=O.453 

Logistic regression test P=0.568N P=O.O29 P=O.491 P=O.453 

Gxhran-Armitage test P=0.471N 

Fisher exact test P=O.O27 P=O.500 P=O.500 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 3/50 (6%) 10/49 (20%) 4/50 (8%) 9/50 (18%) 

Adjusted rate 7.5% 26.1% 11.3% 27.5% 

Terminal rate 2/38 (5%) 8/36 (22%) 2/33 (6%) 7/30 (23%) 

First incidence (days) 689 685 715 570 

Life table test P=O.O63 P=O.O31 P=O.428 P=O.O27 

Logistic regression test P=O.O88 P=O.O31 P=O.475 P=O.O39 

Cochran-Armitage test P=O.147 

Fisher exact test P=O.O33 P=O.500 P=O.O61 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 7/48 (15%) 6/48 (13%) 9/49 (1 8%) 5/48 (10%) 

Adjusted rate 18.3% 16.7% 24.1% 17.9% 

Terminal rate 6/37 (16%) 6/36 (1 7%) 6/33 (18%) 5/28 (18%) 

Life table test 

First incidence (days) 

P=O.517 

728 730 (.r)

P=0.526N P=O.309 

589 

P=0.583N 

730 (T) 

Logistic regression test P=0.533N P=0.564N P=O.388 P=0.615N 

Cochran-Armitage test P=0.374N 

Fisher exact test P=0.500N P=O.410 P=0.379N 

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma 

Overall rate 8/48 (17%) 6/48 (13%) 9/49 (18%) 5/48 (10%) 

Adjusted rate 20.2% 16.7% 24.1% 17.9% 

Terminal rate 6/37 (16%) 6/36 (17%) 6/33 (18%) 5/28 (18%) 

Life table test 

First incidence (days) 

P=0.504N 

689 

P=0.418N 

730 (T) 

P=O.406 

589 

P=0.474N 

730 (T) 

Logistic regression test P=0.429N P=0.427N P=O.501 P=O.452N 

Cochran-Armitage test P=0.288N 

Fisher exact test P=0.387N P=O.519 P=0.276N 

All Organs: Hemangiosarcoma 

Overall rate 4/50 (8%) 1/49 (2%) 2/50 (4%) 0/50 (0%) 

Adjusted rate 10.1% 2.5% 5.6% 0.0% 

First incidence (days) 

Terminal rate 

693 

3/38 (8%) 

685 

On6 (0%) 

707 

1/33 (3%) on0 (0%) -

Life table test P=0.078N P=0.204N P=0.397N P =0.098N 

Logistic regression test P=O.O62N P=0.186N P=0.356N P=0.082N 

Cochran-Armitage test P=0.052N 

Fisher exact test P=0.187N P=0.339N P=0.059N 
230 p-Nitrobenzoic Acid, NTP TR 442 

TABLE D3 

Statistical Analysisof Primary Neoplasms in Female Mice in the 2-Year Feed Studyofp-Nitrobenzoic Acid (continued) 

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

All Organs: Histiocytic Sarcoma 

Overall rate 1/50 (2%) 2/49 (4%) 3/50(6%) 0/50 (0%)

Adjusted rate 7.5% 4.2% 2.6% 0.0% 

Terminal rate 1/38 (3%) OB6 (0%) (3%) 1/33 on0 (0%)

First incidence (days) 624 526 - 730 (T> Life table test P=O.281 P=O.496 P=0.388N P=0.547N 

Logistic regression test P=0.547N P=O.306 P=O.444 P=0.275N 

Cochran-Armitage test P=0.335N 

Fisher exact test P=O.309 P=O.492 P=0.500N 

All Organs: Malignant Lymphoma (Lymphocytic or Mixed) 

Overall rate (18%) 11/499/50 (22%) 9/50 (18%) 7/50 (14%) 

Adjusted rate 

Terminal rate 

21.5% 30.6% 

(18%) (31%)16 6/33 11/6 6383/30

24.0% 20.6% 

(10%) 

First incidence (days) 569 

Life table test P=O.487 P=O.347 P=0.473N 

423 730 (T> 

P=0.586N 

Logistic regression test P=0.458N P=O.598 P=O.378 P=0.344N 

Cochran-Armitage test P=O.262N 

Fisher exact test P=O.382 P=0.393NP=0.602N 

All Organs: Malignant Lymphoma or Histiocytic Sarcoma 

Overall rate 10/50 (20%) 13/49 (27%) 11/50 (22%) 7/50 (14%) 

Adjusted rate 24.0% 33.4% 27.5% 20.6% 

Terminal rate 7/38 (18%) 11/36 (31%) 6/33 (18%) 3/30 (10%) 

First incidence (days) 423 526 569 700 

Life table test P=0.367N P=O.274 P =0.390 P=0.495N 

Logistic regression test P=0.210N P=O.297 P=O.499 P=0.363N 

Cochran-Armitage test' P=0.175N 

Fisher exact test P=O.298 P=O.500 P=0.298N 

All Organs: Benign Neoplasms 

Overall rate 22/50 (44%) 22/49 (45%) 23/50 (46%) 21/50 (42%) 

Adjusted rate 49.8% 53.3% 56.6% 59.5% 

Terminal rate 16/38 (42%) 17/36 (47%) 16/33 (48%) 16/30 (53%) 

First incidence (days) 589 620 480 570 

Life table test P=O.244 P=O.486 P=O.314 P=O.299 

Logistic regression test P=O.452 P=O.543 P=O.471 P=O.481 

Cochran-Armitage test P=0.449N 

Fisher exact test P=O.545 P=O.500 P=0.500N 

AI1 Organs: Malignant Neoplasms 

(46%) 18/50 (36%)

Adjusted 45.6%rate 

Terminal rate 17B818/36 (45%) (50%) 12/33 (36%)09/30

Overall rate 23/50 (46%) 23/50 24/49 (49%) 

51.9% 56.7% 51.9% 

First incidence (days) 569 526 423 

Life table test P=O.387 P=O.407 P=O.472N P=0.527N 

Logistic regression test P=O.USN P=O.560 P=O.463 P=O.l93N 

Cochran-Armitage test P=0.138N 

Fisher exact test P=O.462 P=0.208N P=0.579N
Lesions in Female Mice 231 

TABLED3 

Statistical Analysisof Primary Neoplasms in Female Mice in the 2-Year Feed Studyof g-Nitrobenzoic Acid(continued) 

0 PPm 1,2505,000 ppmppmppm 2,500

All Organs: Benign or Malignant Neoplasms 

Overall rate 36/50 (72%) 38/49 (78%) 35/50 (70%) 33/50 (66%) 

Adjusted 78.5% rate 72.8% 82.5% 75.0% 

Terminal rate 26/38 (68%) 28/36 (78%) 20/33(61%) 21/30 (70%)

First incidence (days) 423 526 480 471 

Life table test P=O.287 P=O.294 P=O.376 P=O.311 

Logistic regression test P=0.211N P=O.347 P=0.390N P=0.507N 

Cochran-Annitage test P=0.198N 

Fisher exact test P=0.343 P=0.500N P=0.333N 

(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 numberof animals necropsied. 

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

Obselved incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposure group incidence are the P values corresponding 

to pairwise comparisons between the controls and that exposure group. The life table test regards neoplasms in animals dying prior to terminal kill 

as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher 

exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

e Not applicable; no neoplasms in animal group 
232 p-Nitrobenzoic Acid, NTP TR 442 

TABLED4 

Historical Incidence of Lung Neoplasms in Untreated Female B6C3Fl Micea 

Incidence in Controls 

Study Alveolar/bronchiolar Alveolar/bronchiolar Alveolar/bronchiolar 

Adenoma Carcinoma Adenoma or Carcinoma 

Historical Incidence at Southern Research Institute 

Benzyl Acetate 

C.I. Pigment Red 23 

C.I. Pigment Red 3 

Ethylene Glycol 

Nitrofurantoin 

o-Nitroanisole 

Polysorbate 80 

Rhodamine 6G 

Roxarsone 

1/50 

1/50 

3/50 

0150 

2/50 

4/50 

3/50 

3/50 

1/50 

0150 

0150 

1/50 

1/50 ' 

1/50 

2/50 

0150 

1/50 

2/50 

1/50 

1/50 

4/50 

1/50 

3/50 

6/50 

3/50 

4/50 

3/50 

Total 181450 (4.0%) 81450 (1.8%) 26/450 (5.8%) 

Standard deviation 2.7% 1.6% 3.4% 

Range 0%-8% 0%-4% 2%-12% 

Overall Historical Incidence 

Total 7811,371 (5.7%) 3011,371(2.2%) 106/1,371 (7.7%) 

Standard deviation 4.9% 2.3% 5.0% 

Range 0%-24% 0%-8% 2%-26% 

a Data as of 20 August 1992 
Lesions in Female Mice 233 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acida 

Disposition Summary 

Animals initially in study 

ZIMonrh intaim+wahdon 

Early deaths 

Accidental deaths 

Moribund 

Natural deaths 

SuMvors 

Terminal sacrifice 

Missing 

Animals examined microscopically 

15-Month Interim Evaluation 

Alimentary System 

Liver 

Angiectasis 

Basophilic focus 

Eosinophilic focus 

Inflammation, subacute 

Karyomegaly 

Necrosis 

Vacuolization cytoplasmic 

Bile duct, cyst 

Mesentery 

Cyst

Fat, hemorrhage 

Fat, inflammation, suppurative 

Fat, necrosis 

Pancreas 

Acinus, atrophy 

Stomach, forestomach 

Hyperplasia 

Endocrine System 

Adrenal cortex 

Hyperplasia 

X-zone, vacuolization cytoplasmic 

Pituitary gland 

Pars distalis, hyperplasia 

Thyroid gland 

Follicle, cyst 

0 PPm 

60 

10 

9 

3 

38 

60 

1 (10%) 

1 (10%) 

1 (10%) 

(1)

1 (100%) 

1 (100%) 

(10) 

(10)

1 (10%) 

(10)

1 (10%) 

1,250 ppm 

60 

10 

8 

5 

36 

1 

59 

1 (10%) 

(10) 

4 (40%) 

(10) 

(10) 

2,500 ppm 

60 

10 

1 

12 

4 

33 

60 

1 (10%) 

1 (10%) 

(1) 

' (10)

1 (10%) 

1 (10%) 

(9) 

(10) 

5,000 ppm 

60 

10 

2 

10 

8 

30 

60 

(10)

1 (10%) 

1 (10%) 

2 (20%) 

(10) 

1 (10%) 

' (10)

2 (20%) 

(10) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
234 p-Nitrobenzoic Acid, NTP TR 442 

~ TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 

(continued) 

0 PPm 

15-Month Interim EvuZuation (continued) 

Genital System 

Ovary 

Angiectasis 

Cyst

Follicle, hemorrhage 

Uterus 

Dilatation 

Hyperplasia, cystic 

Hematopoietic System 

Lymph node 

Iliac, hyperplasia, lymphoid 

Lymph node, mesenteric 

Necrosis 

Spleen 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, lymphoid 

Integumentary System 

Skin 

Inflammation, suppurative 

Dennis, inflammation, subacute 

Urinary System 

Kidney 

Infiltration cellular, lymphocyte 

Mineralization 

Renal tubule, casts 

Renal tubule, regeneration 

(10)

2 (20%) 

(10) 

8 (80%) 

(9) 

(10) 1 

1 (10%) 

(10) 

1 (10%) 

(10) 

2 (20%) 

4 (40%) 

1,250.ppm 

(10) 

4 (40%) 

(10)

1 (10%) 

10 (100%) 

(10) 

(10) 

(10) 

(10) 

1 (10%) 

1 (10%) 

2,500 ppm 

(10) 

3 (30%) 

1 (10%) 

(10) 

2 (20%) 

9 (90%) 

(10)

1 (10%) 

(10)

1 (10%) 

1 (10%) 

1 (10%) 

3 (30%) 

5,000 ppm 

(10) 

1 (10%) 

(10)

2 (20%) 

9 (90%) 

(10) 

(10)

1 (10%) 

1 (10%) 

Systems Examined Wgh No Leswm Observed 

Cardiovascular System 

General Body System 

Musculoskeletal System 

Nervous System 

Respiratory System 

Special Senses System 
Lesions in Female Mice 235 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof p-NitrobenzoicAcid 

(continued) 

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

2-Year Study 

Alimentary System 

Gallbladder (50) (48) (47) (48)

Dilatation 1 (2%) 

Inflammation, subacute 1 (2%) 

Intestine small, jejunum (49) (46) (46)

Peyer’s patch, hyperplasia, lymphoid 

Peyer’s patch, necrosis 1 (2%) 

Liver (50) (49) (50)

Angiectasis 1 (2%) 1 (2%) 

Basophilic focus 1 (2%) 5 (10%) 1 (2%) 1 (2%) 

Clear cell focus 1 (2%) 

Developmental malformation 1 (2%) 

Eosinophilic focus 4 (8%) 5 (10%) 6 (12%) 2 (4%) 

Fatty change, focal 1 (2%) 

Fibrosis 1 (2%) 

Hematopoietic cell proliferation 1 (2%) 3 (6%) 1 (2%) 2 (4%) 

Infiltration cellular, lymphocyte 1 (2%) 

Inflammation, subacute 2 (4%) 2 (4%) 

Mineralization 1 (2%) 1 (2%) 

Mixed cell focus 3 (6%) 3 (6%) 1 (2%) 

Necrosis 1 (2%) 3 (6%) 2 (4%) 2 (4%) 

Thrombosis 1 (2%) 

Vacuolization cytoplasmic 2 (4%) 2 (4%) 1 (2%) 1 (2%) 

Bile duct, hyperplasia 1 (2%) 

Hepatocyte, hypertrophy, diffuse 1 (2%) 

Mesentery (4) (8) (5) (6)

Inflammation, subacute 2(25%) 1 (20%) 1 (17%) 

Pigmentation, hemosiderin 1 (25%) 

Polyarteritis 1 (13%) 

Fat, hemorrhage 1 (17%) 

Fat, inflammation, granulomatous 1 (25%) 

Fat, inflammation, subacute 1 (17%) 

Fat, necrosis 3 (75%) 4 (50%) 3 (60%) 1 (17%) 

Pancreas (50) (47) (49) (49)

Basophilic focus 1 (2%) 

Congestion 1 (2%) 

Edema 1 (2%) 1 (2%) 2 (4%) 

Inflammation, suppurative 1 (2%) 

Acinus, atrophy 1 (2%) 3 (6%) 

Acinus, depletion secretory 1(2%) 2 (4%) 3 (6%) 3 (6%) 

Duct, cyst 2 (4%) 1 (2%) 2 (4%) 3 (6%) 

Duct, degeneration 1 (2%) 

Duct, inflammation, chronic 1 (2%) 

Salivary glands (50) (49) (50) (50) 

Cytoplasmic alteration 1 (2%) 

Stomach, forestomach (50) (49) (49) (49) 

Diverticulum 1 (2%) 

Hyperplasia 7 (14%) 4 (8%) 4 (8%) 1 (2%) 

Inflammation, subacute 2 (4%) 3 (6%) 1 (2%) 

Polyarteritis 1 (2%) 

Ulcer 1 (2%) 
236 p-Nitrobenzoic Acid, NTP TR 442 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

%Year Study (continued) 

Alimentary System (continued) 

Stomach, glandular 

Crst 

Erosion 

Hyperplasia 

Inflammation, chronic 

Polyarteritis 

Cardiovascular System 

Hart 

Hemorrhage 

Infiltration cellular, histiocyte 

Inflammation, subacute 

Coronary artery, amyloid deposition 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Hematopoietic cell proliferation 

Hyperplasia 

Hypertrophy 

Vacuolization cytoplasmic 

Spindle cell, hyperplasia 

X-zone, vacuolization cytoplasmic 

Adrenal medulla 

Hyperplasia 

Islets, pancreatic 

Hyperplasia 

Pituitary gland 

Pars distalis, angiectasis 

Pars distalis, cyst 

Pars distalis, hyperplasia 

Pars intermedia, hyperplasia 

Thyroid gland 

Ccell, hyperplasia 

Follicle, cyst 

Follicle, degeneration 

Follicular cell, hyperplasia 

General Body System 

None 

0 PPm 

(49)

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

(49)

1 (2%) 

1 (2%) 

3 (6%) 

2 (4%) 

(49) 

2 (4%) 

(50) 

(4) 

10 (21%) 

1 (2%) 

(50) 

17 (34%) 

1,250 ppm 

(49)

3 (6%) 

2 (4%) 

1 (2%) 

1 (2%) 

(49) 

(48)

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

(48) 

(49)

1 (2%) 

(48)

1 (2%) 

1 (2%) 

10 (21%) 

(49) 

1 (2%) 

14 (29%) 

2,500 ppm 

(49)

4 (8%) 

1 (2%) 

1 (2%) 

(50) 

(50)

2 (4%) 

1 (2%) 

1 (2%) 

(50) 

(49) 

(49)

1 (2%) 

1 (2%) 

14 (29%) 

(50)

1 (2%) 

3 (6%) 

24 (48%) 

5,000 ppm 

(49)

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50)

2 (4%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 

(50) 

(48)

1 (2%) 

9 (19%) 

(50) 

2 (4%) 

1 (2%) 

8 (16%) 
Lesions in Female Mice 237 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

2-Year Study (continued) 

Genital System 

Clitoral gland 

Inflammation, subacute 

Duct, cyst 

o-=Y 

Abscess 

Atrophy 

Cyst 

Hemorrhage 

Inflammation, subacute 

Inflammation, suppurative 

Pigmentation, hemosiderin 

Oviduct 

Cyst

Uterus 

Angiectasis 

Atrophy 

Cyst

Dilatation 

Hemorrhage 

Hyperplasia, cystic 

Inflammation, subacute 

Thrombosis 

Vagina 

Inflammation, granulomatous 

Hematopoietic System 

Bone marrow 

Atrophy 

Hypercellularity 

Myeloid cell, depletion cellular 

Lymph node 

Axillary, hyperplasia, lymphoid 

Iliac, hyperplasia, lymphoid 

Inguinal, hyperplasia, lymphoid 

Mediastinal, edema 

Mediastinal, hyperplasia, lymphoid 

Mediastinal, inflammation, subacute 

Pancreatic, hyperplasia, lymphoid 

Renal, hyperplasia 

Renal, hyperplasia, lymphoid 

Renal, inflammation, subacute 

Lymph node, mandibular 

Angiectasis 

Cyst

Hyperplasia, lymphoid 

Infiltration cellular, mast cell 

0 PPm 

(50) 

3 (6%) 

(50)

2(4%) 

17(34%) 

1 (2%) 

1 (2%) 

7(14%) 

3(6%) 

43(86%) 

5(10%) 

2 (25%) 

1 (13%) 

3(38%) 

1 (13%) 

4(50%) 

1 (2%) 

1 (2%) 

1,250 ppm 

(49) 

9 (18%) 

(48)

2(4%) 

9(19%) 

1 (2%) 

(49)

1 (2%) 

1 (2%) 

10 (20%) 

1 (2%) 

45(92%) 

1 (2%) 

(49) 

1 (2%) 

(10)

1 (10%) 

2(20%) 

2(20%) 

(49)

2(4%) 

1 (2%) 

1 (2%) 

2,500 ppm 

11 (22%) 

(49) 

1 (2%) 

15 (31%) 

(50) 

1 (2%) 

2(4%) 

6(12%) 

48(96%) 

(1)

1 (100%) 

(48)

1 (2%) 

1 (2%) 

(13) 

2 (15%) 

1 (8%) 

1 (8%) 

1 (2%) 

5,000 ppm 

(49)

1 (2%) 

11 (22%) 

(48)

6(13%) 

14(29%) 

5 (10%) 

1 (2%) 

(50)

1 (2%) 

2(4%) 

4 (8%) 

37 (74%) 

1 (2%) 

(50) 

4 (8%) 

1 (2%) 

(15) 

3(20%) 

3 (20%) 

1 (7%) 

1 (7%) 

2(13%) 

3(20%) 

1 (7%) 

(49) 

2(4%) 
238 p-Nitrobenzoic Acid, NTP TR 442 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

%Year Study (continued) 

Hematopoietic System (continued) 

Lymph node, mesenteric 

Angiectasis 

Cyst

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Spleen 

Atrophy 

Congestion 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Necrosis 

Pigmentation, hemosiderin 

Thymus 

Atrophy 

Integumentary System 

Mammary gland 

Hyperplasia, lobular 

Duct, cyst 

Skin 

Parakeratosis 

Dermis, inflammation, subacute 

Dermis, subcutaneous tissue, inflammation, 

subacute 

Epidermis, necrosis 

Subcutaneous tissue, edema 

Subcutaneous tissue, hemorrhage 

Subcutaneous tissue, inflammation, subacute 

Musculoskeletal System 

Bone 

Osteopetrosis 

Cranium, fibrous osteodystrophy 

Femur, fibrous osteodystrophy 

Nervous System 

Brain 

Compression 

Cyst

Vacuolization cytoplasmic 

Meninges, infiltration cellular 

0 PPm 

(50)

