### 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 Resear ch 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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

# C ONTRIBUTORS

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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 3 

# P EER 

# R EVIEW

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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

# T ABLE OF 

# C ONTENTS

A BSTRACT



### ................................ ................................ ................................ ................................ ........................... 5
I NTRODUCTION



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

................................ ........................... 11 Disposition and Metabolism

................................ ................................ ................................ ........................ 12 Toxicity

................................ ................................ ................................ ................................ ......................... 15 Study Rationale and Design

................................ ................................ ................................ ......................... 19
M ATERIALS AND 

### M ETHODS



### ................................ ................................ ................................ .............................. 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
R ESULTS



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

................................ ................................ .............................. 31
D ISCUSSION



### ................................ ................................ ................................ ................................ ......................... 53
R EFERENCES



### ................................ ................................ ................................ ................................ ....................... 61
A PPENDIXES 

### 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-1O - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 5 

# A BSTRACT

o - Nitrotoluene o - Toluidine Hydrochloride



# HCl

Molecular Formula C 7 

### H 7 

### NO 2 

### C 7 

### H 9 

### N 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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 rec overy 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 i n 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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochlo ride (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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 - N ITROTOLUENE / O - T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 11 

# I NTRODUCTION

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 -Tolui dine 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 P

450 

## -dependent process (deBethizy and Rickert, 1984). o -Nitrobenzyl alcoh

ol, 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 -a cetylcysteine (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 bil e 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 -nitrobenzy l alcohol in the liver by a cytochrome

-P 450 

## -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; RickertO -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 dep endent 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 -Hydrox

ylaminobenzyl 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 14 

## C -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 [ 14 

## C]- 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



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

-Ni trotoluene; (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 -Aminobenzy l) Glutathione; (K) S -( o -Aminobenzyl) - N

-acetylcysteineO -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 15 

# Toxicity
Oral LD 50 

## 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 LD 50 

## of 2,462

mg/kg has been reported for an unspecified strain of mice (Christensen and Luginbyhl, 1974). A TC LO 

## (toxic concentration, low) of 700 g/m 3 

## 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 B6C3F 1 

## 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 B6C3F 1 

## 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 LD

50 

## 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 B6C3F 1 

## mice were administered o -toluidine hydrochloride in feed at nine concentrations ranging from 1,000 to 50,000

ppm (NCI, 1979a). Seven we eks 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.



## C ARCINOGENICITY 

## 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 -dini trotoluene. 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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). B6C3F 1 

## 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 B6C3F 1 

## 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 B6C3F 1 

## mice (NCI, 1979a). In NCI studies, groups of 50 male a nd 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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).



## G ENETIC 

## T OXICITY 

## 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 i n 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 -hou r 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 depe ndent 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 d amage 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 B6C3F 1 

## mice. Rats were given an intraperitoneal injection of 400 mg/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 B6C3F 1 

## 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 21



# M ATERIALS AND 

# M ETHODS Procurement and Characterization of o -Nitrotoluene and o

-Toluidine Hydrochloride
Two lots o f o -nitrotoluene (Lots 111OCJ and 08506MV) were obtained from the Aldric h Chemical Company (Metuchen, NJ). A single lot of o -toluidi ne hydrochloride (Lot 308828/1 891) was obtained from SAF Bulk Chem icals (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 perf ormed 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 Standa rd Spectra

). The results of elemental analyses for carbon, hydrogen, and nitrogen agreed with theoretical values for o -nitrotoluene. Karl Fischer ana lysis indicated 0.021% ± 0.003% water. Functional group titration, by nitro reduction wit h titanium (III) chloride followed by back titration with s tandardized ferric ammonium sulfate, indicated a purity of 99% ± 1%. Thin-laye r chromatography by two solvent systems indicated a major spot only in each system. Two ga s chromatographic

systems with flame ionization detection (FID) showed a major peak and n o impurities with relative areas equal to or greater than 0.38%.

