National Toxicology Program

Toxicity Report Series

Number 44

NTP Technical Report

on Comparative Toxicity and Carcinogenicity Studies of

o-Nitrotoluene and

o-Toluidine Hydrochloride

(CAS Nos. 88-72-2 and 636-21-5)

Administered in Feed

to Male F344/N Rats

Michael R. Elwell, D.V.M., Ph.D., Study Scientist

National Toxicology Program

Post Office Box 12233

Research Triangle Park, NC 27709

NIH Publication 96-3936

March 1996

United States Department of Health and Human Services

Public Health Service

National Institutes of Health
Note to the Reader

The National Toxicology Program (NTP) is made up of four charter agencies of the United States Department of Health and

Human Services (DHHS):

the National Cancer Institute (NCI) of the National Institutes of Health;

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

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

the National Institute for Occupational Safety and Health (NIOSH) of the Centers for Disease Control.

In July 1981, the Carcinogenesis Bioassay Testing Program was transferred from NCI to NIEHS. NTP coordinates the

relevant Public Health Service programs, staff, and resources that are concerned with basic and applied research and with

biological assay development and validation.

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.

NTP designs and conducts studies to characterize and evaluate the toxicologic potential of selected chemicals in laboratory

animals (usually two species, rats and mice). Chemicals selected for NTP toxicology studies are chosen primarily on the

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

Study Report are based only on the results of these NTP studies. Extrapolation of these results to other species, and

quantitative risk analyses for humans, require wider analyses beyond the purview of these studies. Selection per se is not

an indicator of a chemical's toxic potential.

The study described in this toxicity study report was performed under the direction of NIEHS and was conducted in

compliance with NTP laboratory health and safety requirements. The study met or exceeded all applicable federal, state,

and local health and safety regulations. Animal care and use were in accord and compliance with the Public Health Service

Policy on Humane Care and Use of Animals.

Single copies of this report are available without charge, while supplies last, from the NTP Central Data Management

(telephone number 919/541-3419).

NTP Central Data Management

MD E1-02

NIEHS

Post Office Box 12233

Research Triangle Park, NC 27709
National Toxicology Program

Toxicity Report Series

Number 44

NTP Technical Report

on Comparative Toxicity and Carcinogenicity Studies of

o-Nitrotoluene and

o-Toluidine Hydrochloride

(CAS Nos. 88-72-2 and 636-21-5)

Administered in Feed

to Male F344/N Rats

Michael R. Elwell, D.V.M., Ph.D., Study Scientist

National Toxicology Program

Post Office Box 12233

Research Triangle Park, NC 27709

NIH Publication 96-3936

March 1996

United States Department of Health and Human Services

Public Health Service

National Institutes of Health
2 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

CONTRIBUTORS

This NTP report on the toxicity studies of o-nitrotoluene and o-toluidine hydrochloride is based primarily on

26-week feed studies that began in March 1992 and ended in June 1992 at Battelle Columbus Operations,

Columbus, OH.

National Toxicology Program

Evaluated experiment, interpreted results, and reported findings

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

Study Scientist

John R. Bucher, Ph.D.

Leo T. Burka, Ph.D.

Rajendra S. Chhabra, Ph.D.

June K. Dunnick, Ph.D.

Joel Mahler, D.V.M.

H. B. Matthews, Ph.D.

Kristine L. Witt, M.S.

Oak Ridge Associated Universities

Battelle Columbus Operations

Principal contributors

Perry J. Kurtz, D.V.M., Ph.D.,

Principal Investigator

Bruce A. Trela, Ph.D.

Allen W. Singer, D.V.M.

Experimental Pathology Laboratories, Inc.

Provided pathology quality assessment

Suzanne Botts, D.V.M., Ph.D.

Analytical Sciences, Inc.

Provided statistical analyses

Richard W. Morris, M.S.

Sarah Rosenblum, M.S.

NTP Pathology Working Group

Evaluated slides and prepared pathology report

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

Pathology Associates, Inc.

Suzanne Botts, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

John Cullen, V.M.D., Ph.D.

North Carolina State University

J.-P. Descoteaux, D.V.M., Ph.D.

University of Quebec

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

National Toxicology Program

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

National Toxicology Program

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

National Toxicology Program

Patrick Kramme, D.V.M., Ph.D.

University of Wisconsin

Joel Mahler, D.V.M.

National Toxicology Program

Laboratory of Experimental Pathology

Morphometric analysis of liver foci

Thai T. Ton

NIEHS

Biotechnical Services, Inc.

Provided toxicity report preparation

Daphne D. Lambright, Ph.D.,

Principal Investigator

C. Michael Bailey, B.S. Pharm.

Susan R. Gunnels, M.A.

Sophonia A. Roe, B.S.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 3

PEER REVIEW

The draft report on the toxicity studies of o-nitrotoluene and o-toluidine hydrochloride was evaluated by the

reviewers listed below. These reviewers serve as independent scientists, not as representatives of any

institution, company, or governmental agency. In this capacity, reviewers determine if the design and

conditions of this NTP study are appropriate and ensure that the toxicity study report presents the experimental

results and conclusions fully and clearly.

Douglas E. Rickert, Ph.D.

Glaxo Research Institute

Glaxo, Inc.

Research Triangle Park, NC

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

National Cancer Institute

Frederick, MD
4 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE OF CONTENTS

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

INTRODUCTION................................................................................................................................................... 11

Physical and Chemical Properties, Production, Use, and Exposure ........................................................... 11

Disposition and Metabolism ........................................................................................................................ 12

Toxicity......................................................................................................................................................... 15

Study Rationale and Design......................................................................................................................... 19

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

Procurement and Characterization of o-Nitrotoluene

and o-Toluidine Hydrochloride.................................................................................................................... 21

Preparation and Analysis of Dose Formulations.......................................................................................... 22

Preparation of Antibiotic Mixture................................................................................................................ 25

Toxicity Study Designs ................................................................................................................................ 25

Statistical Methods....................................................................................................................................... 29

Quality Assurance ........................................................................................................................................ 30

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

26-Week Feed Studies in Male F344/N Rats .............................................................................................. 31

DISCUSSION......................................................................................................................................................... 53

REFERENCES....................................................................................................................................................... 61

APPENDIXES

Appendix A Summary of Lesions.......................................................................................................A-1

Appendix B Organ Weights and Organ-Weight-to-Body-Weight Ratios .........................................B-1

Appendix C Efficacy and Acute Toxicity of Antibiotic Mixture

(Pilot Studies).................................................................................................................C-1
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 5

ABSTRACT

o-Nitrotoluene o-Toluidine Hydrochloride

HCl

Molecular Formula C7H7NO2 C7H9N HCl

CAS Number 88-72-2 636-21-5

Molecular Weight 137.13 143.62

Synonyms 2-nitrotoluene 2-toluidine hydrochloride

o-methylnitrobenzene o-aminotoluene hydrochloride

nitrotoluol methylaniline

nitrophenylmethane

o-Nitrotoluene and o-toluidine hydrochloride are structurally related chemicals that are suspected

and demonstrated animal carcinogens, respectively. The metabolic potential of the gastrointestinal

flora is considered an important factor in o-nitrotoluene-induced toxicity and involves the reduction

of the nitro group to the corresponding amine (forming o-toluidine). These studies were designed

to 1) compare the target organ toxicities of o-nitrotoluene and o-toluidine hydrochloride

administered in feed at approximately equimolar doses (5,000 ppm) to male F344/N rats for 13

or 26 weeks, 2) determine the potential progression or reversibility of toxic or proliferative lesions

following chemical withdrawal (stop-exposure) for 13 weeks after 13 weeks of exposure, and 3)

examine the effect of antibiotic-altered gastrointestinal flora on the toxicity and/or carcinogenicity

of o-nitrotoluene.

o-Nitrotoluene and o-toluidine hydrochloride caused mesothelial hyperplasia and mesothelioma

in male rats after 13 or 26 weeks of dietary exposure. The incidence of mesothelioma was greater

and the latency was less in rats administered o-nitrotoluene than in rats administered o-toluidine

hydrochloride. Additionally, o-nitrotoluene caused testicular degeneration in rats. Effects of

o-nitrotoluene administration in the liver included progressive, irreversible increases in liver weight

and irreversible increases in the incidences of cytoplasmic vacuolization and oval-cell hyperplasia.
6 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

 Placental glutathione S-transferase (PGST)-positive foci of cellular alteration occurred in the liver

after 13 weeks of o-nitrotoluene exposure, and the number and size (as reflected by the volume

fraction) of foci were increased after 26 weeks of continuous exposure. During the recovery

period, the number of PGST-positive foci in rats in the stop-exposure group decreased slightly, but

the size of the foci continued to increase. After 26 weeks, cholangiocarcinoma occurred in 2 of

20 rats in the stop-exposure group and 1 of 20 rats in the continuous-exposure group administered

o-nitrotoluene. In contrast, liver effects in rats administered o-toluidine hydrochloride consisted

of minimal hemosiderin accumulation in Kupffer cells; the incidence of this lesion in the stopexposure group decreased during the recovery period. o-Toluidine hydrochloride caused fewer

and much smaller PGST-positive foci than those caused by o-nitrotoluene.

o-Nitrotoluene caused an accumulation of hyaline droplets in the renal tubule epithelium; this

accumulation did not increase in severity with continued exposure and completely regressed during

the recovery period. Exposure to o-toluidine hydrochloride did not cause hyaline droplet

accumulation but did cause an accumulation of hemosiderin pigment in renal tubule epithelium.

 This change progressed in severity during the 26-week continuous-exposure study but decreased

in severity in the stop-exposure study during the recovery period.

Exposure to o-nitrotoluene or o-toluidine hydrochloride caused increased incidences of

hematopoiesis, hemosiderin accumulation, and capsular fibrosis in the spleen. In rats administered

o-toluidine, spleen effects were much more prominent and were also reflected by congestion and

markedly increased spleen weights. During the recovery period for the stop-exposure groups

administered either compound, incidences of hemosiderin accumulation and hematopoiesis were

decreased, but the capsular fibrosis did not resolve. Hyperplasia of the transitional epithelium in

the urinary bladder was observed only in rats administered o-toluidine hydrochloride; this lesion

did not increase in severity after 26 weeks of continuous exposure and completely regressed in the

stop-exposure group during the recovery period. Alteration of the gastrointestinal flora by daily

gavage administration of antibiotics did not affect the pattern or severity of toxicity at any site or

the development of mesothelioma in rats exposed to o-nitrotoluene, although cholangiocarcinomas

that occurred in three rats with the normal flora did not occur in groups with the altered intestinal

flora. Subsequent studies determined that the antibiotic regimen used was effective only in

reducing the gut population of aerobic microorganisms and had little effect on obligate anaerobes,

which are thought to play a major role in nitro group reduction.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 7

In summary, these studies confirmed the target organs and compared the relative toxicity for

o-nitrotoluene and o-toluidine hydrochloride administered to male F344/N rats at equimolar

concentrations in feed. With the exception of the spleen toxicity observed with each chemical, but

more prominently with o-toluidine hydrochloride, morphologic effects of exposure to each of these

chemicals in the testis/epididymis, liver, kidney, and urinary bladder were different. The results

of these studies demonstrate the somewhat greater relative carcinogenic potential of o-nitrotoluene

compared to o-toluidine hydrochloride after 13 or 26 weeks of administration based on the

occurrence of mesothelioma and cholangiocarcinoma. The apparently lower potency of o-toluidine

hydrochloride relative to o-nitrotoluene in the induction of mesothelioma suggests that simple

intestinal reduction of the nitro group may not be sufficient for carcinogenic activity in the

mesothelium.
8 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

Summary of the Comparative Toxicity and Carcinogenicity Studies

of o-Nitrotoluene and o-Toluidine Hydrochloride in Male F344/N Rats

Control

(0 ppm)

o-Nitrotoluene

(5,000 ppm)

o-Toluidine

Hydrochloride

(5,000 ppm)

Body weights

Less than controls at

all time points

Less than controls at

all time points

Lesions

13 weeks Kidney: regeneration

(3/10)

Spleen: hematopoietic

cell proliferation (2/10)

Kidney: hyaline droplet

accumulation (20/20);

regeneration (16/20)

Liver: cytoplasmic

vacuolization (20/20);

oval cell hyperplasia

(20/20)

Spleen: hematopoietic

cell proliferation

(20/20); hemosiderin

pigmentation (20/20);

capsule, fibrosis (1/20)

Testis/epididymis: 

degeneration (20/20)

Kidney: pigmentation

(20/20)

Liver: hemosiderin

pigmentation (20/20);

cytoplasmic

vacuolization (1/20)

Spleen: congestion

(20/20); hematopoietic

cell proliferation

(20/20); hemosiderin

pigmentation (20/20);

thrombosis (3/20);

capsule, fibrosis

(20/20)

Testis/epididymis: 

degeneration (1/20)

Urinary bladder: 

transitional epithelium,

hyperplasia (10/20)

Stop-exposure Kidney: protein casts

(3/10); regeneration

(7/10)

Spleen: hematopoietic

cell proliferation (3/10);

hemosiderin

pigmentation (3/10)

Kidney: protein casts

(20/20); regeneration

(19/20)

Liver: cytoplasmic

vacuolization (20/20);

oval cell hyperplasia

(20/20);

cholangiocarcinoma

(2/20)

Spleen: hematopoietic

cell proliferation

(18/20); hemosiderin

pigmentation (17/20);

capsule, fibrosis (7/20)

Testis/epididymis: 

degeneration (20/20);

mesothelial

hyperplasia (2/20);

mesothelioma (5/20)

Kidney: protein casts

(6/20); pigmentation

(20/20); regeneration

(10/20)

Liver: hemosiderin

pigmentation (11/20);

cytoplasmic

vacuolization (1/20)

Spleen: congestion

(20/20); hematopoietic

cell proliferation (1/20);

hemosiderin

pigmentation (18/20);

capsule, fibrosis

(20/20); capsule,

lymphatic angiectasis

(15/20)

Testis/epididymis: 

degeneration (2/20);

mesothelioma (2/20)
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 9

Summary of the Comparative Toxicity and Carcinogenicity Studies

of o-Nitrotoluene and o-Toluidine Hydrochloride in Male F344/N Rats (continued)

Control

(0 ppm)

o-Nitrotoluene

(5,000 ppm)

o-Toluidine

Hydrochloride

(5,000 ppm)

Lesions

26 weeks Kidney: protein casts

(3/10); regeneration

(7/10)

Spleen: hematopoietic

cell proliferation (3/10);

hemosiderin

pigmentation (3/10)

Kidney: hyaline droplet

accumulation (20/20);

protein casts (20/20);

regeneration (19/20)

Liver: cytoplasmic

vacuolization (20/20);

oval cell hyperplasia

(20/20);

cholangiocarcinoma

(1/20)

Spleen: hematopoietic

cell proliferation

(20/20); hemosiderin

pigmentation (20/20);

capsule, fibrosis (3/20)

Testis/epididymis: 

degeneration (20/20);

mesothelial

hyperplasia (2/20);

mesothelioma (7/20)

Kidney: protein casts

(1/20); pigmentation

(20/20); regeneration

(5/20)

Liver: hemosiderin

pigmentation (20/20)

Spleen: congestion

(20/20); hematopoietic

cell proliferation

(20/20); hemosiderin

pigmentation (20/20);

thrombosis (2/20);

capsule, fibrosis

(20/20); capsule,

lymphatic angiectasis

(6/20)

Testis/epididymis: 

degeneration (2/20);

mesothelial

hyperplasia (1/20)

Urinary bladder: 

transitional epithelium,

hyperplasia (17/20)
10 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 11

INTRODUCTION

Physical and Chemical Properties, Production, Use,

and Exposure

o-Nitrotoluene is synthesized by the nitration of toluene with a mixture of nitric acid and sulfuric

acid and subsequent separation by fractional distillation (Merck Index, 1983). o-Nitrotoluene is

a yellowish liquid at room temperature and is virtually insoluble in water but soluble in alcohol,

benzene, and ether. This flammable liquid has a specific gravity of 1.16 g/mL at 20 C, a melting

point of -10 C, a boiling point of 222 C, and a vapor pressure of 0.15 mm Hg at 20 C

(Mackison et al., 1981; Merck Index, 1983).

o-Nitrotoluene is used as a chemical intermediate and in the synthesis of dyestuffs and explosives

(Beard and Noe, 1981). The U.S. production of o-nitrotoluene has been estimated to be 28 million

pounds in 1981 (Dunlap, 1981) and from 1 to 10 million pounds in 1984 (Chemicals in Commerce

Information System, 1984). The annual consumption of o-nitrotoluene has been estimated at

10 to 12 million pounds (Howard et al., 1976). More recent figures are not available. Human

exposure to o-nitrotoluene occurs primarily through occupational sources. According to the

National Institute of Occupational Safety and Health (NIOSH, 1990), 4,354 people were exposed

to nitrotoluenes in the workplace (the isomer or isomers were not indicated).

o-Toluidine hydrochloride is prepared by the amination of toluene with methylhydroxylamine or

hydroxylammonium salts in the presence of aluminum trichloride (Merck Index, 1983), and then

converted to the aniline hydrochloride salt. o-Toluidine hydrochloride, a white, crystalline powder,

is very soluble in water and ethanol but insoluble in benzene and diethyl ether. The melting point

for this compound is 243 C. When heated to decomposition, o-toluidine hydrochloride emits

toxic fumes of hydrochloric acid and nitrogen oxides (USDHHS, 1991).

o-Toluidine and its hydrochloride salt are used in the manufacture of various dyes, including azo

pigment, triarylmethane, sulfur, and indigo (USDHHS, 1991). o-Toluidine is also used as a

photographic dye and as an antioxidant in the manufacture of rubber (Stanford Research Institute,

1976).
12 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

In 1983, U.S. production of o-toluidine was between 11 and 21 million pounds, and 34 companies

were identified as o-toluidine suppliers (Chem Sources USA, 1983). The United States imported

35,700 pounds of o-toluidine and 992 pounds of its hydrochloride salt in 1983 (USEPA, 1984).

 More recent figures are not available.

The primary routes of potential human exposure to o-toluidine and its hydrochloride salt are

through occupational exposure via inhalation and dermal contact (USDHHS, 1991). The National

Occupational Exposure Survey, conducted by NIOSH from 1981 to 1983, estimated that

13,053 workers were potentially exposed to o-toluidine in the workplace (NIOSH, 1995).

Disposition and Metabolism

In hepatocytes isolated from F344 rats, o-nitrotoluene was converted to nitrobenzyl alcohol via a

cytochrome P450-dependent process (deBethizy and Rickert, 1984). o-Nitrobenzyl alcohol, the

principal metabolite of o-nitrotoluene, was primarily conjugated with glucuronic acid (28% of the

metabolized nitrotoluene); a smaller percentage (3%) of metabolized o-nitrotoluene was oxidized

to o-nitrobenzoic acid.

Following oral administration of 200 mg o-nitrotoluene per kilogram body weight to male F344

rats, 86% of the dose was eliminated in the urine within 72 hours (Chism et al., 1984). The major

urinary metabolites of o-nitrotoluene were S-(2-nitrobenzyl)-N-acetylcysteine (12%), o-nitrobenzyl

glucuronide (14%), and a metabolite tentatively identified as a sulfur-containing conjugate of

o-toluidine (16%).

For male F344 rats, 29% of an oral dose (200 mg/kg) of o-nitrotoluene was excreted in the bile

after 12 hours (Chism and Rickert, 1985). o-Nitrobenzyl glucuronide was 78% of the

o-nitrotoluene metabolites in bile. The proposed scheme for the metabolism of o-nitrotoluene is

presented in Figure 1. o-Nitrotoluene is first converted to o-nitrobenzyl alcohol in the liver by a

cytochrome-P450-dependent process. The subsequent conjugation of o-nitrobenzyl alcohol with

glucuronic acid is mediated by hepatic UDP-glucuronosyltransferases. o-Aminobenzyl alcohol is

formed from o-nitrobenzyl glucuronide by the hydrolytic and reductive activities of gastrointestinal

flora. Bacterial nitroreductase activity in the gastrointestinal tract appears to be essential for the

genotoxicity/carcinogenicity of many nitroaromatic compounds (Doolittle et al., 1983; Rickert
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 13

et al., 1986a; Rickert, 1987; deBethizy and Hayes, 1994). Following systemic reabsorption, the

amino group may be oxidized by hepatic microsomal enzymes to the corresponding

hydroxylamine. The final activation step for o-nitrotoluene appears to be dependent upon

sulfotransferase (Rickert et al., 1986b). Either o-aminobenzyl alcohol or o-hydroxylaminobenzyl

alcohol may be conjugated with sulfate. o-Aminobenzyl sulfate is expected to be a highly reactive

alkylating agent (Chism et al., 1984). o-Hydroxylaminobenzyl alcohol is thought to conjugate with

sulfate to yield an unstable N-sulfate which decomposes to an electrophilic nitrenium ion (Chism

et al., 1984; Rickert, 1987; Chism and Rickert, 1989).

Following the subcutaneous administration of a single dose (400 mg/kg) of 14C-radiolabeled

o-toluidine hydrochloride to male CDF rats, the primary urinary metabolites were identified as the

sulfate and glucuronide conjugates of 4-amino-m-cresol and N-acetyl-4-amino-m-cresol (Son et al.,

1977). Following a single oral administration of [14C]-o-toluidine hydrochloride (methyl carbon

labeled) (500 mg/kg) to male Sprague-Dawley rats, greater than 92% of the radiolabel was

eliminated in the urine within 24 hours (Cheever et al., 1980). Metabolism was primarily on the

aromatic ring; greater than 50% of the administered dose was eliminated as phenol conjugates.