3 (6%) 

1 (2%) 

1 (2%) 

2 (4%) 

(50) 

1 (2%) 

12 (24%) 

12 (24%) 

1 (2%) 

(50)

1 (2%) 

(50)

1 (2%) 

(50) 

1 (2%) 

(50) 

2 (4%) 

(49)

1 (2%) 

1 (2%) 

1,250 ppm 

(49)

4 (8%) 

1 (2%) 

(49) 

18 (37%) 

12 (24%) 

1 (2%) 

(49) 

(49)

1 (2%) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

3 (6%) 

2 (4%) 

(49) 

1 (2%) 

2,500 ppm 

(48)

5 (10%) 

1 (2%) 

(49) 

14(29%) 

9 (18%) 

1 (2%) 

(48)

1 (2%) 

(50) 

1 (2%) 

(50) 

2 (4%) 

1 (2%) 

(49)

1 (2%) 

3(6%) 

(50)

4 (8%) 

1 (2%) 

5,000 ppm 

(48)

5 (10%) 

2 (4%) 

(50)

2 (4%) 

16(32%) 

13 (26%) 

1 (2%) 

(46)

4 (9%) 

(49) 

2 (4%) 

(50) 

3 (6%) 

(50) 

1 (2%) 

5 (10%) 

(50) 
Lesions in Female Mice 239 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

(continued) 

2-YeW study (continued) 

Respiratory System 

Lung 

Congestion 

Edema 

Hemorrhage 

Infiltration cellular, lymphocyte 

Infiltration cellular, histiocyte 

Inflammation, subacute 

Alveolar epithelium, hyperplasia 

Mediastinum, inflammation, subacute 

Nose 

Glands, cyst 

Lumen, fungus 

Special Senses System 

Eye

Atrophy 

Cataract 

Inflammation, subacute 

Cornea, inflammation, subacute 

Harderian gland 

Inflammation, chronic 

Urinary System 

Kidney 

Fibrosis 

Glomerulosclerosis 

Infarct 

Infiltration cellular, lymphocyte 

Inflammation, subacute 

Metaplasia, osseous 

Mineralization 

Polyarteritis 

Capsule, fibrosis 

Glomerulus, amyloid deposition 

Renal tubule, casts 

Renal tubule, degeneration 

Renal tubule, dilatation 

Renal tubule, hyperplasia 

Renal tubule, pigmentation 

Renal tubule, regeneration 

Urinary bladder 

Dilatation 

0 PPm 

2 (4%) 

3 (6%) 

(50)

1 (2%) 

(2) 

1 (50%) 

1 (50%) 

1 (50%) 

(3) 

(50) 

4 (8%) 

2 (4%) 

3 (6%) 

1 (2%) 

15 (30%) 

4 (8%) 

1 (2%) 

1 (2%) 

1 (2%) 

18 (36%) 

(50) 

1,250 ppm 

1 (2%) 

1 (2%) 

2 (4%) 

3 (6%) 

1 (2%) 

(49) 

1 (2%) 

(1)

1 (100%) 

(2)

1 (50%) 

(49)

1 (2%) 

3 (6%) 

1 (2%) 

I (14%) 

1 (2%) 

2 (4%) 

14(29%) 

(49)

1 (2%) 

2,500 ppm 

(50)

1 (2%) 

(50) 

1 (2%) 

3 (6%) 

2 (4%) 

7 (14%) 

1 (2%) 

1 (2%) 

12 (24%) 

(49)

1 (2%) 

5,000 ppm 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

5 (10%) 

1 (2%) 

2 (4%) 

4 (8%) 

4 (8%) 

1 (2%) 

9 (18%) 

(50) 
240 p-Nitrobenzoic Acid, NTP TR 442 
241 

APPENDIX E 

GENETIC TOXICOLOGY 

SALMONELLA TypHIMuRluM MUTAGENICITY TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l . 242

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS . . . . . . .. . . . . . . . .. ... . . . . . . . . . . 242 

MOUSEPERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . ......... . . . . .. . . . 243 

RESULTS .................... .............................................. ... 244 

TABLE El Mutagenicity of p-Nitrobenzoic Acid in Salmoneua typhimurium . . . . . . . . . . . . . . . l . . . 245 

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

by p-Nitrobenzoic Acid . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l . . . l . . l .. . 247 

TABLEE3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

by p-Nitrobenzoic Acid . . . .. .. . .. .. l . . . . . . . . . . . . . . . . .. . . . . . . . . . ... l l . . . . . 249 

TABLEE4 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes 

Following Administration of p-Nitrobenzoic Acid in Feed for 13Weeks . . . . . . . . . . .. . 250 
242 p-Nitrobenzoic Acid, NTP TR 442 

GENETIC TOXICOLOGY 

SALMONELLATYPHIMURIM MUTAGENICITY TESTPROTOCOL

Testing was performed as reported by Zeiger et al. (1987). p-Nitrobenzoic acid was sent to the laboratory 

as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella 

typhimuriurn 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 1-histidine and d-biotin was added, and the 

contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidineindependent 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 at least five doses 

ofp-nitrobenzoic acid. The high dose was limited to 3,333 pg/plate. All positive trials were repeated 

under the conditions that elicited the positive response. 

In this test, a positive response is defined as a reproducible, dose-related increase in histidine-independent 

(revertant) colonies in any one straidactivation combination. An equivocal response is defined as an 

increase in revertants that is not dose related, not reproducible, or is of insufficient magnitude to support 

a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies 

was observed following chemical treatment. There was no minimum percentage or fold increase required 

for a chemical to the judged positive or weakly positive. 

CHINESEHAMSTER OVARYCELLCYTOGENETICSPROTOCOLS 

Testing was performed as reported by Galloway et al. (1985) and Zeiger et al. (1987). p-Nitrobenzoic acid 

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 ofp-nitrobenzoic acid; the high dose was limited by toxicity. A single flask per dose 

was used, and tests yielding equivocal or positive results were repeated. 

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 26 hours with 

p-nitrobenzoic acid in McCoy's 5A medium supplemented with fetal bovine serum, 1-glutamine, and 

antibiotics. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the 

medium containingp-nitrobenzoic acid was removed and replaced with fresh medium plus BrdU and 

Colcemid, and incubation was continued for approximately 2 hours. Cells were then harvested by mitotic 

shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were 

incubated with p-nitrobenzoic acid, serum-free medium, and S9 for approximately 2 hours. The medium 

was then removed and replaced with medium containing serum and BrdU and no p-nitrobenzoic acid, and 

incubation proceeded for an additional 26 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 chemical-induced cell cycle delay was seen in the 

absence of S9 at doses of 498 pg/mL and above, incubation time was lengthened for these cultures 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 
Genetic Toxicology 243 

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. 

ChromosomalAberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium 

with p-nitrobenzoic acid for 18.5 to 19.5 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 withp-nitrobenzoic acid and S9 for 2 hours, after which the treatment medium was 

removed and the cells were incubated for 10.5 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: because cell cycle delay was 

anticipated in the absence of S9, the incubation period was extended. 

Cells were selected for scoring on the basis of good morphology and completeness of karyotype 

(21 2 2 chromosomes). All slides were scored blind and those from a single test were read by the same 

person. Generally, 200 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 (PsO.05) difference for one dose point and a significant 

trend (PsO.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 etal., 1987). Ultimately, the trial calls 

were based on a consideration of the statistical analyses as well as the biological information available to 

the reviewers. 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL 

A detailed discussion of this assay can be found in MacGregor et al. (1990). Peripheral blood samples 

were obtained from male and female B6C3Fl mice at the end of the 13-week study. Smears were 

immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye 

mixture of Hoechst 332Wpyronin Y (MacGregor et al., 1983), and coded. Slides were scanned to 

determine the frequency of micronuclei in 10,000 normochromatic erythrocytes (NCEs) in each of 

10 animals per dose group. The criteria of Schmid (1976) were used to define micronuclei, with the 

additional requirement that the micronuclei exhibit the characteristic fluorescent emissions of DNA (blue 

with 360 nm and orange with 540 nm ultraviolet illumination); the minimum size limit was approximately 

one-twentieth the diameter of the NCE cell. 

The frequency of micronucleated cells among NCEs was analyzed by a statistical software package (ILS, 

1990) which employed a one-tailed trend test across dose groups and a t-test for pairwise comparisons of 

each dose group to the concurrent control. 
244 p-Nitrobenzoic Acid, NTP TR 442 

RESULTS 

p-Nitrobenzoic acid, tested in a preincubation protocol at concentrations of 1 to 3,333 pglplate, with and 

without induced rat or hamster S9, was mutagenic in strain TAlOO (Table El; Zeiger et al., 1987). No 

mutagenicity was detected in strains TA1535, TA1537, or TA98, with or without S9. 

In cytogenetic tests with cultured CHO cells, p-nitrobenzoic acid induced significant increases in SCEs 

(Table E2; Zeiger et al., 1987) and Abs (Table E3; Zeiger et aL, 1987) at dose levels which induced cell 

cycle delay in the absence of S9; no increases in either endpoint were observed in the presence of S9. In 

the SCE test without S9, doses ranging from 498 to 1,000 pglmL produced positive responses, induced cell 

cycle delay, and required use of an extended harvest protocol to allow accumulation of sufficient cells for 

metaphase analysis. Doses producing positive responses in the Abs assay without S9 ranged from 875 to 

1,750 pg/mLp-nitrobenzoic acid. A s with the SCE test, cell harvest was delayed to permit a sufficient 

number of cells to progress to metaphase for analysis. 

Despite the positive results obtained in the in vitro studies, results of a single NTP in vivo genotoxicity 

study were negative. In this study, the frequencies of micronucleated normochromatic erythrocytes in the 

peripheral blood of male and female mice were found to be unaffected by administration of p-nitrobenzoic 

acid in feed for 13weeks (Table E4). 
Genetic Toxicology 245 

TABLEEl 

Mutagenicity of p-Nitrobenzoic Acid in Salmonella typhimuriuma 

Revertants/plateb 

Strain Dose -s9 +lo%hamster S9 

(@plate)Trial 1 Trial 2 Trial 1 Trial 2 Trial 3 

TAlOO 0.0 98 f 5.8106 f 13.9144 f 11.5 140 f 7.8 146 f 14.4 

1.0 

3.3 95 f 15.6 

10.0 83 f 4.4 121 f 9.6 159 f 11.0 86 f 5.8 

33.0 126 f 6.1118 f 6.7214 f 17.2 173 f 7.6 109 f 12.0 

100.0 169 f 12.8146 f 7.8 241 f 13.8 228 f 5.9 151 f 16.2 

333.0 Toxic 283 f3291.3 f 7.4 321 f 17.0 244 f 5.6 

1,000.0 

3,333.0 

Trial summary 

Positive control' 

Strain Dose 

bglPlaW 

TAlOO 0.0 

(continued) 1.0 

3.3 

10.0 

33.0 

100.0 

333.0 

1,000.0 

3,333.0 

Trial summary 

Positive control 

Toxic 

Weakly Positive 

1,417 f 47.1 

Trial 1 

130 f 10.5 

164 f 12.1 

232 f 17.1 

274 f 9.5 

Toxic 

17 f 7.2 

Positive 

1,675 f 99.1 

5 f 2.9 

Positive Positive 

765 f 76.9 1,987 f 60.5 

Revertantdplate 

+lo%rat S9 

Trial 2 

163 f 5.2 

168 f 3.7 

182 f 9.2 

224 f 9.0 

351 & 15.6 

501 f 19.4 

Positive 

2,329 f 49.4 

542 f 29.7 

Positive 

1,688 k 111.4 

Trial 3 

133 f 10.2 

116 f 8.2 

126 f 1.2 

169 f 5.8 

227 f 2.7 

401 f 26.5 

Positive 

2,086 f 106.3 

373 f 65.2 

Positive 

1,314 f 35.7 
246 p-Nitrobenzoic Acid, NTP TR 442 

TABLEEl 

Mutagenicity of p-Nitrobenzoic Acid in Salmonella typhimurium (continued) 

Strain Dose -s9 +lo% hamster S9 +lo%rat S9 

(IrglplaW Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 

~~ 

TAl535 0.0 11 f 1.2 16 f 0.9 8 f 0.7 11 f 0.7 

1.o 

3.3 6 f 1.3 

10.05 f 1.3 5 f 1.5 

33.0 7 f 0.7 14 f 1.27 f 0.9 16 f 1.2 

100.0 9 f 0.7 17 f 1.5 8 f 1.2 15 f 0.9 

333.0 7 f 1.8 3 f 0.7 7 f 1.2 4 f 0.9 

1,Ooo.o 5 * 1.5 1 f 1.0 2 f 1.5 

3,333.0 , 0 * 0.0 0 f 0.0 

Trial summary Negative Negative Negative Negative 

Positive control 319 f 66.5 145 f 9.7 148 f 12.8 115 f 13.2 

TAl537 0.0 8 f 1.2 6 f 0.9 18 f 0.9 14 2 0.7 

1.o 4 f 1.5 

3.3 5 f 0.9 

10.0 10 f 0.7 5 f 2.7 

33.0 5 f 1.7 5 +. 0.6 14 f 0.6 14 * 3.0 

100.0 8 f 0.3 6 f 0.9 17 f 2.3 16 f 1.5 

333.0 0 f 0.3 15 f 0.3 10 f 3.9 

1,000.0 1 f 0.6 10 f 0.9 3 f 1.7 

3,333.0 4 f 0.7 1 f 0.3 

Trial summary Negative Negative Negative Negative 

Positive control 228 f35.3 166 2 75.8 335 f 42.9 323 f 44.3 

TA98 0.0 21 f 1.2 28 f 1.2 17 2 4.7 16 f 14 2.0 f 0.3

1.0 

3.3 22 2 0.9 18 f 2.6 

10.0 25 f 4.0 18 f 2.0 25 f 5.2 

33.022 

100.0 

f 2.9 30 f 1.5 20 f 2.2 24 f 2.9 24 f 3.1 

22 f 0.3 25 f 5.8 20f 4.0 20 f 6.5 14 f 0.3

333.0 21 f 0.9 20 f 3.3 18 f 3.6 8 f 2.0 19 f 3.5 

1,000.0 16 +. 0.3 19 f 3.8 10 f 2.9 

3,333.0 18 f 1.8 17 f 3.0 

Trial summary Negative Negative Negative Negative Equivocal 

Positive control 535 f 50.4 . 1,484 f 80.3 1,323f 73.5 1,837 f 135.5 1,793 f 49.9 

a Study performed at Case Western Reserve University. The detailed protocol and these data are presented in Zeiger et al. (1987). 

Revertants are presented as mean f standard error from three plates. 

2-Aminoanthracene was used on all strains in the presence of S9. In the absence of metabolic activation, 4-nitro-ophenylenediamine was tested on TA98, sodium azide was tested on TAlOO and TA1535, and 9-aminoacridine was tested on 

TA1537. 
Genetic Toxicology 247 

TABLEE2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by p-Nitrobenzoic Acida 

No. of scEs/ Relative SCEs/ 

Dose Total Chromo- N. of Chromo- SCEs/ Hrs in Chromosome 

Compound @g/mL) Cells somes SCEs some Cell BrdU (Wb 

-s9 

Trial 1 

Summary:Weakpositive 

Dimethylsulfoxide 

398 50

Mitomycin-C 

0.001 50 1,050 584 0.55 11.7 25.7 46.04 

0.010 5 105 245 2.33 49.0 25.7 512.65 

p-Nitrobenzoic acid 

0.38 1,045 8.0 25.7 

58.3 50 1,031 382 0.37 -2.72 7.6

8.6 

25.7 

175.0 50 1,041 429 0.41 8.20 25.7 

11.4 44.88; 

1,750.0 0 31.3 

P<0.001d 

Trial 2 

Summary:Positive 

Dimethylsulfoxide 

583.0 50 1,033 570 0.55 31.3' 

50 1,044 380 0.36 

Mitomycin-C 

0.001 

0.010 

50 

5 

1,042 

105 

580 

251 

0.55 

2.39 

11.6 

50.2 

25.5 

25.5 

52.93 

556.76 

p-Nitrobenzoic acid 

498 50 1,034 505 0.48 10.1 32.4' 34.18* 

753 50 1,045 604 0.57 12.1 32.4' 58.80* 

1,000 50 1,049 629 0.59 12.6 32.4' 64.74* 

1.510 0 

P<O.OOl 
248 p-Nitrobenzoic Acid, NTP TR 442 

TABLEE2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by p-Nitrobenzoic Acid (continued) 

No. of SCEs/ Relative SCEs/ 

Dose Total Chromo- No. of Chromo- SCEs/ Hrs in Chromosome 

Compound @g/mL)somes Cells SCEs some Cell BrdU (%) 

+s9 

Summary:Negative 

Dimethylsulfoxide 

50 1,041 346 0.33 6.9 25.5 

Cyclophosphamide . 

0.4 25 519 312 0.60 12.5 25.5 80.87 

2.0 5 105 157 1.49 31.4 25.5 349.87 

p-Nitrobenzoic acid 

175 50 1,044 393 0.37 7.9 25.5 13.26 

583 50 1,037 328 0.31 6.6 25.5 -4.84 

1,750 50 1,027 364 0.35 7.3 25.5 6.64 

P=O.485 

* Positive (F'50.01) a Study performed at Litton Bionetics, Inc. The protocol is presented in detail by Galloway et al. (1985); data published in Zeiger 

et uL (1987). SCE = sister chromatid exchange;BrdU = bromodeoxyuridine. 

SCEs/chromosome in treated cells versus SCEs/chromosomein solvent control cells. 

Because of chemical-induced cell cycle delay, incubation time was lengthened to ensure sufficient metaphasecells at harvest. * Significance of relative SCEs/chromosome tested by the linear regression trend testvs. log of the dose. 
Genetic Toxicology 249 

TABLEE3 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by p-Nitrobenzoic Acida 

200 3.0 8

Mitomycin-C 

0.05 50 22 

0.08 25 37 

p-Nitrobenzoic acid 

875 200 21 

1,313 200 30 

1,750 50 20 

Trial 2 - Harvest time: 21.5 hoursb 

Summary:Positive 

Dimethylsulfoxide 

-s9 +s9 

DoseTtal No. of Abs/ Cells Dose Ttal No. of Abs/ Cells 

(IrglmL) Cells Abs with Cell Abs (IrglmL) Cells Abs with Cell Abs

(%) (%I 

Trial 1 - Harvesttime: 20.5 hoursb Trial 1 - Harvest time: 12.5 hours 

Summary:Positive Summary:Equivocal 

Dimethylsulfoxide Dimethylsulfoxide 

0.04 103.5

0.44 

1.48 

0.11 

0.15 

0.40 

30.0 

56.0 

8.5: 

12.5* 

26.0* 

P<0.001C 

Cyclophosphamide 

7.5 

37.5 

p-Nitrobenzoic acid 

875 

1,313 

1,750 

Trial 2 - Harvest time: 

Summary:Negative 

Dimethylsulfoxide 

200 0.05 

200 30 0.15 12.0 

25 38 1.52 84.0 

200 13 0.07 5.5 

200 14 0.07 5.5 

200 24 0.12 9.0' 

P=O.O15 

12.5 hours 

0.51 0.51 

Mitomycin-C Cyclophosphamide 

0.05 200 48 0.24 15.0 7.5 200 16 0.08 7.0 

0.08 25 28 1.12 48.0 37.5 25 12 0.48 36.0 

p-Nitrobenzoic acid p-Nitrobenzoic acid 

439.5 200 3 0.02 1.5 1,249 200 2 0.01 1.0

879.0 200 7 0.04 3.5. 1,505 200 10 0.05 4.5* 

1,313.0 200 17 0.09 7.5* 1,750 200 3 0.02 1.5 

PCO.001 P =0.052 

* Positive (PsO.05) 

a Study performed at Litton Bionetics, Inc. A detailed presentation of the technique for detecting chromosomal aberrations is found 

in Galloway et aL (1985); data published in Zeiger et al. (1987). Abs = aberrations. 

Because of chemical-induced cell cycle delay, incubation time prior to addition of Colcemid was lengthened to ensure sufficient 

metaphase cells at harvest. 

Significance of percent cells with aberrations tested by the linear regression trend testvs. log of the dose. 

200 0.01 200 0.01 
250 p-Nitrobenzoic Acid, NTP TR 442 

TABLEE4 

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Administration 

of p-Nitrobenzoic Acid in Feed for 13 Weeksa 

Dose (PPm) 

Male 

0 

1,250 

2,500 

5,000 

10,000 

20,000 

Female 

0 

1,250 

2,500 

5,000 

10,000 

20,000 

Micronucleated Normochromatic 

Erythrocytes (%)b 

0.1412 f 0.0132 

0.1466 f 0.0119 

0.1313 f 0.0096 

0.1600 f 0.0111 

0.1250 & 0.0119 

0.1492 f 0.0092 

0.1031 f 0.0127 

0.0992 f 0.0062 

0.0997 f 0.0086 

0.1146 2 0.0078 

0.1453 & 0.0238 

0.1032 f 0.0058 

Pairwise 

Significance 

0.365 

0.750 

0.114 

0.869 

0.300 

P=O.424 

0.590 

0.583 

0.242 

0.009 

0.497 

P=O.215 

a Ten thousand normochromatic erythrocytes scored per animal. A detailed description of the protocol is found in MacGregor et at! 