Lot 08506MV was identifi ed 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 th

e bo iling point and density of Lot 1110CJ. Infrared, ultraviolet/visible, and NMR spectra wer e consistent with the structure of o -nitrotoluene, with literature refer ences ( 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 accele rated stability study was performed by MRI on Lot 1110CJ of o -nitrotoluene. Ga

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

s t ored protected from light at temperatures up to 60 C. At the study laboratory, o -nitrotoluene

### 22 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## was stored in sealed amber glass bottles at approximately 25 C. The study laboratory reanalyzed the bulk chemical (Lot 08506MV) by gas chromatog raphy with FID before the start of the studies, durin g the studies, and again after the studies ended; no degradation of o -nitrotoluene wa

s observed.

Chemical a nalyses of o- toluidine hydrochloride were performed by SAF Bulk Chemicals o n Lot 308828/1 891. The chemical, a white, crystalline powder with lumps, was identified a s
o -toluidi ne hydrochloride. An infrared spectrum was consistent with the structure of o -toluidine hydrochlorid

e and with a literature reference. The results of elemental analyses for carbon , hydrogen, and nitrogen agreed with theoretical values for o -toluidine hydrochloride

. Ar gentometric

titration (chloride determination) indicated a purity of 100% for o -toluidin e hydrochloride. At the study laboratory, o -toluidine hy

drochloride was stored in sealed amber glass bottl es at approximately 25 C. The study laboratory reanalyzed the bulk chemical by hig h performance liquid chromatography (HPLC) befor e 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 -tolui

dine 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 or o -toluidine hydrochloride to a small portion of feed. Additional feed

was added unti

l the desired premix weight was reached. Then, the premix was blended with a weighed amo unt of feed in a Patterson-Kelley twin-shell blender (East Stroudsburg, PA) fo r 15 minutes, with the intensifier bar on for the first 5 minutes.

MRI pe rformed homogeneity and stability studies on a feed mixture containing 0.5 m g
o -nitroto luene

(Lot 1110CJ) per gram of feed. o -Nitrotoluene was mixed with NIH-07 Ope n Formula Diet by the method described previously. After feed blending, a sample was taken from each o f three blender ports, and the samples were extracted with ethanol and analyzed fo r
o -nitrotoluene by H

PLC. 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 23



## for 1, 2, 3, 4, or 7 days. All samples were analyzed by HPLC. Feed samples stored in seale d 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 los es of o -nitrotoluene. Samples analyzed after 1 day in the rat cage showed a loss of 6.3% o -n itrotoluene, and, after 7 days of storage, samples showed

a loss of 1 8.7% o -nitrotoluene. These losses were in addition to the approximatel y 12% o -nitrotoluene lost during feed preparation.

The s tudy laboratory performed homogeneity and stability studies on a feed mixture containin g 5 mg o -nitrotoluene/g feed (5,000 ppm) (Lot 085 06MV). o -Nitrotoluene was mixed with NIH-07 Open Formula Diet by the method desc ribed 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.
Samp les of the 5 mg/g o -nitrotoluene-feed mixture were stored in sealed amber glass bottles a t -20 C, 5 C, or room te

mperature for 28 days or in an animal feeder, open to air and light, for 1 or 4 days. All samples were analyzed b y gas chromatography with FID. Samples stored in sealed containers at -20 and 5 C, protected from lig ht, were stable for at least 28 days. Samples stored in sealed containers at room temperature, protected from light, showed statistically significan t losse s 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 a t approximately 5 C f

or 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 -Nit rotoluene-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 do sing (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 -nitr otoluene, respectively. These losses are

### 24 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



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

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

The s tudy laboratory performed homogeneity and stability studies on a feed mixture containin g 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 extracte d with methanol and analyzed for o -tolui dine 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 -tol uidine 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 i n
sea led containers in the dark at -20 or 5 C were stable for 28 days. The feed sample stored at

room temp erature for 28 days contained 90% of the o -toluidine hydrochloride detected in th e sample before storage.
At the study la

boratory, 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 th e
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 mix tures were prepared once every 3 weeks. Samples of the feed mixtures pre pared on 3 March, 14 April, 6 May, 7 July, and 18 August 1992, were collecte d before dosing and analyzed for o -toluidine hydrochloride content by HPLC. The animal roo m 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 th e samples showing 15% and 35% losses, contamination with animal hair and feces contributed to an indeterminate amount of the loss in each sample. Differ ences in water content between the feed used to p repare the standards and the feed removed from the animal rooms accounted for 3% of the loss in each sample.O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 deionize d water. No chemical analyses of the neat chemicals or dose formulations were required. Th e efficacy and acute toxicity of t he antibiotic mixture were evaluated in non-GLP pilot studies. The results of these studies are presented in Appendix C. 