 No metabolites of o-toluidine hydrochloride derived from oxidation of the methyl group were

detected even though o-aminobenzoic acid could have been detected at levels as low as 0.1% of

the administered dose. Approximately 40% of the administered dose was excreted as the parent

compound.
14 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

FIGURE 1 Proposed Metabolic Scheme for o-Nitrotoluene: (A) o-Nitrotoluene;

(B) o-Nitrobenzyl Alcohol; (C) o-Nitrobenzyl Alcohol Glucuronide;

(D) o-Aminobenzyl Alcohol; (E) o-Hydroxylaminobenzyl Alcohol; (F) Unstable

N-Sulfate; (G) Nitrenium Ion; (H) o-Aminobenzyl Alcohol Sulfate; (I) Carbonium

Ion; (J) S-(o-Aminobenzyl) Glutathione;

(K) S-(o-Aminobenzyl)-N-acetylcysteine
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 15

Toxicity

Oral LD50 values for o-nitrotoluene in unspecified strains of rats have been reported to be

2,144 mg/kg (Hanavan, 1975 personal communication, E.I. duPont de Nemours and Co.) and

891 mg/kg (Christensen and Luginbyhl, 1974). An oral LD50 of 2,462 mg/kg has been reported

for an unspecified strain of mice (Christensen and Luginbyhl, 1974). A TCLO (toxic concentration,

low) of 700 g/m3

 has been reported in humans following inhalation exposure to o-nitrotoluene

(Sweet, 1983).

The National Toxicology Program (NTP) conducted 13-week feed studies of o-nitrotoluene in

F344/N rats and B6C3F1 mice (NTP, 1992; Dunnick et al., 1994). In these studies, groups of 10

male and 10 female animals of each species were exposed for 13 consecutive weeks to

o-nitrotoluene at concentrations of 0, 650, 1,250, 2,500, 5,000, and 10,000 ppm in NIH-07 diet.

 These doses delivered approximately 45 to 700 mg of o-nitrotoluene per kilogram of body weight

to rats and 100 to 700 mg of o-nitrotoluene per kilogram of body weight to mice. During the

13-week studies, there were no treatment-related deaths. An exposure-dependent decrease in

average feed consumption was evident in all treatment groups of male and female rats and mice.

 Mean body weights of exposed male and female rats and mice were also lower than those of the

controls.

In male and female F344/N rats and B6C3F1 mice, several organ weights were affected by

13 weeks of o-nitrotoluene treatment. In male rats, the livers were grossly enlarged and the testes

were reduced in size. Absolute liver and relative liver and kidney weights increased with

increasing exposure level in male rats. In addition, statistically significant, exposure-related

reductions in testicular and epididymal weights were apparent in the 5,000 and 10,000 ppm

groups. Results of clinical pathology evaluations from male and female rats revealed a

methemoglobinemia and regenerative anemia which increased in severity with increasing exposure

level (NTP, 1992; Dunnick et al., 1994).

Microscopic lesions in male and female mice were restricted to degeneration and metaplasia of the

olfactory epithelium in the nasal cavity. Several histopathologic changes were observed in male

and female rats. Male rats had hyaline droplet accumulation, testis degeneration, liver

vacuolization, oval cell hyperplasia, and mesothelioma of the epididymis (3/10 in the 5,000 ppm
16 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

group). Male and female rats had increased incidences of splenic hematopoiesis and pigment

accumulation in the red pulp and splenic capsule thickening.

The oral LD50 of o-toluidine hydrochloride was reported as 2,951 mg/kg in Osborne-Mendel rats

(Lindstrom et al., 1969), 900 mg/kg in Sprague-Dawley rats (Jacobson, 1972), and 15 mg/kg in

an unspecified strain of mice (IARC, 1982). o-Toluidine has reportedly induced

methemoglobinemia in mice (Nomura, 1977), rats (Lunkin, 1967), and humans (Ellenhorn and

Barceloux, 1988).

In 7-week studies sponsored by the National Cancer Institute (NCI), groups of five male and five

female F344 rats and B6C3F1 mice were administered o-toluidine hydrochloride in feed at nine

concentrations ranging from 1,000 to 50,000 ppm (NCI, 1979a). Seven weeks of exposure were

followed by 1 week of control diet administration. Mortality was observed in male and female rats

at the 50,000 ppm level. Exposure-related decreases in group mean body weights were evident

in male and female rats and mice. Microscopically, small amounts of renal and splenic

pigmentation were observed in male and female rats in the 12,500 ppm groups. In male and

female mice exposed to 50,000 ppm, pigment deposition was evident in the spleen, kidneys, and

liver.

CARCINOGENICITY

There have been no carcinogenicity studies performed for any of the mononitrotoluene isomers.

 The carcinogenicity of dinitrotoluene, a structural analogue of nitrotoluene, has been demonstrated

in long-term studies.

In a 2-year dosed-feed study sponsored by the Chemical Industry Institute of Toxicology (CIIT),

a technical grade of dinitrotoluene (tDNT) was demonstrated to be carcinogenic in male and

female F344 rats (CIIT, 1979). The tDNT was composed primarily of 2,4-dinitrotoluene and

2,6-dinitrotoluene. Rats received an estimated dose of 0, 3.5, 14, or 35 mg tDNT per kilogram

of body weight per day. After 1 year, all males and 50% of females in the 35 mg/kg groups had

developed hepatocellular carcinomas. There was a high incidence of hepatocellular carcinoma in

the 14 mg/kg group after 2 years. Long-term administration of tDNT also resulted in the formation

of cholangiocarcinomas which were presumably derived from the bile duct epithelium.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 17

In a 78-week bioassay sponsored by NCI, male and female F344 rats were administered 0.008%

or 0.02% 2,4-dinitrotoluene (96% pure) in feed for 78 weeks (NCI, 1978a). B6C3F1 mice were

similarly administered 0.008% or 0.04% 2,4-dinitrotoluene in feed. Exposure to 2,4-dinitrotoluene

was terminated after 78 weeks and animals were maintained on the control diet for an additional

26 weeks (rats) or 13 weeks (mice). Under the conditions of this bioassay, 2,4-dinitrotoluene was

not hepatocarcinogenic, although there was an increased incidence of benign skin fibromas in

exposed male rats and mammary gland fibroadenomas in female rats given 0.02% There was no

evidence of carcinogenicity in male or female B6C3F1 mice.

Popp and Leonard (1983) determined, in a series of 1-year dosed feed studies, that 7 or 14 mg/kg

2,6-dinitrotoluene (99.9% pure) was hepatocarcinogenic, while 27 mg/kg 2,4-dinitrotoluene

(99.9% pure) was not. Based on these results and the lack of hepatocarcinogenicity for

2,4-dinitrotoluene in the aforementioned NCI study (NCI, 1978a), the reported

hepatocarcinogenicity of tDNT (CIIT, 1979) appears to be primarily due to its 2,6-substituted

isomer. Studies with the structurally related compound, toluidine (which exists as ortho-, meta-,

and para-isomers), also suggest that the ortho-substituted compound is a more potent carcinogen

than the meta- or para-substituted isomers (Weisburger et al., 1978).

NCI sponsored 2-year bioassays of o-toluidine hydrochloride in F344 rats and B6C3F1 mice (NCI,

1979a). In NCI studies, groups of 50 male and 50 female rats and mice were given o-toluidine

hydrochloride in feed at concentrations of 3,000 or 6,000 ppm for rats and 1,000 or 3,000 ppm for

mice for 101 to 104 weeks. Relatively high, exposure-related mortality was observed in male and

female rats by the end of the study. Mortality in mice did not appear to be significantly affected

by treatment. Mean body weights in male and female rats and mice were lower than those of the

controls at the end of the studies.

When compared to controls, rats given o-toluidine hydrochloride in feed had increased incidences

of benign and malignant tumors (sarcomas) of the spleen in males and females, mesotheliomas of

the abdominal cavity or scrotum in males (control, 0%; 3,000 ppm, 24%; 6,000 ppm, 12%), and

transitional-cell carcinomas of the urinary bladder in females (0%, 20%, 47%) (NCI, 1979a). 

o-Toluidine hydrochloride exposure also resulted in increased incidences of fibromas of the

subcutaneous tissue in males (0%, 56%, 55%) and fibroadenomas or adenomas of the mammary

gland in females (36%, 40%, 71%).
18 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

In mice given o-toluidine hydrochloride, there were increased incidences of hemangiosarcomas in

males (5%, 4%, 24%) and of hepatocellular carcinomas or adenomas in females (0%, 8%, 26%)

(NCI, 1979a).

GENETIC TOXICITY

The genetic toxicity data for o-nitrotoluene are summarized in NTP Toxicity Report 23 (NTP,

1992). Results from genetic toxicity tests were mixed. Negative results were obtained in a

Salmonella typhimurium mutation assay (Haworth et al., 1983) and a test for induction of

chromosomal aberrations in cultured Chinese hamster ovary cells (Galloway et al., 1987). Positive

results were reported for o-nitrotoluene in a test for induction of sister chromatid exchanges in

cultured Chinese hamster ovary cells (Galloway et al., 1987) and in an assay for induction of

unscheduled DNA synthesis in hepatocytes of male rats treated in vivo (NTP, 1992). 

o-Nitrotoluene administered intraperitoneally did not induce micronucleated polychromatic

erythrocytes in rat or mouse bone marrow (NTP, unpublished). In the intraperitoneal study, the

highest dose tested in rats was 2,500 mg/kg (single injection) and in mice the highest dose was

three 400 mg/kg injections, administered at 24-hour intervals.

Because activation of o-nitrotoluene by intestinal bacteria is necessary to induce a positive

unscheduled DNA synthesis response, the S. typhimurium assay, performed in the absence of a

specialized protocol which employs reductive metabolism, would not be expected to yield a

positive response. Also, the metabolic pathways that generate the active metabolite of

o-nitrotoluene appear to be species and sex specific. Therefore, detection of the mutagenic activity

of o-nitrotoluene is highly dependent upon the test system and the protocol employed.

The genotoxicity of o-toluidine has recently been reviewed in detail (Danford, 1991). o-Toluidine

was genotoxic in a variety of in vitro and in vivo assays, although the experimental conditions for

detecting genotoxicity were rather stringent, particularly with regard to the metabolic activation

system used for the in vitro studies (Danford, 1991). Positive results were reported for o-toluidine

in the following assay systems: bacterial mutation tests (Tanaka et al., 1980; Kada, 1981;

Trueman, 1981; Venitt and Crofton-Sleigh, 1981; Zeiger and Haworth, 1985), yeast aneuploidy

tests (Parry and Sharp, 1981; Parry and Eckardt, 1985), Drosophila somatic recombination assays

(Vogel, 1985; Würgler et al., 1985), in vitro cytogenetics assays (Perry and Thomson, 1981;
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 19

Danford, 1985; Darroudi and Natarajan, 1985; Gulati et al., 1985; Ishidate and Sofuni, 1985; Lane

et al., 1985; Palitti et al., 1985; Priston and Dean, 1985; van Went, 1985), and DNA damage

assays (Martin and McDermid, 1981; Cesarone et al., 1982; Barrett, 1985; Bradley, 1985; Douglas

et al., 1985; Glauert et al., 1985; Martin and Campbell, 1985). Results from these assays,

however, were not uniform among all testing laboratories, and appear to be protocol dependent.

 Experimental parameters such as S9 source and concentration, length of exposure time to

o-toluidine, dose, and cell line or type were important in determining whether a particular test was

positive or negative.

Results of in vivo mouse bone marrow micronucleus assays with o-toluidine were negative

(Salamone et al., 1981; Tsuchimoto and Matter, 1981). However, results of recent NTP

investigations suggest that species differences may play an important role in the induction of

micronucleated normochromatic erythrocytes (NTP, unpublished). These studies showed that

o-toluidine induced micronuclei in bone marrow cells of male F344/N rats, but not male B6C3F1

mice. Rats were given an intraperitoneal injection of 400mg/kg o-toluidine at 24-hour intervals

for a total of three injections, and bone marrow was examined 24 hours after the final injection.

 Mice were administered a single injection of 400 mg/kg o-toluidine, and bone marrow samples

were taken 24 hours after dose administration. These treatments were the maximum nonlethal

dose that could be administered to each species, as determined by preliminary range finding tests.

 In another study, o-toluidine hydrochloride was shown to induce sister chromatid exchanges but

not chromosomal aberrations or micronuclei in bone marrow cells of male B6C3F1 mice (McFee

et al., 1989). In this test, 150 to 600 mg/kg o-toluidine hydrochloride was administered to the mice

as a single intraperitoneal injection.

The genetic toxicity test data for o-toluidine indicate that the compound has genotoxic activity and

detection of this activity is highly dependent on the assay system and protocol employed. The

genotoxic activity demonstrated in several assays has not been strong; thus, o-toluidine would

perhaps best be described as a weak mutagen.

Study Rationale and Design

o-Nitrotoluene and o-toluidine hydrochloride are high-production, structurally related compounds.

 In a 13-week dosed-feed study, three male rats given 5,000 ppm of o-nitrotoluene developed

mesothelioma of the epididymis, a neoplasm that had not previously been identified in a 13-week
20 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

toxicity study (NTP, 1992; Dunnick et al., 1994). In a 2-year carcinogenicity study with

o-toluidine hydrochloride, there were increased treatment-related incidences of several tumor types

in F344 rats, including mesothelioma of the abdominal cavity or scrotum; potential toxic and

carcinogenic effects were not evaluated at 13 weeks (NCI, 1979a).

The NTP studies reported here were designed to confirm the carcinogenic effects of o-nitrotoluene

and to compare the potential toxicity and carcinogenicity of o-nitrotoluene and o-toluidine

hydrochloride with respect to the development, progression, and regression of treatment-related

effects during 13-week or 26-week exposure. Because metabolism and genotoxicity studies of

o-nitrotoluene have indicated that gastrointestinal flora may play an obligatory role in the metabolic

activation (reduction of the nitro group to the amine function) and genotoxicity of this compound,

additional groups were included in an attempt to determine the effect of altered gastrointestinal

flora on the toxicity or carcinogenicity of o-nitrotoluene.

Based on earlier studies, the concentration chosen for these studies, 5,000 ppm, was expected to

cause a marked decrease in the body weight gains of rats receiving o-nitrotoluene. It was

recognized that this lower weight gain might inhibit the expression of neoplasms; however, this

dose was effective in producing mesothelioma in the study reported by Dunnick et al. (1994) and

was therefore chosen for the present comparative study.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 21

MATERIALS AND METHODS

Procurement and Characterization

of o-Nitrotoluene and o-Toluidine Hydrochloride

Two lots of o-nitrotoluene (Lots 111OCJ and 08506MV) were obtained from the Aldrich

Chemical Company (Metuchen, NJ). A single lot of o-toluidine hydrochloride (Lot 308828/1 891)

was obtained from SAF Bulk Chemicals (Ronkonkoma, NY). Lot 111OCJ of o-nitrotoluene was

used for chemistry methods development and stability studies. Lot 08506MV of o-nitrotoluene

and Lot 308828/1 891 of o-toluidine hydrochloride were used for the 26-week studies.

Chemical analyses of o-nitrotoluene were performed by Midwest Research Institute (MRI; Kansas

City, MO). For Lot 1110CJ, infrared, ultraviolet/visible, and nuclear magnetic resonance (NMR)

spectra were consistent with the structure of o-nitrotoluene and with a literature reference (Sadtler

Standard Spectra). The results of elemental analyses for carbon, hydrogen, and nitrogen agreed

with theoretical values for o-nitrotoluene. Karl Fischer analysis indicated 0.021% ± 0.003% water.

Functional group titration, by nitro reduction with titanium (III) chloride followed by back titration

with standardized ferric ammonium sulfate, indicated a purity of 99% ± 1%. Thin-layer

chromatography by two solvent systems indicated a major spot only in each system. Two gas

chromatographic systems with flame ionization detection (FID) showed a major peak and no

impurities with relative areas equal to or greater than 0.38%.

Lot 08506MV was identified as o-nitrotoluene (a clear, pale yellow liquid) by MRI with infrared,

ultraviolet, and NMR spectroscopy. A boiling point of 221.6 C and a density (d ) of 1.156 g/mL 23.7

23.4

(n=3) for Lot 08506MV agreed with a literature reference (Merck Index, 1983) and with the

boiling point and density of Lot 1110CJ. Infrared, ultraviolet/visible, and NMR spectra were

consistent with the structure of o-nitrotoluene, with literature references (Sadtler Standard Spectra;

Aldrich, 1985), and with the spectra of Lot 1110CJ. Concomitant analyses of Lot 08506MV and

Lot 1110CJ by gas chromatography with FID showed a purity of 99.8% ± 0.3% for Lot 08506MV

relative to Lot 1110CJ.

An accelerated stability study was performed by MRI on Lot 1110CJ of o-nitrotoluene. Gas

chromatographic analyses with FID indicated that bulk o-nitrotoluene was stable for 2 weeks when

stored protected from light at temperatures up to 60 C. At the study laboratory, o-nitrotoluene
22 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

was stored in sealed amber glass bottles at approximately 25 C. The study laboratory reanalyzed

the bulk chemical (Lot 08506MV) by gas chromatography with FID before the start of the studies,

during the studies, and again after the studies ended; no degradation of o-nitrotoluene was

observed.

Chemical analyses of o-toluidine hydrochloride were performed by SAF Bulk Chemicals on

Lot 308828/1 891. The chemical, a white, crystalline powder with lumps, was identified as

o-toluidine hydrochloride. An infrared spectrum was consistent with the structure of o-toluidine

hydrochloride and with a literature reference. The results of elemental analyses for carbon,

hydrogen, and nitrogen agreed with theoretical values for o-toluidine hydrochloride.

Argentometric titration (chloride determination) indicated a purity of 100% for o-toluidine

hydrochloride. At the study laboratory, o-toluidine hydrochloride was stored in sealed amber glass

bottles at approximately 25 C. The study laboratory reanalyzed the bulk chemical by high

performance liquid chromatography (HPLC) before the start of the studies, during the studies, and

again after the studies ended; no degradation of o-toluidine hydrochloride was observed.

Preparation and Analysis of Dose Formulations

o-Nitrotoluene or o-toluidine hydrochloride was mixed with NIH-07 Open Formula Diet (Zeigler

Bros., Inc., Gardners, PA) in meal form. A premix was prepared by adding an accurately weighed

amount of o-nitrotoluene oro-toluidine hydrochloride to a small portion of feed. Additional feed

was added until the desired premix weight was reached. Then, the premix was blended with a

weighed amount of feed in a Patterson-Kelley twin-shell blender (East Stroudsburg, PA) for

15 minutes, with the intensifier bar on for the first 5 minutes.

MRI performed homogeneity and stability studies on a feed mixture containing 0.5 mg

o-nitrotoluene (Lot 1110CJ) per gram of feed. o-Nitrotoluene was mixed with NIH-07 Open

Formula Diet by the method described previously. After feed blending, a sample was taken from

each of three blender ports, and the samples were extracted with ethanol and analyzed for

o-nitrotoluene by HPLC. Homogeneity of the feed mixture was confirmed. Approximately 12%

o-nitrotoluene was lost during feed preparation. 

Samples of the 0.5 mg/g o-nitrotoluene-feed mixture were stored in sealed containers in the dark

at room temperature, 5 C, or -20 C for 7, 14, or 21 days or in a rat cage, open to air and light,
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 23

for 1, 2, 3, 4, or 7 days. All samples were analyzed by HPLC. Feed samples stored in sealed

containers in the dark at 5 C were stable for 3 weeks. Feed samples stored in the rat cage for all

time periods showed statistically significant loses of o-nitrotoluene. Samples analyzed after 1 day

in the rat cage showed a loss of 6.3% o-nitrotoluene, and, after 7 days of storage, samples showed

a loss of 18.7% o-nitrotoluene. These losses were in addition to the approximately

12% o-nitrotoluene lost during feed preparation.

The study laboratory performed homogeneity and stability studies on a feed mixture containing

5 mg o-nitrotoluene/g feed (5,000 ppm) (Lot 08506MV). o-Nitrotoluene was mixed with NIH-07

Open Formula Diet by the method described previously. After feed blending, a sample was taken

from each of three blender ports, and the samples were extracted with ethanol and analyzed for

o-nitrotoluene by HPLC. Homogeneity of the feed mixture was confirmed. Approximately 2%

o-nitrotoluene was lost during feed preparation.

Samples of the 5 mg/g o-nitrotoluene-feed mixture were stored in sealed amber glass bottles at

-20 C, 5 C, or room temperature for 28 days or in an animal feeder, open to air and light, for 1

or 4 days. All samples were analyzed by gas chromatography with FID. Samples stored in sealed

containers at -20 and 5 C, protected from light, were stable for at least 28 days. Samples stored

in sealed containers at room temperature, protected from light, showed statistically significant

losses at 7, 14, and 28 days. Samples stored in an animal feeder, open to air and light, exhibited

a loss of 3.4% after 1 day and 10.0% after 4 days. The loss after 4 days was statistically significant

and was likely due to evaporation of o-nitrotoluene from the open feeders.

At the study laboratory, o-nitrotoluene feed mixtures were stored in sealed containers at

approximately 5 C for up to 28 days. On Day 7 of the study, o-nitrotoluene-feed mixtures were

stored at room temperature because of refrigerator malfunction.

In the animal rooms, clean feed jars with fresh feed were provided to all rats at least weekly.

o-Nitrotoluene-feed mixtures were prepared once every 2 weeks for the first 14 weeks and once

every 3 weeks thereafter. Samples of the o-nitrotoluene-feed mixtures prepared on 22 April and

20 May 1992 were taken before dosing and after dosing (from the animal rooms) and analyzed for

o-nitrotoluene by gas chromatography with FID. The animal room samples of the 22 April and

20 May 1992 mixtures showed losses of 6% and 9% o-nitrotoluene, respectively. These losses are
24 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

consistent with the results of prestart stability studies, which showed a loss of approximately 10%

o-nitrotoluene after storage in open containers for 4 days.