(1990). 

Data presented as mean f standard error. Pairwise comparison of treated group to concurrent control by Student’s t-test. 

One-tailed trend test performed across all doses(ILS, 1990). 
251 

APPENDIX F 

ORGAN WEIGHTS 

AND ORGAN-MTEIGHT-TO-BODY-WEIGHTRATIOS 

TABLEF1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 14-Day Feed Study of p-Nitrobenzoic Acid .............................. 252 

TABLEF2 Organ Weights and Organ-Weight-to-Body-Weight Ratiosfor Rats 

in the 13-Week Feed Studyof p-Nitrobenzoic Acid ............................. 254 

TABLEF3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

at the 15-Month Interim Evaluation in the 2-Year Feed Study 

ofp-Nitrobenzoic Acid .................................................. 256 

TABLE F4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 14-Day Feed Study of p-Nitrobenzoic Acid .............................. 257 

TABLEF5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 13-Week Feed Study of p-Nitrobenzoic Acid ............................. 259 

TABLEF6 Organ Weights and Organ-Weight-to-Bodyweight Ratiosfor Mice 

at the 15-Month Interim Evaluation in the 2-Year Feed Study 

of p-Nitrobenzoic Acid .................................................. 261 
252 p-Nitrobenzoic Acid, NTP TR 442 

TABLEF1 

Organ Weights and Organ-Weight-to-Body-WeightRatios for Rats in the 14-Day Feed Study of p-Nitrobenzoic Acida 

0 PPm 2,500 ppm 5,000 ppm 10,000 ppm 20,000 ppm 40,000 ppm 

Male 

n 5 5 5 5 5 5 

Necropsy body wt 210 f 4 205 f 6 198 f 3 173 2 4** 127 f 5** 110 2 6** 

Brain 

Absolute 1.866 f 0.013 1.840 f 0.041 1.847 f 0.015 1.785 f 0.017: 1.727 f 0.013** 1.674 f 0.032** 

Relative 8.89 f 0.14 8.99 f 0.12 9.34 f 0.12 10.32 f 0.16** 13.68 f 0.47:: 15.33 f 0.58** 

Heart 

Absolute 0.856 f 0.019 0.859 f0.061 0.843 f 0.024 0.777 f 0.030 0.649 f 0.022** 0.550 f 0.041** 

Relative 4.08 f 0.09 4.18 2 0.22 4.26 f 0.11 4.49 f 0.15 5.13 f 0.20** 4.99 f 0.25'. 

R. Kidney 

Absolute 1,005 f 0.031 1.073 f 0.068 1.093 f 0.043 0.992 f 0.045 0.722 f 0.029** 0.631 f 0.032** 

Relative 4.79 f 0.11 5.22 f 0.19 5.52 f 0.19** 5.72 r 0.13** 5.69 f 0.12** 5.75 f 0.17** 

Liver 

Absolute 12.380 f 0.156 12.272 f 0.420 12.555 f 0.502 11.831 A 0.452 7.733 f 0.313** 6.145 f 0.268** 

Relative 58.97 2 0.62 59.86 f 0.51 63.39 k 1.93 68.25 5 1.31'. 60.96 f 1.09 56.11 5 2.14 

Absolute 

Lungs 

1.204 f 0.041 1.321 f 0.092 1.350 f 0.059 1.325 f 0.081 0.908 f 0.033** 0.881 f 0.022** 

Relative 5.73 & 0.15 6.43 f 0.30 6.82 f 0.30* 7.65 f 0.42** 7.16 f 0.07** 8.08 & 0.40** 

Spleen 

Absolute 0.533 f 0.017 0.556 2 0.020 0.553 f 0.018 0.640 f 0.038* 0.856 f 0.051** 0.779 f 0.018** 

Relative 2.54 f 0.10 2.71 f 0.06 2.80 f 0.09 3.69 f 0.19** 6.73 f OX** 7.14 f 0.30** 

R. Testis 

Absolute 1.208 ? 0.039 1.156 f 0.050 1.147 f 0.016 1.100 f 0.027 0.866 & 0.062** 0.775 f 0.063** 

Relative 5.76 f 0.19 5.64 f 0.17 5.80 f 0.12 6.35 f 0.08 6.79 f 0.24** 7.00 f 0.30** 

Absolute 

Thymus 

0.450 f 0.029 0.382 f 0.022 0.402 f 0.018 0.306 f 0.016** 0.158 f 0.023** 0.076 f 0.008** 

Relative 2.15 f 0.18 1.88 2 0.16 2.03 f 0.07 1.77 f 0.09 1.26 f OM** 0.69 f 0.07** 
Organ Weight Analyses 253 

TABLEF1 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 14-Day Feed Studyof p-Nitrobenzoic Acid 

(continued) 

0 PPm 2,500 ppm 5,000 ppm 10,000 ppm 20,000 ppm 40,000 ppm 

Female 

n 5 5 5 5 5 5 

Necropsy body wt 140 f 3 139 f 4 134 f 3 123 f 3** 95 & 4** 91 f 4** 

Brain 

Absolute 1.750 f 0.018 1.719 f 0.016 1.878 f 0.105 1.680 f 0.012 1.633 f 0.007 1.638 f 0.026 

Relative 12.48 f 0.20 12.38 f 0.42 14.07 f 1.09 13.73 f 0.33 17.29 f 0.65** 18.07 f 0.62** 

Heart 

Absolute 0.653 f 0.019 0.623 f 0.022 0.635 f 0.017 0.625 f 0.022 0.473 f 0.027**0.477 f 0.034** 

Relative 4.65 2 0.12 4.48 f 0.12 4.74 f 0.10 5.10 f 0.11 4.97 f 0.15 5.21 f 0.17** 

R. Kidney 

Absolute 0.734 f 0.010 0.704 f 0.015 0.694 f 0.026 0.654 f 0.022*0.507 f 0.022** 0.510 f 0.020** 

Relative 5.23 f 0.08 5.06 f 0.07 5.17 f 0.18 5.33 f 0.095.33 f 0.10 5.60 f 0.05* 

Liver 

Absolute 7.497 f 0.312 7.018 f 0.308 7.256 f 0.158 7.100 f 0.415 5.221 f 0.365** 5.228 f 0.231** 

Relative 53.34 f 1.52 50.34 f 1.47 54.16 f 1.55 57.75 f 2.12 54.81 f 2.39 57.41 f 1.45 

Lungs 

Absolute 0.968 f 0.026 0.988 f 0.043 1.021 f 0.023 0.911 f 0.0330.778 f 0.031**0.737 f 0.027** 

Relative 6.89 f 0.09 7.12 f 0.35 7.61 f 0.15 7.42 f 0.16 8.21 -r 0.29**8.14 f 0.43** 

Spleen 

Absolute 0.404 f 0.008 0.395 f 0.014 0.406 f 0.011 0.522 f 0.023** 0.516 f 0.032** 0.485 f 0.038** 

Relative 2.88 f 0.10 2.84 f 0.13 3.03 f 0.07 4.26 & 0.17** 5.44 f 0.33** 5.32 & 0.35** 

Thymus 

Absolute 0.346 f 0.010 0.361 f 0.020 0.331 f 0.007 0.295 f 0.012* 0.146 f 0.011** 0.110 f 0.028** 

Relative 2.47 2 0.09 2.60 f 0.17 2.47 f 0.09 2.42 f 0.131.54 f 0.10** 1.17 f 0.26** 

* Significantly different (PSO.05) from the control group by Williams’or Dunnett’s test 

** PSO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean f standard error) 
254 p-Nitrobenzoic Acid, NTP TR 442 

TABLEF2 

Organ Weights and Organ-Weight-to-Bodyweight Ratiosfor Rats in the 13-Week Feed Study of p-NitrobenzoicAcida 

0 PPm 630 ppm 1,250 ppm 2,500 ppm 5,000 ppm 10,000 ppm 

Male 

n 10 10 10 10 10 10 

Necropsy bodywt 362 f 5 367 f 5 348 f 6 347 f 4 330 f 7** 271 f 3** 

Brain 

Absolute 1.943 f 0.018 1.923 f 0.0211.925 f 0.019 1.941 f 0.022 1.882 f 0.026 1.863 f 0.018* 

Relative 5.38 f 0.07 5.24 f 0.05 5.55 f 0.11 5.59 f 0.06 5.73 f 0.15* 6.88 f 0.11** 

Heart 

Absolute 1.156 f 0.027 1.114 f 0.0201.107 f 0.038 1.110 f 0.015 1.039 f 0.019**0.918 f 0.021** 

Relative 3.20 f 0.07 3.04 f 0.05 3.18 f 0.09 3.20 f 0.043.15 f 0.073.39 f 0.07 

R. Kidney 

Absolute 1.284 f 0.032 1.331 f 0.029 1.297 f 0.031 1.341 f 0.024 1.318 f 0.027 1.118 f 0.013** 

Relative 3.55 f 0.05 3.62 2 0.05 3.73 f 0.06* 3.86 f 0.06** 4.00 f 0.05** 4.13 f 0.05** 

Liver 

Absolute 12.899 f 0.312 13.802 f 0.241 12.765 f 0.256 13.263 f 0.218 12.713 f 0.252 12.131 f 0.185* 

Relative 35.63 f 0.40 37.61 f 0.40* 36.72 f 0.37. 38.18 f 0.42.. 38.54 f 0.44** 44.80 f 0.67** 

Lungs 

Absolute 1.693 f 0.077 2.003 f 0.1361.741 f 0.111 1.734 f 0.067 1.792 f 0.112 1.522 f 0.075 

Relative 4.68 f 0.19 5.45 f 0.36 5.01 f 0.31 4.99 f 0.17 5.46 f 0.39 5.63 f 0.29* 

Spleen 

Absolute 0.785 f 0.027 0.822 f 0.0230.776 f 0.015 0.800k 0.0110.806 f 0.016 1.073 f 0.023** 

Relative 2.17 f 0.06 2.24 f 0.05 2.23 f 0.03 2.30 f 0.022.45 f 0.07**3.96 f 0.09** 

R. Testis 

Absolute 1.488 f 0.024 1.478 f 0.0321.455 f 0.027 1.435 f 0.026 1.373 f 0.020**0.408 f0.024** 

Relative 4.12 f 0.04 4.03 f 0.08 4.18 f 0.03 4.13 f 0.064.17 f 0.061.50 f 0.08" 

Absolute 

Thymus 

0.333 f 0.020 0.355 f 0.0220.312 f 0.015 0.314 f 0.0140.304 f 0.0180.233 f 0.011** 

Relative 0.92 f 0.04 0.97 f 0.06.90 k 0.04 0.90 f 0.03 0.92 f 0.05 0.86 f 0.04 
Organ Weight Analyses 255 

TABLEF2 

Organ Weights and Organ-Weight-to-Body-WeightRatios for Rats in the 13-Week Feed Study of p-Nitrobenzoic Acid 

(continued) 

~~ ~ 

0 PPm 630ppm 1,250 ppm 2,500 ppm 5,000 ppm 10,000 ppm 

Female 

n 10 10 10 10 10 10

Necropsy body wt 205 f 3 206 f 3 202f3 201 f 3 185 f 3': 171 f 2** 

Brain 

Absolute 1.789 f 0.025 1.822 f 0.015 1.805 f 0.013 1.797 -C 0.026 1.805 f 0.011 -1.780 f 0.027 

Relative 8.72 f 0.13 8.86 f 0.14 8.95 f 0.16 8.94 f 0.11 9.79 f 0.12:: 10.41 f 0.20:: 

HtXrt 

Absolute 0.705 f 0.011 0.714 f 0.017 0.703 f 0.018 0.711 f 0.017 0.691 f 0.010 0.656 f 0.012: 

Relative 3.44 f 0.05 3.48 f 0.10 3.48 f 0.07 3.54 f 0.08 3.74 f 0.05:: 3.83 f 0.06:: 

R. Kidney 

Absolute 0.763 f 0.015 0.747 f 0.016 0.736 f 0.015 0.717 f 0.017:0.683 f 0.014:: 0.681 f 0.007** 

Relative 3.72 f 0.08 3.63 f 0.06 3.64 f 0.06 3.56 f0.05 3.70 f 0.053.98 f 0.04:' 

Liver 

Absolute 6.582 f 0.104 7.030 f 0.138 6.626 f 0.125 6.675 f 0.114 6.257 f 0.101 6.187 f0.104: 

Relative 32.10 f 0.56 34.14 f 0.48 32.79 f 0.54 33.20 f 0.42 33.89 2 0.34: 36.16 f 0.69:: 

Lungs 

Absolute 1.157 f0.052 1.124 f 0.042 1.139 f 0.050 1.255 f 0.105 1.098 f 0.031 1.007 f 0.034 

Relative 5.62 f 0.18 5.46 f 0.20 5.65 f 0.27 6.22 f 0.49 5.94 f 0.13 5.88 f 0.17 

Spleen 

Absolute 0.524 f 0.020 0.522 f 0.007 0.498 f 0.016 0.545 f 0.017 0.549 f 0.0060.676 f 0.011** 

Relative 2.55 f0.10 2.54 f 0.04 2.46 f 0.07 2.71 f 0.05 2.98 ? 0.05:: 3.95 f 0.05:: 

Thymus 

Absolute 0.277 f 0.011 0.287 f 0.009 0.280 f 0.009 0.295 f 0.016 0.266 f 0.012 0.2412 f 0.008* 

Relative 1.35 f 0.05 1.40 f 0.06 1.38 f 0.05 1.46 f 0.07 1.44 f 0.06 1.41 f 0.04 

* Significantly different (P50.05) from the control group by Williams' or Dunnett's test 

** P50.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean f standard error). 
256 p-Nitrobenzoic Acid, NTP TR 442 

TABLEF3 

Organ Weights and Organ-Weight-to-Body-Weight Ratiosfor Rats at the 15-Month Interim Evaluation 

in the 2-Year Feed Studyof p-Nitrobenzoic Acida 

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

Male 

n 10 9 10 10 

Necropsy body wf 483 f 7 488 f 9 490 -t 8 475 -c 5 

R. Kidney 

Absolute 1.697 f 0.045 1.720 k 0.042 1.700 -t 0.037 1.744 f 0.042 

Relative 3.51 f 0.07 3.52 f 0.07 3.47 f 0.07 3.67 f 0.08 

Liver 

Absolute 17.192 f 0.398 16.933 f 0.554 16.238 -t 0.393 17.289 f 0.353 

Relative 35.58 f 0.44 34.64 f 0.76 33.14 f 0.69* 36.37 2 0.67 

Spleen 

Absolute 1.ils f 0.058 1.112 f 0.069 1.065 f 0.031 1.046 f 0.020 

Relative 2.31 f 0.12 2.28 f 0.13 2.18 f 0.07 2.20 k 0.04 

Female 

n 10 10 10 10 

Necropsy body wt 297 f 7 297 f 7 280 2 5 260 f 4** 

R. Kidney 

Absolute 1.000 f 0.026 0.984 f 0.016 0.920 f 0.019** 0.897 f 0.017** 

Relative 3.37 f 0.07 3.32 f 0.05 3.29 f 0.05 3.46 f 0.06 

Liver 

Absolute 9.103 f 0.257 9.575 k 0.280 8.842 k 0.174 9,480 k 0.279 

Relative 30.69 f 0.63 32.17 f 0.30 31.60 f 0.56 36.49 f 0.81** 

Spleen 

Absolute 0.512 f 0.021 0.585 f 0.024* 0.583 f 0.018’ 0,671 f 0.022** 

Relative 1.73 f 0.07 1.97 f 0.07* 2.08 f 0.05** 2.59 f 0.08** 

* Significantly different (P50.05) from the control group by Williams’ or Dunnett’s test 

** PSO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean k standard error). 
Organ Weight Analyses 257 

TABLEF4 

Organ Weights and Organ-Weight-to-Bodyweight Ratiosfor Mice in the 14-Day Feed Study of p-Nitrobenzoic Acida 

0 PPm 2,500 ppm 5,000 ppm 10,000 ppm 20,000 ppm 40,000 ppm 

Male 

n 5 5 5 5 5 2 

Necropsy body wt 19.5 f 0.9 19.1 f 0.7 18.4 f 0.519.0 f 0.417.9 f 0.5 18.0 f 0.4 

Brain 

Absolute 0.469 f 0.008 0.455 f 0.006 0.447 f 0.008 0.457 f 0.007 0.455 f 0.003 0.469 f 0.020 

Relative 24.30 f 1.00 23.92 f 0.98 24.42 f 0.66 24.02 f 0.35 25.51 f 0.81 26.12 f 0.60 

Heart 

Absolute 0.159 f 0.011 0.149 f 0.006 0.154 f 0.007.151 f0.005.153 k 0.008.142 f 0.006 

Relative 8.17 f 0.44 7.77 f 0.14 8.37 f 0.24 7.96 f 0.398.56 5 0.317.89 f 0.46 

R. Kidney 

Absolute 0.246 f 0.012 0.210 f 0.016 0.185 f 0.007;; 0.205 f 0.010 0.196 k 0.009; 0.226 & 0.013 

Relative 12.74 f 0.78 10.94 f 0.63 10.06 f 0.31* 10.76 f 0.60 11.03 f 0.74 12.58 f 0.48 

Liver 

Absolute 1.334 2 0.046 1.111f 0.054 1.084 f 0.025 1.265 f 0.027 1.337 k 0.031 1.518 f 0.069; 

Relative 68.77 f 1.78 58.08 f 1.51 59.07 f 0.93 66.46 f 1.35 75.00 k 2.54; 84.53 f 2.20;. 

Lungs 

Absolute 0.216 f 0.009 0.192 f 0.011 0.192 f 0.007 0.203 f 0.011 0.226 5 0.007 0.227 f 0.051 

Relative 11.22 f 0.67 10.00 f0.26 10.48 f0.41 10.70 f 0.61 12.70 k 0.76 12.57 f 2.57 

Spleen 

Absolute 0.058 f 0.002 0.060 & 0.002 0.055 f 0.0020.062 f 0.003 0.060 f 0.0060.048 f 0.005 

Relative 2.99 f 0.10 3.12 f 0.09 3.02 f 0.023.26 f 0.133.35 f0.28 2.64 f 0.20 

R. Testis 

Absolute 0.100 f 0.004 0.103 f 0.004 0.100 f 0.005 0.100 f 0.0030.097 f 0.005.093 & 0.005 

Relative 5.18 f 0.17 5.42 f 0.29 5.47 f 0.235.27 f 0.085.41 f 0.175.15 f 0.15 

Thymus 

Absolute 0.065 f 0.004 0.051 f 0.006 0.057 f 0.0050.067 f 0.0100.039 +. 0.006;0.037 f 0.012; 

Relative 3.36 f 0.30 2.70 f 0.31 3.14 f 0.323.57 f 0.562.16 f 0.31;2.05 f 0.68 
258 p-NitrobenzoicAcid, NTP TR 442 

TABLEF4 

Organ Weights and Organ-Weight-to-Body-Weight Ratiosfor Mice in the 14-Day Feed Study of p-Nitrobenzoic Acid 

(continued) 

0 PPm 2,500 5,000 ppmppm10,000 20,000 ppmpm40,000 ppm

Female 

n 5 5 5 5 5 3 

Necropsy bodywt 16.8 f 0.7 16.5 f 0.4 15.9 f 0.4 15.3 f 0.2* 14.6 f 0.3** 15.3 f 0.7' 

Brain 

Absolute 0.468 f 0.007 0.444 f 0.008 0.453 f 0.007 0.441 f 0.003* 0.443 & 0.008* 0.446 f 0.011' 

Relative 28.00 f 0.85 27.00 f 0.69 28.48 2 0.44 28.86 f 0.22 30.32 f 0.60 29.36 k'1.90 

Heart 

Absolute 0.140 f 0.005 0.126 f 0.005 0.120 f 0.003** 0.115 f 0.005** 0.111 f 0.004'. 0117 f 0.012**

Relative 8.39 k 0.27 7.65 f 0.21756 f 0.30* 7.50 f 0.31 7.59 f 0.14 7.66 f 0.49

R.Kidney 

Absolute 0.197 f 0.013 0.143 f 0.006** 0.144 f 0.005** 0.143 f 0.009** 0.151 f 0.012** 0.164 f 0.010 

Relative 11.76 f 0.76 8.70 f 0.28" 9.06 f 0.19** 9.33 -C 0.53* 10.27 f 0.72 10.76 f 0.29 

Liver 

Absolute 1.128 f 0.059 0.967 f 0.025 1.001 f 0.026 1.130 f 0.008 1.180 f 0.040 1.243 f 0.070 

Relative 67.10 f 1.42 58.71 f 0.89 63.05 f 2.45 73.92 f 1.11* 80.58 f 2.11** 81.68 f 4.96.. 