# Toxicity Study Designs B ASE 

## S TUDIES 

## Male F344/N r

ats used in these studies were obtained from Charles River Breeding Laboratories (R aleigh, 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 col lected from 10 sentinel rats at the start of the studies and w

ere 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 r

eceived 5,000 ppm o -nitrotoluene or o -toluidine hydrochloride in feed for 13 or 26 weeks. The dose selected for admin istration of o -nitrotoluene was based on the previous 13-week study where a concentration of 5,000 ppm in the diet caused me sothelioma 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 amoun t (5,000 ppm) of o -toluidine hydrochloride. o -Nitrotoluene was administered to 60 rats with normal gastrointes

tinal 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, on e 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 ny statin) 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## mixture due to a technician oversight. The missed treatment likely had minimal impact o n microbial growth, the for

mation 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 contr ol group (normal and altered gastrointestinal flora) and 20 rats from each exposur e group (normal and altered gastrointestinal flora) were killed for the 13-week interim evaluation. Follo wing

the 13-week administration of o -nitrotoluene or o -toluidine hydrochloride and th e 13-week maintenance period on the control diet, 20 rats from each exposure group (normal and altered gastrointestinal flora) were killed for t he stop-exposure evaluation. Following the 26-week administration of o -nitrotoluene or o -toluidine hydrochloride, 20 rats from each norma l gastrointestinal flora exposure group were killed for the 26-week evaluation. Ten rats from each control grou p (normal and altered gastrointestinal flora) were killed at 26 weeks for the stop

exposure and 26-week evaluations.
Rats were housed five per cage, and NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA) in meal f

orm and water (Columbus, OH) were available ad libitum

. Feed consumption was measured weekly. Animal rooms were maintained at 72 ± 3 F and 50% ± 15% relativ e humidity, with at least 10 room air change s per hour. Fluorescent light was provided for 12 hours per day.
Necropsies, with limited tissue collect ion, were performed on all rats exposed to o -nitrotoluene or

o -tolu idine hydrochloride for 13 weeks, 13 weeks with 13 weeks of recovery, or 26 weeks. The epi didymis,

right kidney, liver, spleen, and right testis of each rat were weighed. Organs an d tissues were examined for gross lesions a nd fixed in 10% neutral buffered formalin. Tissues to be examine d microscopically were trimmed, embedded in paraffin, sectioned, and stained wit h hemato xylin

and eosin. For all paired organs (i.e., adrenal gland, kidney, ovary), samples fro m each organ were examined.Osmium staining for the presence of fat was performed on formalinfix ed samples of liver from control and o -nitrotoluene-exposed rats. Proliferating cell nuclea r antige n staining of formalin-fixed, paraffin-embedded samples of cholangiocarcinoma were also performed (Greenwell et al. , 1991). Histopat hologic evaluations were performed on all rats at the 13-week interim evaluation and the end of the studies. Tissues ex amined microscopically are listed in Table 1.O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 27



## Upon completion of the laboratory pathologist's histopathologic evaluation, the slides, paraffi n bl ocks, 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

re viewed and evaluated by the NTP Pathology Working Group (PWG); the final diagnose s represent a consensus of contractor pathologists an d the PWG. Details of these review procedures have been described by Maronpot and Boorman (1982) and Boorman et al . (1985).



## S UPPLEMENTAL 

## E VALUATIONS 

## At the 13-week interim evaluation, cecal specimens were collected from five rats per group in the contro l groups (normal and altered gastrointestinal flora) and o -nitrotoluene groups (normal and altere d gastrointestinal flora). The samples were diluted with sterile phosphate buffered salin e (1:100 on a weight:weight basis) and mixed for 4 minutes with a Model 400 Stomacher La b 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. Plate s containing

30 to 300 colonies were counted manually. If no plates fell within this range, counts were estimated by standard microbiologi cal procedures. Counts were reported as colony-forming units per gram of sample analyzed.