The study laboratory performed homogeneity and stability studies on a feed mixture containing

5 mg o-toluidine hydrochloride/g feed (5,000 ppm). o-Toluidine hydrochloride was mixed with

NIH-07 Open Formula Diet by the method described previously. After blending, a sample was

taken from each of three blender ports, and the samples were extracted with methanol and analyzed

for o-toluidine hydrochloride by HPLC. Homogeneity of the feed mixture was confirmed.

Approximately 9% o-toluidine hydrochloride was lost during feed preparation.

Samples of the 5 mg/g o-toluidine hydrochloride-feed mixture were stored in sealed containers in

the dark at room temperature, -20 C or 5 C for 7, 14, or 28 days or in an animal feeder, open

to air and light, for 1 or 4 days. All samples were analyzed by HPLC. Feed samples stored in

sealed containers in the dark at -20 or 5 C were stable for 28 days. The feed sample stored at

room temperature for 28 days contained 90% of the o-toluidine hydrochloride detected in the

sample before storage.

At the study laboratory, o-toluidine hydrochloride feed mixtures were stored in sealed containers

at approximately 5 C, with one exception, for up to 28 days. From Day 1 to Day 7 of the study,

the o-toluidine hydrochloride feed mixtures were improperly stored at room temperature. Based

on the results of the stability study, storage at room temperature was expected to decrease the

o-toluidine concentration by 5% to 10%.

In the animal rooms, clean feed jars with fresh feed were provided to all rats at least weekly.

o-Toluidine hydrochloride feed mixtures were prepared once every 3 weeks. Samples of the feed

mixtures prepared on 3 March, 14 April, 6 May, 7 July, and 18 August 1992, were collected

before dosing and analyzed for o-toluidine hydrochloride content by HPLC. The animal room

samples collected from feeders 4 or 5 days after preparation on 6 May, 7 July, and 18 August 1992

mixtures showed losses of 0%, 15%, and 35% o-toluidine hydrochloride, respectively. For the

samples showing 15% and 35% losses, contamination with animal hair and feces contributed to

an indeterminate amount of the loss in each sample. Differences in water content between the feed

used to prepare the standards and the feed removed from the animal rooms accounted for 3% of

the loss in each sample.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 25

Preparation of Antibiotic Mixture

Tetracycline hydrochloride, neomycin sulfate, and nystatin were obtained from Sigma Chemical

Co. (St. Louis, MO). Weighed amounts of each antibiotic (in powdered form) were combined,

apportioned into sealed containers in daily allotments, and stored at approximately 0 C. Before

dosing, the antibiotic mixture was reconstituted by adding the appropriate volume of deionized

water. No chemical analyses of the neat chemicals or dose formulations were required. The

efficacy and acute toxicity of the antibiotic mixture were evaluated in non-GLP pilot studies. The

results of these studies are presented in Appendix C.

Toxicity Study Designs

BASE STUDIES

Male F344/N rats used in these studies were obtained from Charles River Breeding Laboratories

(Raleigh, NC). Rats were 36 days old at receipt, were quarantined for 9 days, and were 45 days

old at the start of the studies. Blood samples were collected from 10 sentinel rats at the start of the

studies and were analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al.,

1989a,b); all results were negative. Additional details concerning the study design are provided

in Table 1.

All exposed male rats received 5,000 ppm o-nitrotoluene or o-toluidine hydrochloride in feed for

13 or 26 weeks. The dose selected for administration of o-nitrotoluene was based on the previous

13-week study where a concentration of 5,000 ppm in the diet caused mesothelioma in 3 of 10 rats.

At that dose, there was no effect on survival, but body weights were 7% less than those of controls.

For the comparison study, rats were administered an approximately equimolar amount

(5,000 ppm) of o-toluidine hydrochloride. o-Nitrotoluene was administered to 60 rats with normal

gastrointestinal flora and to 40 rats with altered gastrointestinal flora. o-Toluidine hydrochloride

was administered to 60 rats with normal gastrointestinal flora. There were two control groups with

20 unexposed rats each, one with normal gastrointestinal flora and one with altered flora. Rats in

the altered gastrointestinal flora groups received a single gavage dose of an antibiotic mixture (20

mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin) in deionized water daily

for 6 days before the start of the study and daily for 13 weeks thereafter. This treatment was based

on the results of a pilot study (Appendix C) that demonstrated a reduction of aerobic flora greater

than 97% compared to controls. On one day during Week 12, rats did not receive the antibiotic
26 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

mixture due to a technician oversight. The missed treatment likely had minimal impact on

microbial growth, the formation of o-nitrotoluene-derived metabolites, or any associated toxicity.

Following the 13-week administration of o-nitrotoluene or o-toluidine hydrochloride, 10 rats from

each control group (normal and altered gastrointestinal flora) and 20 rats from each exposure

group (normal and altered gastrointestinal flora) were killed for the 13-week interim evaluation.

Following the 13-week administration of o-nitrotoluene or o-toluidine hydrochloride and the

13-week maintenance period on the control diet, 20 rats from each exposure group (normal and

altered gastrointestinal flora) were killed for the stop-exposure evaluation. Following the 26-week

administration of o-nitrotoluene or o-toluidine hydrochloride, 20 rats from each normal

gastrointestinal flora exposure group were killed for the 26-week evaluation. Ten rats from each

control group (normal and altered gastrointestinal flora) were killed at 26 weeks for the stopexposure and 26-week evaluations.

Rats were housed five per cage, and NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners,

PA) in meal form and water (Columbus, OH) were available ad libitum. Feed consumption was

measured weekly. Animal rooms were maintained at 72 ± 3 F and 50% ± 15% relative

humidity, with at least 10 room air changes per hour. Fluorescent light was provided for 12 hours

per day.

Necropsies, with limited tissue collection, were performed on all rats exposed to o-nitrotoluene or

o-toluidine hydrochloride for 13 weeks, 13 weeks with 13 weeks of recovery, or 26 weeks. The

epididymis, right kidney, liver, spleen, and right testis of each rat were weighed. Organs and

tissues were examined for gross lesions and fixed in 10% neutral buffered formalin. Tissues to be

examined microscopically were trimmed, embedded in paraffin, sectioned, and stained with

hematoxylin and eosin. For all paired organs (i.e., adrenal gland, kidney, ovary), samples from

each organ were examined. Osmium staining for the presence of fat was performed on formalinfixed samples of liver from control and o-nitrotoluene-exposed rats. Proliferating cell nuclear

antigen staining of formalin-fixed, paraffin-embedded samples of cholangiocarcinoma were also

performed (Greenwell et al., 1991). Histopathologic evaluations were performed on all rats at the

13-week interim evaluation and the end of the studies. Tissues examined microscopically are listed

in Table 1.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 27

Upon completion of the laboratory pathologist's histopathologic evaluation, the slides, paraffin

blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match,

and wet tissue audit. The slides, individual animal data records, and pathology tables were sent

to an independent pathology laboratory where quality assessment was performed. Results were

reviewed and evaluated by the NTP Pathology Working Group (PWG); the final diagnoses

represent a consensus of contractor pathologists and the PWG. Details of these review procedures

have been described by Maronpot and Boorman (1982) and Boorman et al. (1985).

SUPPLEMENTAL EVALUATIONS

At the 13-week interim evaluation, cecal specimens were collected from five rats per group in the

control groups (normal and altered gastrointestinal flora) and o-nitrotoluene groups (normal and

altered gastrointestinal flora). The samples were diluted with sterile phosphate buffered saline

(1:100 on a weight:weight basis) and mixed for 4 minutes with a Model 400 Stomacher Lab

Blender. Three serial dilutions were made for each sample and plated in triplicate on MacConkey

pour plates. The plates were incubated aerobically at 35 to 37 C for 18 to 24 hours. Plates

containing 30 to 300 colonies were counted manually.If no plates fell within this range, counts

were estimated by standard microbiological procedures. Counts were reported as colony-forming

units per gram of sample analyzed.

Additional sections of liver were prepared and stained to determine and quantitate placental

glutathione S-transferase (PGST)-positive foci of cellular alteration (Hsu et al., 1981). Livers from

9 or 10 rats in the 13-week interim and 26-week continuous-exposure groups were evaluated for

PGST-positive foci. Image analyses of PGST-positive foci were performed to determine the

number of foci per cubic centimeter of liver and the volume fraction (expressed as a percentage)

of liver occupied by PGST-positive foci (Ton et al., 1995).
28 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE 1 Experimental Design and Materials and Methods in the 26-Week Feed Studies

of o-Nitrotoluene and o-Toluidine Hydrochloride

EXPERIMENTAL DESIGN

Study Laboratory

Battelle Columbus Operations, Columbus, OH

Strain and Species

Male F344/N rats

Animal Source

Charles River Breeding Laboratories, Raleigh, NC

Size of Study Groups

13-week interim:

 Normal gastrointestinal flora groups: 10 control rats; 20 rats (o-nitrotoluene); 20 rats (o-toluidine hydrochloride)

 Altered gastrointestinal flora groups: 10 control rats; 20 rats (o-nitrotoluene)

Stop-exposure:

 Normal gastrointestinal flora groups: 20 rats (o-nitrotoluene); 20 rats (o-toluidine hydrochloride)

 Altered gastrointestinal flora groups: 10 control rats; 20 rats (o-nitrotoluene); 20 rats (o-toluidine hydrochloride)

26-week study:

 Normal gastrointestinal flora groups: 10 control rats; 20 rats (o-nitrotoluene); 20 rats (o-toluidine hydrochloride)

Route of Administration

Feed

Exposure Concentrations/ Duration

13-Week interim groups:

 0 ppm or 5,000 ppm o-nitrotoluene or o-toluidine hydrochloride for 13 weeks

Stop-exposure groups:

 0 ppm or 5,000 ppm o-nitrotoluene or o-toluidine hydrochloride for first 13 weeks; 0 ppm for final 13 weeks

26-Week continuous-exposure groups:

 0 ppm or 5,000 ppm o-nitrotoluene or o-toluidine hydrochloride for 26 weeks

Date of First Exposure

6 March 1992

Date of Last Exposure/ Necropsy

13-Week interim: 8 June 1992

Study termination: 8 September 1992 (stop-exposure study groups), 9 September 1992 (26-week continuous-exposure

groups)

Type and Frequency of Observation

Rats were observed twice daily. Body weights and clinical signs were recorded weekly and at necropsy. Feed consumption

was measured weekly.

Necropsy and Histologic Examinations

Complete necropsies were performed on all rats exposed to o-nitrotoluene or o-toluidine hydrochloride for 13 weeks,

13 weeks with 13 weeks of recovery, or 26 weeks. The right kidney, liver, spleen, right testis, and epididymis of all control

rats and 10 of 20 rats from each exposure group were weighed. Histopathologic evaluations were performed on all rats at

the 13-week interim evaluation and at the end of the studies. The following tissues were examined in all groups: 

epididymides (three portions of each), gross lesions, liver, right kidney, spleen, and testes. The urinary bladder was also

examined in rats administered o-toluidine hydrochloride for 13 or 26 weeks.

Supplemental Evaluations

At the 13-week interim, cecal specimens were collected from five rats per group in the control groups (normal and altered

gastrointestinal flora) and o-nitrotoluene groups (normal and altered gastrointestinal flora); microbial colony counts were

taken for each specimen.

At the 13-week interim and the end of the 26-week continuous and stop-exposure periods, liver samples were collected from

9 or 10 rats per group in the control groups (normal and altered gastrointestinal flora) and o-nitrotoluene groups (normal and

altered gastrointestinal flora); tissue was stained for placental glutathione S-transferase-positive foci.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 29

TABLE 1 Experimental Design and Materials and Methods in the 26-Week Feed Studies

of o-Nitrotoluene and o-Toluidine Hydrochloride (continued)

ANIMAL MAINTENANCE

Time Held Before Studies

9 days

Age When Studies Began

45 days

Age When Killed

19 weeks (13-week interim); 32 weeks (stop-exposure and 26-week continuous-exposure)

Method of Animal Distribution

Animals were distributed randomly into groups of approximately equal initial mean body weight.

Diet

NIH-07 Open Formula Diet (Zeigler Bros., Inc., Gardners, PA) in meal form and water (City of Columbus), available

ad libitum.

Animal Room Environment

Rats were housed five per cage. In the animal room, the temperature was maintained at 72 ± 3 F and relative humidity at

50% ± 15%, with at least 10 room air changes per hour. Fluorescent light was provided for 12 hours per day.

Statistical Methods

ANALYSIS OF LESION INCIDENCES

The Fisher exact test (Armitage, 1971; Gart et al., 1979), a procedure based on the overal

proportion of lesion-bearing animals, was used to evaluate histopathologic lesion data.

ANALYSIS OF CONTINUOUS VARIABLES

Organ and body weight data, which have approximately normal distributions, were analyzed with

the parametric multiple comparison procedures of Dunnett (1955). The number of PGST-positive

foci per cubic centimeter of liver and the volume fraction of liver occupied by PGST-positive foci

were analyzed with Wilcoxon's rank sum test (Conover, 1971).

Before analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were

examined by NTP personnel. Implausible values, extreme values from animals that were suspected

of being sick due to causes other than treatment, and values that the study laboratory indicated as

being inadequate due to technical problems were eliminated from the analysis.
30 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

Quality Assurance

The animal studies of o-nitrotoluene and o-toluidine hydrochloride were performed in compliance

with United States FDA Good Laboratory Practices regulations (21 CFR, Part 58). The Quality

Assurance Unit of Battelle Columbus Operations performed audits and inspections of protocols,

procedures, data, and reports throughout the course of the studies.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 31

RESULTS

26-Week Feed Studies in Male F344/N Rats

All rats in the 13-week interim, stop-exposure, and 26-week continuous-exposure groups survived

until the scheduled evaluations (Table 2). Average daily fed consumption was 12% to 18% lower

than that by controls for rats in the o-nitrotoluene-exposed groups and 5% to 9% lower than that

by controls for rats in the o-toluidine hydrochloride-exposed groups (Table 3). Feed consumption

was similar in the corresponding control and exposed rats in the normal and altered gastrointestinal

flora groups. Mean body weight gains of rats in all exposed groups were lower than those of the

controls after 13 or 26 weeks of exposure. At the 13-week interim, mean body weight gains were

42% (altered flora) and 44% (normal flora) lower for rats exposed to o-nitrotoluene and 21%

lower for rats exposed to o-toluidine hydrochloride than those of the respective controls (Table 2

and Figure 2). In the 26-week continuous-exposure groups, mean body weight gains were 53%

lower for rats exposed to o-nitrotoluene and 27% lower for rats exposed to o-toluidine

hydrochloride than the control value (Table 2 and Figure 3). Rats in the stop-exposure groups had

slightly greater mean body weight gains than rats continuously exposed for 26 weeks but still

weighed less than the controls (Table 2 and Figure 4). There were no treatment-related clinical

signs in the 13-week or 26-week stop- or continuous-exposure groups.
32 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

Table 2 Survival and Body Weights of Male F344/N Rats 

in the 26-Week Feed Studies of o-Nitrotoluene 

and o-Toluidine Hydrochloride

Final Weight

Mean Body Weight (grams) Relative to

Group Survival )

1

Initial Final Change Controls2 (%

13-WEEK INTERIM

Normal Gastrointestinal Flora

 Control (0 ppm) 10/10 157 331 175

 o-Nitrotoluene (5,000 ppm) 10/10 163 261 98 79

 o-Toluidine HCl (5,000 ppm) 10/10 155 293 139 89

Altered Gastrointestinal Flora

 Control (0 ppm) 10/10 157 314 158

 o-Nitrotoluene (5,000 ppm) 10/10 153 245 92 78

STOP-EXPOSURE

Normal Gastrointestinal Flora

 Control (0 ppm) 10/10 159 382 224

 o-Nitrotoluene (5,000 ppm) 10/10 159 297 139 78

 o-Toluidine HCl (5,000 ppm) 10/10 155 342 187 89

Altered Gastrointestinal Flora

 Control (0 ppm) 10/10 158 379 221

 o-Nitrotoluene (5,000 ppm) 10/10 159 273 115 72

26-WEEK CONTINUOUS-EXPOSURE

Normal Gastrointestinal Flora

 Control (0 ppm) 10/10 159 382 224

 o-Nitrotoluene (5,000 ppm) 10/10 159 265 106 69

 o-Toluidine HCl (5,000 ppm) 10/10 151 314 163 82

Number surviving at 13 or 26 weeks/number of animals per exposure group. 1

(Exposure group mean/control group mean) x 100. 2
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 33

Table 3 Feed and Estimated Compound Consumption 

by Male F344/N Rats in the 26-Week Feed Studies 

of o-Nitrotoluene and o-Toluidine Hydrochloride

Feed Compound

Consumption Consumption

Group (g/day) (mg/kg/day) 1 2

13-WEEK INTERIM

Normal Gastrointestinal Flora

 Control (0 ppm) 14.9

 o-Nitrotoluene (5,000 ppm) 12.2 293

 o-Toluidine HCl (5,000 ppm) 13.5 301

Altered Gastrointestinal Flora

 Control (0 ppm) 14.3

 o-Nitrotoluene (5,000 ppm) 12.0 296

STOP-EXPOSURE

Normal Gastrointestinal Flora

 Control (0 ppm) 14.7

 o-Nitrotoluene (5,000 ppm) 12.9 293

 o-Toluidine HCl (5,000 ppm) 13.9 304

Altered Gastrointestinal Flora

 Control (0 ppm) 14.5

 o-Nitrotoluene (5,000 ppm) 12.7 293

26-WEEK CONTINUOUS-EXPOSURE

Normal Gastrointestinal Flora

 Control (0 ppm) 14.7

 o-Nitrotoluene (5,000 ppm) 12.3 292

 o-Toluidine HCl (5,000 ppm) 13.4 285

Average of group mean feed consumption values measured at Weeks 1-14 for rats in the 13-week interim evaluation or 1

Weeks 1-27 for rats in the stop-exposure evaluation and 26-week study.

Average of group mean compound consumption values measured at Weeks 1-14 for rats in the 13-week interim 2

evaluation or Weeks 2-27 for rats in the stop-exposure evaluation and 26-week study.
34 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

FIGURE 2 Body Weights of Male F344/N Rats Exposed to o-Nitrotoluene

or o-Toluidine Hydrochloride in Feed for 13 Weeks

FIGURE 3 Body Weights of Male F344/N Rats Exposed to o-Nitrotoluene

or o-Toluidine Hydrochloride in Feed for 26 Weeks
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 35

Normal Gastrointestinal Flora

Altered Gastrointestinal Flora

FIGURE 4 Body Weights of Male F344/N Rats Exposed to o-Nitrotoluene

or o-Toluidine Hydrochloride in Feed for 13 Weeks

and Allowed to Recover for 13 Weeks
36 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

Chemical-related effects were present in the testis and epididymis, liver, kidney, and spleen of rats

exposed to o-nitrotoluene or o-toluidine hydrochloride and in the urinary bladder of rats exposed

to o-toluidine hydrochloride. Because of the variation in the character, severity, and potential for

progression or reversal of these lesions, the effects of o-nitrotoluene and o-toluidine hydrochloride

for each target organ in the 13-week interim and stop-exposure groups (normal and altered

gastrointestinal flora) and 26-week continuous-exposure group are described separately. Organ

weight changes presented in Table 4 are discussed with the chemical-related histopathology

findings for each organ.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 37

TABLE 4 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for Male F344/N Rats in the 26-Week Feed Studies of o-Nitrotoluene

and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2

Control o-Nitrotoluene o-Toluidine Control o-Nitrotoluene

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM

n 10 10 10 10 10

Necropsy body wt 345 ± 5 261 ± 2** 298 ± 4** 318 ± 5 247 ± 4**

Right kidney

Absolute 1.130 ± 0.016 1.058 ± 0.023 1.043 ± 0.026* 1.034 ± 0.023 1.032 ± 0.022

Relative 3.27 ± 0.03 4.05 ± 0.06** 3.50 ± 0.05** 3.25 ± 0.03 4.19 ± 0.08**

Liver

Absolute 12.056 ± 0.235 13.844 ± 0.316** 12.671 ± 0.206 10.140 ± 0.237 13.201 ± 0.400**

Relative 34.91 ± 0.31 53.08 ± 1.07** 42.57 ± 0.36** 31.86 ± 0.36 53.53 ± 1.55**

Spleen

Absolute 0.730 ± 0.010 0.789 ± 0.021 2.212 ± 0.056** 0.648 ± 0.010 0.729 ± 0.015**

Relative 2.12 ± 0.03 3.02 ± 0.06** 7.43 ± 0.16** 2.04 ± 0.02 2.96 ± 0.08**

Right testis

Absolute 1.593 ± 0.027 1.186 ± 0.028** 1.508 ± 0.020* 1.536 ± 0.030 1.114 ± 0.025**

Relative 4.61 ± 0.03 4.55 ± 0.08 5.07 ± 0.07** 4.83 ± 0.07 4.51 ± 0.08*

Epididymis

Absolute 0.477 ± 0.006 0.331 ± 0.013** 0.420 ± 0.009** 0.469 ± 0.011 0.321 ± 0.007**

Relative 1.38 ± 0.02 1.27 ± 0.04* 1.41 ± 0.03 1.48 ± 0.05 1.30 ± 0.03**

STOP-EXPOSURE 3

n 10 10 10 10 10

Necropsy body wt 389 ± 5 304 ± 6** 351 ± 9** 388 ± 5 281 ± 3**

Right kidney

Absolute 1.379 ± 0.036 1.326 ± 0.027 1.255 ± 0.047* 1.274 ± 0.029 1.371 ± 0.022*

Relative 3.55 ± 0.08 4.36 ± 0.04** 3.56 ± 0.05 3.29 ± 0.07 4.88 ± 0.07**

Liver

Absolute 14.405 ± 0.257 18.186 ± 0.301** 13.910 ± 0.532 13.929 ± 0.390 19.089 ± 0.416**

Relative 37.09 ± 0.69 59.89 ± 1.08** 39.58 ± 1.13 35.88 ± 0.75 67.98 ± 1.59**

Spleen

Absolute 0.806 ± 0.017 0.818 ± 0.017 1.130 ± 0.048** 0.739 ± 0.020 0.748 ± 0.012

Relative 2.08 ± 0.05 2.70 ± 0.06** 3.23 ± 0.16** 1.90 ± 0.04 2.66 ± 0.05**

Right testis

Absolute 1.615 ± 0.027 0.893 ± 0.077** 1.496 ± 0.025 1.585 ± 0.017 0.821 ± 0.090**

Relative 4.16 ± 0.06 2.93 ± 0.25** 4.28 ± 0.12 4.09 ± 0.07 2.91 ± 0.31**

Epididymis

Absolute 0.484 ± 0.006 0.288 ± 0.018** 0.432 ± 0.014 0.469 ± 0.008 0.254 ± 0.020** 4

Relative 1.25 ± 0.01 0.95 ± 0.06** 1.23 ± 0.02 1.21 ± 0.02 0.90 ± 0.07** 4
38 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE 4 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for Male F344/N Rats in the 26-Week Feed Studies of o-Nitrotoluene

and o-Toluidine Hydrochloride (continued)

Normal Gastrointestinal Flora

Control o-Nitrotoluene o-Toluidine

(0 ppm) (5,000 ppm) (5,000 ppm)

26-WEEK CONTINUOUS-EXPOSURE

n 10 10 10

Necropsy body wt 389 ± 5 269 ± 4** 316 ± 3**

Right kidney

Absolute 1.379 ± 0.036 1.304 ± 0.029 1.257 ± 0.024*

Relative 3.55 ± 0.08 4.86 ± 0.11** 3.99 ± 0.09**

Liver

Absolute 14.405 ± 0.257 20.054 ± 0.385** 14.426 ± 0.315

Relative 37.09 ± 0.69 74.68 ± 1.21** 45.71 ± 0.88**

Spleen

Absolute 0.806 ± 0.017 0.877 ± 0.026 2.845 ± 0.097**

Relative 2.08 ± 0.05 3.27 ± 0.10** 9.01 ± 0.26**

Right testis

Absolute 1.615 ± 0.027 0.740 ± 0.055** 1.528 ± 0.020

Relative 4.16 ± 0.06 2.76 ± 0.21** 4.84 ± 0.05**

Epididymis

Absolute 0.484 ± 0.006 0.244 ± 0.017** 0.435 ± 0.007**

Relative 1.25 ± 0.01 0.91 ± 0.06** 1.38 ± 0.02

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given 1

as mg organ weight/g body weight (mean ± standard error).