Lungs 

Absolute 0.214 f 0.014 0.182 f 0.012 0.165 2 0.005* 0.177 f 0.010 0.184 f 0.012 0.197 f 0.006 

Relative 12.67 f 0.29 11.01 f 0.63 10.40 f 0.29* 11.56 f 0.56 12.59 f 0.74 13.02 f 1.01 

Spleen 

Absolute 0.065 f 0.005 0.063 f 0.005 0.061 f 0.003 0.058 f 0.004 0.051 f 0.006* 0.050 f 0.005 

Relative 3.86 f 0.19 3.81 f 0.26 3.80 f 0.13 3.79 f 0.26 3.43 f 0.37 3.26 f 0.20 

Thymus 

Absolute 0.082 f 0.006 0.071 f 0.0080.069 f 0.0090.046 f 0.004** 0.035 f 0.005** 0.038 f 0.002** 

Relative 4.88 f 0.36 4.29 f 0.42 4.38 f 0.652.98 f 0.29** 2.35 f 0.34** 2.49 f 0.22** 

* Significantly different (PSO.05) from the control group by Williams' or Dunnett's test 

** P50.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean fstandard error) 
Organ Weight Analyses 259 

TABLEF5 

Organ Weights and Organ-Weight-to-Body-Weight Ratiosfor Mice in the 13-Week Feed Study of p-Nitrobenzoic Acida 

1,250 ppm 2,500 ppm 5,000 ppm 10,000 ppm 20,000 ppm 

Male 

n 10 10 10 10 10 10 

Necropsy body wt 34.0 f 0.7 31.7 f 0.6* 31.8 f 1.2 30.8 f 0.8**27.6 f 0.4**24.1 f 0.6** 

Brain 

Absolute 0.466 f 0.007 0.481 f 0.012 0.487 f 0.009 0.494 f 0.008 0.482 rt 0.012 0.465 f 0.005 

Relative 13.78 f 0.36 15.20 f 0.43* 15.51 f 0.57** 16.11 f 0.44** 17.50 f 0.44** 19.35 f 0.44** 

Heart 

Absolute 0.160 f 0.007 0.157 f 0.003 0.156 f 0.005 0.155 f 0.0040.137 f 0.004**0.126 f 0.006** 

Relative 4.71 f 0.16 4.96 f 0.05 4.94 f 0.18 5.03 f 0.104.98 f 0.115.23 f 0.27 

R. Kidney 

Absolute 0.295 f 0.007 0.310 f 0.008 0.316 f 0.010 0.302 k 0.0120.264 f 0.011*0.211 f 0.005** 

Relative 8.74 f 0.33 9.76 f 0.17' 10.01 f 0.25** 9.78 f 0.25* 9.57 f 0.358.77 f 0.16 

Liver 

Absolute 1.431 f 0.050 1.416 f 0.048 1.470 f0.071 1.524 f 0.050 1.346 f 0.032 1.130 f 0.047** 

Relative 42.09 f 1.02 44.58 f 1.05 46.22 2 1.21** 49.47 f 1.11** 48.81 f 1.08** 46.68 f 0.92** 

Lungs 

Absolute 0.203 f 0.006 0.227 f 0.010 0.221 f 0.013 0.217 f 0.0120.222 f 0.0180.216 f 0.012 

Relative 6.00 f 0.23 7.21 f 0.38 7.05 f 0.51 7.07 f 0.398.02 f 0.61**9.04 f 0.66** 

Spleen 

Absolute 0.071 f 0.002 0.071 f 0.003 0.072 f 0.004 0.077 f 0.005 0.075 f 0.005 0.052 f 0.004** 

Relative 2.08 f 0.05 2.23 rt 0.09 2.26 f 0.08 2.50 f 0.14* 2.73 rt 0.17* 2.11 f 0.13 

R. Testis 

Absolute 0.124 f 0.001 0.123 f 0.002 0.139 f 0.003 0.120 f 0.003b 0.127 f 0.006 0.097 f 0.002** 

Relative 3.65 f 0.09 3.88 f 0.08 4.41 f 0.19* 3.89 f O.lO*b 4.60 f 0.19** 4.05 f 0.11** 

Thymus 

Absolute 0.055 f 0.003 0.048 f 0.003 0.051 f 0.006 0.051 f 0.005 0.041 f 0.003 0.044 f 0.004 

Relative 1.60 f 0.07 1.51 f 0.09 1.58 f 0.17 1.63 f 0.14 1.48 f 0.13 1.78 f 0.12 
260 p-Nitrobenzoic Acid, NTP TR 442 

TABLEF5 

Organ Weights and Organ-Weight-to-Body-WeightRatios for Mice in the 13-Week Feed Study of p-Nitrobenzoic Acid 

(continued) 

0 PPm 1,250 2,500 ppmppm5,000 10,000 ppmpm20,000 ppm

Female 

n 10 9 10 10 10 10 

Necropsy bodywt 26.4 f 0.6 26.2 f 0.4 26.8 f 0.6 25.2 f 0.3 22.8 f 0.3** 20.6 f 0.4** 

Brain 

Absolute 0.520 f 0.012 0.495 f 0.011 0.511 f 0.010 0.500 f 0.010 0.499 f 0.011 0.473 f 0.011** 

Relative 19.77 f 0.45 18.95 f 0.60 19.16 f 0.50 19.85 f 0.51 21.96 f OM** 23.05 f 0.71** 

Heart 

Absolute 0.143 f 0.004 0.128 f 0.004 0.139 f 0.005 0.131 f 0.004b 0.125 f 0.003*0.123 f 0.008** 

Relative 5.45 f 0.19 4.89 & 0.12 5.18 f 0.15 5.19 f 0.16b 5.51 f 0.16 5.98 f 0.40

R. Kidney 

Absolute 0.212 f 0.009 0.212 f 0.007 0.226 f 0.008 0.208 f 0.005 0.188 f 0.005* 0.171 f 0.005** 

Relative 8.06 f 0.31 8.10 

*0.28 8.45 f 0.23 8.25 f 0.20 8.25 f 0.20 8.29 f 0.19

Liver 

Absolute 1.345 f 0.057 1.167 f 0.098 1.401 f 0.049 1.272 f 0.037 1.230 f 0.041 1.095 f 0.024** 

Relative 50.95 f 1.45 44.82 f 3.86 52.30 f 1.17 50.40 f 1.16 54.05 f 1.59 53.23 f 0.69 

Lungs 

Absolute 0.198 f 0.012 0.200 f 0.007 0.207 f 0.011 0.241 f 0.025 0.223 f 0.0130.239 f 0.012 

Relative 7.49 f 0.40 7.65 f 0.34 7.76 f 0.36 9.50 f 0.88** 9.81 f 0.53**11.56 f 0.42** 

Spleen 

Absolute 0.090 f 0.005 0.090 f 0.004 0.103 f 0.005 0.095 f 0.004 0.084 f 0.0070.061 f 0.005** 

Relative 3.41 f 0.16 3.45 f 0.14 3.85 f 0.14 3.75 f 0.14 3.68 f 0.26 2.95 f 0.21 

Thymus 

Absolute 0.065 f 0.005 0.054 f 0.003 0.064f 0.004 0.049 f 0.002* 0.056 f 0.005* 0.043 f 0.003.. 

Relative 2.46 f 0.16 2.07 f 0.13 2.40 f 0.14 1.94 f 0.10 2.47 f 0.25.06 f 0.14

l Significantly different (PSO.05) from the control group by Williams’or Dunnett’s test 

** PSO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratiosare given as mg organ weight/g body weight 

(mean f standard error). 

n=9 
Organ Weight Analyses 261 

TABLEF6 

Organ Weights and Organ-Weight-to-Body-Weight Ratiosfor Mice at the 15-Month Interim Evaluation 

in the 2-Year Feed Studyof p-NitrobenzoicAcida 

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

Male 

n 10 10 10 10 

Necropsy body wt 47.8 f 1.5 44.3 f 1.7 47.9 & 1.6 46.2 f 1.5 

R. Kidney 

Absolute 0.397 f 0.015 0.356 f 0.007 0.379 f 0.013 0.363 f 0.014 

Relative 8.32 & 0.24 8.11 & 0.26 7.94 & 0.24 7.86 f 0.23 

Liver 

Absolute 2.083 f 0.157 2.427 & 0.377 2.001 & 0.129 1.906 & 0.098 

Relative 43.40 & 2.57 57.08 rt 11.55 41.64 rt 1.88 41.05 & 0.93 

Spleen 

Absolute 0.085 f 0.013 0.081 f 0.009 0.082 f 0.006 0.072 f 0.005 

Relative 1.78 f 0.24 1.90 f 0.31 1.73 f 0.15 1.55 f 0.09 

Female 

n 10 10 9 10 

Necropsy body wt 48.9 f 1.2 46.3 & 2.3 43.1 f 1.5* 38.9 f 1.0** 

R. Kidney 

Absolute 0.272 f 0.005 0.275 f 0.005 0.268 f 0.008 0.279 & 0.021 

Relative 5.59 ? 0.16 6.06 f 0.28 6.29 f0.32 7.32 & 0.78* 

Liver 

Absolute 1.734 f 0.111 1.714 f 0.038 1.662 & 0.045 1.649 f 0.049 

Relative 35.54 f 2.22 37.66 f 1.60 38.95 f 1.55 42.58 f 1.56** 

Spleen 

Absolute 0.108 f 0.007 0.093 f 0.003 0.116 f 0.012 0.103 f 0.004 

Relative 2.21 f 0.14 2.08 & 0.17 2.80 f 0.44 2.67 f 0.13 

* Significantly different (PSO.05) from the control group by Williams’ or Dunnett’s test 

** PSO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean & standard error). 
262 p-Nitrobenzoic Acid, NTP TR 442 
263 

APPENDIX G 

HEMATOLOGY AND CLINICAL CHEMISTRY RESULTS 

TABLE G1 Hematology Data for Rats in the 14-Day Feed Study 

of p-Nitrobenzoic Acid .................................................. 264 

TABLE 62 Hematology and Clinical Chemistry Data for Special Study Rats 

in the 13-Week Feed Study of p-Nitrobenzoic Acid ............................. 266 

TABLE63 Hematology Data for Rats at the 15-Month Interim Evaluation 

in the 2-Year Feed Study of p-Nitrobenzoic Acid .............................. 270 

TABLE 64 Hematology Data for Mice in the 14-Day Feed Study 

of p-Nitrobenzoic Acid .................................................. 271 

TABLE65 Hematology Data for Mice at the 15-Month Interim Evaluation 

in the 2-Year Feed Study of p-Nitrobenzoic Acid .............................. 273 
264 p-Nitrobenzoic Acid, NTP TR 442 

TABLEG1 

Hematology Datafor Rats in the 14-Day Feed Studyofp-Nitrobenzoic Acida 

~~ 

0 PPm 2,500 ppm 5,000 ppm 10,000 ppm 20,000 ppm 40,000 ppm 

Male 

n 5 5 5 5 5 5 

Hematocrit (%) 

43.7 f 0.8 44.2 f 0.5 44.1 f 0.6 42.3 f 1.0 36.9 f 0.6** 37.4 f 0.9'* 

Hemoglobin (g/dL) 

15.5 f 0.2 15.5 f 0.1 15.3 f 0.2 14.8 f 0.3 13.5 f 0.3** 13.5 f 0.3** 

Erythrocytes (106/pL) 

8.13 f 0.16 8.09 f 0.10 8.22 f 0.10 7.77 f 0.15 6.51 f 0.18** 6.22 f 0.31** 

Mean cell volume (fL) 

53.8 f 0.5 54.8 f 1.0 53.6 * 0.4 54.6 f 0.8 56.6 f 0.8. 60.6 f2.0** 

Reticulocytes (106/bL) 

0.2 f0.0 0.4 f 0.1 0.4 f O.O* 0.5 f 0.1** 0.9 f 0.1** 1.3 f 0.2** 

Leukocytes (103/bL) 

9.84 f0.57 9.42 2 0.51 9.38 f 1.14 10.84 f 0.73 19.44 _+ 1.38** 15.78 f 1.62** 

Segmented neutrophils (103/bL) 

1.16 f 0.20 1.24 f 0.25 1.31 f 0.16 1.73 & 0.16 3.48 f0.92** 2.58 f 0.39** 

Lymphocytes (103/bL) 

8.36 f0.57 7.95 f 0.58 7.90 f 1.08 8.95 f 0.69 15.66 f 0.78** 12.86 f 1.22* 

Monocytes (103/bL) 

0.25 f0.03 0.19 f 0.05 0.16 f 0.03 0.17 f 0.04 0.25 f 0.09 0.34 f0.13 

Eosinophils (103/&L) 

0.06 f 0.03 0.04 f 0.02 0.02 f 0.02 0.00 f o.oo* 0.05 f 0.05 0.00 fo.oo* 

Nucleated erythrocytes (103/bL) 

0.12 * 0.05 0.06 -C 0.04 0.17 f0.08 2.88 f 0.84** 37.14 f 4.60** 47.76 f 6.91** 

Methemoglobin (I hemoglobin) 

0.14 f 0.05 0.11 f 0.01 0.27 f 0.01 0.38 f 0.13 3.06 f 0.20** 2.48 f 0.18** 
Hematology and Clinical Chemistry 265 

TABLEG1 

Hematology Data for Rats in the 14-Day Feed Studyof p-NitrobenzoicAcid (continued) 

0 PPm 2,500 ppm 5,000 ppm 10,000 ppm 20,000 ppm 40,000 ppm 

Female 

n 5 5 5 5 5 5 

Hematocrit (%) 

44.7 f 0.5 45.0 f 0.3 44.8 ? 0.4 40.6 f 0.7* 37.8 f 0.6** 38.5 f 1.2** 

Hemoglobin (g/dL) 

15.8 f 0.2 16.0 f 0.1 15.8 f 0.2 14.4 f 0.2. 13.4 f 0.2** 13.7 f 0.4** 

Erythrocytes (106/pL) 

8.27 f 0.12 8.36 f 0.08 8.45 f 0.10 7.47 f 0.11: 7.12 f 0.10:: 7.24 f 0.32* 

Mean cell volume (E) 

54.0 f 0.3 53.8 f 0.4 53.2 f 0.2 54.4 f 0.5 53.2 f 0.8 53.2 k 0.7 

Reticulocytes (106/pL) 

0.2 f 0.0 0.3 & O.O* 0.2 & 0.0 0.8 f 0.1** 0.7 f 0.1** 0.3 f 0.1* 

Leukocytes (103/pL) 

10.38 f 0.64 10.42 k 0.64 10.50 f 1.07 10.28 k 0.70 13.50 f 1.25 10.92 f 0.79 

Segmented neutrophils(103/pL) 

1.35 f 0.12 1.48 f 0.28 1.46 + 0.14 1.47 f 0.07 2.14 f 0.25* 1.81 f 0.33 

Lymphocytes (103/pL) 

8.69 f 0.82 8.64f 0.48 8.66 f 0.96 8.50 k 0.69 11.04 f 1.18 8.89 2 0.80 

Monocytes (103/pL) 

0.29 f 0.07 0.26 f 0.10 0.33 f 0.08 0.26 f 0.07 0.31 f 0.10 0.22 f 0.10 

Eosinophils (103/pL) 

0.05 f 0.04 0.04 f 0.02 0.04 f 0.03 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 

Nucleated erythrocytes(103/bL) 

0.10 f 0.04 0.14 f 0.08 0.06 f 0.02 2.42 f 0.45* 9.03 f 2.38** 12.26 f 5.35** 

Methemoglobin (% hemoglobin) 

0.09 f 0.02 0.16 f 0.02. 0.35 f 0.02.. 0.53 f 0.03** 1.63 f 0.13** 1.27 f 0.15** 

** PSO.01 

* Significantly different (PSO.05) from the control group by Dunn’s or Shirley’s test 

a Mean f standard error 
266 p-Nitrobenzoic Acid, NTP TR 442 

TABLE62 

Hematology and Clinical Chemistry Data for Special Study Rats in the 13-Week Feed Study 

of p-Nitrobenzoic Acida 

~ 

0 PPm 630 ppm 2,500 ppm 10,000 ppm 

Male 

n 10 10 10 10 

Hematology 

Hematocrit (%) 

Day 7 42.8 f 0.9 42.0 f 0.5 43.9 f 0.6 43.7 f OSb 

Day 30 48.0 f 0.7 46.6 f 0.8 46.6 f 0.9 44.5 k 0.7** 

Day 60 45.6 f 0.3' 45.1 f 0.6' 44.8 f 0.7' 43.6 f OS* 

Day 90 45.0 f 0.7 44.3 * 0.5 43.7 f 0.6 42.9 f 0.6 

Hemoglobin (g/dL) 

Day 7 15.1 f 0.1 15.1 f 0.2 15.5 f 0.2 15.4 f 0.2b 

Day 30 16.9 f 0.2 16.6 f 0.2 16.5 f 0.2 15.5 f 0.1** 

Day 60 16.8 2 0.2' 16.8 f 0.3' 16.8 f 0.3' 15.5 f 0.2:. 

Day 90 16.5 f 0.2 16.6 f 0.2 16.4 f 0.2 15.5 f O.P* 

Elythrocytes (106/bL) 

Day 7 7.00 f 0.15 6.92 f 0.11 7.11 f 0.12 7.18 f 0.08b 

Day 30 9.12 f 0.13 8.84 f 0.12 8.89 f 0.15 7.83 f 0.13** 

Day 60 9.62 f 0.09' 9.28 f 0.12.' 9.46 f 0.13' 8.32 f 0.17** 

Day 90 9.37 f 0.13 9.09 f 0.14 9.08 f 0.13 8.18 f 0.12** 

Mean cell volume (a) 

Day 7 61.2 f 1.1 60.9 f 1.3 61.9 f 1.0 60.9 f O.Sb 

Day 30 52.5 f 0.4 52.6 f 0.3 52.5 f 0.4 56.8 f 0.4** 

Day 60 47.4 f 0.2= 48.6 f 0.3*' 47.4 f 0.2' 52.5 f 0.7:: 

Day 90 48.0 f 0.2 48.7 f 0.3* 48.2 f 0.3 52.4 f 0.3** 

Mean cell hemoglobin (pg) 

Day 7 21.7 f 0.5 21.9 f 0.4 21.9 f 0.4 21.4 f 0.3b 

Day 30 18.6 f 0.2 18.8 f 0.1 18.7 f 0.3 19.9 f 0.2** 

Day 60 17.5 f 0.2' 18.1 f 0.1*' 17.7 f 0.2' 18.6 f 0.2:: 

Day 90 17.6 f 0.1 18.2 f 0.2* 18.1 f 0.2* 18.9 f 0.2** 

Mean cell hemoglobin concentration (g/dL) 

Day 7 35.5 f 0.8 36.0 f 0.2 35.4 f 0.2 35.2 f 0.3b 

Day 30 35.3 2 0.4 35.7 f 0.4 35.6 f 0.6 35.0 f 0.4 

Day 60 36.9 f 0.4' 37.2 f 0.4' 37.4 f 0.4' 35.6 f 0.3* 

Day 90 36.7 f 0.4 37.4 f 0.4 37.7 f 0.5 36.1 f 0.3 

Reticulocytes (106/bL) 

Day 7 0.16 f 0.02 0.15 f 0.02 0.17 f 0.02 0.23 f 0.03*b 

Day 30 0.07 f 0.01 0.11 f 0.01:: 0.09 f 0.01 0.26 f 0.03.' 