Additio nal

sections of liver were prepared and stained to determine and quantitate placenta l glutathione S -transferase (PGST)-positive foci of cellular alter ation (Hsu et al. , 1981). Livers from 9 or 10 rats in t

he 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 th e number of foc

i 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 A NALYSIS OF 

## L ESION 

## I NCIDENCES 

## The Fis her 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.



## A NALYSIS OF 

## C ONTINUOUS 

## V ARIABLES 

## Organ and body weight dat a, which have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dun nett (1955). The number of PGST-positive foci per cubic centimeter of liver a nd the volume fraction of liver occupied by PGST-positive foci were analyzed with Wilcoxon's rank sum test (Conover, 1971).

Before anal ysis, extreme values identified by the outlier test of Dixon and Massey (1951) wer e examined by NTP personnel. Implausible values, extreme val ues 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Assura nce Unit of Battelle Columbus Operations performed audits and inspections of protocols, procedures, data, and reports throughout the course of the studies.O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 31 

# R ESULTS
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 fe d consumption was 12% to 18% lower than th at 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 hyd rochloride-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 exposu re. 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 expo

sed 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 -toluidin e hydrochloride than the control value (Table 2 and F igure 3). Rats in the stop-exposure groups had sli ghtly greater mean body weight gains than rats continuously exposed for 26 weeks but stil l 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Controls (% 2

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. 2O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 WeeksO -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## Chemical-related effects were present in the testis and epididymis, liver, kidney, and spleen of rats exposed to o -nitr

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

hloride. 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 altere d gastrointestinal

flora) and 26-week continuous-exposure group are described separately. Organ weig ht changes presented in Table 4 are discussed with the chemical-related histopatholog y findings for each organ.O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochloride 1
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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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
O r gan 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 do se of 20 mg tetracycline hydrochloride, 40 mg neomycin sulfate, and 4 mg nystatin daily for 2 14 consecutive weeks. Rats w ere 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 39



## T ESTIS AND 

## E PIDIDYMIS



## o - Nitrotoluene

: The absolute and relative weights of the right testis and epididymis of rats from all ex posed groups at 13 and 26 weeks (normal and altered gastrointestinal flora) were generally signific antly

less than those of controls. At 26 weeks, the testes of all rats in the stop- an d continu ous-exposure

groups were grossly reduced in size relative to those of the controls. Th e ri ght testis and epididymis weights of rats in the stop-exposure groups (normal and altere d gastrointestina

l 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 tho se of controls were a reflection of mild testicular degeneration seen microscopicall y (Table 5). Degeneration consisted of a loss of spermatogenic epithelium and a reduction in th e diameter of t

he seminiferous tubules. In some tubules, there was an almost complete absence of germi nal epithelium and the remaining cells were primarily Sertoli cells; dense aggregates o f mineral deposition were seen in s ome tubules. The distribution of degeneration was multifocal to di ffuse in both testes. In the 26-week continuous-exposure group, the average severity o f 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 degeneratio n and the average severity of this lesio n was slightly greater than that present in the 13-week interim group. Interstitial cel l hyperplasia was present in a few rats fro m the stop- and continuous-exposure groups at 26 weeks. This consisted of a min

imal 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 vaginal is 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 fl

ora. 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 prol iferation 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 an d consi sted of cuboidal or polyhedral-shaped cells forming solid areas with papillary or villou s proje ctions that extended into the peritoneal space. A dense, fibrous core was present in mos t

### 40 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -Tol uidine

Hydrochloride:

There were no gross lesions in the 13-week interim and 26-wee k stop- and continuous-exposure groups. In all o -tol uidine hydrochloride groups, absolute testis and epididymis weights were slig htly less than those of the controls and were considered secondary to body-weight reduction (Table 4). Degene ration of seminiferous tubules was a unilateral testicular lesion present in 5% to 10% of rats from each exp osed group (Table 5). At 26 weeks, 2 of 20 rats had epididymal mesothe

lioma (stop-exposure group) and one rat had epididymal mesothelial cell hyperplasia (cont

inuous-exposure group). The morphology of these lesions was the same as that in rats exposed to o -nitrotoluene.O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochloride 1
Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2
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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## L IVER