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily for 2

14 consecutive weeks.

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 13 weeks. 3

n=9. 4

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

** Significantly different (P 0.01) from the control group by Dunnett's test.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 39

TESTIS AND EPIDIDYMIS

o-Nitrotoluene: The absolute and relative weights of the right testis and epididymis of rats from

all exposed groups at 13 and 26 weeks (normal and altered gastrointestinal flora) were generally

significantly less than those of controls. At 26 weeks, the testes of all rats in the stop- and

continuous-exposure groups were grossly reduced in size relative to those of the controls. The

right testis and epididymis weights of rats in the stop-exposure groups (normal and altered

gastrointestinal flora) continued to decrease during the recovery period and were similar to those

of rats treated with continuous exposure (Table 4). Lower testis weights and grossly smaller testes

than those of controls were a reflection of mild testicular degeneration seen microscopically

(Table 5). Degeneration consisted of a loss of spermatogenic epithelium and a reduction in the

diameter of the seminiferous tubules. In some tubules, there was an almost complete absence of

germinal epithelium and the remaining cells were primarily Sertoli cells; dense aggregates of

mineral deposition were seen in some tubules. The distribution of degeneration was multifocal to

diffuse in both testes. In the 26-week continuous-exposure group, the average severity of

degeneration was greater than that seen in the 13-week interim group. In the stop-exposure group,

there was no morphological evidence for resolution of degeneration and the average severity of this

lesion was slightly greater than that present in the 13-week interim group. Interstitial cell

hyperplasia was present in a few rats from the stop- and continuous-exposure groups at 26 weeks.

This consisted of a minimal focal increase in the amount of interstitial cells between degenerative

seminiferous tubules.

Mesothelial cell hyperplasia and/or mesothelioma occurred on the mesothelial surface of the tunica

vaginalis on the testis and epididymis of rats in the stop- and continuous-exposure groups.

Mesothelioma occurred in 2 of 20 rats from the 13-week interim group (altered gastrointestinal

flora). By 26 weeks, an increased incidence of mesothelioma occurred in rats from the continuousexposure group (7/20) and the stop-exposure groups with normal (5/20) or altered (8/20)

gastrointestinal flora. Focal hyperplasia of the epididymal mesothelium was also seen in five rats

from these three groups examined at 26 weeks (Table 5). Hyperplasia was a focal or multifocal

proliferation of one to two layers of basophilic-staining cuboidal cells on the mesothelial surface

of the epididymis. Mesotheliomas had broad-based attachment to the tunica vaginalis and

consisted of cuboidal or polyhedral-shaped cells forming solid areas with papillary or villous

projections that extended into the peritoneal space. A dense, fibrous core was present in most
40 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

neoplasms, and mitotic figures were common features of these neoplasms (Plates 1, 2, 3, and 4).

There was no indication that these neoplasms had metastasized to distant sites.

o-Toluidine Hydrochloride: There were no gross lesions in the 13-week interim and 26-week

stop- and continuous-exposure groups. In all o-toluidine hydrochloride groups, absolute testis and

epididymis weights were slightly less than those of the controls and were considered secondary to

body-weight reduction (Table 4). Degeneration of seminiferous tubules was a unilateral testicular

lesion present in 5% to 10% of rats from each exposed group (Table 5). At 26 weeks, 2 of 20 rats

had epididymal mesothelioma (stop-exposure group) and one rat had epididymal mesothelial cell

hyperplasia (continuous-exposure group). The morphology of these lesions was the same as that

in rats exposed to o-nitrotoluene.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 41

TABLE 5 Selected Lesions of the Testis and Epididymis 

in Male F344/N Rats in the 26-Week Feed Studies

of o-Nitrotoluene and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora2

ne Control o-Nitrotoluene Control o-Nitrotoluene o-Toluidi 3

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM

Testis 10 20 20 10 20 4

Degeneration 0 20 ** (2.0) 1 (1.0) 0 20 ** (1.6) 5

Epididymis 10 20 20 10 20

Mesothelial hyperplasia 0 0 0 0 2 (1.0)

Mesothelioma 0 0 0 0 2

STOP-EXPOSURE 6

Testis 10 20 20 10 20

Degeneration 0 20 ** (2.4) 2 (4.0) 0 20 ** (3.1)

Interstitial cell, hyperplasia 0 11 ** (1.0) 0 0 7 * (1.0)

Tunic, mesothelioma 0 2 0 0 4

Epididymis 10 20 20 10 20

Mesothelial hyperplasia 0 2 (1.0) 0 0 1 (2.0)

Mesothelioma 0 4 2 0 8 *

Testis or epididymis 10 20 20 10 20

Mesothelioma 0 5 2 0 8 *

26-WEEK CONTINUOUS-EXPOSURE

Testis 10 20 20

Degeneration 0 20 ** (2.9) 2 (2.5)

Interstitial cell, hyperplasia 0 3 (1.0) 0

Bilateral, hemangioma 0 1 0

Tunic, mesothelioma 0 2 0

Epididymis 10 20 20

Mesothelial hyperplasia 0 2 (1.0) 1 (1.0)

Mesothelioma 0 7 * 0

For nonneoplastic lesions, average severity (in parentheses) is based on the number of animals with lesions: 1=minimal, 1

2=mild, 3=moderate, and 4=marked.

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily 2

for 14 consecutive weeks.

For statistical analysis, control rats in the normal and altered gastrointestinal flora groups were pooled. 3

Number of animals with organ examined microscopically. 4

Number of animals with lesion. 5

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 6

13 weeks.

* Significantly different (P 0.05) from the control group by Fisher's exact test.

** Significantly different (P 0.01) from the control group by Fisher's exact test.
42 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

LIVER

o-Nitrotoluene: At the 13-week interim evaluation, rats in both o-nitrotoluene-exposed groups

(normal and altered gastrointestinal flora) had grossly enlarged livers and significantly greater liver

weights than the controls (Table 4). At 26 weeks, the increased size of the liver was apparent

grossly, and liver weights had increased with continuous exposure. The liver weights in the two

stop-exposure groups (normal and altered gastrointestinal flora) also continued to increase during

the 13-week recovery period, and the increases were similar to those seen with continuous

exposure at the end of 26 weeks.

Oval cell hyperplasia and cytoplasmic vacuolization were present in the livers of all exposed rats

(Table 6). Oval cell hyperplasia consisted of a minimal increase in the number of cells with a scant

amount of visible cytoplasm and oval-shaped nuclei. Proliferation of these oval cells was most

prominent in the periportal region of hepatic lobules. This change was of minimal severity, was

present in all exposed groups (normal and altered gastrointestinal flora), and did not increase in

severity in the continuous-exposure group or regress in the stop-exposure group. Cytoplasmic

vacuolization consisted of hepatocytes containing one to several vacuoles of varying sizes. This

was generally a diffuse change but was more prominent in the centrilobular to midzonal area of

the hepatic lobule. Vacuoles stained positive for the presence of lipid. The severity of this change

was minimal to mild at the 13-week interim (normal and altered gastrointestinal flora groups) and

increased to a moderate severity at 26 weeks. In the stop-exposure groups, this lesion did not

regress and was similar in severity to that seen in the continuous-exposure group (Table 6)

(Plates 5 and 6).

Foci of cellular alteration based on placental glutathione S-transferase (PGST)-positive staining of

hepatocytes occurred with o-nitrotoluene exposure by 13 weeks and increased in number and size

(as reflected by volume fraction) with continuous exposure for 26 weeks (Table 7). In the stopexposure groups (normal and altered gastrointestinal flora) at the end of the recovery period, the

number of foci was less than that in the corresponding 13-week interim groups but remained

significantly greater than that in controls. The size of the foci continued to increase in

o-nitrotoluene-exposed rats in both normal and altered gastrointestinal flora groups during the

recovery period. Areas representing PGST-positive foci generally could not be recognized in the

routine hematoxylin/eosin-stained sections. Although a few PGST-positive foci corresponded to
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 43

areas of cytoplasmic vacuolization in hepatocytes, most of the cells affected by vacuolization did

not stain positive for PGST.

At 26 weeks, three focal lesions were identified by gross examination of the liver at necropsy. Two

rats from the stop-exposure group (normal gastrointestinal flora) had a mass (1 centimeter in the

greatest dimension) located in the right posterior lobe. A third rat from the continuous-exposure

group had a pale focal area (4 mm by 4 mm) in the caudal lobe. Microscopically, these expansile

masses were identified as cholangiocarcinomas that consisted of large, irregularly shaped bile ducts

separated by dense, fibrous connective tissue. The ducts were lined by pleomorphic columnar to

low cuboidal, hyperchromatic epithelium; many of these ductular lining cells stained positive for

proliferating cell nuclear antigen. The lumen of some of these atypical ducts contained a mucous

exudate. Cholangiocarcinomas were not seen in the altered gastrointestinal flora groups (Plates

7 and 8).

o-Toluidine Hydrochloride: There were no gross findings and no treatment-related effects on liver

weights. Accumulation of hemosiderin pigment in Kupffer cell cytoplasm was present in all

o-toluidine hydrochloride groups at 13 and 26 weeks and was a minimal, reversible change

(Table 6). At each time point, the incidence of this lesion was significantly greater than that in the

controls.

At 26 weeks, the number of PGST-positive foci was significantly greater in the o-toluidine

hydrochloride exposure group than in controls. However, the number was significantly lower than

that seen with 26 weeks of exposure to o-nitrotoluene (Table 7), and the volume fraction was

significantly less than that seen with o-nitrotoluene.
44 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE 6 Selected Lesions of the Liver in Male F344/N Rats 

in the 26-Week Feed Studies of o-Nitrotoluene 

and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora2

ne Control o-Nitrotoluene Control o-Nitrotoluene o-Toluidi 3

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM

Liver 10 20 20 10 20 4

Pigmentation, hemosiderin 0 0 20 ** (1.0) 0 0

5

Vacuolization, cytoplasmic 0 20 ** (1.8) 1 (1.0) 0 20 ** (1.5)

Bile duct, hyperplasia 2 (1.0) 0 1 (1.0) 0 0

Oval cell, hyperplasia 0 20 ** (1.0) 0 0 20 ** (1.0)

STOP-EXPOSURE6

Liver 10 20 20 10 20

Pigmentation, hemosiderin 0 0 11 ** (1.0) 0 0

Vacuolization, cytoplasmic 0 20 ** (2.9) 1 (1.0) 0 20 ** (3.0)

Bile duct, hyperplasia 4 (1.0) 0 ** 5 (1.0) 4 (1.0) 0 **

Oval cell, hyperplasia 0 20 ** (1.0) 0 0 20 ** (1.0)

Cholangiocarcinoma 0 2 0 0 0

26-WEEK CONTINUOUS-EXPOSURE

Liver 10 20 20

Pigmentation, hemosiderin 0 0 20 ** (1.1)

Vacuolization, cytoplasmic 0 20 ** (3.0) 0

Bile duct, hyperplasia 4 (1.0) 0 ** 10 (1.1)

Oval cell, hyperplasia 0 20 ** (1.0) 0

Cholangiocarcinoma 0 1 0

For nonneoplastic lesions, average severity (in parentheses) is based on the number of animals with lesions: 1=minimal, 1

2=mild, 3=moderate, and 4=marked.

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily 2

for 14 consecutive weeks.

For statistical analysis, control rats in the normal and altered gastrointestinal flora groups were pooled. 3

Number of animals with liver examined microscopically. 4

Number of animals with lesion. 5

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 6

13 weeks.

** Significantly different (P 0.01) from the control group by Fisher's exact test.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 45

TABLE 7 Placental Glutathione S-Transferase-Positive Foci in the Liver

of Male F344/N Rats in the 26-Week Feed Studies of o-Nitrotoluene

and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora2

ne Control o-Nitrotoluene Control o-Nitrotoluene o-Toluidi 3

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM

Foci/cm tissue 6 ± 6 445 ± 78** — 0 ± 0 609 ± 176** 3 4

Mean volume (mm ) 0.002 ± 0.000 0.049 ± 0.006** — 0.000 ± 0.000 0.034 ± 0.013** 3

STOP-EXPOSURE5

Foci/cm tissue 17 ± 2 181 ± 43** — 10 ± 10 458 ± 148**

3 6

Mean volume (mm ) 0.000 ± 0.000 0.085 ± 0.023** — 0.000 ± 0.000 0.115 ± 0.031**

3

26-WEEK CONTINUOUS-EXPOSURE

Foci/cm3 tissue 17 ± 2 961 ± 91** 145 ± 61** — —

Mean volume (mm3) 0.000 ± 0.000 0.446 ± 0.101** 0.017 ± 0.004** — —

Ten rats were evaluated from each group at each time point. Data are given as mean ± standard error. 1

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin 2

daily for 14 consecutive weeks.

For statistical analysis, control rats in the normal and altered gastrointestinal flora groups were pooled. 3

Not examined or not applicable. 4

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 5

13 weeks.

n=9. 6

** Significantly different (P 0.01) from the normal or altered gastrointestinal flora control groups by Wilcoxon's rank sum

test.

Significantly different (P 0.05) from exposed rats evaluated at 13 weeks by Wilcoxon's rank sum test.

Significantly different (P 0.01) from exposed rats evaluated at 13 weeks by Wilcoxon's rank sum test.
46 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

KIDNEY AND URINARY BLADDER

o-Nitrotoluene: There were no treatment-related gross lesions or consistent effects on kidney

weights in the o-nitrotoluene-exposed groups (Table 4). Slight increases in relative kidney weights

were attributed to decreased mean body weights in exposed groups. At 13 weeks, there was an

accumulation of hyaline droplets in the renal tubule of exposed rats in the normal or altered

gastrointestinal flora groups. The incidence, but not the severity, of renal tubule regeneration was

increased only in rats exposed to o-nitrotoluene in the normal flora group in the 13-week interim

study (Table 8). In the stop-exposure groups (normal and altered gastrointestinal flora), the

accumulation of hyaline droplets completely regressed, but both stop-exposure normal and altered

gastrointestinal flora groups exposed to o-nitrotoluene had a similar slight increase in the incidence

and severity of lesions including foci of renal tubule epithelial regeneration and dilated renal

tubules containing eosinophilic-staining protein casts. With continued exposure to o-nitrotoluene

for 26 weeks, the hyaline droplet accumulation was still present but was not increased in severity.

The incidence and severity of other renal lesions typically associated with nephropathy

(regeneration and protein casts) were greater than those in the controls but did not differ from those

seen in the stop-exposure groups.

No gross or microscopic effects were identified in the urinary bladder of rats from any

o-nitrotoluene-exposure group.

o-Toluidine Hydrochloride: There were no gross kidney lesions in any o-toluidine hydrochlorideexposed group; kidney weights of exposed rats were lower than those of the controls, but this

difference was considered secondary to overall reductions in body weights of exposed rats

(Table 4). Microscopic lesions in exposed rats were limited to increased hemosiderin pigment

accumulation in the cytoplasm of renal tubule epithelium (Table 8). Hemosiderin accumulation

was of mild severity in the 13-week interim and 26-week continuous-exposure groups. The

severity of this lesion was slightly reduced in the stop-exposure group.

There were no gross lesions in the urinary bladders of rats from any o-toluidine hydrochlorideexposed group. Mild diffuse hyperplasia of the uroepithelium was present in o-toluidine

hydrochloride-exposed rats in the 13-week interim and 26-week continuous-exposure groups

(Table 8). The average severity of this lesion did not increase with longer exposure. Hyperplasia

was not observed in rats from the stop-exposure groups, indicating that the lesion was reversible

(Plates 9 and 10).
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 47

TABLE 8 Selected Lesions of the Kidney and Urinary Bladder 

in Male F344/N Rats in the 26-Week Feed Studies 

of o-Nitrotoluene and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora2

ne Control o-Nitrotoluene Control o-Nitrotoluene o-Toluidi 3

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM

Kidney 10 20 20 10 20 4

Hyaline droplet accumulation 0 20 ** (2.0) 0 0 20 ** (1.1) 5

Pigmentation 0 0 20 ** (2.0) 0 0

Regeneration 3 (1.0) 16 * (1.0) 7 (1.0) 3 (1.0) 2 (1.0)

Urinary bladder 10 20 10 20

Inflammation 0 0 2 (2.0) 0 0

Transitional epithelium,

 hyperplasia 0 0 10 ** (2.2) 0 0

STOP-EXPOSURE 6

Kidney 10 20 20 10 20

Hyaline droplet accumulation 0 0 0 0 0

Casts, protein 3 (1.0) 20 ** (1.2) 6 (1.0) 0 20 **

(1.1)

Pigmentation 0 0 20 ** (1.1) 0 0

Regeneration 7 (1.0) 19 (1.8) 10 (1.0) 6 (1.0) 20 **

(1.8)

26-WEEK CONTINUOUS-EXPOSURE

Kidney 10 20 20

Hyaline droplet accumulation 0 20 ** (1.6) 0

Casts, protein 3 (1.0) 20 ** (1.0) 1 (1.0)

Pigmentation 0 0 20 ** (2.2)

Regeneration 7 (1.0) 19 (1.1) 5 (1.0)

Urinary bladder 10 20 18

Transitional epithelium,

 hyperplasia 0 0 17 ** (2.0)

For nonneoplastic lesions, average severity (in parentheses) is based on the number of animals with lesions: 1=minimal, 1

2=mild, 3=moderate, and 4=marked.

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily 2

for 14 consecutive weeks.

For statistical analysis, control rats in the normal and altered gastrointestinal flora groups were pooled. 3

Number of animals with organ examined microscopically. 4

Number of animals with lesion. 5

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 6

13 weeks.

* Significantly different (P 0.05) from the control group by Fisher's exact test.

** Significantly different (P 0.01) from the control group by Fisher's exact test.
48 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

SPLEEN

o-Nitrotoluene: In the 13-week interim groups (normal and altered gastrointestinal flora) exposed

to o-nitrotoluene, absolute and relative spleen weights were generally slightly but significantly

greater than those of the controls (Table 4). In the 26-week continuous- and stop-exposure groups,

spleen weights were slightly greater than those of the controls; however, only the relative spleen

weights were significantly greater than the control value.

There were no grossly visible changes in the spleen. Microscopic findings were limited to

increased incidences and severity of hematopoiesis and hemosiderin accumulation in the splenic

red pulp of rats in the 13-week interim and 26-week continuous-exposure groups relative to the

controls (Table 9). Minimal focal areas of fibrosis in the spleen capsule were also present. After

26 weeks of continuous o-nitrotoluene exposure, hematopoiesis and hemosiderin accumulation

were slightly more severe than at 13 weeks. There was no evidence of a reduction in the severity

or incidence of the spleen effects in the stop-exposure group.

o-Toluidine Hydrochloride: At the 13-week interim evaluation, the spleens of all o-toluidine

hydrochloride-exposed rats were grossly enlarged, and the absolute and relative spleen weights

were significantly greater than those of the controls (Table 4). After 26 weeks of continuous

o-toluidine hydrochloride exposure, the spleens were also enlarged, but the spleen weights were

only slightly greater than those seen at the 13-week interim evaluation. In the o-toluidine

hydrochloride stop-exposure groups, the spleens did not appear grossly enlarged, but the absolute

and relative spleen weights were significantly greater than those of the controls (Table 4).