Day 60 0.09 f 0.02' 0.08 f 0.01' 0.11 f 0.01' 0.20 f 0.03:: 

Day 90 0.08 * 0.01 0.06 * 0.01 0.09 k 0.01 0.15 f 0.02: 

Leukocytes (103/bL) 

Day 7 5.48 f 0.39 5.60 f 0.14 5.81 f 0.41 5.30 f 0.33b 

Day 30 7.98 f 0.29 7.55 f 0.24 7.59 f 0.28 9.35 f 0.45 

Day 60 7.06 f 0.49' 7.41 f 0.51' 7.17 f 0.22' 7.34 f 0.39 

Day 90 7.46 f 0.55 7.34 f 0.54 8.14 f 0.46 8.@0f 0.69 

Segmented neutrophils (103/pL) 

Day'7 0.49 f 0.06 0.56 f 0.07 0.60 f 0.05 0.76 f 0.07**b 

Day 30 0.90 f 0.09 0.92 f 0.08 1.09 f 0.12 2.01 & 0.12** 

Day 60 1.44 f 0.20' 1.31 2 0.11' 1.24 2 0.16' 1.82 f 0.12 

Day 90 1.54 f 0.07 1.61 -+ 0.13 1.83 f 0.15 2.31 f 0.20** 
Hematology and Clinical Chemistry 267 

TABLE62 

Hematology and Clinical Chemistry Data for Special Study Rats in the 13-Week Feed Study 

of p-Nitrobenzoic Acid(continued) 

Male (continued) 

n 

Hematology (continued) 

Lymphocytes (103/fiL) 

Day 7 

Day 30 

Day 60 

Day 90 

Monocytes (103/bL) 

Day 7 

Day 30 

Day 60 

Day 90 

Eosinophils (103/fiL) 

Day 7 

Day 30 

Day 60 

Day 90 

Heinz bodies (% RBC) 

Day 7 

Day 30 

Day 60 

Day 90 

Methemoglobin (% hemoglobin) 

Day 7 

Day 30 

Day 60 

Day 90 

Clinical Chemistry 

Alkaline phosphatase (IUL) 

Day 7 

Day 30 

Day 60 

Day 90 

Alanine aminotransferase (IUL) 

Day 7 

Day 30 

Day 60 

Day 90 

Sorbitol dehydrogenase (IUL) 

Day 7 

Day 30 

Day 60 

Day 90 

0 PPm 

10 

4.91 f 0.39 

6.95 f 0.27 

5.40 f0.43' 

5.86 f 0.52 

0.06 f 0.02 

0.13 f 0.03 

0.10 f 0.03' 

0.01 f 0.01 

0.01 f 0.01 

0.05 f 0.02 

0.11 f0.04' 

0.07 f 0.02 

0.22 f 0.03 

0.10 & 0.02 

0.09 f 0.04' 

0.10 f 0.02 

0.39 f 0.06 

0.49 k 0.21 

0.72 f 0.09' 

0.79 f 0.09 

326 f 7 

252 f 11 

146 f 6 

129 & 9 

26*1 

40 f 2 

42 & 2 

42 f 2 

63 & 6 

83 f 9 

81 f 6 

65 f 6 

630 ppm 

10 

4.96 f 0.13 

6.40 +. 0.23 

5.98 f 0.47' 

5.61 f 0.47 

0.04 f 0.02 

0.12 f 0.03 

0.06 f 0.02' 

0.02 f 0.01 

0.05 f 0.02 

0.10 f0.02 

0.09 & 0.02' 

0.12 f 0.04 

0.26 f 0.03 

0.19 & 0.04 

0.17 f 0.09' 

0.05 f 0.02 

0.43 f 0.08 

1.21 f 0.24* 

0.59 & 0.06' 

0.66 +. 0.09 

313 2 5 

259 2 8 

170 2 15 

117 f 4 

28fl 

35 f 1 

43 f 2 

41 f 1 

64 zk 7 

70 & 6 

68 & 4 

56 f 7 

2,500 ppm 

10 

5.11 & 0.40 

6.37 f 0.30 

5.76 f 0.20' 

6.22 f 0.45 

0.06 f 0.02 

0.08 f 0.03 

0.08 & 0.03' 

0.02 f 0.01 

0.05 f 0.02 

0.05 f 0.03 

0.09 f 0.02' 

0.09 f 0.02 

0.23 f 0.03 

0.08 & 0.03 

0.26 f0.07'' 

0.15 f 0.03 

0.39 f 0.12 

1.92 f 0.40** 

0.69 f 0.10 

0.89 & 0.13 

328 f 6 

247 f 5 

137 f 4 

111 & 3 

30 f 1 

34 & 1* 

40 f 2 

42 f 1 

63 f 7 

70 f 5 

70 f 5 

57 f 6 

10,000 ppm 

10 

4.46 f 0.27b 

7.22 f 0.37 

5.47 f 0.35 

5.64 f0.65 

0.03 f O.OZb 

0.10 f 0.03 

0.03 f 0.02* 

0.00 f0.00 

0.04 f O.OZb 

0.02 f 0.01 

0.03 f 0.02* 

0.06 f 0.02 

0.40 f 0.05**b 

0.37 k 0.06** 

0.48 f 0.06** 

0.47 f 0.12** 

1.04 f0.22* 

4.03 f 0.18;; 

3.20 f 0.25.. 

3.33 f0.25** 

283 f 4** 

191 ? 6** 

116 f 4** 

101 k 4** 

33 f 2** 

40 f 1 

49 f 6 

39 f 2 

61 f 7 

67 f 5 

73 f 6 

49 f 6* 
268 p-Nitrobenzoic Acid, NTP TR 442 

TABLE62 

Hematology and Clinical Chemistry Data for Special Study Rats in the 13-WeekFeed Study 

ofp-Nitrobenzoic Acid (continued) 

0 PPm 630 ppm 2,500 ppm 10,000 ppm 

Female 

n 10 10 10 10 

Hematology 

Hematocrit (%) 

Day 7 42.3 f 0.5 43.2 rt 0.9 44.7 f 1.1 43.8 f 0.7 

Day 30 46.8 f 0.4 46.4 f 0.7 45.5 f 0.3* 44.0 f 0.7** 

Day 60 45.2 f 0.4 45.7 rt 0.4 45.6 f 0.5 42.5 f OS** 

Day 90 44.3 f 0.6 43.4 rt 0.6 44.0 f 0.5 43.0 f 0.6 

Hemoglobin (g/dL) 

Day 7 15.5 f 0.2 15.6 2 0.2 15.9 f 0.2 15.6 f 0.2 

Day 30 16.8 f 0.1 16.9 f 0.2 16.7 f 0.1 15.7 f 0.2** 

Day 60 16.9 f 0.2 16.9 f 0.1 16.7 f 0.1 15.5 f 0.1** 

Day 90 16.6 f 0.1 16.3 2 0.2 16.5 0.1 15.9 f 0.1** 

Erythrocytes (106/bL) 

Day 7 7.33 f 0.13 7.56 f 0.18 7.72 f 0.21 7.74 f 0.20 

Day 30 8.77 f 0.08 8.73 f 0.17 8.62 f 0.06 8.01 f 0.14** 

Day 60 9.03 f 0.12 9.08 f 0.11 9.05 f 0.09 8.15 & 0.08** 

Day 90 8.65 f 0.11 8.39 f 0.12 8.67 f 0.09 8.27 f 0.10 

Mean cell volume (E) 

Day 7 57.9 2 0.7 57.3 +- 0.6 58.0 f 0.7 56.8 f 0.7 

Day 30 53.5 f 0.3 53.4 f 0.3 52.9 f 0.4 55.0 f 0.4* 

Day 60 50.0 f 0.5 50.3 f 0.3 50.5 f 0.2 52.2 f 0.3** 

Day 90 51.3 f 0.4 51.8 2 0.3 50.6 f 0.4 51.9 f 0.3 

Mean cell hemoglobin (pg) 

Day 7 21.2 f 0.3 20.7 +. 0.3 20.6 f 0.4 20.3 f 0.3 

Day 30 19.2 f 0.1 19.3 k 0.2 19.4 f 0.1 19.6 f 0.2 

Day 60 18.7 f 0.1 18.6 +. 0.1 18.5 f 0.2 19.0 f 0.2 

Day 90 19.2 f 0.1 19.5 f 0.2 19.0 f 0.2 19.3 f 0.2 

Mean cell hemoglobin concentration (g/dL) 

Day 7 36.7 f 0.2 36.2 k 0.4 35.6 f 0.5. 35.7 f 0.3' 

Day 30 36.0 f 0.2 36.3 k 0.4 36.8 f 0.1 35.7 f 0.3 

Day 60 37.3 f 0.2 36.9 e 0.2 36.7 f 0.3 36.4 f 0.3. 

Day 90 37.5 k 0.3 37.6 f 0.3 37.5 f 0.4 37.1 f 0.4 

Reticulocytes (106/bL) 

Day 7 0.1 f 0.0 0.1 f 0.0 0.1 f 0.0 0.2 'fo.o** 

Day 30 0.1 f 0.0 0.0 f 0.0 0.1 f 0.0 0.2 f o.o** 

Day 60 0.1 f 0.0 0.1 f 0.0 0.1 f 0.0 0.2 f O.O*f 

Day 90 0.1 -+ 0.0 0.1 r 0.0 0.0 f o.o* 0.1 2 0.0 

Leukocytes (103/bL) 

Day 7 5.36 f 0.42 6.04 f 0.40 5.65 f 0.24 6.63 f 0.38* 

Day 30 6.95 f 0.30 6.53 f 0.31 6.19 f 0.42 8.82 f 0.31** 

Day 60 5.43 f 0.26 5.60 f 0.30 5.58 f 0.35 6.09 f 0.54 

Day 90 6.64 f 0.35 6.44 f 0.44 5.58 f 0.37 7.14 f 0.90 

Segmented neutrophils (103/bL) 

Day 7 0.62 f 0.07 0.72 f 0.11 0.63 f 0.06 0.78 f 0.14 

Day 30 0.72 f 0'15 0.77 f 0.09 0.86 f 0.13 1.45 f 0.13** 

Day 60 0.99 f 0.08 0.93 f 0.13 1.08 f 0.16 1.22 f 0.11 

Day 90 1.46 f 0.14 1.65 f 0.19 1.28 f 0.12 1.40 f 0.17 
Hematology and Clinical Chemistry 269 

TABLE62 

Hematology and Clinical Chemistry Data for Special Study Rats in the 13-Week Feed Study 

of p-Nitrobenzoic Acid(continued) 

Female (continued) 

n 

Hematology (continued) 

Lymphocytes (103/pL) 

Day 7 

Day 30 

Day 60 

Day 90 

Monocytes (103/pL) 

Day 7 

Day 30 

Day 60 

Day 90 

Eosinophils (103/pL) 

Day 7 

Day 30 

Day 60 

Day 90 

Heinz bodies (% RBC) 

Day 7 

Day 30 

Day 60 

Day 90 

Methemoglobin (% hemoglobin) 

Day 7 

Day 30 

Day 60 

Day 90 

Clinical Chemistxy 

Alkaline phosphatase(IUL) 

Day 7 

Day 30 

Day 60 

Day 90 

Alanine aminotransferase(IUL) 

Day 7 

Day 30 

Day 60 

Day 90 

Sorbitol dehydrogenase (IUL) 

Day 7 

Day 30 

Day 60 

Day 90 

0 PPm 

10 

4.65 f 0.35 

5.95 f 0.27 

4.35 f 0.26 

5.07 f 0.33 

0.04 f 0.02 

0.10 f 0.04 

0.03 f 0.02 

0.02 f 0.01 

0.06 f 0.02 

0.07 f 0.03 

0.05 f 0.02 

0.09 f 0.02 

0.18 f 0.04 

0.10 f 0.03 

0.11 f 0.02 

0.08 f 0.03 

0.48 f 0.10 

1.24 f 0.23 

0.64 f 0.11' 

0.78 f 0.10' 

240 f 7 

183 f 7 

105 f 5 

75 f 2 

2621 

32 f 2 

45 2 3 

33 f 1 

70 f 4 

58 f 5 

72 & 2 

52 f6 

630 ppm 

10 

5.24 f 0.41 

5.68 f 0.27 

4.51 f 0.21 

4.69 f 0.33 

0.03 f 0.02 

0.06 k 0.02 

0.07 f 0.03 

0.01 f 0.01 

0.05 f 0.02 

0.05 f 0.02 

0.11 2 0.02 

0.12 f 0.02 

0.19 f 0.04 

0.09 f 0.03 

0.12 f 0.04 

0.11 f 0.04 

0.56 f 0.12 

2.34 f 0.66 

0.85 f 0.08 

0.86 f 0.06 

235 f 6 

169 f 6 

109 f 5 

77 f 4 

26f1 

31 f 1 

37 f 2 

34 f 1 

55 f 6 

59 f 4 

69 f 5 

61 f 8 

2,500 ppm 

10 

4.87 f 0.19 

5.20 f 0.32 

4.36 f 0.24 

4.18 f 0.32 

0.07 f 0.02 

0.06 f 0.03 

0.07 f 0.03 

0.02 f 0.01 

0.10 f 0.02 

0.08 f 0.03 

0.08 2 0.03 

0.09 f 0.02 

0.13 f 0.05 

0.11 f 0.03 

0.16 f 0.03 

0.08 f 0.02 

0.36 f 0.10 

0.62 f 0.24 

0.73 f 0.09' 

0.95 f 0.14 

238 f 5 

191 f 6 

107 f 3 

88 f 3** 

30 f 2 

33 2 2 

47 f 7 

34 f 1 

58 f 4 

74 f 7 

73 f 6 

54 f 6 

10,000 ppm 

10 

5.66 2 0.34* 

7.23 f 0.29 

4.74 f 0.48 

5.64 f 0.77 

0.04 f 0.02 

0.10 f 0.03 

0.07 f 0.03 

0.01 f 0.01 

0.04 f 0.02 

0.06 f 0.02 

0.05 f 0.02 

0.03 f 0.02' 

0.37 f 0.03.. 

0.24 f 0.03** 

0.37 f 0.15** 

0.25 f 0.16** 

1.29 f 0.19** 

3.10 f 0.21** 

1.92 f 0.21** 

2.08 f 0.15** 

208 f 5** 

164 f 5 

102 f 4 

101 f 5** 

37 f 2** 

47 f 2** 

63 f 10 

43 f 2** 

60 f 8 

69 f 7 

76 f 5 

52 k 4 

* Significantly different (PSO.05) from the control group by Dunn's or Shirley's test 

** PSO.01 

a Mean f standard error 

n=8 ' n=9 
270 p-Nitrobenzoic Acid, NTP TR 442 

TABLE63 

Hematology Data for Rats at the 15-Month Interim Evaluation in the 2-Year Feed Study 

of p-Nitrobenzoic Acida 

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

Male \ 

n 10 8 10 10 

Hematocrit (%) 47.1 f 0.7 48.1 f 0.6 48.6 f 0.6 47.1 f 0.8 

Hemoglobin (g/dL) 15.5 f 0.2 15.9 f 0.2 16.0 f 0.2 15.4 f 0.2 

Mean cell volume (L) 

Erythrocytes (106/bL) 

53.5 * 0.2 

8.81 f 0.10 

53.3 f 0.5 

9.04 f 0.15 

53.0 f 0.5 

9.18 f 0.12* 

51.2 f 0.3;' 

9.21 f 0.12. 

Mean cell hemoglobin (pg) 17.6 f 0.2 17.7 k 0.2 17.5 f 0.2 16.7 f 0.1** 

Mean cell hemoglobin 

concentration (g/dL) 32.9 f 0.3 33.1 f 0.3 32.9 f 0.2 32.6 f 0.2 

Platelets (IO~/~L) 602.1 f 14.6 579.6 f 18.2 593.7 f 13.2 647.3 f 15.1 

Reticulocytes (106/pL) 0.2 f 0.0 0.2 f 0.0 0.1 f 0.0 0.1 f 0.ob 

Leukocytes (103/pL) 8.55 f 0.57 8.65 f 0.41 8.58 f 0.45 10.55 f OM* 

Lymphocytes (lO%L) 

Segmented neutrophils (103/bL) 

5.93 f 0.34 

2.24 f 0.36 

5.99 f 0.35 

2.20 f 0.24 

5.50 * 0.40 

2.73 f 0.36 

6.88 f 0.32 

2.67 f 0.23b 

Atypical lymphocytes (103/bL) 0.02 f 0.01 0.06 f0.03 0.02 f 0.01 0.02 f 0.02 

Monocytes (103/bL) 0.21 f 0.06 0.28 f 0.09 0.18 f 0.05 0.15 f 0.03 

Eosinophils (103/bL) 0.14 f 0.03 0.14 f 0.04 0.16 f 0.03 0.08 & 0.03 

Nucleated erythrocytes (103/bL) 0.09 & 0.02 0.14 -C 0.03 0.18 f 0.04 0.20 f 0.03* 

Methemoglobin (% hemoglobin) 0.22 f 0.02 0.24 f 0.02b 0.26 f 0.02 0.28 f 0.02. 

Female 

n 10 10 9 10 

Hematocrit (%) 46.3 f 0.3 44.9 ? 0.4* 45.8 f 0.4 44.1 f 0.4** 

Hemoglobin (g/dL) 16.0 f 0.2 15.5 -C 0.2 15.7 f 0.1 14.9 f 0.2'* 

Mean cell volume (fL) 

Erythrocytes (106/pL) 

56.3 f 0.3 

8.24 f 0.06 

56.1 k 0.6 

8.00 f 0.12 

55.8 f 0.3 

8.20 f 0.07 

56.3 k 0.3 

7.84 f 0.07** 

Mean cell hemoglobin (pg) 19.4 f 0.1 19.4 f 0.1 19.2 f 0.1; 19.0 f 0.1** 

Mean cell hemoglobin 

concentration (g/dL) 34.5 f 0.2 34.6 f 0.3 34.4 f 0.2 33.8 f 0.1. 

Platelets (~o~/~L) 560.3 f 16.8 510.1 f 29.2 576.7 & 12.7 652.3 f 21.8** 

Leukocytes (103/fiL) 

Reticulocytes (lO'/bL) 

4.09 f 0.16 

0.2 f 0.0 

4.58 f 0.40 

0.3 -C 0.1 

4.52 f 0.22 

0.3 f 0.0 0.3 f 0.0 

6.05 f 0.49** 

Segmented neutrophils (103/gL) 1.11 f 0.08 1.42 f 0.12* 1.10 f 0.08 1.87 f OX* 

Methemoglobin (% hemoglobin) 

Nucleated erythrocytes (103/bL) 

Eosinophils (103/bL) 

Monocytes (103/bL) 

Atypical lymphocytes (103/bL) 

Lymphocytes (103/bL) 

0.26 f 0.03 

0.09 f 0.04 

0.05 f 0.02 

0.02 f 0.02 

0.02 f 0.02 

2.86 2 0.12 

0.26 -c 0.02 

0.19 f 0.04b 

0.09 f 0.02 

0.01 f 0.01 

0.03 f 0.02 

3.02 f 0.34 

0.28 & 0.03' 

0.18 f 0.05 

0.07'f 0.02 

0.00 f0.00 

0.01 f 0.01 

3.32 f 0.21 

0.31 k 0.03 

0.42 f 0.08** 

0.09 & 0.02 

0.03 f 0.02 

0.02 * 0.01 

4.06 f OB** 

Significantly different (PSO.05) from the control group by Dunn's or Shirley's test 

** PSO.01 

a Mean f standard error 

n=9 n=10 
Hematology and Clinical Chemistry 271 

TABLE64 

Hematology Data for Mice in the 14-Day Feed Study of p-Nitrobenzoic Acida 

0 PPm 1,250ppm 5,000 ppm 10,000ppm 20,000 ppm 40,000 ppm 

Male 

n 5 4 5 4 5 2 

Hematocrit (%) 

47.1 f 1.4 48.8 f 1.6 50.5 f 0.8 48.3 f 0.5 50.3 f 1.5 49.8 f 1.0 

Hemoglobin (g/dL) 

15.8 f 0.4 16.3 f 0.1 16.7 f 0.1 16.3 f 0.1 15.8 f 0.3 16.1 f 0.2 

Erythrocytes (106/pL) 

Mean cell volume (a) 

47.8 f 0.6 

9.89 f 0.41 

48.0 f 0.0 

10.18 f 0.32 

49.2 f 0.6 

10.29 f 0.14 

46.8 f 0.5 

10.21 f 0.13 

48.8 f 0.4 

10.31 f 0.30 

48.5 f 0.5 

10.24 f 0.10 

Reticulocytes (106/pL) 

0.3 f 0.0 0.2 f 0.0 0.2 f 0.1 0.3 f 0.1 0.5 f 0.1 0.3 f 0.0 

Leukocytes (103/pL) 

4.60 5 0.68 5.13 f 0.48 5.38 f 0.38 4.40 f 0.84 3.10 f 0.38 3.70 f 1.00 

Segmented neutrophils (103/pL) 

0.55 f 0.07 0.44 f 0.08 0.60 f 0.12 0.37 ? 0.07 0.62 5 0.03 0.53 f 0.23 

Lymphocytes (103/pL) 

3.87 f 0.59 4.51 f 0.48 4.62 f 0.28 3.92 f 0.75 2.43 f 0.37 3.02 f 0.70 

Monocytes (103/pL) 

0.13 f 0.08 0.09 f 0.03 0.08 f 0.02 0.06 f 0.03 0.04 f 0.02 0.05 f 0.00 

Eosinophils (103/pL) 

0.05 f 0.05 0.09 f 0.04 0.08 f 0.04 0.05 f 0.03 0.02 f 0.01 0.11 f 0.08 

0.06 f 0.04 

Methemoglobin (% hemoglobin) 

0.00 f 0.00 0.02 f O.Olb 0.00 f o.ooc 0.05 f 0.03 -d
272 p-Nitrobenzoic Acid, NTP TR 442 

TABLE64 

Hematology Data for Mice in the 14-Day Feed Study of p-Nitrobenzoic Acid (continued) 

0 PPm 1,250 ppmppm 5,000 10,000 ppm 20,000 ppm 40,000 ppm

Female 

n 5 5 5 5 5 3 

Hematocrit (%) 

48.5 f 1.0 48.3 -t 1.2 48.6 f 0.9 49.2 f 1.5 46.7 f 0.9 46.6 f 1.2 

Hemoglobin (g/dL) 

16.3 f 0.3 16.0 -+ 0.2 16.4 f 0.1 16.1 f 0.4 15.4 f 0.1* 15.3 f 0.3 

Erythrocytes (106/pL) 