## o - Nitrotoluene:

At the 13-week interim evaluation, rats in both o -nitrotoluene-exposed group s (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 apparen t grossly, and liver weights had increased with continuous exposure. The liver weights in the two stop-exposure groups (normal an d altered gastrointestinal flora) also continued to increase during the 13-week recovery period, and the increases were similar to those seen with continuou s 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 inc rease in the number of cells with a scant

amoun t of visible cytoplasm and oval-shaped nuclei. Proliferation of these oval cells was mos t 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

sev erity in the continuous-exposure group or regress in the stop-exposure group. Cytoplasmi c 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 positiv e 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 no t reg ress 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 p lacental glutathione S -transferase (PGST)-positive staining of hepatocytes occurred with o -nitrotolu ene exposure by 13 weeks and increased in number and size (a s 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 numbe r of foci was less than that in the corresponding 13-week interim groups but remaine d significantl

y greater than that in controls. The size of the foci continued to increase i n
o -n itrotoluene-exposed

rats in both normal and altered gastrointestinal flora groups during th e recovery period. Are

as representing PGST-positive foci generally could not be recognized in the routine hemato

xylin/eosin-stained sections. Although a few PGST-positive foci corresponded toO -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 43



## areas of cyto

plasmic 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 examinati on of the liver at necropsy. Two rats from t he stop-exposure group (normal gastrointestinal flora) had a mass (1 centimeter in the greatest dimen

sion) 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 cubo idal, hyperchromatic epithelium; many of these ductular lining cells stained positive for proliferating cell nucle

ar 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 weig hts. Accumulation of hemosiderin pigment in Kupffer cell cytoplasm was present in al l

o -toluidine hydrochloride groups at 13 and 26 weeks and was a minimal, reversible chang e (Table 6). At each time point, the i ncidence 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 -toluidin e hydrochloride exposure group than in controls. H owever, the number was significantly lower than tha t seen with 26 weeks of exposure to o -nitrotoluene (Table 7), and the volume fraction wa s significantly less than that seen with o -nitrotoluene.

### 44 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochloride 1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2
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-EXPOSURE 6
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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochloride 1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2
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-EXPOSURE 5
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/cm tissue 17 ± 2 961 ± 91 ** 145 ± 61**

— —

3 Mean volume (mm ) 0.000 ± 0.000 0.446 ± 0.101 ** 0.017 ± 0.004**

— — 3
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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## K IDNEY AND 

## U RINARY 

## B LADDER



## o -Nitr otoluene: There were no treatment-related gross lesions or consistent effects on kidne y 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 accumulati on of hyaline droplets in the renal tubule of exposed rats in the normal or altere d gastrointestinal flora groups. The incidence, but not the severity, of renal tubule regeneration was increased o

nly 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), th e accumulation of hyaline droplets completely r egressed, but both stop-exposure normal and altered gastrointestinal flora groups exposed to o -nitrotoluene

had a similar slight increase in the incidence and severi ty of lesions including foci of renal tubule epithelial regeneration and dilated rena l tubules containing eosino

philic-staining protein casts. With continued exposure to o -nitrotoluene for 26 weeks, the hyaline dr oplet accumulation was still present but was not increased in severity. The in cidence and severity of other renal lesions typically associated with nephropath y (regeneration and protein casts) were greater than those in the contro ls but did not differ from those seen in the stop-exposure groups.

No gross or m

icroscopic effects were identified in the urinary bladder of rats from an y
o -nitrotoluene-exposure group.

o -Toluidine Hydrochloride: There were no g

ross kidney lesions in any o -toluidine hydrochlorideex posed group; kidney weights of exposed rats were lower than those of the controls, but thi s differenc e was considered secondary to overall reductions in body weights of exposed rat s (Table 4). Microscopic lesions in exposed rats were limited to increased hemosiderin pigmen t accumulation

in the cytoplasm of renal tubule epithelium (Table 8). Hemosiderin accumulation

was of mil d severity in the 13-week interim and 26-week continuous-exposure groups. Th e 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 hydrochloride

expose d group. Mild diffuse hyperplasia of the uroepithelium was present in o -toluidin e hydro chloride-exposed

rats in the 13-week interim and 26-week continuous-exposure group s (Table 8). The average se

verity of this lesion did not increase with longer exposure. Hyperplasia was not o bserved in rats from the stop-exposure groups, indicating that the lesion was reversible (Plates 9 and 10).O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochloride 1
Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2
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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## S PLEEN