The spleens of all o-toluidine hydrochloride-exposed rats were distinguished grossly from the

spleens of the control group by the presence of white, granular plaques on the capsular surface.

Small (2 mm diameter) fluid-filled cysts were attached to the capsule in five rats. Focal, thickened

areas of capsular fibrosis seen microscopically corresponded to the irregular plaques observed

grossly on the capsular surface (Table 9). Within the areas of fibrosis were infiltrates of

lymphocytes and hematopoietic cells; the more extensively dilated lymphatic vessels in the capsule

(capsular angiectasis) corresponded to the cysts observed grossly. Microscopic findings in the

continuous- and stop-exposure groups included increases in hematopoietic cell proliferation and

hemosiderin concentration above the background amounts seen in the spleens of control rats. The

amount of blood in sinusoids (congestion) of the red pulp in exposed rats was also greater than that

in the controls (Table 9) (Plates 11 and 12).
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 49

With the exception of a slight increase in the severity of capsular fibrosis, the spleen changes that

occurred with 26 weeks of o-toluidine hydrochloride exposure were similar to those seen at 13

weeks. In the stop-exposure groups, a slight recovery of the exposure-related spleen lesions was

suggested by the reduction in the severity of congestion and by the lower incidence of

hematopoiesis.
50 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE 9 Selected Lesions of the Spleen in Male F344/N Rats 

in the 26-Week Feed Studies of o-Nitrotoluene 

and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora2

ne Control o-Nitrotoluene Control o-Nitrotoluene o-Toluidi 3

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM

Spleen 10 20 20 10 20 4

Congestion 0 0 20 ** (2.0) 0 0

5

Hematopoietic cell proliferation 2 (1.0) 20 ** (1.4) 20 ** (2.0) 0 20 ** (1.8)

Pigmentation, hemosiderin 0 20 ** (1.9) 20 ** (1.6) 0 20 ** (1.6)

Thrombosis 0 0 3 (1.0) 0 0

Capsule, fibrosis 0 1 (1.0) 20 ** (1.9) 0 1 (1.0)

STOP-EXPOSURE 6

Spleen 10 20 20 10 19

Congestion 0 0 20 ** (1.0) 0 0

Hematopoietic cell proliferation 3 (1.0) 18 ** (1.3) 1 (1.0) 1 (1.0) 19 ** (1.8)

Pigmentation, hemosiderin 3 (1.0) 17 ** (1.4) 18 ** (1.0) 5 (1.0) 19 ** (1.4)

Capsule, fibrosis 0 7 * (1.0) 20 ** (2.2) 0 1 (1.0)

Capsule, lymphatic, angiectasis 0 0 15 ** (1.7) 0 0

26-WEEK CONTINUOUS-EXPOSURE

Spleen 10 20 20

Congestion 0 0 20 ** (2.0)

Hematopoietic cell proliferation 3 (1.0) 20 ** (1.8) 20 ** (1.9)

Pigmentation, hemosiderin 3 (1.0) 20 ** (2.5) 20 ** (1.4)

Thrombosis 0 0 2 (1.0)

Capsule, fibrosis 0 3 (1.0) 20 ** (2.7)

Capsule, lymphatic, angiectasis 0 0 6 (1.3)

For nonneoplastic lesions, average severity (in parentheses) is based on the number of animals with lesions: 1=minimal, 1

2=mild, 3=moderate, and 4=marked.

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily 2

for 14 consecutive weeks.

For statistical analysis, control rats in the normal and altered gastrointestinal flora groups were pooled. 3

Number of animals with spleen examined microscopically. 4

Number of animals with lesion. 5

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 6

13 weeks.

* Significantly different (P 0.05, o-nitrotoluene; P=0.065, o-toluidine hydrochloride) from the control group by Fisher's exact

test.

** Significantly different (P 0.01) from the control group by Fisher's exact test.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 51

The results from the microbial analysis of cecal samples obtained at necropsy from the 13-week

interim groups are presented in Table 10. Daily administration of antibiotics by gavage over 14

weeks resulted in a significant (P=0.041) reduction in gastrointestinal flora counts, as established

under aerobic incubation conditions. The slight reductions in gastrointestinal flora counts with

o-nitrotoluene exposure in the normal gastrointestinal flora group or the combination effect of

o-nitrotoluene with antibiotics in the altered gastrointestinal flora group were not statistically

significant. However, this suggests that o-nitrotoluene in the feed may have contributed slightly

to the inhibitory effect of antibiotic administration on bacterial growth in the intestine. The effect

of antibiotics on cecal flora counts is similar to that seen in the pilot study (Appendix C) concluded

after 7 days of antibiotic dosing.

TABLE 10 Gastrointestinal Flora Counts from Selected Male F344/N Rats

at the 13-Week Interim Evaluation in the 26-Week Studies 

of o-Nitrotoluene and o-Toluidine Hydrochloride1

Gastrointestinal Flora Counts

Individual Group Average

Normal Gastrointestinal Flora Groups

 Control 9.0 x 10 2.6 x 10 2 5

5.5 x 104

1.7 x 105

9.5 x 105

1.3 x 105

 o-Nitrotoluene 1.3 x 10 6.8 x 10 4 4

 (5,000 ppm) 4.9 x 104

1.0 x 105

3.6 x 104

1.4 x 105

Altered Gastrointestinal Flora Groups 2

 Control 1.5 x 10 2.9 x 10 *

3 4

7.0 x 102

1.3 x 104

1.2 x 103

1.3 x 105

 o-Nitrotoluene 1.5 x 10 6.2 x 10 4 3

 (5,000 ppm) 7.7 x 103

7.8 x 103

3.3 x 102

1.0 x 102

Cecal samples were collected from five randomly selected rats per group. Counts are given in colony-forming units per 1

gram (CFU/g).

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily 2

for 14 consecutive weeks.

* Significantly different (P=0.041) from normal flora control based on a two-way analysis of variance on log-transformed

flora counts.
52 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44
PLATE 1

Testis of a male rat administered 5,000 ppm o-nitrotoluene in feed

for 13 weeks. Seminiferous tubules show focal mineralization

(arrows) and degeneration (arrowheads) with the loss of germinal

epithelium. Less severely affected tubules are seen in the upper

right portion of the plate. H&E, 60+.

PLATE 3

Epididymis of a male rat administered 5,000 ppm o-nitrotoluene for

13 weeks and evaluated at 26 weeks. A mesothelioma (arrows) is

attached to the surface of the epididymis (E). Detail of the area in

brackets is shown in Plate 4. H&E, 40+.

PLATE 2

Portion of the epididymis (E) and tunica vaginalis (T) of a male rat

administered 5,000 ppm o-nitrotoluene in feed for 13 weeks. Note

the presence of focal hyperplasia (arrows) of the mesothelium. 

H&E, 300+.

PLATE 4

Higher magnification of bracketed area of the epididymis in Plate

3. Two blunt, papillary masses extend from the surface of the

epididymis. Polyhedral-shaped neoplastic cells form an irregular

villous pattern (arrows) on the surface of the neoplasm. 

H&E, 185+.
PLATE 5

Liver of a male rat administered 5,000 ppm o-nitrotoluene in feed

for 26 weeks. Prominent cytoplasmic vacuolization (arrows) can

be seen in enlarged hepatocytes. H&E, 150+.

PLATE 7

Cholangiocarcinoma in the liver of a male rat administered

5,000 ppm o-nitrotoluene in feed for 13 weeks and evaluated at

26 weeks. An expansile mass of fibrous tissue and atypical biliary

ducts (arrows) have displaced hepatic lobules. H&E, 60+.

PLATE 6

Liver of a control male rat for comparison with Plate 5. H&E,

150+.

PLATE 8

Higher magnification of the biliary epithelium in Plate 7. Ductal

structures containing numerous mitotic figures are surrounded by

a fibrous stroma (arrows). H&E, 300+.
PLATE 9

Urinary bladder of a male rat administered 5,000 ppm o-toluidine

hydrochloride in feed for 26 weeks. There is hyperplasia

characterized by an increased thickness of the transitional

epithelium (between arrows). H&E, 300+.

PLATE 11

Spleen of a male rat administered 5,000 ppm o-toluidine

hydrochloride in feed for 26 weeks. The spleen capsule is

thickened (arrows) and the red pulp shows increased congestion

and hematopoiesis (P). H&E, 75+.

PLATE 10

Urinary bladder of a control male rat. Compare the thickness of

the transitional epithelial layer to that in Plate 9. H&E, 300+.

PLATE 12

Spleen of a control male rat. Compare the spleen capsule thickness

(arrow) and the red pulp with those in Plate 11. Note the

distinction between the red and white pulp is more evident because

of the less extensive congestion and hematopoiesis than that seen

in Plate 11. H&E, 75+.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 53

DISCUSSION

The 13- and 26-week studies of two structurally similar chemicals, o-nitrotoluene and o-toluidine

hydrochloride, in F344/N rats demonstrated several differences in carcinogenic potency and in the

site, severity, and potential for regression or progression of treatment-related toxic effects.

Bacterial reduction of the nitro group to the amine by the gastrointestinal flora is considered to be

an important step in metabolism of nitroaromatic chemicals including o-nitrotoluene (Doolittle

et al., 1983; Rickert et al., 1986a). For this reason, groups of rats treated with antibiotics were

included in these studies in an attempt to determine the effect of a reduced gastrointestinal flora

on the carcinogenic potential of o-nitrotoluene, although this treatment regimen was subsequently

demonstrated to be less effective than desired. Because of the numerous differences observed

between o-nitrotoluene and o-toluidine hydrochloride with respect to their carcinogenicity and

toxicity in rats, the effects of chemical exposure and altered intestinal flora are discussed

separately.

MESOTHELIOMA

Mesothelial cells form a single layer of epithelium on a delicate fibrous stroma that lines the

peritoneal and pleural cavities. In the F344 rat, spontaneous and treatment-related proliferative

lesions (hyperplasia and mesothelioma) of the peritoneal mesothelial cells are most commonly

observed on the peritoneal surface lining the testis, epididymis, and scrotal sac (Hall, 1990).

Although more malignant and invasive forms of mesothelioma may be disseminated throughout

the abdominal cavity and metastasize to lymph nodes in the thorax, the early or smaller neoplasms

are typically limited to the surface of the epididymis and testis. In a previous 2-year study of

o-toluidine hydrochloride, mesothelioma occurred in 24% of male rats exposed to a dietary

concentration of 3,000 ppm (NCI, 1979a). It could not be determined if mesothelioma occurred

in shorter studies used to set doses for the 2-year study. o-Nitrotoluene also caused a smal

number of mesotheliomas in male rats in a 13-week study (NTP, 1992). Because of this common

target-tissue effect and its structural similarity to o-nitrotoluene, groups of rats were exposed to

approximately equimolar amounts of o-nitrotoluene or o-toluidine hydrochloride in this study to

compare the relative potency of these chemicals with respect to the onset and development of

proliferative lesions in the mesothelium. The studies reported here confirm a chemical-related

increase in the incidence of mesothelioma in male rats after 13 or 26 weeks of exposure to

o-nitrotoluene and the development of a smaller number of mesotheliomas in rats exposed to
54 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

o-toluidine hydrochloride. After only 13 weeks of exposure to o-nitrotoluene, mesothelioma or

mesothelial hyperplasia occurred in a few rats. However, in the 26-week stop- and continuousexposure studies, 20 of 60 rats (33%) exposed to o-nitrotoluene developed mesothelioma.

Mesothelioma has not previously occurred in control rats from 13-week studies, but is a

spontaneously occurring neoplasm in 1% of control male rats from 2-year studies (NTP, 1992).

Mesothelial hyperplasia, a potential preneoplastic lesion, also occurred in rats exposed to

o-nitrotoluene but not in the controls. The incidences and morphology of the mesotheliomas were

similar in rats from the stop- and continuous-exposure groups. The development of hyperplasia

and mesothelioma in both the stop- and continuous-exposure studies indicates that a relatively

short-term exposure to o-nitrotoluene or o-toluidine hydrochloride is sufficient to produce this

carcinogenic effect.

TESTIS

Significantly lower testis weights and greater incidences of degeneration of the seminiferous tubule

germinal epithelium than those in the controls occurred in all rats exposed to o-nitrotoluene. These

effects increased in severity with continuous exposure and showed no evidence of recovery in the

stop-exposure groups. In some rats with the testicular degeneration, a minimal hyperplasia of the

interstitial (Leydig) cells was also present. These testicular effects were not a feature of o-toluidine

hydrochloride exposure. One or two rats in each o-toluidine hydrochloride-exposed group had

unilateral testicular degeneration. However, unilateral degeneration of the testes is generally

considered to be a nonspecific finding and has been seen occasionally in control F344 rats from

13-week studies (Dixon et al., 1995). The relationship of testicular degeneration and the

development of mesothelioma on the adjacent serosa is unclear. Testicular degeneration is a

common treatment-related effect with chemical exposure in rats and, in many instances, this lesion

has not been associated with an increase in mesothelioma. Glycidol, a multisite, multisex,

multispecies carcinogen (NTP, 1990), produced degeneration of the testes and mesothelioma.

However, cytembena (NTP, 1981) produced a high incidence of mesothelioma but did not cause

testicular degeneration.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 55

LIVER

After 13 weeks of exposure too-nitrotoluene, liver weights were slightly greater than those of the

controls, and morphologic alterations included cytoplasmic vacuolization and oval cell hyperplasia.

In both the stop- and continuous-exposure groups, the liver weights of rats exposed to

o-nitrotoluene continued to increase relative to those of the controls. In the stop- and continuousexposure groups, cytoplasmic vacuolization increased in severity and oval cell hyperplasia

persisted. Oval cell hyperplasia is a chemical-induced, putative preneoplastic lesion that is seen

in rats that subsequently develop cholangiofibrosis and cholangiocarcinoma (Maronpot et al.,

1991). Chemicals causing oval cell hyperplasia may also increase the incidence of hepatocellular

adenoma and carcinoma (Maronpot et al., 1991; Stenbäck et al., 1994). Oval cells are precursor

cells in cholangiocellular and undifferentiated liver tumors of rats (Steinberg et al., 1994). Oval

cell hyperplasia was seen after 13 and 26 weeks of exposure to o-nitrotoluene and did not regress

in the stop-exposure groups. With o-nitrotoluene exposure, three rats developed focal masses

considered to be cholangiocarcinoma. In the rat, cholangiofibrosis, cholangiofibroma, and

cholangiocarcinoma represent a progression of chemical-induced lesions often preceded by oval

cell hyperplasia (Bannash and Gössner, 1994). Morphologic features of cholangiofibrosis and the

benign and malignant cholangial tumors are similar. However, stop-exposure and transplantation

studies have shown the persistence and potentially progressive nature of these chemical-induced

proliferative lesions (Maronpot et al., 1991).

In addition to oval cell hyperplasia, cytoplasmic vacuolization with associated hepatocyte and liver

enlargement and PGST-positive foci of cellular alteration were significantly increased in

o-nitrotoluene-exposed groups at 13 and 26 weeks. PGST-positive foci are considered to be a

preneoplastic change in the liver (Sato et al., 1984; Satoh et al., 1985). PGST-positive foci have

been induced by a variety of unrelated carcinogenic chemicals including 2-acetylaminofluorene

(Stenbäck et al., 1994) and estrogen (Ogawa et al., 1995). With hepatocarcinogens,

PGST-positive staining is seen in individual cells, foci, and neoplasms (Hendrich et al., 1987;

Dragan et al., 1994). When chemical exposure to o-nitrotoluene is stopped, the number of foci

decreases during the 13-week recovery period but the volume fraction increases. After 26 weeks

of continuous exposure to o-nitrotoluene, the number and volume fraction of foci were

approximately 2-fold and 10-fold greater, respectively, compared to those in the 13-week exposure

group. In the stop-exposure groups (normal and altered gastrointestinal flora) the number of foci

decreased to approximately half of that seen at 13 weeks, but the volume fraction of foci continued
56 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

to increase during the recovery period. This partial regression of foci of alteration, persistence of

oval cell hyperplasia, and the presence of a small number of cholangiocellular neoplasms at 26

weeks suggests the potential for a strong carcinogenic response in the liver of rats administered

o-nitrotoluene over a longer period of time. In previous 2-year studies of dinitrotoluene, a

chemical structurally related to o-nitrotoluene, there were increases in hepatocellular and

cholangiocarcinoma (Rickert et al., 1987).

In contrast to o-nitrotoluene exposure, the only liver effect seen with o-toluidine hydrochloride

exposure was a minimal accumulation of hemosiderin pigment in the cytoplasm of the Kupffer

cells lining the hepatic sinusoids. This was considered to be secondary to the increased erythrocyte

destruction. Immunohistochemical staining of liver sections demonstrated an increase in

PGST-positive foci in the o-toluidine hydrochloride group relative to controls after 26 weeks of

continuous exposure. However, the number and size of these foci were significantly smaller than

that seen in the liver of rats exposed to o-nitrotoluene. In a 2-year study of o-toluidine

hydrochloride there was no evidence of a carcinogenic effect in the liver of rats (NCI, 1979a).

KIDNEY AND URINARY BLADDER

Hyaline droplet accumulation was present in the renal tubule epithelium and appeared

morphologically similar in rats continuously exposed to o-nitrotoluene for 13 or 26 weeks. The

absence of this finding in the stop-exposure groups demonstrates the reversibility of this effect.

However, other kidney lesions (renal tubule epithelium regeneration and protein casts) typically

associated with spontaneous nephropathy of rats were slightly increased in incidence and severity

in the stop-exposure groups compared to the controls, suggesting incomplete resolution of renal

toxicity. Previous studies have shown that the hyaline droplet formation in the kidney of male rats

exposed to o-nitrotoluene is the result of a dose-related increase in the concentration of - 2μ

globulin in the kidney (Dunnick et al., 1994). A variety of structurally dissimilar chemicals also

causing an increase in -globulin are typically associated with an increase in the severity of 2μ

lesions associated with spontaneous nephropathy; with chronic exposure, an increased incidence

of renal tubule neoplasms is sometimes observed (Baetcke et al., 1991). The only renal effect of

o-toluidine hydrochloride was an increase in hemosiderin pigment in tubule epithelium. The

incidence of pigment accumulation increased with exposure duration and decreased in the

stop-exposure study. This pigment accumulation was most likely secondary to the increased
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 57

erythrocyte destruction reflected by the spleen changes in these rats. In 2-year studies of otoluidine hydrochloride, there was no carcinogenic effect in the kidney (NCI, 1979a).

There were no treatment-related effects in the urinary bladder of rats exposed to o-nitrotoluene.

In the urinary bladder of rats continuously exposed to o-toluidine hydrochloride for 13 or

26 weeks, there was a mild, diffuse hyperplasia of the transitional epithelium. There was no

evidence of treatment-related urinary calculus formation. Complete regression of the hyperplasia

occurred in the stop-exposure group. In the 2-year study, continuous exposure to o-toluidine

hydrochloride caused an increase in hyperplasia and neoplasia of the bladder in male and female

rats. Other structurally similar chemicals including o-nitroanisole (NTP, 1993),

4-chloro-o-phenylenediamine (NCI, 1978b), 4-amino-2-nitrophenol (NCI, 1978c), o-anisidine

(NCI, 1978d), and p-cresidine (NCI, 1979b) also caused hyperplasia and neoplasia of the urinary

bladder epithelium in 2-year studies.

SPLEEN

Increased spleen weights and incidences of hematopoiesis and hemosiderin accumulation were

observed in rats exposed to o-nitrotoluene. These findings are consistent with results from

previous studies where o-nitrotoluene has been shown to increase methemoglobin levels and cause

a mild regenerative anemia (NTP, 1992). In the present study, there was little difference in the

severity of these effects between the 13- and 26-week continuous-exposure groups. The spleen

toxicity seen with o-toluidine hydrochloride exposure was more severe than that caused by

o-nitrotoluene. Spleens appeared grossly enlarged and discolored, and spleen weights were twoto threefold greater than those of the controls. These findings corresponded to the increased

incidences of congestion, hemosiderin, hematopoiesis and capsular fibrosis seen microscopically.

Studies of other aromatic amines (Chhabra et al., 1990; NTP, 1993) have shown similar effects

on the hematopoietic system. Spleen changes (congestion, hemosiderin accumulation, fibrosis, and

increased hematopoiesis) have been attributed to the denaturation of hemoglobin, Heinz body

formation, and increased erythrocyte destruction in the spleen. o-Toluidine hydrochloride binds

with hemoglobin (DeBord et al., 1992) and causes anemia and methemoglobin formation in rats

(IARC, 1982). In the 2-year study of o-toluidine hydrochloride, there was a treatment-related

increase in the incidence of sarcomas of the spleen (NCI, 1979a). This increased incidence of

sarcomas of the spleen has also been seen in 2-year studies of other aromatic amines (Goodman

et al., 1984; Chhabra et al., 1991).
58 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

ALTERED GASTROINTESTINAL FLORA DIET

There was little evidence that the attempt to reduce gastrointestinal bacteria affected the

carcinogenic potential or the pattern and severity of o-nitrotoluene toxicity in male rats. The

incidence of mesothelioma and the severity and pattern of toxic lesions in the liver (increased

weight, cytoplasmic vacuolization, oval cell hyperplasia, PGST-positive foci), kidney (hyaline

droplet accumulation), testes (degeneration), and spleen (hematopoiesis, hemosiderin, capsular

fibrosis) were generally similar in the o-nitrotoluene groups (normal and altered intestinal flora).