9.99 f 6.26 10.06 -t 0.27 10.17 f 0.21 10.34 f 0.47 9.71 f 0.21 9.61 f 0.28 

Mean cell volume (a) 

48.4 f 0.4 48.0 -t 0.0 47.8 f 0.6 47.6 f 0.9 48.2 f 0.4 48.3 f 0.3 

Reticulocytes (106/bL) 

0.3 f 0.0 0.2 f 0.0 0.2 * 0.0 0.3 f O.lb 0.3 f 0.1 0.3 f 0.0 

Leukocytes (103/pL) 

3.80 f 0.67 6.70 f 0.50 6.58 rt 0.31 5.16 f 0.92 3.40 f 0.27 5.30 f 1.40 

Segmented neutrophils (103/bL) 

0.37 f 0.14 0.59 -t 0.11 0.85 f 0.15* 0.69 f 0.12b 0.81 f 0.18* 0.61 f 0.40 

Lymphocytes (103/bL) 

3.37 f 0.61 5.80 f 0.54 5.43 f 0.34 4.41 f l.Olb 2.56 f 0.20 3.30 f 1.76 

Monocytes (103/bL) 

0.01 f 0.01 0.05 f 0.01 0.17 f 0.05 0.13 f O.O.Sb 0.01 f. 0.01 0.11 f 0.07 

Eosinophils (103/bL) 

0.05 f 0.02 0.26 +- 0.10 0.02 f 0.02 0.10 f 0.03b 0.02 f 0.01 0.18 f 0.12 

Methemoglobin (% hemoglobin) 

0.01 f 0.01 0.07 f 0.04 0.16 f 0.04 0.00 f 0.ooc 0.27 f 0.16 0.15 f 0.02 

l Significantly different (PsO.05) from the control groupby Dunn’s or Shirley’stest a Mean f standard error 

n=4 

n=3 

n=O; no data reported 
Hematology and Clinical Chemistry 273 

TABLE65 

Hematology Data for Mice at the 15-Month Interim Evaluation in the 2-Year Feed Study 

of p-Nitrobenzoic Acida 

Male 

n 

Hematocrit (%) 

Hemoglobin (g/dL) 

Erythrocytes (106/pL) 

Mean cell volume (fL) 

Mean cell hemoglobin (pg) 

Mean cell hemoglobin 

concentration (g/dL) 

Platelets (~o~/~L) 

Reticulocytes (106/pL) 

Leukocytes (103/pL) 

Segmented neutrophils (103/pL) 

Lymphocytes (103/pL) 

Atypical lymphocytes (103/pL) 

Monocytes (103/pL) 

Eosinophils (103/pL) 

Nucleated erythrocytes (103/pL) 

Methemoglobin (% hemoglobin) 

Female 

n 

Hematocrit (%) 

Hemoglobin (g/dL) 

Erythrocytes (106/pL) 

Mean cell volume(fL) 

Mean cell hemoglobin (pg) 

Mean cell hemoglobin 

concentration (g/dL) 

Platelets (103/p~) 

Reticulocytes (106/pL) 

Leukocytes (103/pL) 

Segmented neutrophils (103/pL) 

Lymphocytes (103/pL) 

Atypical lymphocytes (103/pL) 

Monocytes (103/pL) 

Eosinophils (103/pL) 

Nucleated erythrocytes (103/~L) 

Methemoglobin (% hemoglobin) 

* Significantly different (PSO.05) from 

a Mean f standard error 

n=7 ' n=9 

" n=8 

2,500 ppm 

10 

47.6 f 0.5 

16.2 f 0.1 

10.04 f 0.16 

47.5 f 0.7 

16.2 f 0.2 

34.1 f 0.3 

1,310 f 57 

0.2 f 0.0' 

3.79 f 0.17d 

0.96 f O.OSd 

2.89 f 0.22' 

0.00 f 0.00' 

0.00 f 0.ooc 

0.13 f 0.03' 

0.00 f 0.ooc 

0.16 f 0.02' 

9 

47.1 f 0.7 

16.2 f 0.2 

10.11 f 0.10 

46.8 f 0.3 

16.0 f 0.2 

34.4 f 0.4 

1,074.2 f 61.7 

0.2 f 0.0 

4.19 f 0.54 

1.32 k 0.40 

2.74 f 0.21 

0.00 f 0.00 

0.02 f 0.02 

0.12 f 0.04 

0.00 f 0.00 

0.12 f 0.02 

5,000 ppm 

9 

48.3 f 0.5 

16.3 f 0.1* 

10.16 t 0.14 

47.6 f 0.5 

16.1 t 0.2 

33.9 f 0.3 

1,309 f 30 

0.2 f 0.0 

4.26 f 0.34 

1.21 f 0.15 

2.90 f 0.25 

0.00 f 0.00 

0.01 f0.01 

0.13 f 0.04 

0.00 f 0.00 

0.14 f 0.02 

10 

45.2 f 0.5 

15.7 f 0.1 

9.59 f 0.12 

47.3 f 0.3 

16.4 f 0.2 

34.8 f 0.2 

1,032.6 f 44.9 

0.2 f0.0' 

3.81 5 0.30 

1.00 f 0.13 

2.73 f 0.22 

0.00 f 0.00 

0.00 f 0.00 

0.07 f 0.02 

0.00 f 0.00 

0.11 f 0.02 

0 PPm 

9 

47.1 f 0.5 

16.0 f 0.1 

9.94 f 0.16 

47.3 f 0.4 

16.1 f 0.3 

34.0 f 0.4 

1,279 k 53 

0.1 f 0.ob 

4.35 f 0.29d 

1.13 f OXd 

3.14 f 0.21d 

0.00 f 0.ood 

0.00 f 0.00d 

0.08 f OMd 

0.00 f o.ood 

0.16 f 0.03d 

10 

46.3 f 0.7 

16.0 f 0.2 

9.83 f 0.22 

47.2 f 0.5 

16.3 f 0.2 

34.5 f 0.2 

947.4 f 42.9 

0.2 f 0.0' 

3.30 f 0.33 

0.92 2 0.15 

2.29 & 0.23 

0.00 t 0.00 

0.01 & 0.01 

0.10 & 0.02 

0.00 f 0.00 

0.16 t 0.03 

1,250 ppm 

10 

48.2 f 0.9 

16.2 f0.4 

10.12 f 0.30 

47.8 f 0.5 

16.1 f 0.3 

33.7 f 0.4 

1,405 f 68 

0.2 f 0.0' 

4.54 f 0.21 

1.40 f 0.11 

3.06 f 0.24 

0.00 f0.00 

0.01 f 0.01 

0.09 f 0.02 

0.00 f 0.00' 

0.17 f 0.02 

9 

46.6 f 0.5 

16.0 f 0.1 

9.81 f 0.11 

47.6 f 0.3 

16.3 f 0.1 

34.4 f 0.3 

1,060.8 f 37.1 

0.2 f 0.0 

3.62 ? 0.31 

1.08 f 0.18 

2.43 2 0.14 

0.00 f 0.00 

0.00 f 0.00 

0.11 f 0.04 

0.00 f 0.00 

0.13 f 0.02 

the control group by Shirley's test 
274 p-Nitrobenzoic Acid, NTP TR 442 
l l 

275 

APPENDIX H 

CHEMICAL CHARACTERIZATION AND 

DOSE FORMULATION STUDIES 

PROCUREMENT AND CHARACTERIZATION OF ~-NITROBENZOICACID . . . . . . ..., . , . . . . . . . . . . . 276 

PREPARATION AND ANALYSISOF DOSE FORMULATIONS . . . . . . . . . . . . , , .. . . . . . . . . . . . . . . . . . . . . 277

FIGURE H1 Infrared Absorption Spectrum of p-Nitrobenzoic Acid . . . . . . . . . . . . . . . . . .. .. . . . . . 278 

FIGURE H2 Nuclear Magnetic Resonance Spectrum of p-Nitrobenzoic Acid . .. . .. . . . . . . . . . . . . . 279 

TABLE H1 Preparation and Storage of Dose Formulations in the Feed Studies 

of p-Nitrobenzoic Acid . . . . . . . . . . . . . . . ...,, , l . . . . . . . . . . . . . . . . . . . . . . l .. . . 280 

TABLE H2 Results of Analysis of Dose Formulations Administered 

to Rats and Mice in the 14-Day Feed Studies of p-Nitrobenzoic Acid . . . . . . . . . . . . . . 280 

TABLE H3 Results of Analysis of Dose Formulations Administered 

to Rats and Mice in the 13-Week Feed Studies of p-Nitrobenzoic Acid . . . . . . . . . . . . . 281 

TABLEH4 Results of Analysis of Dose Formulations Administered 

to Rats and Mice in the 2-Year Feed Studies of p-Nitrobenzoic Acid , , , . , , , . , . . . . . . 282 

TABLE H5 Results of Referee Analysis of Dose Formulations 

in the 13-Week and 2-Year Feed Studies of p-Nitrobenzoic Acid . . . . . . . . . . . . . . . . . . 286 
276 p-Nitrobenzoic Acid, NTP TR 442 

CHEMICAL CHARACTERIZATION AND 

DOSE FORMULATION STUDIES 

PROCUREMENT AND CHARACTERIZATION 

OF ~NITROBENZOIC ACID 

p-Nitrobenzoic acid was obtained from E.I. du Pont de Nemours and Company, Inc. (Wilmington, DE), in 

one lot (40) which was used throughout the studies. Identity, purity, and stability analyses were conducted 

by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO). Reports on 

analyses performed in support ofp-nitrobenzoic acid studies are on file at the National Institute of 

Environmental Health Sciences. 

The chemical, a light yellow, crystalline solid, was identified as p-nitrobenzoic acid by infrared, ultraviolet/ 

visible, and nuclear magnetic resonance spectroscopy. All spectra were consistent with those expected for 

the structure and with the literature spectra (Sadtler Standard Spectra) of p-nitrobenzoic acid (Figures H1 

and H2). The observed melting point of 239.7' to 241.2' C was consistent with the literature reference 

(Mack Index, 1983). 

The purity of p-nitrobenzoic acid was determined by elemental analyses, Karl Fischer water analysis, 

functional group titration, thin-layer chromatography, and high-performance liquid chromatography. 

Functional group titration was performed by dissolving a sample ofp-nitrobenzoic acid in methanol and 

titrating with 0.1 N aqueous sodium hydroxide. The titration was monitored potentiometrically using a 

combination pH/mV electrode filled with 4 M potassium chloride. Thin-layer chromatography was 

performed on Silica Gel 60 F-254 plates using two solvent systems: A) to1uene:ethyl acetate:glacial acetic 

acid (70:25:5) and B) diethylamine:methanolfl,N-dimethylformamide(48:40:12). The reference standard 

used was 10 pg of 1-nitronaphthalene (1 pL of a 10pg/pL solution in methanol). Visualization was 

accomplished with ultraviolet light (254 and 366 nm) and a spray of 5% titanous chloride in 1N 

hydrochloric acid. High-performance liquid chromatography was performed using a Fisher Scientific 

Resolvex C,, column (250 x 4.6mm ID) and a solvent system of water with 1% (v/v) phosphoric 

acid:methanol with 1% phosphoric acid (61:39). The flow rate was 1.0 mL/minute. Detection was with 

ultraviolet light at 254 nm. 

Elemental analysis for carbon, hydrogen, and nitrogen were in agreement with the theoretical values for 

p-nitrobenzoic acid. Karl Fischer analysis indicated 0.08% rt 0.01% water. Functional group titration 

indicated a purity of 100.1% rt 0.4%. Thin-layer chromatography using system A detected one major spot 

and one slight trace impurity; using system B, one major spot and one trace impurity were detected. 

High-performance liquid chromatography indicated no impurities with areas greater than 0.1% relative to 

the major peak area. The overall purity was determined to be greater than 99%. 

Stability studies were performed by the analytical chemistry laboratory. High-performance liquid 

chromatography was performed using the system described above except with a solvent ratio of 52:48. 

These studies indicated that p-nitrobenzoic acid was stable as a bulk chemical when stored in the dark for 

2 weeks at temperatures up to 60" C. The study laboratory stored the bulk chemical in sealed containers, 

protected from light, at room temperature. Purity and stability were monitored during the 2-year study by 

high-performance liquid chromatography and functional group titration. No degradation of the bulk 

chemical was observed. 
Chemical Characterization and Dose Formulations 277 

PREPARATION AND ANALYSIS OF DOSEFORMULATIONS 

The dose formulations were prepared weekly by mixingp-nitrobenzoic acid and feed to give the required 

concentrations (Table Hl). Mixtures were made by preparing ap-nitrobenzoic acid/feed premix with a 

spatula, which was then blended with feed in a twin shell blender for 15 minutes using an intensifier bar 

for the initial 5 minutes. Formulations were stored in doubled sealed plastic bags at -22" C or less for up 

to 3 weeks. 

Homogeneity and stability studies of the dose formulations were performed by the analytical chemistry 

laboratory. For the homogeneity studies at the 400 ppm concentration, aliquots were extracted with 

methanol containing 0.5% phosphoric acid and centrifuged. Aliquots of the extracts were mixed with an 

internal standard solution (propiophenone diluted with mobile phase). High-performance liquid 

chromatography was then performed using a Brownlee RP-18 column and a mobile phase of 

methano1:water:phosphoricacid (425750.5) at a flow rate of 1.0 mL/minute. Homogeneity was confirmed 

and the stability of the dose formulations was confirmed for at least 3 weeks when stored in the dark at 

room temperature. Dose formulations open to air and light were stable for 1week. 

Periodic analyses of the dose formulations ofp-nitrobenzoic acid were conducted at the study laboratory 

and analytical chemistry laboratory using high-performance liquid chromatography. Dose formulations 

were analyzed once during the 14-day studies and were within 10% of the target concentrations 

(Table H2). Dose formulations for the 13-week studies were analyzed prestudy, during week 1, at study 

mid-point, and at the final mix (Table H3). During the 2-year studies, the dose formulations were 

analyzed approximately every 2 months (Table H4). All dose formulations were within 10% of the target 

concentrations during the 13-week studies; 95% (160/168) of the formulations were within 10% of the 

target concentration during the 2-year studies. Results of the periodic referee analyses performed by the 

analytical chemistry laboratory were in good agreement with the results obtained by the study laboratory 

(Table H5). 
278 p-Nitrobenzoic Acid, NTP TR 442 

O=O 

I 

0 

I 

0

z, 

FIGUREH1 

Infrared Absorption Spectrum of p-NitrobenzoicAcid 
Stalt of Sweep >H+ 

0 

9 2 1 0 PPm (6) lo 

Lock Pos. -

PPm SpectrumAmp1 3x 100 SweepTime 5 min Nucleus Proton Sample 840342 Operator BJH 

p-NitrobenzoicAcid 

Lock Power - mG Lot No.:40 

Filter 0.1 Sweep Widthsec 10 ppm ZeroRef. TMS Balch No.: 01 Date 12-19-84

Decouple Pos. - PPm Task No.: BS/CV-1424 

DecouplingPower -mG RF Power 0.05 mGof EndSweep 0 ppm Sample Temp.-C Solvent DMSO-d6 Spectrum No. 330N N 
280 p-Nitrobenzoic Acid, NTP TR 442 

14-Day Studies 13-Week Studies 2-Year Studies 

Preparation 

Premixprepared was mixing bySame as 14-day Doses 14-day studies. wereas studies. were Same Doses 

p-nitrobenzoic withandacid feed a prepared every 2weeks.weekly. prepared 

spatula; premix and feed were then 

layered in a twin shell blender and 

mixed for 15 minutes with the 

intensifier bar on for the first 

5 minutes. Doses were prepared at 

study initiation. 

Lot Number 

40 40 40 

Maximum Storage Time 

3 weeks 3 weeks 2weeks 

Storage 

In double, sealed plastic bags at Same as 14day studies Same as 14-day studies 

-22"C or less for 2 weeks 

Study Laboratory 

Microbiological Associates, Same as 14day studies Southern Research Institute, 

Incorporated, Bethesda, MD Birmingham,AL 

Analytical Chemistry Laboratory 

Midwest Research Institute, Same as 14-day studies Same as 14-day studies 

Kansas City,MO 

TABLEH2 

Results of Analysis of Dose Formulations Administered to Rats and Mice 

in the 14-Day Feed Studiesof p-Nitrobenzoic Acid 

Target Determined Difference 

Date Prepared Date Analyzed Concentration Concentrationa from Target 

(PPm) (PPm) (%) 

6 December 19859-10 December 1985 2,500 2,580 +3 

5,000 4,990 0 

10,000 9,520 -5 

20,000 20,700 +4 

40.000 37.000 -7 

a Results of duplicateanalyses 
Chemical Characterization and Dose Formulations 281 

TABLEH3 

Results of Analysis of Dose Formulations Administered to Rats and Mice 

in the 13-Week Feed Studiesof p-Nitrobenzoic Acid 

Date Prepared Date Analyzed ConcentrationConcentrationa Targetfrom 

Difference Determined Target 

(PPm) (PPm) (%) 

Rats 

5 May 1986 6-8 May 1986 

630 

630 

630 

660d 

702' 

703b 

+5 

+10 

+10 

12 May 1986 12-14May 1986 

10,000 

5,000 

2,500 

1,250 

630 

9,710 

4,930 

2,470 

1,330 

677 

-3 

-1 

-1 

+6 

+7 

25 June 1986 26-30 June 1986 

10,000 

5,000 

2,500 

1,250 

630 

10,300 

5,290 

2,470 

1,270 

582 

+3 

+6 

-1 

+2 

-8 

6 August 1986 7-9 August 1986 

10,000 

5,000 

2,500 

1,250 

630 

9,980 

5,200 

2,440 

1,180 

578 

+4 

-2 

-6 

-8 

0 

Mice 

5 May 1986 6-8 May 1986 

20,000 

20,000 

20,000 

20,700d 

19,900' 

19,500b 

+3 

-1 

-3 

12 May 1986 12-14May 1986 

20,000 

10,000 

5,000 

2,500 

1,250 

19,600 

9,710 

4,930 

2,470 

1,330 

-2 

-3 

-1 

-1 

+6 

25 June 1986 26-30 June 1986 

20,000 

10,000 

5,000 

2,500 

1,250 

20,000 

10,300 

5,290 

2,470 

1,270 

+3 

+6 

-1 

+2 

0 

6 August 1986 7-9 August 1986 

20,000 

10,000 

5,000 

2,500 

1,250 

20,800 

9,980 

5,200 

2,440 

1,180 

+4 

-2 

-6 

+4 

0 

a Results of duplicate analyses 

Sample taken from top right of blender ' Sample taken from top left of blender 

Sample taken from bottom of blender 
282 p-Nitrobenzoic Acid, NTP TR 442 

TABLEH4 

Results of Analysis of Dose Formulations Administered to Rats and Mice 

in the 2-Year Feed Studies of p-NitrobenzoicAcid 

Determined Target Difference 

Date Prepared Date Targetfrom AnalyzedConcentrationConcentrationa

(PP@ (PP@ (W 

27 April 1988b 27-29 April 1988 1,250 1,270' +2 

1,250 1,220d -2 

1,250 1,130e -10 

5,000 5,150' +3 

5,000 5,080d +2 

5,000 5.000e 0 

4 May 1988b 4-6 May 1988 1,250 1,240c -1 

1,250 1,300d +4 

1,250 1,320e +6 

1,250 1,380' +10 

1,250 1,290d +3 

1,250 1,230e -2 

4 May 1988f 4-6 May 1988 1,250 1,270 +1 

1,250 1,300 +4 

2,500 2,630 +5 

2,500 2,540 +2 

5,000 5,420 +8 

5,000 5,260 +5 

1819881988 18-20 MayMay 1,250 1,190 -5

1,250 1,250 0 

1,250 1,240 -1 

1,250 1,280 +2 

2,500 2,500 0 

2,500 2,580 +3 

2,500 2,540 +2 

2,500 2,600 +4 

5,000 5,180 +4 

5,000 5,320 +6 

5,000 5,210 +4 

5,000 5,150 +3 

6 July 1988 7-11 July 1988 1,250 1,310 +5 

1,250 1,260 +1 

1,250 1,260 +1 

1,250 1,310 +5 

2,500 2,530 +1 

2,500 2,540 +2 

2,500 2,620 +5 

2,500 2,580 +3 

5,000 5,070 +1 

5,000 5,140 +3 

5,000 5,020 0 

5,000 5,270 +5 
Chemical Characterization and Dose Formulations 283 

TABLEH4 

Results of Analysis of Dose Formulations Administered to Rats and Mice 

in the 2-Year Feed Studiesof p-Nitrobenzoic Acid (continued) 