## o -Nitrotoluene:

In the 13-week interim groups (normal and altered gastrointestinal flora) exposed to o -nitro toluene, absolute and relative spleen weights were generally slightly but significantl y greater than those of the controls (Table 4). In the 26-week conti nuous- 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 t o 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). Mi

nimal focal areas of fibrosis in the spleen capsule were also present. After 26 w eeks of continuous o -nitrotoluene exposure, hematopoiesis and hemosiderin accumulatio n were slightly more sev

ere 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 -toluidin e hydrochl oride-exposed

rats were grossly enlarged, and the absolute and relative spleen weight s
were significantly greater than those of the controls (Table 4). After 26 weeks of continuou s
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 -toluidin e hydrochloride stop-exposure group s, 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 s pleens of all o -toluidine hydrochloride-exposed rats were distinguished grossly from th e spleens of the control group by the presence of white, granular plaques on the capsular surface. Small (2 mm diameter) fluid-filled cysts were attach ed to the capsule in five rats. Focal, thickened

areas of capsular fibrosis seen microscopically corresponded to the irregular plaques observe d grossly on the capsular surface (Table 9). Within the areas of fibrosis were infiltrates o f lymphocytes and hematopoietic cells; the more ex tensively dilated lymphatic vessels in the capsule (capsul ar angiectasis) corresponded to the cysts observed grossly. Microscopic findings in th e conti nuous- and stop-exposure groups included increases in hematopoietic cell proliferation and hemosiderin concentration above the backgrou nd amounts seen in the spleens of control rats. The amount of blood in sinusoids (congestion) of the red pulp in ex posed rats was also greater than that in the controls (Table 9) (Plates 11 and 12).O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 49



## With the exceptio

n of a slight increase in the severity of capsular fibrosis, the spleen changes that

occurre d with 26 weeks of o -toluidine hydrochloride exposure were similar to those seen at 1 3 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 o f hematopoiesis.

### 50 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochloride 1

Normal Gastrointestinal Flora Altered Gastrointestinal Flora 2
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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 wit h

o -nitrotoluene

exposure in the normal gastrointestinal flora group or the combination effect o f

o -nitrotoluene

with antibiotics in the altered gastrointestinal flora group were not statisticall y significant.

However, this suggests that o -nitrotoluene in the feed may have contributed slightly to the inhibitory effec

t of antibiotic administration on bacterial growth in the intestine. The effect of antibiotics on cecal flora counts is similar to that s een 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 Hydrochloride 1
Gastrointestinal Flora Counts Individual Group Average Normal Gastrointestinal Flora Groups
Control 9.0 x 10 2.6 x 10 2 5 5.5 x 10 4 1.7 x 10 5 9.5 x 10 5 1.3 x 10 5

o -Nitrotoluene 1.3 x 10 6.8 x 10 4 4 (5,000 ppm) 4.9 x 10 4 1.0 x 10 5 3.6 x 10 4 1.4 x 10 5 Altered Gastrointestinal Flora Groups 2
Control 1.5 x 10 2.9 x 10 * 3 4 7.0 x 10 2 1.3 x 10 4 1.2 x 10 3 1.3 x 10 5

o -Nitrotoluene 1.5 x 10 6.2 x 10 4 3 (5,000 ppm) 7.7 x 10 3 7.8 x 10 3 3.3 x 10 2 1.0 x 10 2 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44PLATE 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 va ginalis (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, pap illary 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 expan sile mass of fibrous tissue and atypical b iliary 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 b iliary 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 53 

# D ISCUSSION

The 13- and 26-week studies of two structurally similar chemicals, o -nitrotoluene and o -toluidine hydrochloride, in F344/N rats demonstra ted several differences in carcinogenic potency and in the site, sev erity, and potential for regression or progression of treatment-related toxic effects

. Bacterial reduction of the nit ro group to the amine by the gastrointestinal flora is considered to be

an i mportant step in metabolism of nitroaromatic chemicals including o -nitrotoluene (Doolittl

e 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 demo nstrated

to be less effective than desired. Because of the numerous differences observe d bet ween o -nitrotoluene and o -toluidine hydrochloride with respect to their carcinogenicity an d toxicity in rats, the effects of chemical exposure and altered intestinal flora are discusse d separately.