The only effect that may be related to the altered gastrointestinal flora diet is the absence of

cholangiocarcinomas, which were seen in 3 of 40 rats administered o-nitrotoluene in the normal

diet. However, the altered gastrointestinal flora did not prevent increased liver weight or hepatic

toxicity, reflected by the presence of cytoplasmic vacuolization, PGST-positive foci, and oval cell

hyperplasia. Oval cell hyperplasia is considered to be a morphologic change preceding and

associated with the development of cholangial neoplasms. Based on these findings, the short

duration of the study, and the small number of these liver neoplasms seen in the normal

gastrointestinalflora group, it is unlikely that the attempt to alter gastrointestinal flora prevented

the development of cholangiocarcinoma.

Previous short-term studies have shown the importance of the gastrointestinal flora in producing

adverse effects of o-nitrotoluene in the liver of rats (Doolittle et al., 1983; Rickert et al., 1987).

Gavage administration of o-nitrotoluene caused unscheduled DNA synthesis in primary hepatocyte

cultures from male rats (Doolittle et al., 1983). This effect of o-nitrotoluene was not seen in

similarly treated, bacteria-free rats. Gardner and Renwick (1978) demonstrated a reduction in the

bacterially mediated reduction of nitrobenzoic acids in rats receiving a mixture of antibiotics

similar to that used in the current studies, but with the bacteriostat bacitracin rather than the

antifungal agent nystatin. Although a significant alteration and reduction of the gastrointestinal

flora was observed with daily administration of antibiotics in this study, this was only demonstrated

for aerobes, and it was subsequently pointed out during a review of an earlier draft of this

document that the obligate anaerobes may have been less affected by the antibiotics used. This

would have resulted in an insufficient alteration of flora to affect the hydrolytic and reductive

activity of the intestinal bacteria on the glucuronidated forms of the o-nitrotoluene secreted in the

bile. Also, although bacterial counts from cecal contents of dosed rats were significantly reduced

at 13 weeks compared to the controls, the numbers of bacteria in the altered gastrointestinal flora

diet group were higher than those observed after one week of antibiotic treatment (Appendix C),
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 59

and this increase in resistant bacteria may have been sufficient to adequately reduce the chemical

conjugates in the intestine.

In an effort to further determine the efficacy of the antibiotic mixture used to reduce

gastrointestinal flora, the pilot study described in Appendix C was repeated, and cultures were

plated and enumerated under conditions appropriate for determining both aerobic and anaerobic

bacteria. The results of the second pilot study extended the findings of the initial study by

demonstrating a shift in the types of aerobic bacteria present, with a decline in enteric Gramnegative rods but an increase in several other bacteria, including Streptococcus sp. The second

pilot study also demonstrated a complete lack of efficacy in reducing the gastrointestinal load of

obligate anaerobic bacteria. Therefore, no conclusions concerning the involvement of

gastrointestinal flora in the pathogenesis of o-nitrotoluene-induced lesions could be drawn from

these studies.

In summary, the results of these studies have confirmed the carcinogenicity of o-nitrotoluene based

on the development of a high incidence of mesotheliomas and a small number of

cholangiocarcinomas in male rats after only 13 or 26 weeks of exposure. The increased incidence

of mesothelioma indicates the relative greater carcinogenic potential or decrease in tumor latency

for o-nitrotoluene compared to an equivalent dose and exposure time for o-toluidine hydrochloride.

While the chemicals are similar in structure, basic differences exist in the way o-nitrotoluene and

o-toluidine hydrochloride are metabolized, e.g., methyl group oxidation (o-nitrotoluene) verses

aromatic ring oxidation (o-toluidine hydrochloride). It may be that the metabolite responsible for

the increased incidence of mesotheliomas may be a relatively minor metabolite of o-toluidine

hydrochloride and a major one for o-nitrotoluene. Although metabolites of both compounds are

excreted primarily in the urine, the renal effects seen with o-nitrotoluene and the urinary bladder

hyperplasia with o-toluidine hydrochloride suggest differences in the toxicity of the urinary

metabolites of each chemical. These findings, plus the presence of testicular degeneration,

hepatotoxicity, and cholangiocarcinoma only in o-nitrotoluene groups and the relatively greater

spleen toxicity seen in the o-toluidine hydrochloride groups, indicate that the important toxic and

carcinogenic effects of o-nitrotoluene may not be attributed primarily to the intestinal tract

reduction of the nitro group to the amine.
60 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 61

REFERENCES

THE ALDRICH LIBRARY OF FT-IR SPECTRA (1985). 1st ed. (C. J. Pouchert, Ed.), p. 1392A. Aldrich

Chemical Company, Milwaukee, WI.

ARMITAGE, P. (1971). Statistical Methods in Medical Research. John Wiley and Sons, New York.

BAETCKE,K. P.,RODGERS,I. S., HARD,G.C., MCGAUGHY, R. E., AND TAHAN, L. M. (1991). Alpha - 2u

globulin: Association with chemically induced renal toxicity and neoplasia in the male rat .

Report No. EPA/625/3-91/019F. Risk Assessment Forum, U.S. Environmental Protectio n

Agency, Washington, DC.

BANNASCH, P., AND GÖSSNER, W. (EDS.) (1994). Pathology of Neoplasia and Preneoplasia in Rodents.

EULEP Color Atlas, pp. 43-53. Schattauer, Stuttgart. 

BARRETT, R. H. (1985). Assays for unscheduled DNA synthesis in HeLa S3 cells. In Progress in

Mutation Research. Volume 5. Evaluation of Short-Term Tests for Carcinogens (J. Ashby, F. J. de Serres,

M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E. Matter, and M. D. Shelby, Eds.), pp. 347-352.

Elsevier Science Publishers, Amsterdam.

BEARD, R. R., AND NOE, J. T. (1981). Aromatic nitro and amino compounds. In Patty's Industrial

Hygiene and Toxicology, 3rd ed. (G. D. Clayton and F. E. Clayton, Eds.), Vol. 2A, pp. 2413-2490.

John Wiley and Sons, New York.

BOORMAN, G. A., MONTGOMERY, C. A., JR., EUSTIS, S. L., WOLFE, M. J., MCCONNELL, E. E., AND

HARDISTY, J. F. (1985). Quality assurance in pathology for rodent carcinogenicity studies. I n

Handbook of Carcinogen Testing (H. A. Milman and E. K. Weisburger, Eds.), pp. 345-357. Noyes

Publications, Park Ridge, NJ.

BOORMAN,G.A., HICKMAN,R. L.,DAVIS,G. W., RHODES, L. S., WHITE, N. W., GRIFFIN, T. A., MAYO,

J., AND HAMM,T.E.,JR. (1986). Serological titers to murine viruses in 90-day and 2-year studies.

In Complications of Viral and Mycoplasmal Infections in Rodents 

to Toxicology Research and Testing (T. E. Hamm, Jr., Ed.), pp. 11-23. Hemisphere, New York.
62 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

BRADLEY, M. O. (1985). Measurement of DNA single-strand breaks by alkaline elution in ra t

hepatocytes. In Progress in Mutation Research. Volume 5. Evaluation of Short-Term Tests for

Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E. Matter,

and M. D. Shelby, Eds.), pp. 353-357. Elsevier Science Publishers, Amsterdam.

CESARONE,C. F.,BOLOGNESI,C., AND SANTI, L. (1982). Evaluation of damage to DNA after in vivo

exposure to different classes of chemicals. Arch. Toxicol. (Suppl. 5), 355-359.

CHEEVER,K. L.,RICHARDS,D.E., AND PLOTNICK,H. B. (1980). Metabolism of ortho-, meta-, and paratoluidine in the adult male rat. Toxicol. Appl. Pharmacol. 56, 361-369.

CHEM SOURCES USA (1983). 24th ed., pp. 658-659. Directories Publishing Company, Ormon d

Beach, FL.

CHEMICAL INDUSTRY INSTITUTE OF TOXICOLOGY (CIIT) (1979). Chemical Safety Studies,

Dinitrotoluene Final Report Docket No. 12362. CIIT, Research Triangle Park, NC.

CHEMICALS IN COMMERCE INFORMATION SYSTEM (1984).

CHHABRA, R. S., THOMPSON, M., ELWELL, M. R., AND GERKEN, D. K. (1990). Toxicity of

p-chloroaniline in rats and mice. Food Chem. Toxicol. 28, 717-722.

CHHABRA, R. S., HUFF, J. E., HASEMAN, J. K., ELWELL, M. R., AND PETERS, A. C. (1991).

Carcinogenicity of p-chloroaniline in rats and mice. Food Chem. Toxicol. 29, 119-124.

CHISM, J. P., AND RICKERT, D. E. (1985). Isomer- and sex-specific bioactivation o f

mononitrotoluenes. Role of enterohepatic circulation. Drug Metab. Dispos. 13, 651-657.

CHISM,J. P., AND RICKERT, D. E. (1989). In vitro activation of 2-aminobenzyl alcohol and 2-amino6-nitrobenzyl alcohol, metabolites of 2-nitrotoluene and 2,6-dinitrotoluene. Chem. Res. Toxicol.

2, 150-156.

CHISM, J. P., TURNER, M. J., JR., AND RICKERT, D. E. (1984). The metabolism and excretion of

mononitrotoluenes by Fischer 344 rats. Drug Metab. Dispos. 12, 596-602.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 63

CHRISTENSEN, H. E., AND LUGINBYHL, T. L. (EDS.) (1974). The Toxic Substances List. HEW

Publication No. (NIOSH) 74-134. National Institute for Occupational Safety and Health ,

Cincinnati, OH.

CODE OF FEDERAL REGULATIONS (CFR) 21, Part 58.

CONOVER, W. J. (1971). Practical Nonparametric Statistics. John Wiley and Sons, New York.

DANFORD, N. (1985). Tests for chromosome aberrations and aneuploidy in the Chinese hamste r

fibroblast cell line CH1-L. In Progress in Mutation Research. Volume 5. Evaluation of Short-Term Tests

for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E .

Matter, and M. D. Shelby, Eds.), pp. 397-411. Elsevier Science Publishers, Amsterdam.

DANFORD, N. (1991). The genetic toxicology of ortho-toluidine. Mutat. Res. 258, 207-236.

DARROUDI, F., AND NATARAJAN, A. T. (1985). Cytostatic drug activity in plasma, a bioassay fo r

detecting mutagenicity of directly and indirectly acting chemicals, an evaluation of 20 chemicals.

Mutat. Res. 143, 263-269.

DEBETHIZY,J.D., AND HAYES,J.R. (1994). Metabolism. A determinant of toxicity. In Principles and

Methods of Toxicology, 3rd ed. (A. W. Hayes, Ed.), pp. 59-100. Raven Press, New York.

DEBETHIZY, J. D., AND RICKERT, D. E. (1984). Metabolism of nitrotoluenes by freshly isolate d

Fischer 344 rat hepatocytes. Drug Metab. Dispos. 12, 45-50.

DEBORD, D. G., SWEARENGIN, T. F., CHEEVER, K. L., BOOTH-JONES, A. D., AND WISSINGER, L. A.

(1992). Binding characteristics of ortho-toluidine to rat hemoglobin and albumin. Arch. Toxicol.

66, 231-236.

DIXON,D.,HEIDER,K., AND ELWELL, M. R. (1996). Incidence of nonneoplastic lesions in historical

control male and female Fischer 344 rats from 90-day toxicity studies. Toxicol. Pathol. (in press).

DIXON, W. J., AND MASSEY, F. J., JR. (1951). Introduction to Statistical Analysis, 1st ed., pp. 145-147.

McGraw-Hill Book Company, New York.
64 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

DOOLITTLE,D.J., SHERRILL,J. M., AND BUTTERWORTH, B. E. (1983). Influence of intestinal bacteria,

sex of the animal, and position of the nitro group on the hepatic genotoxicity of nitrotoluen e

isomers in vivo. Cancer Res. 43, 2836-2842.

DOUGLAS,G.R.,BLAKEY,D.H., LIU-LEE, V. W., BELL, R. D. L., AND BAYLEY, J. M. (1985). Alkaline

sucrose sedimentation, sister-chromatid exchange and micronucleus assays in CHO cells. I n

Progress in Mutation Research. Volume 5. Evaluation of Short-Term Tests for Carcinogenesis (J. Ashby, F. J.

de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E. Matter, and M. D. Shelby, Eds.) ,

pp. 359-366. Elsevier Science Publishers, Amsterdam.

DRAGAN,Y. P.,CAMPBELL,H.A.,BAKER,K.,VAUGHAN,J., MASS, M., AND PITOT, H. C. (1994). Focal

and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats.

Carcinogenesis 15, 2587-2591.

DUNLAP, K. L. (1981). Nitrobenzene and nitrotoluenes. In Kirk-Othmer Encyclopedia of Chemical

Technology, 3rd ed., Vol. 15, pp. 925-932. John Wiley and Sons, New York.

DUNNETT, C. W. (1955). A multiple comparison procedure for comparing several treatments with

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

DUNNICK, J. K., ELWELL, M. R., AND BUCHER, J. R. (1994). Comparitive toxicities of o-, m-, and pnitrotoluene in 13-week feed studies in F344 rats and B6C3F mice. Fundam. Appl. Toxicol. 22, 1

411-421.

ELLENHORN, M.J., AND BARCELOUX, D. G. (1988). Airborne toxins. In Medical Toxicology: Diagnosis

and Treatment of Human Poisoning, pp. 813-894. Elsevier, New York.

GALLOWAY, S. M.,ARMSTRONG, M.J.,REUBEN, C., COLMAN, S., BROWN, B., CANNON, C., BLOOM, A.

D., NAKAMURA, F., AHMED, M., DUK, S., RIMPO, J., MARGOLIN, B. H., RESNICK, M. A.,

ANDERSON, B., AND ZEIGER, E. (1987). Chromosome aberrations and sister chromati d

exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ. Mol. Mutagen.

10 (Suppl. 10), 1-175.

GARDNER, D. M., AND RENWICK, A. G. (1978). The reduction of nitrobenzoic acids in the rat .

Xenobiotica 8, 679-690.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 65

GART, J. J., CHU, K. C., AND TARONE, R. E. (1979). Statistical issues in interpretation of chroni c

bioassay tests for carcinogenicity. J. Natl. Cancer Inst. 62, 957-974.

GLAUERT, H. P., KENNAN, W. S., SATTLER, G. L., AND PITOT, H. C. (1985). Assays to measure the

induction of unscheduled DNA synthesis in cultured hepatocytes. In Progress in Mutation Research.

Volume 5. Evaluation of Short-Term Tests for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M.

Ishidate, Jr., B. H. Margolin, B. E. Matter, and M. D. Shelb y, Eds.), pp. 371-373. Elsevier Science

Publishers, Amsterdam.

GOODMAN, D. G., WARD, J. M., AND REICHARDT, W. D. (1984). Splenic fibrosis and sarcomas in

F344 rats fed diets containing aniline hydrochloride, p-chloroaniline, azobenzene, o-toluidine

hydrochloride, 4,4 -sulfonyldianiline, or D & C Red No. 9. JNCI 73, 265-273.

GREENWELL, A., FOLEY, J. F., AND MARONPOT, R. R. (1991). An enhancement method for

immunohistochemical staining of proliferating cell nuclear antigen in archival rodent tissues .

Cancer Lett. 59, 251-256.

GULATI, D. K., SABHARWAL, P. S., AND SHELBY, M. D. (1985). Tests for the induction of

chromosomal aberrations and sister chromatid exchanges in cultured Chinese hamster ovar y

(CHO) cells. In Progress in Mutation Research. Volume 5. Evaluation of Short-Term Tests for

Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E. Matter,

and M. D. Shelby, Eds.), pp. 413-426. Elsevier Science Publishers, Amsterdam.

HALL,W.C. (1990). Peritoneum, retroperitoneum, mesentery, and abdominal cavity. 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. 63-70. Academic Press, San Diego.

HAWORTH, S., LAWLOR, T., MORTELMANS, K., SPECK, W., AND ZEIGER, E. (1983). Salmonella

mutagenicity test results for 250 chemicals. Environ. Mutagen. 5 (Suppl. 1), 3-142.

HENDRICH, S., CAMPBELL, H. A., AND PITOT, H. C. (1987). Quantitative stereological evaluation of

four histiochemical markers of altered foci in multistage hepatocarcinogensis in the rat .

Carcinogenesis 8, 1245-1250.
66 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

HOWARD, P. H., SANTODONATO, J., SAXENA, J., MALLING, J. E., AND GRENINGER, D. (1976).

Investigation of selected potential environmental contaminants: Nitroaromatics. Prepared for

EPA Contract No. 68-01-2999, Report No. EPA-560/2-76-010.

HSU, S.-M., RAINE, L., AND FANGER, H. (1981). The use of antiavidin antibody and avidin-biotin -

peroxidase complex in immunoperoxidase technics. Am. J. Clin. Pathol. 75, 816-821.

INTERNATIONAL AGENCY FOR RESEARCH ON CANCER (IARC) (1982). Aromatic amines. In Some

Aromatic Amines, Anthraquinones and Nitroso Compounds, and Inorganic Fluorides Used in Drinking-Water

and Dental Preparations. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to

Humans, Vol. 27, pp. 157-175. IARC, Lyon.

ISHIDATE, M., JR., AND SOFUNI, T. (1985). The in vitro chromosomal aberration test using Chinese

hamster lung (CHL) fibroblast cells in culture. In Progress in Mutation Research. Volume 5.

Evaluation of Short-Term Tests for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr.,

B. H. Margolin, B. E. Matter, and M. D. Shelby, Eds.), pp. 427-432. Elsevier Science Publishers,

Amsterdam.

JACOBSON, K. H. (1972). Acute oral toxicity of mono- and di-alkyl ring-substituted derivatives o f

aniline. Toxicol. Appl. Pharmacol. 22, 153-154.

KADA,T. (1981). The DNA-damaging activity of 42 coded compounds in the rec-assay. In Progress

in Mutation Research. Volume 1. Evaluation of Short-Term Tests for Carcinogens (F. J. de Serres and J.

Ashby, Eds.), pp. 175-182. Elsevier/North-Holland, New York.

LANE,A. M., PHILLIPS,B.J., AND ANDERSON, D. (1985). Tests for the induction of sister chromatid

exchanges in Chinese hamster ovary (CHO) cells in culture. In Progress in Mutation Research.

Volume 5. Evaluation of Short-Term Tests for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M.

Ishidate, Jr., B. H. Margolin, B. E. Matter, and M. D. Shelb y, Eds.), pp. 451-455. Elsevier Science

Publishers, Amsterdam.

LINDSTROM, H. V., BOWIE, W. C., WALLACE, W. C., NELSON, A. A., AND FITZHUGH, O. G. (1969).

The toxicity and metabolism of mesidine and pseudocumidine in rats. J. Pharmacol. Exp. Ther.

167, 223-234.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 67

MCFEE,A. F., JAUHAR, P. P., LOWE, K. W., MACGREGOR, J. T., AND WEHR, C. M. (1989). Assays of

three carcinogen/non-carcinogen chemical pairs for in vivo induction of chromosom e

aberrations, sister chromatid exchanges and micronuclei. Environ. Mol. Mutagen. 14, 207-220.

MACKISON, F. W., STRICOFF, R. S., AND PARTRIDGE, L. J., JR. (EDS.) (1981). Occupational Health

Guideline for Nitrotoluene. In NIOSH/OSHA Occupational Health Guidelines for Chemical

Hazards, Vol. 2. DHHS (NIOSH) Publication No. 81-123. U.S. Department of Health an d

Human Services, U.S. Department of Labor, Washington, DC.

MARONPOT,R.R., AND BOORMAN,G.A. (1982). Interpretation of rodent hepatocellular proliferative

alterations and hepatocellular tumors in chemical safety assessment. Toxicol. Pathol. 10, 71-80.

MARONPOT, R. R., GILES, H. D., DYKES, D. J., AND IRWIN, R. D. (1991). Furan-induced hepatic

cholangiocarcinomas in Fischer 344 rats. Toxicol. Pathol. 19, 561-570.

MARTIN, C. N., AND CAMPBELL, J. (1985). Tests for the induction of unscheduled DNA repai r

synthesis in HeLa cells. In Progress in Mutation Research. Volume 5. Evaluation of Short-Term Tests for

Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E. Matter,

and M. D. Shelby, Eds.), pp. 375-379. Elsevier Science Publishers, Amsterdam.

MARTIN, C. N., AND MCDERMID, A. C. (1981). Testing of 42 coded compounds for their ability to

induce unscheduled DNA repair synthesis in HeLa cells. In Progress in Mutation Research.

Volume 1. Evaluation of Short-Term Tests for Carcinogens, (F. J. de Serres and J. Ashby, Eds.),

pp. 533-537. Elsevier/North-Holland, New York.

THE MERCK INDEX (1983). 10th ed. (M. Windholz, Ed.), pp. 953 and 1365. Merck and Company,

Rahway, NJ.

NATIONAL CANCER INSTITUTE (NCI) (1978a). Bioassay of 2,4-Dinitrotoluene for Possibl e

Carcinogenicity (CAS No. 121-14-2). Technical Report Series No. 54. NIH Publication No .

78-1360. U.S. Department of Health, Education, and Welfare, Public Health Service, National

Institutes of Health, Bethesda, MD.
68 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

NATIONAL CANCER INSTITUTE (NCI) (1978b). Bioassay of 4-Chloro-o-phenylenediamine fo r

Possible Carcinogenicity (CAS No. 95-83-0). Technical Report Series No. 63. NIH Publication

No. 78-1313. U.S. Department of Health, Education, and Welfare, Public Health Service ,

National Institutes of Health, Bethesda, MD.