Difference Determined 

Date Prepared Date Concentration Analyzed Concentrationa

7 September 1988 8-12 September 1988 

9 November 198810-11 November 1988 

18January 198919-20 January 1989 

23 January 198923-24 January 1989 

Target 

from Target 

(PPm) (PPd (W 

1,250 1,270 +2 

1,250 1,310 +5 

1,250 1,240 -1 

1,250 1,300 +4 

2,500 2,680 +7 

2,500 2,520 +1 

2,500 2,550 +2 

2,500 2,610 +4 

5,000 4,920 -2 

5,000 5,020 0 

5,000 4,980 0 

5,000 5,200 +4 

1,250 1,200 -4 

1,250 1,300 +4 

1,250 1,340 +7 

1,250 1,240 -1 

2,500 2,640 +6 

2,500 2,480 -1 

2,500 2,570 +3 

2,500 2,600 +4 

5,000 5,160 +3 

5,000 5,140 +3 

5,000 4,990 0 

5,000 4,990 0 

1,250 1,340 +7 

1,250 1,360 +9 

1,250 1,350 +8 

1,250 1,370 +10 

2,500 2,590 +4 

2,500 2,700 +8 

2,500 2,540 +2 

2,500 2,860g +14 

5,000 5,190 +4 

5,000 5,360 +7 

5,000 5,120 +2 

5,000 5,580g +12 

2,500 2,590h +4 

5,000 5.120h +2 
284 p-Nitrobenzoic Acid, NTP TR 442 

TABLEH4 

Results of Analysis of Dose Formulations Administered to Rats and Mice 

in the 2-Year Feed Studiesof p-Nitrobenzoic Acid(continued) 

Difference Determined 

Concentration TargetfromDate Date AnalyzedPrepared Concentrationa 

22 March 198923-24 March 1989 

10 May 1989 11-12May 1989 

17May 1989 18May 1989 

5 July 1989 7 July 1989 

16August 198916-22 August 1989 

Target 

(PPm) (PPm) (94 

1,250 1,250 0 

1,250 1,250 0 

1,250 1,260 +1 

1,250 1,350 +8 

2,500 2,630 +5 

2,500 2,720 +9 

2,500 2,580 +3 

2,500 2,530 +1 

5,000 5,190 +4 

5,000 5,080 +2 

5,000 5,260 +5 

5,000 5,370 +7 

1,250 1,330 +6 

1,250 1,340 +7 

1,250 1,360 +9 

2,500 2,550 +2 

2,500 2,640 +6 

2,500 2,830g +13 

5,000 5,450 +9 

5,000 5,410 +8 

5,000 5,320 +6 

2,500 2,820h +13 

1,250 1,260 +1 

1,250 1,220 -2 

1,250 1,290 +3 

2,500 2,470 -1 

2,500 2,480 -1 

2,500 2,480 -1 

5,000 5,050 +1 

5,000 5,010 0 

5,000 5,070 +1 

1,250 1,230 -2 

1,250 1,240 -1 

1,250 1,240 -1 

2,500 2,520 +1 

2,500 2,500 0 

2,500 2,540 +2 

5,000 5,350 +7 

5,000 4,920 -2 

5,000 5,210 +4 
Chemical Characterization and Dose Formulations 285 

TABLEH4 

Results of Analysis of Dose Formulations Administered to Rats and Mice 

in the 2-Year Feed Studiesof p-NitrobenzoicAcid (continued) 

Difference Determined 

Date Prepared Date Concentration Analyzed Concentrationa Targetfrom 

27 October 198930-31 October 1989 

8 December 198911-12 9, December 1989 1,250

2 February 19905-6 February 1990 

30 March 1990 2-3April 1990 

3 April 1990 4 April 1990 

Target 

(PP@ 

1,250 

1,250 

1,250 

2,500 

2,500 

2,500 

5,000 

5,000 

5,000 

1,250 

1,250 

2,500 

2,500 

2,500 

5,000 

5,000 

5,000 

1,250 

1,250 

1,250 

2,500 

2,500 

2,500 

5,000 

5,000 

5,000 

1,250 

1,250 

1,250 

2,500 

2,500 

2,500 

5,000 

5,000 

5,000 

1,250 

1,250 

1,250 

(PP@ 

1,320 

1,240 

1,310 

2,630 

2,510 

2,580 

5,170 

4,900 

5,160 

1,270 

1,160 

1,230 

2,560 

2,500 

2,530 

4,860 

5,000 

4.980 

1,240 

1,240 

1,390 

2,520 

2,660 

2,470 

5,320 

5,220 

5,020 

1,100g. 

1,480&' 

1,390g 

2,740 

2,470. 

2,590' 

4,600 

5,230 

4,680 

1,280h 

1,zsoh 

1,360' 

(%) 

+6 

-1 

+5 

+5 

0 

+3 

+3 

-2 

+3 

+2 

-7 

-2 

+2 

0 

+1 

-3 

0 

0 

-1 

-1 

+11 

+1 

+6 

-1 

+6 

+4 

0 

-12 

+18 

+11 

+10 

-1 

+4 

-8 

+5 

-6 

+2 

+2 

+9 
286 

i 

p-Nitrobenzoic Acid, NTP TR 442 

TABLEH4 

Results of Analysis of Dose Formulations Administered to Rats and Mice 

in the 2-Year Feed Studies of p-NitrobenzoicAcid (continued) 

Determined Target Difference 

Date Prepared Target from Concentration Concentrationa AnalyzedDate

(PPd (PPm) (W 

27 April 199d30 April 1990 1,250 1,190 -5 

1,250 1,240, -1 

1,250 1,290 +3 

2,500 2,530 +1 

2,500 2,470 -1 

2,500 2,340 -6 

5,000 4,870 -3 

5,000 5,070, +1 

5,000 4,950 -1 

Results of duplicate analyses except where indicated 

Samples not used for dosing 

Sample taken from top right of blender 

Sample taken from top left of blender 

e Sampletakenfrombottomofblender 

Used only for rats 

Sample remixed 

Results of remix 

Results of triplicate analyses 

j used only for mice 

TABLEH5 

Results of Referee Analysis of Dose Formulations in the 13-Week and 2-Year Feed Studies 

of p-Nitrobenzoic Acid 

DeterminedConcentration (ppm) 

Target ConcentrationReferee Study 

Date Prepared (PP4 Laboratoryb Laborato$

13-Week Studies 

12May 1986 630 677 633 f 29 

6August 1986 2,500 2,440 2,410f 40 

2-Year Studies 

4 May 1988 1,250 1,260 1,135f 36 

9 November 1988 5,000 5,140 4,900f 200 

10 May 1989 2,500 2,550 2,590f 30 

27 October 1989 1,250 1,240 1,2102 4 

a Results of duplicateanalyses 

Results of triplicate analyses (mean f standard deviation) 
287 

APPENDIX I 

FEED AND COMPOUND CONSUMPTION 

IN THE 2-YEAR FEED STUDIES 

TABLEI1 Feed and Compound Consumption by Male Rats in the 2-Year Feed Study 

of p-Nitrobenzoic Acid .................................................. 288 

TABLE I2 Feed and Compound Consumption by Female Rats in the 2-Year Feed Study 

of p-Nitrobenzoic Acid .................................................. 289 

TABLEI3 Feed and Compound Consumption by Male Mice in the 2-Year Feed Study 

of p-Nitrobenzoic Acid .................................................. 290 

TABLE I4 Feed and Compound Consumption by Female Mice in the 2-Year Feed Study 

of p-Nitrobenzoic Acid ................................................. 291 
288 p-NitrobenzoicAcid, NTP TR 442 

TABLEI1 

Feed and Compound Consumption by Male Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

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

Body Feed Body Feed Dose/ Body FeedDose/Body Feed Dose/ 

Week 

(@day)a Weight Day(@day)WeightDay Dayb WeightWeight (@day)(@day)

(9) (8) (mglkglday) (SI (mp/k%daY) (€9 (WdaY) 

3 17.4 207 17.6 206 107 17.7 200 222 17.4 198 437 

6 20.7 283 19.5 281 87 19.3 280 172 18.7 262 356 

10 18.8 338 19.0 337 70 19.2 337 142 17.5 315 278 

13 17.2 362 17.2 360 60 16.6 359 116 17.4 338 258 

17 20.2 387 18.6 381 61 17.5 391 112 16.4 366 224 

21 18.3 409 18.9 409 58 18.6 412 113 17.8 389 229 

25 17.3 426 19.0 421 56 18.9 427 110 18.5 400 232 

28 18.3 435 18.0 431 52 18.4 435 106 16.6 410 202 

33 19.1 450 17.4 450 48 18.0 447 101 18.6 426 218 

37 17.3 460 18.4 461 50 18.4 464 99 17.8 442 201 

41 16.2 469 16.2 470 43 17.7 466 95 16.7 446 187 

45 17.2 475 16.9 475 44 17.6 480 91 17.3 459 188 

49 19.0 478 17.1 485 44 18.8 484 97 17.9 464 194 

53 16.2 480 17.4 488 45 17.7 485 91 17.3 463 186 

57 17.8 482 17.5 491 45 16.6 486 86 18.0 464 194 

61 16.0 484 18.4 484 48 17.2 480 89 16.4 461 178 

65 15.8 483 16.4 487 42 17.1 486 88 17.7 466 191 

69 15.7 482 16.6 490 42 15.7 485 81 16.2 466 174 

73 15.2 474 14.3 478 37 14.2 472 75 16.5 458 180 

77 15.7 475 16.1 480 42 16.3 474 86 16.9 465 182 

80 17.0 472 17.1 471 45 16.2 469 87 17.7 458 193 

85 17.0 463 17.6 463 48 16.7 469 89 17.9 459 195 

89 16.6 462 14.6 468 39 15.8 473 84 16.8 462 182 

93 16.7 455 15.6 458 43 14.9 470 79 15.9 457 174 

97 15.2 442 15.6 447 44 14.3 451 79 14.8 447 165 

101 15.8 449 16.3 441 46 16.6 460 90 16.3 444 184 

Mean for weeks 

1-13 18.5 297 18.3 296 81 18.2 294 163 17.7 278 332 

14-52 18.1 443 17.8 443 51 18.2 445 103 17.5 422 208 

53-101 16.2 470 16.4 473 43 16.1 474 85 16.8 459 183 

a Grams of feed consumed per animal per day 

Milligrams of p-nitrobenzoicacid consumed per day per kilogram body weight 
Feed and Compound Consumption 289 

TABLEI2 

Feed and Compound Consumption by Female Rats in the 2-Year Feed Studyof p-Nitrobenzoic Acid 

0 PDm 1,250 RRm 2,500 RRm 5,000 RRm 

Feed Dose/Body Feed Body Feed 

(&day)a WeightWeightWeight (&day)(&day) Dayb Day (&day)Day Weight

Week (9) (9) (mglkg/daY) (€9 (mglk%daY) (9) (w%"aY) 

3 12.1 137 11.7 137 LO7 11.7 134 219 11.8 131 449 

6 12.3 166 12.9 168 96 12.5 164 190 12.1 160 378 

10 11.8 184 11.8 185 80 11.7 183 160 11.3 179 316 

13 10.8 192 11.2 192 73 11.0 188 147 11.2 184 303 

17 10.9 203 10.6 205 64 11.9 193 153 10.1 199 253 

21 11.2 211 11.3 211 67 11.2 207 136 10.8 203 266 

25 11.1 218 10.7 218 61 10.5 212 124 10.5 206 254 

29 11.0 217 11.0 220 62 10.8 216 125 10.5 208 251 

33 10.9 228 10.5 226 58 10.9 221 123 10.7 212 252 

37 11.8 236 11.6 235 62 11.2 227 123 11.1 219 253 

41 10.8 242 10.6 243 55 10.9 234 116 10.6 224 237 

45 11.6 249 11.6 249 58 11.7 242 121 11.4 230 249 

50 11.8 262 12.4 261 59 11.8 254 117 11.7 237 246 

53 12.4 271 12.2 269 57 11.8 262 113 11.6 243 239 

57 12.9 280 12.7 276 58 12.3 267 115 12.1 245 247 

61 12.7 290 12.2 282 54 12.0 273 110 11.8 253 234 

65 11.6 298 12.4 289 53 12.2 280 109 11.8 257 229 

69 12.1 306 12.2 297 51 11.5 286 101 11.3 267 211 

73 11.7 308 10.9 299 46 11.8 290 102 10.7 264 203 

77 12.9 316 13.3 304 55 12.5 292 107 11.8 270 219 

81 13.2 321 13.7 308 55 13.1 298 110 12.6 274 229 

85 13.7 324 13.6 313 54 13.2 296 112 13.3 270 245 

89 12.8 334 12.8 323 49 12.7 306 104 12.6 282 224 

93 12.0 332 12.5 324 48 12.5 305 103 12.5 283 220 

97 12.9 337 12.6 327 48 13.0 316 103 12.6 289 218 

101 12.4 341 13.9 331 52 13.5 314 107 12.8 288 223 

Mean for weeks 

1-13 11.8 170 11.9 170 89 11.7 167 179 11.6 164 362 

14-52 11.2 230 11.1 230 61 11.2 223 126 10.8 215 251 

12.653-101 312 12.7 303 52 12.5 291 107 12.1 268 226 

a Grams of feed consumed per animal per day 

Milligrams of p-nitrobenzoic acid consumed per day per kilogram body weight 
290 p-Nitrobenzoic Acid, NTP TR 442 

TABLEI3 

Feed and Compound Consumption by Male Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 

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

dy Feed Dose/Body Feed Body Feed Dose/ Dose/ Body Feed 

(&day)(%day) Day a Day (glaay)(&day) WeightWeightWeightWeight Dayb

Week (€9 (mg/kg/daY) (9) (SI (“ay) ($9 (m%k%daY)

2 5.1 24.8 4.7 24.3 244 4.8 24.4 491 4.8 23.9 1,001 

6 5.3 28.1 5.2 27.9 231 5.2 27.9 462 6.0 27.3 1,091 

10 5.2 31.1 5.3 30.1 219 4.9 30.8 398 5.3 29.5 893 

13 5.8 33.1 6.0 32.8 228 5.5 33.1 416 6.0 31.7 949 

17 5.0 35.3 5.1 34.9 183 5.2 34.9 375 5.6 33.2 836 

21 4.5 37.2 4.6 36.5 156 4.4 36.4 303 4.8 34.5 698 

25 4.3 38.4 4.6 38.0 151 4.5 37.8 299 4.7 35.3 672 

29 4.0 40.8 4.3 40.2 133 4.3 39.8 272 4.6 37.5 620 

33 4.6 41.9 4.6 41.9 138 4.8 41.6 286 4.8 38.7 620 

37 5.1 43.4 5.1 43.5 145 4.8 43.3 279 5.3 40.8 654 

41 4.4 45.5 4.7 45.1 130 4.5 44.8 254 5.0 41.8 601 

45 4.9 46.1 4.8 45.4 133 4.9 45.4 270 5.0 41.9 599 

49 5.1 47.5 5.0 46.9 135 5.0 46.7 265 5.3 43.3 610 

53 4.6 47.4 4.7 47.3 123 4.6 46.7 247 4.8 43.7 553 

57 5.0 47.6 5.0 46.8 134 4.9 46.3 267 5.5 42.9 640 

61 4.9 47.3 5.0 46.5 135 5.1 46.0 277 5.2 42.8 607 

65 5.0 47.9 5.2 47.5 138 5.3 46.2 285 5.7 43.5 650 

69 4.8 47.8 5.0 48.0 130 5.0 46.3 269 5.1 42.8 592 

73 4.6 49.5 5.1 48.9 131 5.2 48.2 268 5.0 44.4 560 

77 4.8 49.2 5.0 48.8 128 5.1 48.2 265 5.1 44.7 571 

570 81 5.0 49.2 4.8 48.1 125 4.9 47.1 260 5.0 43.5 

85 4.7 48.0 4.6 48.4 120 4.7 47.3 247 4.7 43.6 543 

89 4.8 48.9 4.9 48.8 125 4.8 47.4 256 5.2 43.3 597 

93 5.0 48.0 5.3 49.0 135 5.2 47.5 272 5.3 43.1 619 

97 4.9 46.5 5.1 47.5 134 5.1 46.6 275 5.6 41.8 671 

101 5.0 45.9 5.3 47.7 139 5.4 46.4 289 6.0 41.4 722 

Mean for weeks 

1-13 5.4 29.3 5.3 28.8 230 5.1 29.1 442 5.5 28.1 983 

41.8 4.8 41.4 145 4.7 41.2 289 5.0 38.6 657 

47.9 5.0 47.9 131 5.0 46.9 268 5.2 43.2 607 

a Grams of feed consumed per animal per day 

Milligrams o fp-nitrobenzoicacid consumed per day per kilogram body weight 

14-52 4.7 

53-1014.9 
Feed and Compound Consumption 291 

TABLEI4 

Feed and Compound Consumption by Female Mice in the 2-Year Feed Study of p-Nitrobenzoic Acid 

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

Body Feed Body Feed Dose/ Feed Body Dose/ Feed Body Dose/ 

(%day)a Weight (@day) Weight Dayb(%day) Weight Day(@day) Weight Day 

Week (9) (9) (mg/k%day) (SI (mglkglday) (9) (mdk"ay) 

2 5.2 20.4 4.8 20.2 299 4.6 20.1 571 4.7 19.8 1,198 

6 5.6 24.3 5.9 24.5 301 6.2 24.2 637 6.8 23.3 1,457 

10 5.6 27.1 5.5 27.0 253 5.4 26.1 518 6.2 25.3 1,217 

12 6.7 28.8 6.8 28.5 298 6.6 27.6 595 7.5 26.4 1,417 

16 5.4 31.2 5.7 30.2 234 5.9 29.7 495 7.2 27.4 1,315 

20 4.8 33.4 4.8 32.3 186 4.9 31.5 390 6.0 28.9 1,037 

24 4.3 34.3 5.0 33.7 185 5.1 32.1 400 5.3 29.7 896 

28 4.8 36.8 4.7 35.7 166 5.4 34.8 385 5.7 31.1 922 

32 4.9 39.2 5.4 38.1 178 5.3 37.2 359 5.7 32.9 864 

36 5.9 40.4 5.9 39.7 187 6.0 38.6 390 6.2 34.1 916 

40 5.0 42.6 5.4 42.6 157 5.3 41.0 321 6.0 35.6 843 

44 5.2 44.6 5.4 44.1 154 5.6 42.5 330 5.6 36.4 769 

48 5.6 46.0 5.6 45.7 153 5.6 44.0 316 5.8 38.1 764 

52 5.1 47.0 5.2 46.3 142 5.2 45.0 287 5.5 38.5 708 

56 5.6 46.3 5.6 45.8 153 5.7 43.8 328 7.1 37.0 957 

60 5.3 47.0 5.1 46.9 136 5.4 44.5 305 5.6 37.2 750 

64 5.5 48.8 5.6 48.0 146 5.7 44.7 321 6.5 37.6 859 

68 5.2 48.5 5.5 48.7 140 5.2 45.9 283 5.8 38.0 766 

72 5.2 49.7 5.4 49.5 137 5.6 47.2 299 5.7 39.0 728 

76 5.4 51.2 5.5 51.4 135 5.6 49.0 287 6.0 40.0 748 

80 5.6 52.1 5.3 51.0 130 5.6 48.2 289 6.1 39.9 758 

84 5.1 51.2 5.2 51.0 128 5.4 48.2 278 5.4 40.5 669 

88 5.3 52.0 5.4 50.6 133 5.3 48.1 277 6.2 39.6 789 

93 5.5 50.6 5.8 49.5 147 5.6 47.4 295 6.5 38.5 838 

96 5.4 49.9 5.5 48.5 142 6.0 46.5 322 6.5 39.3 828 

100 5.4 49.8 5.9 48.6 152 6.2 46.2 335 6.9 38.8 887 

104 5.4 48.2 5.9 47.7 155 6.2 45.2 343 6.9 38.8 887 

Mean for weeks 

1-13 5.8 25.2 5.8 25.1 288 5.7 24.5 580 6.3 23.7 1,322 

14-52 5.1 39.6 5.3 38.8 174 5.4 37.6 367 5.9 33.3 903 

53-104 5.4 49.6 5.5 49.0 141 5.7 46.5 305 6.2 38.8 805 

a Grams of feed consumed per animal per day 

Milligrams of p-nitrobenzoic acid consumed per day per kilogram body weight 
292 p-Nitrobenzoic Acid, NTP TR 442 
293 

TABLEJ1 

TABLE52 

TABLE53 

TABLE54 

AND CONTAMINANT LEVELS 

INGREDIENTS, NUTRIENT COMPOSITION, 

APPENDIX J 

IN NIH-07 RAT AND MOUSE RATION 

Ingredients of NIH-07 Rat and Mouse Ration 

Vitamins and Minerals in NIH-07 Rat and Mouse Ration 

Nutrient Compositionof NIH-07 Rat and Mouse Ration 

Contaminant Levels in NIH-07 Rat and Mouse Ration 

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

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

......................... 296 

295 

294 

294 
294 

TABLEJl 

Ingredients of NIH-07 Rat and Mouse Ration" 

Ingredientsb 

Ground #2 yellow shelled corn 

Ground hard winter wheat 

Soybean meal (49% protein) 

Fish meal (60% protein) 

Wheat middlings 

Dried skim milk 

Alfalfa meal (dehydrated, 17% protein) 

Corn gluten meal (60%protein) 

Soy oil 

Dried brewer's yeast 

Dry molasses 

Dicalcium phosphate 

Ground limestone 

Salt 

Premixes (vitamin and mineral) 

a NCI, 1976;NIH, 1978 

p-Nitrobenzoic Acid, NTP TR 442 

~ ~ ~ ~~~ 

Percent by Weight 

24.50 

23.00 

12.00 

10.00 

10.00 

5.00 

4.00 

3.00 

2.50 

2.00 

1.50 

1.25 

0.50 

0.50 

0.25 

Ingredients were ground to pass through a US. Standard Screen No. 16 before being mixed. 