NATIONAL CANCER INSTITUTE (NCI) (1978c). Bioassay of 4-Amino-2-nitrophenol for Possibl e

Carcinogenicity (CAS No. 119-34-6). Technical Report Series No. 94. NIH Publication No .

78-1344. U.S. Department of Health, Education, and Welfare, Public Health Service, National

Institutes of Health, Bethesda, MD.

NATIONAL CANCER INSTITUTE (NCI) (1978d). Bioassay of o-Anisidine Hydrochloride for Possible

Carcinogenicity (CAS No. 134-29-0). Technical Report Series No. 89. NIH Publication No .

78-1339. U.S. Department of Health, Education, and Welfare, Public Health Service, National

Institutes of Health, Bethesda, MD.

NATIONAL CANCER INSTITUTE (NCI) (1979a). Bioassay of o-Toluidine Hydrochloride for Possible

Carcinogenicity (CAS No. 636-21-5). Technical Report Series No. 153. NIH Publication No.

79-1709. U.S. Department of Health, Education, and Welfare, Public Health Service, National

Institutes of Health, Bethesda, MD.

NATIONAL CANCER INSTITUTE (NCI) (1979b). Bioassay of p-Cresidine for Possible Carcinogenicity

(CAS No. 120-71-8). Technical Report Series No. 142. NIH Publication No. 79-1397. U.S .

Department of Health, Education, and Welfare, Public Health Service, National Institutes o f

Health, Bethesda, MD.

NATIONAL INSTITUTE OF OCCUPATIONAL SAFETY AND HEALTH (NIOSH) (1990). National

Occupational Hazard Survey (1972-1974). Cincinnati, OH.

NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH (NIOSH) (1995). National

Occupational Exposure Survey (1981-1983), unpublish ed provisional data as of January 24, 1995.

NATIONAL TOXICOLOGY PROGRAM (NTP) (1981). Carcinogenesis Bioassay of Cytembena (CAS No.

21739-91-3). Technical Report Series No. 207. NIH Publication No. 81-1763. U.S. Department

of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda,

MD and Research Triangle Park, NC.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 69

NATIONAL TOXICOLOGY PROGRAM (NTP) (1990). Toxicology and Carcinogenesis Studies o f

Glycidol (CAS No. 556-52-5) in F344/N Rats and B6C3F Mice (Gavage Studies). Technical 1

Report Series No. 374. NIH Publication No. 90-2829. U.S. Department of Health and Human

Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

NATIONAL TOXICOLOGY PROGRAM (NTP) (1992). Toxicity Studies of o-, m-, and p-Nitrotoluenes

(CAS Nos. 88-72-2, 99-08-1, 99-99-0) Administered in Dosed Feed to F344/N Rats and B6C3F1

Mice. Toxicity Report Series No. 23. NIH Publication No. 93 -3346. U.S. Department of Health

and Human Services, Public Health Service, National Institutes of Health, Research Triangl e

Park, NC.

NATIONAL TOXICOLOGY PROGRAM (NTP) (1993). Toxicology and Carcinogenesis Studies o f

o-Nitroanisole (CAS No. 91-23-6) in F344 Rats and B6C3F Mice (Feed Studies). Technical

1

Report Series No. 416. NIH Publication No. 93-3147. U.S. Department of Health and Human

Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

NOMURA, A. (1977). Studies on sulfhemoglobin formation by various drugs. Nippon Yakurigaku

Zasshi 73, 423-435.

OGAWA, T., HIGASHI, S., KAWARADA, Y., AND MIZUMOTO, R. (1995). Role of reactive oxygen in

synthetic estrogen induction of hepatocellular carcinomas in rats and preventive effect o f

vitamins. Carcinogenesis 16, 831-836.

PALITTI, F., FIORE, M., DE SALVIA, R., TANZARELLA, C., RICORDY, R., FORSTER, R., MOSESSO, P.,

ASTOLFI, S., AND LOPRIENO, N. (1985). Tests for the induction of chromosomal aberrations in

Chinese hamster ovary (CHO) cells in culture. In Progress in Mutation Research. Volume 5.

Evaluation of Short-Term Tests for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr.,

B. H. Margolin, B. E. Matter, and M. D. Shelby, Eds.), pp. 443-450. Elsevier Science Publishers,

Amsterdam.

PARRY, J. M., AND ECKARDT, F. (1985). The induction of mitotic aneuploidy, point mutation an d

mitotic crossing-over in the yeast Saccharomyces cerevisiae strains D61-M and D6. In Progress in

Mutation Research. Volume 5. Evaluation of Short-Term Tests for Carcinogenesis (J. Ashby, F. J. de Serres,

M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E. Matter, and M. D. Shelby, Eds.), pp. 285-295.

Elsevier Science Publishers, Amsterdam.
70 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

PARRY,J. M., AND SHARP,D.C. (1981). Induction of mitotic aneuploidy in the yeast strain D6 by 42

coded compounds. In Progress in Mutation Research. Volume 1. Evaluation of Short-Term Tests for

Carcinogens (F. J. de Serres and J. Ashby, Eds.), pp. 468-480. Elsevier/North-Holland, New York.

PERRY, P. E., AND THOMSON, E. J. (1981). Evaluation of the sister chromatid exchange method in

mammalian cells as a screening system for carc inogens. In Progress in Mutation Research. Volume 1.

Evaluation of Short-Term Tests for Carcinogens (F. J. de Serres and J. Ashby, Eds.), pp. 560-569 .

Elsevier/North-Holland, New York.

PHYSICIANS' DESK REFERENCE (PDR) (1992). 46th ed. Medical Economics Data, Montvale, NJ.

POPP,J.A., AND LEONARD, T. B. (1983). Hepatocarcinogenicity of 2,6-dinitrotoluene (DNT). Proc.

Am. Assoc. Cancer Res. 24, 91. (Abstr.)

PRISTON, R. A. J., AND DEAN, B. J. (1985). Tests for the induction of chromosome aberrations,

polyploidy and sister-chromatid exchanges in rat liver (RL ) cells. In Progress in Mutation Research. 4

Volume 5. Evaluation of Short-Term Tests for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M.

Ishidate, Jr., B. H. Margolin, B. E. Matter, and M. D. Shelb y, Eds.), pp. 387-395. Elsevier Science

Publishers, Amsterdam.

RAO, G. N., HASEMAN, J. K., AND EDMONDSON, J. (1989a). Influence of viral infections on bod y

weight, survival, and tumor prevalence in Fischer 344/NCr rats on two-year studies. Lab. Anim.

Sci. 39, 389-393.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 71

RAO, G. N., PIEGORSCH, W. W., CRAWFORD, D. D., EDMONDSON, J., AND HASEMAN, J. K. (1989b).

Influence of viral infections on body weight, survival, and tumor prevalence of B6C3F 1

(C57BL/6N × C3H/HeN) mice in carcinogenicity studies. Fundam. Appl. Toxicol. 13, 156-164.

RICKERT, D. E. (1987). Metabolism of nitroaromatic compounds. Drug Metab. Rev. 18, 23-53.

RICKERT,D.E.,IRONS,R. D., POPP, J. A., AND GOLDSWORTHY, T. L. (1986a). The effects of diet on

the toxicity of nitroaromatic chemicals in rodents. In New Concepts and Developments in Toxicology

(P. L. Chambers, P. Gehring, and F. Sakai, Eds.), pp. 107-114. Elsevier Science Publishers B.V.

(Biomedical Division), Amsterdam.

RICKERT, D. E., CHISM, J. P., AND KEDDERIS, G. L. (1986b). Metabolism and carcinogenicity o f

nitrotoluenes. Adv. Exp. Med. Biol. 197, 563-571.

RICKERT, D. E., BUTTERWORTH, B. E., AND POPP, J. A. (1987). Dinitrotoluene: Acute toxicity ,

oncogenicity, genotoxicity, and metabolism. CRC Crit. Rev. Toxicol. 13, 217-234.

SADTLER STANDARD SPECTRA. IR No. 4692; UV No. 1292; NMR No. 676M. Sadtler Researc h

Laboratories, Philadelphia, PA.

SALAMONE, M. F.,HEDDLE,J.A., AND KATZ, M. (1981). Mutagenic activity of 41 compounds in the

in vivo micronucleus assay. In Progress in Mutation Research. Volume 1. Evaluation of Short-Term Tests

for Carcinogens (F. J. de Serres and J. Ashby, Eds.), pp. 686-697. Elsevier/North-Holland, New

York.

SATO, K., KITAHARA, A., SATOH, K., ISHIKAWA, T., TATEMATSU, M., AND ITO, N. (1984). The

placental form of glutathione S-transferase as a new marker protein for preneoplasia in ra t

chemical hepatocarcinogensis. Gann 75, 199-202.

SATOH, K., KITAHARA, A., SOMA, Y., INABA, Y., HATAYAMA, I., AND SATO, K. (1985). Purification,

induction, and distribution of placental glutathione transferase: A new marker enzyme fo r

preneoplastic cells in the rat chemical hepatocarcinogenesis. Proc. Natl. Acad. Sci. USA 82, 3964-

3968.
72 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

SON, O. S., WEISS, L., FIALA, E. S., AND WEISBURGER, E. K. (1977). Metabolism of the carcinogen

o-toluidine. Proc. Am. Assoc. Cancer Res. 18, 123. (Abstr.)

STANFORD RESEARCH INSTITUTE (1976). o-Toluidine, Stage II, Chemical Dossier. Stanford Research

Institute, Menlo Park, CA.

STEINBERG, P., STEINBRECHER, R., RADAEVA, S., SCHIRMACHER, P., DIENES, H. P., OESCH, F., AND

BANNASCH, P. (1994). Oval cell lines OC/CDE 6 and OC/CDE 22 give rise to cholangio -

cellular and undifferentiated carcinomas after transformation. Lab. Invest. 71, 700-709.

STENBÄCK, F., GEBHARDT, R., SIRMA, H., GARBAY, J.-M., AND WILLIAMS, G. M. (1994). Sequential

functional and morphological alterations during hepatocarcinogensis induced in rats by feeding

of a low dose of 2-acetylaminofluorene. Toxicol. Pathol. 22, 620-632.

SWEET, D. V. (ED.) (1983). Registry of Toxic Effects of Chemical Substances, 1981-1982 Edition,

Vol. 3, p. 773. DHHS (NIOSH) Publication. U.S. Department of Health and Human Services,

Public Health Service, Centers for Disease Control, Washington, DC.

TANAKA, K.-I., MARUI, S., AND MII, T. (1980). Mutagenicity of extracts of urine from rats treate d

with aromatic amines. Mutat. Res. 79, 173-176.

TON, T. T., ELWELL, M. R., MORRIS, M. W., AND MARONPOT, R. R. (1996). Development and

persistence of PGST-positive foci in livers of male F344 rats exposed to o-nitrotoluene. Cancer

Lett. (in press).

TRUEMAN, R. W. (1981). Activity of 42 coded compounds in the Salmonella reverse mutation test.

In Progress in Mutation Research. Volume 1. Evaluation of Short-Term Tests for Carcinogens (F. J. de Serres

and J. Ashby, Eds.), pp. 343-350. Elsevier/North-Holland, New York.

TSUCHIMOTO,T., AND MATTER,B.E. (1981). Activity of coded compounds in the micronucleus test.

In Progress in Mutation Research. Volume 1. Evaluation of Short-Term Tests for Carcinogens (F. J. de Serres

and J. Ashby, Eds.), pp. 705-711. Elsevier/North-Holland, New York.

UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES (USDHHS) (1991). Sixth Annual

Report on Carcinogens, pp. 382-385.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 73

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY (USEPA) (1984). ETD

Production/Exposure Profile: o-Toluidine; o-Toluidine Hydrochloride. Office of Pesticid e

Programs and Toxic Substances, USEPA, Washington, DC.

VAN WENT, G. F. (1985). The test for sister-chromatid exchanges in Chinese hamster V79 cells in

culture. In Progress in Mutation Research. Volume 5. Evaluation of Short-Term Tests for Carcinogenesis

(J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E. Matter, and M. D .

Shelby, Eds.), pp. 469-477. Elsevier Science Publishers, Amsterdam.

VENITT, S., AND CROFTON-SLEIGH, C. (1981). Mutagenicity of 42 coded compounds in a bacterial

assay using Escherichia coli and Salmonella typhimurium. In Progress in Mutation Research. Volume 1.

Evaluation of Short-Term Tests for Carcinogens (F. J. de Serres and J. Ashby, Eds.), pp. 351-360 .

Elsevier/North-Holland, New York.

VOGEL,E. W. (1985). The Drosophila somatic recombination and mutation assay (SRM) using the

white-coral somatic eye color system. In Progress in Mutation Research. Volume 5. Evaluation of

Short-Term Tests for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H .

Margolin, B. E. Matter, and M. D. Shelby, Eds.), pp. 313-317. Elsevier Science Publishers ,

Amsterdam.

WEISBURGER, E. K., RUSSFIELD, A. B., HOMBURGER, F., WEISBURGER, J. H., BOGER, E.,

VAN DONGEN, C. G., AND CHU, K. C. (1978). Testing of twenty-one environmental aromati c

amines or derivatives for long-term toxicity or carcinogenicity. J. Environ. Pathol. Toxicol. 2,

325-356.

WÜRGLER, F.E.,GRAF, U., AND FREI, H. (1985). Somatic mutation and recombination test in wings

of Drosophila melanogaster. In Progress in Mutation Research. Volume 5. Evaluation of Short-Term Tests

for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E .

Matter, and M. D. Shelby, Eds.), pp. 325-340. Elsevier Science Publishers, Amsterdam.
74 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

ZEIGER, E., AND HAWORTH, S. (1985). Tests with a preincubation modification of th e

Salmonella/microsome assay. In Progress in Mutation Research. Volume 5. Evaluation of Short-Term

Tests for Carcinogenesis (J. Ashby, F. J. de Serres, M. Draper, M. Ishidate, Jr., B. H. Margolin, B. E.

Matter, and M. D. Shelby, Eds.), pp. 187-199. Elsevier Science Publishers, Amsterdam.
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 A-1

APPENDIX A

Summary of Lesions

Table A Summary of the Incidence of Neoplasms and Nonneoplastic Lesions

in Male F344/N Rats in the 26-Week Feed Studies of o-Nitrotoluene

and o-Toluidine Hydrochloride ............................................... A-2
A-2 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE A Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2

Control o-Nitrotoluene o-Toluidine Control o-Nitrotoluene

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

DISPOSITION SUMMARY

Animals initially in study 20 60 60 20 40

13-week interim evaluation 10 20 20 10 20

Stop-exposure evaluation 20 20 10 20 3

Survivors

Terminal sacrifice 10 20 20

Animals examined microscopically 20 60 60 20 40

13-WEEK INTERIM

Alimentary System

Liver (10) (20) (20) (10) (20)

Hepatodiaphragmatic nodule 1 (10%) 1 (5%) 1 (5%)

Inflammation 9 (90%) 20 (100%) 20 (100%) 10 (100%) 20 (100%)

Pigmentation, hemosiderin 20 (100%)

Vacuolization, cytoplasmic 20 (100%) 1 (5%) 20 (100%)

Bile duct, hyperplasia 2 (20%) 1 (5%)

Oval cell, hyperplasia 20 (100%) 20 (100%)

Mesentery (1)

Fat, necrosis 1 (100%)

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System

Epididymis (10) (20) (20) (10) (20)

Hyperplasia 2 (10%)

Inflammation 1 (10%) 1 (5%) 1 (5%)

Mesothelioma malignant 2 (10%)

Testes (10) (20) (20) (10) (20)

Degeneration 20 (100%) 1 (5%) 20 (100%)

Hematopoietic System

Spleen (10) (20) (20) (10) (20)

Congestion 20 (100%)

Hematopoietic cell proliferation 2 (20%) 20 (100%) 20 (100%) 20 (100%)

Pigmentation, hemosiderin 20 (100%) 20 (100%) 20 (100%)

Thrombosis 3 (15%)

Capsule, fibrosis 1 (5%) 20 (100%) 1 (5%)
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 A-3

TABLE A Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride (continued)

Normal Gastrointestinal Flora Altered Gastrointestinal Flora

Control o-Nitrotoluene o-Toluidine Control o-Nitrotoluene

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM (continued)

Integumentary System

None

Musculoskeletal System

None

Nervous System

None

Respiratory System

None

Special Senses System

None

Urinary System

Kidney (10) (20) (20) (10) (20)

Accumulation, hyaline droplet 20 (100%) 20 (100%)

Inflammation 1 (5%)

Pigmentation 20 (100%)

Regeneration 3 (30%) 16 (80%) 7 (35%) 3 (30%) 2 (10%)

Urinary bladder (10) (20) (20) (10) (20)

Inflammation 2 (10%)

Transitional epithelium, hyperplasia 10 (50%)

Systemic Lesions

Multiple organs (10) (20) (20) (10) (20) 4

Mesothelioma malignant 2 (10%)

NEOPLASM SUMMARY

Total animals with primary neoplasms 2

5

Total primary neoplasms 2

Total animals with malignant neoplasms 2

Total malignant neoplasms 2
A-4 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE A Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride (continued)

Normal Gastrointestinal Flora Altered Gastrointestinal Flora

Control o-Nitrotoluene o-Toluidine Control o-Nitrotoluene

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

STOP-EXPOSURE

Alimentary System

Liver (10) (20) (20) (10) (20)

Basophilic focus 1 (5%)

Hepatodiaphragmatic nodule 1 (5%)

Inflammation 10 (100%) 20 (100%) 20 (100%) 10 (100%) 20 (100%)

Pigmentation, hemosiderin 11 (55%)

Vacuolization, cytoplasmic 20 (100%) 1 (5%) 20 (100%)

Bile duct, hyperplasia 4 (40%) 5 (25%) 4 (40%)

Oval cell, hyperplasia 20 (100%) 20 (100%)

Cholangiocarcinoma 2 (10%)

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System

Epididymis (10) (20) (20) (10) (20)

Hyperplasia 2 (10%) 1 (5%)

Inflammation 1 (10%) 1 (5%)

Mesothelioma malignant 4 (20%) 2 (10%) 8 (40%)

Testes (10) (20) (20) (10) (20)

Degeneration 20 (100%) 2 (10%) 20 (100%)

Interstitial cell, hyperplasia 11 (55%) 7 (35%)

Tunic, mesothelioma malignant 2 (10%) 4 (20%)

Hematopoietic System

Spleen (10) (20) (20) (10) (19)

Congestion 20 (100%)

Hematopoietic cell proliferation 3 (30%) 18 (90%) 1 (5%) 1 (10%) 19 (100%)

Pigmentation, hemosiderin 3 (30%) 17 (85%) 18 (90%) 5 (50%) 19 (100%)

Capsule, fibrosis 7 (35%) 20 (100%) 1 (5%)

Capsule, lymphatic, angiectasis 15 (75%)

Integumentary System

None
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 A-5

TABLE A Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride (continued)

Normal Gastrointestinal Flora Altered Gastrointestinal Flora

Control o-Nitrotoluene o-Toluidine Control o-Nitrotoluene

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

STOP-EXPOSURE (continued)

Musculoskeletal System

None

Nervous System

None

Respiratory System

None

Special Senses System

None

Urinary System

Kidney (10) (20) (20) (10) (20)

Casts protein 3 (30%) 20 (100%) 6 (30%) 20 (100%)

Inflammation 2 (20%) 1 (5%) 1 (10%) 3 (15%)

Mineralization 1 (10%)

Pigmentation 20 (100%)

Regeneration 7 (70%) 19 (95%) 10 (50%) 6 (60%) 20 (100%)

Urinary bladder (10) (20) (20) (10) (20)

Systemic Lesions

Multiple organs (10) (20) (20) (10) (20)

Mesothelioma malignant 5 (25%) 2 (10%) 8 (40%)

NEOPLASM SUMMARY

Total animals with primary neoplasms 6 2 8

Total primary neoplasms 7 2 8

Total animals with malignant neoplasms 6 2 8

Total malignant neoplasms 7 2 8
A-6 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE A Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride (continued)

Normal Gastrointestinal Flora

Control o-Nitrotoluene o-Toluidine

(0 ppm) (5,000 ppm) (5,000 ppm)

26-WEEK STUDY

Alimentary System

Liver (10) (20) (20)

Inflammation 10 (100%) 20 (100%) 20 (100%)

Mixed cell focus 1 (5%)

Pigmentation, hemosiderin 20 (100%)

Vacuolization, cytoplasmic 20 (100%)

Bile duct, hyperplasia 4 (40%) 10 (50%)

Oval cell, hyperplasia 20 (100%)

Cholangiocarcinoma 1 (5%)

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System

Epididymis (10) (20) (20)

Hyperplasia 2 (10%) 1 (5%)

Mesothelioma malignant 7 (35%)

Testes (10) (20) (20)

Degeneration 20 (100%) 2 (10%)

Inflammation 1 (5%)

Interstitial cell, hyperplasia 3 (15%)

Bilateral, hemangioma 1 (5%)

Tunic, mesothelioma malignant 2 (10%)

Hematopoietic System

Spleen (10) (20) (20)

Congestion 20 (100%)

Hematopoietic cell proliferation 3 (30%) 20 (100%) 20 (100%)

Pigmentation, hemosiderin 3 (30%) 20 (100%) 20 (100%)

Thrombosis 2 (10%)

Capsule, fibrosis 3 (15%) 20 (100%)

Capsule, lymphatic, angiectasis 6 (30%)

Integumentary System

None
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 A-7

TABLE A Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride (continued)

Normal Gastrointestinal Flora

Control o-Nitrotoluene o-Toluidine

(0 ppm) (5,000 ppm) (5,000 ppm)

26-WEEK STUDY (continued)

Musculoskeletal System

None

Nervous System

None

Respiratory System

None

Special Senses System

None

Urinary System

Kidney (10) (20) (20)

Accumulation, hyaline droplet 20 (100%)

Casts protein 3 (30%) 20 (100%) 1 (5%)

Inflammation 2 (20%) 2 (10%)

Pigmentation 20 (100%)

Regeneration 7 (70%) 19 (95%) 5 (25%)

Urinary bladder (10) (20) (18)

Transitional epithelium, hyperplasia 17 (94%)

Systemic Lesions

Multiple organs (10) (20) (20)

Mesothelioma malignant 7 (35%)

NEOPLASM SUMMARY

Total animals with primary neoplasms 9

Total primary neoplasms 9

Total animals with benign neoplasms 1

Total benign neoplasms 1

Total animals with malignant neoplasms 8

Total malignant neoplasms 8

Number of animals examined microscopically at site and number of animals with lesion. 1

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily for 14 consecutive 2

weeks. Altered gastrointestinal flora groups were only included in the 13-week interim and stop-exposure evaluations.

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 13 weeks. 3

Number of animals with any tissue examined microscopically. 4

Primary neoplasms: all neoplasms except metastatic neoplasms. 5
A-8 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 B-1

APPENDIX B

Organ Weights and

Organ-Weight-to-Body-Weight Ratios

Table B Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride ........ B-2
B-2 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE B Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2

Control o-Nitrotoluene o-Toluidine Control o-Nitrotoluene

(0 ppm) (5,000 ppm) (5,000 ppm) (0 ppm) (5,000 ppm)

13-WEEK INTERIM

n 10 10 10 10 10

Necropsy body wt 345 ± 5 261 ± 2** 298 ± 4** 318 ± 5 247 ± 4**

Right kidney

Absolute 1.130 ± 0.016 1.058 ± 0.023 1.043 ± 0.026* 1.034 ± 0.023 1.032 ± 0.022

Relative 3.27 ± 0.03 4.05 ± 0.06** 3.50 ± 0.05** 3.25 ± 0.03 4.19 ± 0.08**

Liver

Absolute 12.056 ± 0.235 13.844 ± 0.316** 12.671 ± 0.206 10.140 ± 0.237 13.201 ± 0.400**

Relative 34.91 ± 0.31 53.08 ± 1.07** 42.57 ± 0.36** 31.86 ± 0.36 53.53 ± 1.55**

Spleen

Absolute 0.730 ± 0.010 0.789 ± 0.021 2.212 ± 0.056** 0.648 ± 0.010 0.729 ± 0.015**

Relative 2.12 ± 0.03 3.02 ± 0.06** 7.43 ± 0.16** 2.04 ± 0.02 2.96 ± 0.08**

Right testis

Absolute 1.593 ± 0.027 1.186 ± 0.028** 1.508 ± 0.020* 1.536 ± 0.030 1.114 ± 0.025**

Relative 4.61 ± 0.03 4.55 ± 0.08 5.07 ± 0.07** 4.83 ± 0.07 4.51 ± 0.08*

Epididymis

Absolute 0.477 ± 0.006 0.331 ± 0.013** 0.420 ± 0.009** 0.469 ± 0.011 0.321 ± 0.007**

Relative 1.38 ± 0.02 1.27 ± 0.04* 1.41 ± 0.03 1.48 ± 0.05 1.30 ± 0.03**

STOP-EXPOSURE 3

n 10 10 10 10 10

Necropsy body wt 389 ± 5 304 ± 6** 351 ± 9** 388 ± 5 281 ± 3**

Right kidney

Absolute 1.379 ± 0.036 1.326 ± 0.027 1.255 ± 0.047* 1.274 ± 0.029 1.371 ± 0.022*

Relative 3.55 ± 0.08 4.36 ± 0.04** 3.56 ± 0.05 3.29 ± 0.07 4.88 ± 0.07**

Liver

Absolute 14.405 ± 0.257 18.186 ± 0.301** 13.910 ± 0.532 13.929 ± 0.390 19.089 ± 0.416**

Relative 37.09 ± 0.69 59.89 ± 1.08** 39.58 ± 1.13 35.88 ± 0.75 67.98 ± 1.59**

Spleen

Absolute 0.806 ± 0.017 0.818 ± 0.017 1.130 ± 0.048** 0.739 ± 0.020 0.748 ± 0.012

Relative 2.08 ± 0.05 2.70 ± 0.06** 3.23 ± 0.16** 1.90 ± 0.04 2.66 ± 0.05**

Right testis

Absolute 1.615 ± 0.027 0.893 ± 0.077** 1.496 ± 0.025 1.585 ± 0.017 0.821 ± 0.090**

Relative 4.16 ± 0.06 2.93 ± 0.25** 4.28 ± 0.12 4.09 ± 0.07 2.91 ± 0.31**

Epididymis

Absolute 0.484 ± 0.006 0.288 ± 0.018** 0.432 ± 0.014 0.469 ± 0.008 0.254 ± 0.020** 4

Relative 1.25 ± 0.01 0.95 ± 0.06** 1.23 ± 0.02 1.21 ± 0.02 0.90 ± 0.07** 4
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 B-3

TABLE B Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male F344/N Rats

in the 26-Week Feed Studies of o-Nitrotoluene and o-Toluidine Hydrochloride (continued)

Normal Gastrointestinal Flora

Control o-Nitrotoluene o-Toluidine

(0 ppm) (5,000 ppm) (5,000 ppm)

26-WEEK STUDY

n 10 10 10

Necropsy body wt 389 ± 5 269 ± 4** 316 ± 3**

Right kidney

Absolute 1.379 ± 0.036 1.304 ± 0.029 1.257 ± 0.024*

Relative 3.55 ± 0.08 4.86 ± 0.11** 3.99 ± 0.09**

Liver

Absolute 14.405 ± 0.257 20.054 ± 0.385** 14.426 ± 0.315

Relative 37.09 ± 0.69 74.68 ± 1.21** 45.71 ± 0.88**

Spleen

Absolute 0.806 ± 0.017 0.877 ± 0.026 2.845 ± 0.097**

Relative 2.08 ± 0.05 3.27 ± 0.10** 9.01 ± 0.26**

Right testis

Absolute 1.615 ± 0.027 0.740 ± 0.055** 1.528 ± 0.020

Relative 4.16 ± 0.06 2.76 ± 0.21** 4.84 ± 0.05**

Epididymis

Absolute 0.484 ± 0.006 0.244 ± 0.017** 0.435 ± 0.007**

Relative 1.25 ± 0.01 0.91 ± 0.06** 1.38 ± 0.02

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1

weight/g body weight (mean ± standard error).

Rats received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily for 14 consecutive 2

weeks. Altered gastrointestinal flora groups were only included in the 13-week interim and stop-exposure evaluations.

Rats were exposed to o-nitrotoluene or o-toluidine hydrochloride in feed for 13 weeks and allowed to recover for 13 weeks. 3

n=9. 4

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

** Significantly different (P 0.01) from the control group by Dunnett's test.
B-4 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 C-1

APPENDIX C

Efficacy and Acute Toxicity of Antibiotic Mixture

(Pilot Studies)

Materials and Methods .............................................................. C-2

Results ............................................................................ C-4

Table C1 Survival and Body Weights of Male F344/N Rats Following 7 Days

of Antibiotic Administration ................................................... C-5

Table C2 Gastrointestinal Flora Counts of Male F344/N Rats Following 7 Days

of Antibiotic Administration ................................................... C-5

Table C3 Total Aerobic and Anaerobic Plate Counts of Male F344/N Rats 

Following 7 Days of Antibiotic Administration .................................... C-6
C-2 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

EFFICACY AND ACUTE TOXICITY OF

ANTIBIOTIC MIXTURE (PILOT STUDIES)

Materials and Methods

These studies were conducted to evaluate the efficacy and acute toxicity of an antibiotic mixture containin g

tetracycline hydrochloride, neomycin sulfate, and nystatin. In each pilot study, male F344/N rats given the antibiotic

mixture by gavage in water daily for 7 days were evaluated for total gastrointestinal flora counts, body weights, and

signs of acute toxicity. Due to the preliminary nature of this work, these studies were not conducted under stric t

compliance with Good Laboratory Practice regulations.

In the preliminary pilot study, the antibiotic mixture was prepared approximately 1 week before the beginning of the

study. Tetracycline hydrochloride, neomycin sulfate, and nystatin were obtained from Sigma Chemical Co. (St. Louis,

MO). Weighed amounts of each antibiotic (in powdered form) were combined, apportioned into sealed containers

in daily allotments, and stored at approximately 0 C. Before dosing, the antibiotic mixture was reconstituted b y

adding the appropriate amount of deionized water. No chemical analyses of the neat chemicals or dose formulations

were required.

Thirty male F344/N rats were obtained from Charles River Laboratories (Kingston, NY). The rats wer e

approximately 4 to 6 weeks old at receipt and were quarantined for 10 days before the beginning of the study. Blood

samples were collected from three rats and the sera were analyze d for antibody titers to rodent viruses; all results were

negative. Rats were weighed and then randomly distributed into five groups. Four groups contained five rats each

and were designated for antibiotic administration and fecal sample collection. The fifth group contained two rat s

designated for the pre-study collection of cecal contents. Each group of rats was housed in a separate cage. Drinking

water and NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA) in meal form were available ad libitum.

Rats designated for fecal sample collection were given a single daily gavage dose of the antibiotic mixture (20 m g

tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin) for 7 consecutive days. One rat was replaced

on Day 1 due to a suspected gavage error. Rats were observed twice daily for clinical signs of moribundity an d

morbidity. Body weights were recorded 2 days prior to dosing (for randomization), on Day 1 (before dosing), and

on Day 7. Fecal samples were collected on Day 1 (be fore dosing) and on Day 7 (approximately 4 hours after dosing).

Approximately 1 hour before sample collection, rats in each of the four groups were placed in cages (one group per

cage) without hardwood bedding. After sample collection, rats were returned to cages with hardwood bedding. At

the end of the study, rats were killed by CO asphyxiation. 2
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 C-3

Rats designated for the pre-study collection of cecal contents were killed by CO asphyxiation on Day 1, and cecal 2

contents were collected and combined. On Day 7, cecal contents were collected and combined from two rat s

randomly selected from the pool of 20 rats given the antibiotic mixture. All fecal and cecal specimens were placed

in autoclaved, screw-top test tubes and transported to Battelle's Microbiology Department on ice. The samples were

diluted with sterile phosphate buffered saline (1:100 on a weight:weight basis) and mixed for 4 minutes with a Model

400 Stomacher Lab Blender. Three serial dilutions were made for each sample. The samples were plated in triplicate

with MacConkey pour plates and incubated aerobically at 35 to 37 C for 18 to 24 hours. Plates containing 30 to

300 colonies were counted manually. If no plates fell within this range, counts were estimated using standar d

microbiological procedures. Counts were reported as colony-forming units per gram of sample analyzed.

In response to scientific criticism, a second pilot study was conducted after the completion of the 13- and 26-week

base studies to measure changes in obligate anaerobes as well as total gastrointestinal flora counts in cecal samples.

Twenty-one male F344 rats were received from Taconic Laboratories (Germantown, NY). The rats wer e

approximately 4 to 6 weeks old at receipt and were quarantined for 12 days. Blood samples were collected from five

rats and the sera were analyzed for antibody titers to rodent viruses; all results were negative. Rats were weighed and

then randomly distributed into two groups of five rats each; one group was an untreated control group and the second

group received the same antibiotics combination as rats in the preliminary pi lot study. The rats were maintained under

conditions similar to those in the preliminary pilot study.

Four hours after dosing on Day 7, cecal contents were collected from control and dosed rats. The samples fro m

individual rats were diluted with sterile phosphate buffered saline. Three serial dilutions were made for each sample.

The samples were plated in triplicate; aerobic counts were determined with MacConkey agar plates and blood agar

plates (trypticase soy agar plus 5% sheep blood) and anaerobic counts were determined with Brucella agar plates

(Brucella agar plus 5% sheep blood, hemin, and Vitamin K1). The plates were incubated at 35 to 37 C for 24 to

48 hours (aerobic incubation) or for 48 to 72 hours (anaerobic incubation). Plates containing 25 to 250 colonies were

counted manually. If no plates fell within this range, counts were estimated usi ng standard microbiological procedures.

Individual colony types were counted and characterized by colony morphology and Gram stain results; presumptive

bacterial identifications were based on growth on selective media, colony morphology, Gram reaction, distinguishing

characteristics, and aerotolerance versus anaerobic requirements.
C-4 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

Results

No deaths occurred in the studies, and rats display ed no overt signs of toxicity as a result of antibiotic administration.

Group mean body weights and absolute mean body weight gains (relative to Day 1) were increased in all four treated

groups in the preliminary pilot study (Table C1); the final mean body weight of dosed rats in the second pilot study

was slightly lower than that of the controls, due to the low body weight gain of one rat (Table C1).

Following the 7-day antibiotic administration period in the preliminary pilot study, total intestinal microflora wer e

reduced in two of the four fecal samples analyzed (97.68% and 99.63% reductions) (Table C2). The high microflora

counts observed on Day 7 in two of the fecal samples may have been due to coliform contamination from the cage

floor or from the animals. The greatest reduction in intestinal microflora was observed in cecal contents (99.96%).

In the second pilot study, the total microflora counts of dosed rats were similar to those of the controls; however,

shifts in aerobic and anaerobic bacterial types were observed (Table C3). Gamma-hemolytic-presumptive Streptococcus

sp. were increased in dosed rats; presumptive Staphylococcus sp. and enteric Gram-negative rods (i.e., Escherichia sp.,

Citrobacter sp., Enterobacter sp., Klebsiella sp.) were decreased in dosed rats. One Bacillus isolate observed in the controls

(10 CFU/g) was not present in dosed rats, although the range of types and corresponding colony counts of Bacillus 8

were greater in cecal samples from dosed rats. One yeast and two fungal isolates were cultured at low levels in dosed

rats, but none occurred in the controls.

Obligate anaerobes were less affected by antibiotic treatment than aerobes. More diverse colony types of Gram -

negative anaerobes (i.e., Bacteroides sp., Prevotella sp., Fusobacterium sp.) were observed in dosed rats than in the controls,

but these anaerobes grew in a wider range in control rats (10 -10 CFU/g) than in dosed rats (10 -10 CFU/g). Gram- 6 10 8 9

positive anaerobes (i.e., Clostridium sp., Propionibacterium sp., Peptostreptococcus sp.) was detected primarily at 10 CFU/g 8

in controls and 10 CFU/g in dosed rats. 9

Based on the results of the preliminary pilot study, rats in the altered flora groups in the 26-week studies were given

a daily gavage treatment of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin in deionized

water. To evaluate the effectiveness of the antibiotic mixture in the 26-week studies, cecal samples were collected at

the 13-week interim evaluation from five rats in each of the following groups: altered flora control, normal flor a

control, altered flora o-nitrotoluene, and normal flora o-nitrotoluene. There is no indication in the literature or i n

reported safety assessment studies of any carcinogenic potential of the individual antibiotics used in these studie s

(PDR, 1992).
O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44 C-5

TABLE C1 Survival and Body Weights of Male F344/N Rats Following 7 Days

of Antibiotic Administration1

Body Weight

Group Survival2

Initial Final Change

Study 1

1 5/5 141 ± 4 162 ± 9 21

2 5/5 139 ± 3 165 ± 8 26

3 5/5 138 ± 5 162 ± 6 24

4 5/5 139 ± 5 165 ± 6 26

Study 2

Control 5/5 103 ± 3 131 ± 3 28

Dosed 5/5 103 ± 3 127 ± 3 24

Rats in each group received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily for 7 1

consecutive days. Body weights are given in grams (mean ± standard deviation).

Number surviving at 7 days/number of animals per group. 2

TABLE C2 Gastrointestinal Flora Counts of Male F344/N Rats Following 7 Days

of Antibiotic Administration1

Gastrointestinal Flora Counts Percent

Day 12

Day 7 Reduction3

Fecal Sample4

1 3.7 x 10 8.6 x 10 97.68 6 † 4

2 2.5 x 10 1.3 x 10 48.00 5 5

3 4.4 x 10 1.6 x 10 99.64 5 3 † ‡

4 1.4 x 10 8.3 x 10 40.71 6 5

Cecal Contents 6.4 x 10 2.3 x 10 99.96 5 6 † 3 †

Rats, except for two rats designated for pre-study collection of cecal contents, received a single gavage dose of 20 mg tetracycline 1

hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily for 7 consecutive days. Counts are given in colony-forming units per gram

(CFU/g).

Samples were taken before antibiotic administration. 2

[(Day 1 count Day 7 count)/Day 1 count] x 100. 3

Each fecal sample was taken from one cage containing five rats that received the antibiotic mixture. 4

Cecal contents were collected on Day 1 from two rats not given the antibiotic mixture and on Day 7 from two rats randomly selected from the 5

pool of 20 rats given the antibiotic mixture. At each time point, cecal contents were combined before analysis.

Count was estimated using standard microbiological procedures and expressed using scientific notation. †

Small sample. ‡
C-6 O-NITROTOLUENE/O-TOLUIDINE HYDROCHLORIDE, NTP TOXICITY REPORT NUMBER 44

TABLE C3 Total Aerobic and Anaerobic Plate Counts of Male F344/N Rats Following 7 Days

of Antibiotic Administration1

Animal Plate Count (CFU/g)

Number MAC BAP BRU 2

Control Group

1 1.9 × 10 2.3 × 10 6.6 × 10 5 8 9

2 7.5 × 10 6.0 × 10 3.0 × 10 4 8 9

3 6.0 × 10 4.7 × 10 4.7 × 10 4 8 9

4 4.5 × 10 1.4 × 10 2.3 × 10 4 8 9

5 1.6 × 10 8.6 × 10 >5.7 × 10 5 8 11

Dosed Group

6 <100 1.0 × 10 2.7 × 10 10 9

7 <100 7.9 × 10 3.5 × 10 9 9

8 <100 1.0 × 10 3.6 × 10 10 9

9 <100 9.1 × 10 3.2 × 10 9 9

10 1.5 × 10 1.1 × 10 3.7 × 10 2 10 9

Rats in each group received a single gavage dose of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily for 7 1

consecutive days. CFU = colony-forming units; MAC = MacConkey agar; BAP = trypticase soy agar plus 5% sheep blood; BRU = Brucella

agar plus 5% sheep blood, hemin, and Vitamin K1. MAC and BAP plates were used to determine aerobic microflora counts; BRU plates were

used to determine anaerobic microflora counts.

Additional dilutions were required to obtain colony counts for one control rat (animal number 5) and all dosed rats. 2
NTP TECHNICAL REPORTS ON TOXICITY STUDIES

PRINTED AS OF FEBRUARY 1996

Toxicity Chemical Route of Exposure Publication

Report Number Number

1 Hexachloro-1,3-butadiene Dosed Feed 91-3120

2 n-Hexane Inhalation 91-3121

3 Acetone Drinking Water 91-3122

4 1,2-Dichloroethane Drinking Water, Gavage 91-3123

5 Cobalt Sulfate Heptahydrate Inhalation 91-3124

6 Pentachlorobenzene Dosed Feed 91-3125

7 1,2,4,5-Tetrachlorobenzene Dosed Feed 91-3126

8 D & C Yellow No. 11 Dosed Feed 91-3127

9 o-Cresol Dosed Feed 92-3128

m-Cresol

p-Cresol

10 Ethylbenzene Inhalation 92-3129

11 Antimony Potassium Tartrate Drinking Water, I.P. Inject. 92-3130

12 Castor Oil Dosed Feed 92-3131

13 Trinitrofluorenone Dermal, Dosed Feed 92-3132

14 p-Chloro- , , -Trifluorotoluene Gavage (corn oil, a-CD) 92-3133

15 t-Butyl Perbenzoate Gavage 92-3134

16 Glyphosate Dosed Feed 92-3135

17 Black Newsprint Ink Dermal 92-3340

18 Methyl Ethyl Ketone Peroxide Dermal 92-3341

19 Formic Acid Inhalation 92-3342

20 Diethanolamine Drinking Water, Dermal 92-3343

21 2-Hydroxy-4-Methoxybenzophenone Dosed Feed, Drinking Water 92-3344

22 N, N-Dimethylformamide Inhalation 93-3345

23 o-Nitrotoluene Dosed Feed 92-3346

m-Nitrotoluene

p-Nitrotoluene

24 1,6-Hexanediamine Inhalation 93-3347

25 Glutaraldehyde Inhalation 93-3348

26 Ethylene Glycol Ethers Drinking Water 93-3349

27 Riddelliine Gavage 94-3350

28 Tetrachlorophthalic Anhydride Gavage 93-3351

29 Cupric Sulfate Drinking Water, Dosed Feed 93-3352

30 Dibutyl Phthalate Feed 95-3353

31 Isoprene Inhalation 95-3354
NTP TECHNICAL REPORTS ON TOXICITY STUDIES

PRINTED AS OF FEBRUARY 1996 (continued)

Toxicity Chemical Route of Exposure Publication

Report Number Number

32 Methylene Bis(thiocyanate) Gavage 94-3381

33 2-Chloronitrobenzene Inhalation 93-3382

4-Chloronitrobenzene

35 Chemical Mixture of 25 Groundwater Drinking Water 93-3384

Contaminants

36 Pesticide/Fertilizer Mixtures Drinking Water 93-3385

37 Sodium Cyanide Drinking Water 94-3386

38 Sodium Selenate Drinking Water 94-3387

Sodium Selenite

39 Cadmium Oxide Inhalation 95-3388

40 -Bromo- -nitrostyrene Gavage 94-3389

42 1,3-Diphenylguanidine Feed 95-3933

45 Halogenated Ethanes Gavage 96-3935