TABLE52 

Vitamins and Minerals in NIH-07 Rat and Mouse Rationa 

Vitamins 

A 

D3 

K3 

d-a-Tocopheryl acetate 

Choline 

Folic acid 

Niacin 

&Pantothenic acid 

Riboflavin 

Thiamine 

B12 

Pyndoxine 

Biotin 

Minerals 

Iron 

Manganese 

Zinc 

Copper

Iodine 

Cobalt 

a Per ton (2,000 Ib) of finished product 

Amount 

5,500,000IU 

4,600,000IU 

2.8g 

20,000IU 

560.0 g 

2.2g 

30.0 g 

18.0g 

3.4 g 

10.0g 

4,000 kg 

1.7 g 

140.0 mg 

120.0g 

60.0 g 

16.0 g 

4.0 g 

1.4 g 

0.4 g 

Source 

Stabilized vitaminA palmitate or acetate 

D-activated animal sterol 

Menadione 

Choline chloride 

d-Calcium pantothenate 

Thiamine mononitrate 

F'yndoxine hydrochloride 

d-Biotin 

Iron sulfate 

Manganous oxide 

Zinc oxide 

Copper sulfate 

Calcium iodate 

Cobalt carbonate 
Feed Analyses 295 

TABLE53 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

Nutrient 

Protein (% by weight) 

Crude fat (% by weight) 

Crude fiber (% by weight) 

Ash (% by weight) 

Amino Acids (% of total diet) 

Arginine 

Cystine 

Glycine 

Histidine 

Isoleucine 

Leucine 

Lysine 

Methionine 

Phenylalanine 

Threonine 

Tryptophan 

'Qrosine 

Valine 

Essential Fatty Acids (% of total diet) 

Linoleic 

Linolenic 

Vitamins 

Vitamin A (IU/kg) 

Vitamin D (IU/kg) 

a-Tocopherol (ppm) 

Thiamine (ppm) 

Riboflavin (ppm) 

Niacin (ppm) 

Pantothenic acid (ppm) 

F'yndoxine (ppm) 

Folic acid (ppm) 

Biotin (ppm) 

Vitamin B,, (ppb) 

Choline (ppm) 

Minerals 

Calcium (%) 

Phosphorus (%) 

Potassium (%) 

Chloride (%) 

Sodium (%) 

Magnesium (%) 

Sulfur (%) 

Iron (PP4 

Manganese (ppm) 

Zinc (ppm) 

Copper (PPm)

Iodine (ppm) 

Chromium (ppm) 

Cobalt (ppm) 

Mean f Standard 

Deviation 

22.90 f 1.03 

5.31 f0.26 

3.64 f 0.55 

6.73 f 0.31 

1.287 f 0.084 

0.306 f 0.075 

1.160 f 0.050 

0.580 f 0.024 

0.917 f 0.034 

1.972 f 0.052 

1.273 f 0.051 

0.437 f 0.115 

0.994 f 0.125 

0.896 f 0.055 

0.223 f 0.160 

0.677 2 0.105 

1.089 f 0.057 

2.389 f 0.233 

0.277 f 0.036 

6,554 f 1,288 

4,450 f 1,382 

36.92 f 9.32 

19.96 2 2.88 

7.92 f 0.93 

100.95 f 25.92 

30.30 f 3.60 

9.25 f 2.62 

2.51 f 0.64 

0.267 f 0.049 

40.14 f 20.04 

3,068 f 314 

1.26 f0.13 

0.96 f 0.05 

0.887 f 0.067 

0.526 f 0.092 

0.315 f 0.344 

0.168 f 0.008 

0.274 f 0.063 

356.2 f 90.0 

92.24 f 5.35 

58.14 f 9.91 

11.50 f 2.40 

3.70 k 1.14 

1.71 f 0.45 

0.797 f 0.23 

Number of Samples 

26 

26 

26 

26 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

9 

9 

26 

4 

9 

26 

10 

9 

10 

10 

10 

10 

10 

9 

26 

26 

8 

8 

10 

10 

10 

10 

10 

10 

10 

10 

9 

6 

Range 

21.30 - 4.80 - 2.80 - 6.12 -

1.100 - 0.181 -

1.060 - 0.531 - 0.867 - 1.850 - 1.200-

0.306 - 0.665 - 0.824 -

0.107 -

0.564 - 0.962 -

1.830-

0.210 -

4,100 -

3,000 - 22.5 -

15.0 -

6.10 -

65.0 - 23.0 -

5.60 -

1.80 -

0.19 -

10.6 -

2,400 -

0.90 - 0.88 -

0.772 -

0.380 -

0.258 -

0.151 -

0.208 -

255.0 -

81.70 -

46.10 -

8.090 -

1.52 -

0.85 -

0.490 -

24.60 

5.90 

4.80 

7.27 

1.390 

0.400 

1.220 

0.608 

0.965 

2.040 

1.370 

0.699 

1.110 

0.985 

0.671 

0.794 

1.170 

2.570 

0.320 

9,190 

6,300 

48.9 

28.0 

9.00 

150.0 

34.6 

14.0 

3.70 

0.35 

65.0 

3,430 

1.55 

1.10 

0.971 

0.635 

0.370 

0.180 

0.420 

523.0 

99.40 

81.60 

15.39 

5.83 

2.09 

1.150 
296 p-Nitrobenzoic Acid, NTP TR 442 

TABLE54 

Contaminant Levels in NIH-07 Rat and Mouse Ration 

~~ 

Mean 2 Standard 

Deviationa Range Number of Samples 

Contaminants 

Arsenic (ppm) 0.25 f 0.17 0.05 - 0.60 26 

Cadmium (ppm) <0.10 26 

(PPW 0.26 k 0.18 0.10 - 0.90 26 

Mercury (ppm)b 0.04 f 0.01 0.02 - 0.08 26 

Selenium (ppm) 0.34 k 0.10 0.15 - 0.55 26 

Aflatoxins (ppb) <5.0 26 

Nitrate nitrogen (ppm)" 15.21 f 5.00 0.30 - 22.0 26 

Nitrite nitrogen (ppm)" 0.19 k 0.14 CO.10 -0.60 26 

BHA (PPNd 1.49 k 0.70 C2.00 - 3.00 26 

BHT (PPW 1.25 k 0.64 C 1.00 - 3.00 26 

Aerobic plate count (CFU/g)e 115,769 k 92,211 25,000 - 380,000 26 

Coliform (MPN/g)f 31.96 f 29.95 <3.00 - 93 26 

E. coli (MPN/g) 3.35 k 1.20 C3.00 - 9.00 26 

Total nitrosoamines (ppb)g 7.63 f 2.90 2.00 - 13.70 26 

N-Nitrosodimethylamine (ppb)g 5.33 * 2.38 1.00- 11.00 26 

N-Nitrosopyrrolidine (ppb)g 2.30 f 1.23 1.00-4.70 26 

Pesticides (ppm) 

II-BHC~ co.01 26 

&BHC co.02 26 

v-BHC <0.01 26 

6-BHC co.01 26 

Heptachlor <0.01 26 

Aldrin co.01 26 

Heptachlor epoxide <0.01 26 

DDE <0.01 26 

DDD co.01 26 

DDT co.01 26 

HCB <0.01 26 

Mirex <0.01 26 

Methoxychlor co.05 26 

Dieldrin co.01 26 

Endrin co.01 26 

Telodrin co.01 26 

Chlordane co.05 26 

Toxaphene <0.1 26 

Estimated PCBs <0.2 26 

Ronnel co.01 26 

Ethion co.02 26 

Trithion <0.05 26 

Diazinon <0.1 26 

Methyl parathion c0.02 26 

Ethyl parathion co.02 26 

Malathion 0.22 f 0.28 0.05 - 1.29 26 

Endosulfan I co.01 26 

Endosulfan I1 <0.01 26 

Endosulfan sulfate ~0.03 26 
Feed Analyses 297 

TABLE54 

Contaminant Levels in NIH-07 Rat and Mouse Ration (continued) 

a For values less than the limit of detection, the detection limit is given as the mean. 

The lot milled 03 September 1986 contained 0.08 ppm; all other lots were less than or equal to the detection limit. 

Sources of contamination: alfalfa, grains, and fish meal 

Sources of contamination: soy oil and fish meal 

e CFLJ= colonyformingunits 

MPN = most probable number 

g All values were corrected for percent recovery. 

BHC is hexachlorocyclohexaneor benzene hexachloride 
298 p-Nitrobenzoic Acid, NTP TR 442 
299 

APPENDIX K 

SENTINEL ANIMAL PROGRAM 

METHODS............ . . ........... . ..... ...... . ........ . . . ... . . . . . ............ 300 
300 p-Nitrobenzoic Acid, NTP TR 442 

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 all 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. 

Rats 

For the 2-year study, 15 male and 15 female rats were selected at the time of randomization and allocation 

of the animals to the various study groups. Sera were obtained from five male and five female sentinel 

rats at 6, 12, and 18 months into the study. Serum for the 24-month screening was obtained from five 

high-dose males and five mid-dose females. Blood from each collection was processed appropriately, 

shipped to Microbiological Associates (Bethesda, MD), and screened for the following: 

Method of Analvsis Time of Analvsis 

ELISA 

PVM (pneumonia virus of mice) 6, 12, 18, and 24 months 

Sendai 

sialodacryoadenitis virus) 

RCV/SDA (rat coronavirusl 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

KRV (Kilham rat virus) 

H-1 (Toolan’s H-1 virus) 

Hemagglutination Inhibition 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

Mice 

For the 13-week study, samples were obtained from five male and five female controls at the end of the 

study. These samples were processed appropriately and were submitted to Microbiological Associates for 

viral titer screening. The following tests were performed: 

Method of Analvsis Time of Analvsis 

Complement Fixation 

LCM(lymphocytic choriomeningitis virus) Study termination 

ELISA 

CARB (cilia-associated respiratory bacillus) Study termination 

Ectromelia virus Study termination 

GDVII (mouse encephalomyelitis virus) Study termination 

MHV (mouse hepatitis virus) Study termination 

Mouse adenoma virus Study termination 

Mycoplasma arthritidk Study termination 

Mycoplasma pulmonis Study termination 

PVM Study termination 

Reovirus 3 Study termination 

Sendai Study termination 
Sentinel Animal Program 301 

Mice (continued) 

Method of Analvsis 

Hemagglutination Inhibition 

K (papovavirus) 

MVM (minute virus of mice) 

Polyoma virus 

Immunofluorescence Assay 

Time of Analvsis 

Study termination 

Study termination 

Study termination 

EDIM (epizootic diarrhea of infant mice) Study termination

For the 2-year study, 15 male and 15 female mice were selected at the time of randomization and 

allocation of the animals to the various study groups. Sera were obtained from as many as five male and 

five female sentinel mice at 6, 12, and 18 months into the study. Serum for the 24-month screening was 

obtained from five high-dose males and five high-dose females. Blood from each collection was processed 

appropriately, shipped to Microbiological Associates, and screened for the following: 

Method of Analvsis 

ELISA 

Ectromelia virus 

GDVII 

LCM 

MHV 

MVM 

Mouse adenoma virus 

PVM 

Reovirus 3 

Sendai 

Hemagglutination Inhibition 

K 

Polyoma virus 

Immunofluorescence Assay 

EDIM 

LCM 

MVM 

All test results were negative. 

Time of Analysis 

6, 12, 18,and 24 months 

6, 12, 18, and 24 months 

18 and 24 months 

6, 12, 18,and 24 months 

6 and 12 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6 and 12 months 

18 and 24 months 
NATIONAL TOXICOLOGY PROGRAM TECHNICAL REPORTS 

PRINTED AS OF NOVEMBER 1994 

' TR No. CHEMICAL 

201 2,3,7,8-Tetrachlorodibenzop-dioxin(Dermal) 

206 1,2-Dibromo-3-chloropropane 

207Cytembena 

208 FD & C Yellow No. 6 

209 2,3,7,8-Tetrachlorodibenzop-dioxin(Gavage) 

210,2-Dibromoethane 

211C.I.AcidOrange 10 

212Di(2-ethylhexy1)adipate 

213ButylBenzyl Phthalate 

214Caprolactam 

215Bisphenol A 

21611-AminoundecanoicAcid 

217 Di(2-ethylhexyl)phthalate 

219 2,6-Dichloro-p-phenylenediamine 

220C.I.AcidRed 14 

221LocustBeanGum 

222C.I.DisperseYellow 3 

223Eugenol 

224TaraGum 

225 D & C Red No. 9 

226C.I.SolventYellow14 

227GumArabic 

228VinylideneChloride 

229 Guar Gum 

230Agar 

231StannousChloride 

232Pentachloroethane 

2332-BiphenylamineHydrochloride 

234AllylIsothiocyanate 

235 Zearalenone 

236 D-Mannitol 

237 1,1,1,2-Tetrachloroethane 

238Ziram 

239 Bis(2-chloro-1-methy1ethyl)ether 

240 PropylGallate 

242Diallyl Phthalate (Mice) 

243Trichlorethylene(RatsandMice) 

244PolybrominatedBiphenylMixture 

245Melamine 

246ChrysotileAsbestos(Hamsters) 

247L-AscorbicAcid 

2484,4'-MethylenedianilineDihydrochloride 

249AmositeAsbestos(Hamsters) 

250 BenzylAcetate 

2512,4- & 2,6-TolueneDiisocyanate 

252GeranylAcetate 

253AllylIsovalerate 

254Dichloromethane(MethyleneChloride) 

2551,2-Dichlorobenzene 

257DiglycidylResorcinol Ether 

259EthylAcrylate 

261Chlorobenzene 

2631,2-Dichloropropane 

266Monuron 

2671,2-PropyleneOxide 

269Telone IW (1,3-Dichloropropene) 

271HCBlueNo. 1 

272Propylene 

TR No. 

273 

274 

275 

276 

277 

278 

279 

280 

281 

282 

284 

285 

287 

288 

289 

291 

293 

294 

295 

296 

298 

299 

300 

301 

303 

304 

305 

306 

307 

308 

309 

310 

311 

312 

313 

314 

315 

316 

317 

318 

319 

320 

321 

322 

323 

324 

325 

326 

327 

328 

329 

330 

331 

332 

333 

334 

335 

CHEMICAL 

Trichloroethylene (Four Rat Strains) 

Tris(2-ethy1hql)phosphate 

2-Chloroethanol 

8-Hydroxyquinoline 

Tremolite 

2,6-Xylidine 

Amosite Asbestos 

Crocidolite Asbestos 

HC Red No. 3 

Chlorodibromomethane 

Diallylphthalate (Rats) 

C.I. Basic Red 9 Monohydrochloride 

Dimethyl Hydrogen Phosphite 

1,3-Butadiene 

Benzene 

Isophorone 

HC Blue No. 2 

Chlorinated Trisodium Phosphate 

Chrysotile Asbestos (Rats) 

Tetrakis(hydroxymethy1)phosphonium Sulfate & 

Tetrakis(hydroxymethy1)phosphonium Chloride 

Dimethyl Morpholinophosphoramidate 

C.I. Disperse Blue 1 

3-Chloro-2-methylpropene 

o-Phenylphenol 

4-Vinylcyclohexene 

Chlorendic Acid 

Chlorinated Paraffins (CB, 43% chlorine) 

Dichloromethane (Methylene Chloride) 

Ephedrine Sulfate 

Chlorinated Paraffins (C,,, 60% chlorine) 

Decabromodiphenyl Oxide 

Marine Diesel Fuel and JP-5 Navy Fuel 

Tetrachloroethylene (Inhalation) 

n-Butyl Chloride 

Mirex 

Methyl Methacrylate 

Oxytetracycline Hydrochloride 

1-Chloro-2-methylpropene 

Chlorpheniramine Maleate 

Ampicillin Trihydrate 

1,4-Dichlorobenzene 

Rotenone 

Bromodichloromethane 

Phenylephrine Hydrochloride 

Dimethyl Methylphosphonate 

Boric Acid 

Pentachloronitrobenzene 

Ethylene Oxide 

Xylenes (Mixed) 

Methyl Carbamate 

1.2-Epoxybutane 

4-He&lr&orcinol 

Malonaldehyde, Sodium Salt 

2-Mercaptobenzothiazole 

N-Phenyl-2-naphthylamine 

2-Amino-5-nitrophenol

C.I. Acid Orange 3 
NATIONAL TOXICOLOGY PROGRAM TECHNICAL REPORTS 

PRINTED AS OF NOVEMBER 1994 (CONT.) 

TR No. 

336 

337 

338 

339 

340 

341 

342 

343 

344 

345 

346 

347 

348 

349 

350 

351 

352 

353 

354 

355 

356 

357 

358 

359 

360 

361 

362 

363 

364 

365 

366 

367 

368 

369 

370 

371 

372 

373 

374 

375 

376 

377 

378 

379 

380 

381 

382 

384 

385 

386 

CHEMICAL 

Penicillin VK 

Nitrofurazone 

Erythromycin Stearate 

2-Amino-4-nitrophenol 

Iodinated Glycerol 

Nitrofurantoin 

Dichlorvos 

Benzyl Alcohol 

Tetracycline Hydrochloride 

Roxarsone 

Chloroethane 

D-Limonene 

a-Methyldopa Sesquihydrate 

Pentachlorophenol 

Tribromomethane 

p-Chloroaniline Hydrochloride 

N-Methylolacrylamide 

2,4-Dichlorophenol 

Dimethoxanc 

Diphenhydramine Hydrochloride 

Furosemide 

Hydrochlorothiazide 

Ochratoxin A 

8-Methoxypsoralen 

N,N-Dimethylaniline 

Hexachloroethane 

4-Vinyl-1-cyclohexene Diepoxide 

Bromoethane (Ethyl Bromide) 

Rhodamine 6G (C.I. Basic Red 1) 

Pentaerythritol Tetranitrate 

Hydroquinone 

Phenylbutazone 

Nalidixic Acid 

a-Methylbenzyl Alcohol 

Benzofuran 

Toluene 

3,3-Dimethoxybenzidine Dihydrochloride 

Succinic Anhydride 

Glycidol 

Vinyl Toluene 

Allyl Glycidyl Ether 

o-Chlorobenzalmalononitrile 

Benzaldehyde 

2-Chloroacetophenone 

Epinephrine Hydrochloride 

d-Carvone 

Furfural 

1,2,3-Trichloropropane 

Methyl Bromide 

Tetranitromethane 

TR No. 

387 

388 

389 

390 

391 

392 

393 

394 

395 

396 

397 

398 

399 

400 

401 

402 

403 

404 

405 

406 

407 

408 

409 

410 

411 

412 

413 

414 

415 

416 

417 

418 

419 

420 

421 

422 

423 

424 

425 

426 

427 

428 

430 

431 

432 

433 

434 

437 

440 

443 

CHEMICAL 

Amphetamine Sulfate 

Ethylene Thiourea 

Sodium Azide 

3,3'-Dimethylbenzidine Dihydrochloride 

Tris(2-chloroethyl) Phosphate 

Chlorinated Water and Chloraminated Water 

Sodium Fluoride 

Acetaminophen 

Probenecid 

Monochloroacetic Acid 

C.I. Direct Blue 15 

Polybrominated Biphenyls 

Titanocene Dichloride 

2,3-Dibromo-l-propanol 

2,4-Diaminophenol Dihydrochloride 

Furan 

Resorcinol 

5,5-Diphenylhydantoin 

C.I. Acid Red 114 

y-Butyrolactone 

C.I. Pigment Red 3 

Mercuric Chloride 

Quercetin 

Naphthalene 

C.I. Pigment Red 23 

4,4-Diamino-2,2-stilbenedisulfonicAcid 

Ethylene Glycol 

Pentachloroanisole 

Polysorbate 80 

o-Nitroanisole 

p-Nitrophenol 

p-Nitroaniline 

HC Yellow 4 

Triamterene 

Talc 

Coumarin 

Dihydrocoumarin 

o-Benzyl-p-chlorophenol 

Promethazine Hydrochloride 

Corn Oil, Safflower Oil, and Tricaprylin 

Turmeric Oleoresin 

Manganese (11) Sulfate Monohydrate 

C.I. Direct Blue 218 

Benzyl Acetate 

Barium Chloride Dihydrate 

Tricresyl Phosphate 

1,3-Butadiene 

Hexachlorocyclopentadiene 

Ozone and Ozone/NNK 

Oxazepam 

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(and while supplies last) from the NTP Central Data Management, NIEHS, P.O. Box 12233, MD AO-01, Research Triangle Park, NC 

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NIH Publication No. 95-3358 

December 1994