## M ESOTHELIOMA 

## Mes othelial

cells form a single layer of epithelium on a delicate fibrous stroma that lines th e peritoneal and pleural cavities. In the F344 rat, spontaneous and treatment-related proliferative lesions (hyperplasia and mesothelioma) of the peritoneal mesothelial cells are most commonl y 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 l ymph 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 o f

o -t oluidine

hydrochloride, mesothelioma occurred in 24% of male rats exposed to a dietar y concentration o

f 3,000 ppm (NCI, 1979a). It could not be determined if mesothelioma occurred in sho rter 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

a p proximately equimolar amounts of o -nitrotoluene or o -toluidine hydrochloride in this study to

com pare the relative potency of these chemicals with respect to the onset and development o f prol iferative

lesions in the mesothelium. The studies reported here confirm a chemical-relate d increas e in the incidence of mesothelioma in male rats after 13 or 26 weeks of exposure t o
o -nitrotoluene and the development of a smaller number of mesotheliomas in rats exposed t o

### 54 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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.

Meso thelioma

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). Mesothelia

l hyperplasia, a potential preneoplastic lesion, also occurred in rats exposed t o
o -nitrotoluene but not in the controls. The incidences and morphology of the mesotheliomas were

s i milar in rats from the stop- and continuous-exposure groups. The development of hyperplasia and mesothe lioma in both the stop- and continuous-exposure studies indicates that a relativel y short-te rm exposure to o -nitrotoluene or o -toluidine hydrochloride is sufficient to produce thi s carcinogenic effect.



## TESTIS 

## Significantly lower testis weights and greater incidenc es 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 severi

ty with continuous exposure and showed no evidence of recovery in the stop-exposure groups. In som e rats with the testicular degeneration, a minimal hyperplasia of the interstitial (Leydig) cells was also present. These testicula r effects were not a feature of o -toluidine hydroc hloride

exposure. One or two rats in each o -toluidine hydrochloride-exposed group ha d unilateral testicular degeneration. However, unilateral degeneration of the testes is generall y 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 th e developme

nt of mesothelioma on the adjacent serosa is unclear. Testicular degeneration is a common treatment-related effect with chemical exp osure in rats and, in many instances, this lesion has not been associated with an increase in mesothelioma. Glycidol, a multisite, multisex , multispe cies carcinogen (NTP, 1990), produced degeneration of the testes and mesothelioma

. However, cytemben

a (NTP, 1981) produced a high incidence of mesothelioma but did not cause testicular degeneration.O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 55



## L IVER 

## After 13 weeks of exposure to o -nitrotoluene, liver weights were slightly greater than those of the controls, and morphologic alterations included cytoplasmic v acuolization and oval cell hyperplasia. In both the stop- and continuous-exposure groups, the liver weights of rats exposed t o
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 hyperplasi a 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 causi

ng oval cell hyperplasia may also increase the incidence of hepatocellular adenoma and carcinoma (Ma ronpot et al. , 1991; Stenbäck et al. , 1994). Oval cells are precursor

ce lls in cholangiocellular and undifferentiated liver tumors of rats (Steinberg et al. , 1994). Oval cell hyperplasia was seen af ter 13 and 26 weeks of exposure to o -nitrotoluene and did not regress in the sto p-exposure groups. With o -nitrotoluene exposure, three rats developed focal masse s considered

to be cholangiocarcinoma. In the rat, cholangiofibrosis, cholangiofibroma, an d chola ngiocarcinoma 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 cholang ial tumors are similar. However, stop-exposure and transplantation stu dies have shown the persistence and potentially progressive nature of these chemical-induced proliferative lesions (Maronpot et al. , 1991).
In addition to oval cell hyperplasia, cytoplasm ic vacuolization with associated hepatocyte and liver enlarge ment

and PGST-positive foci of cellular alteration were significantly increased i n
o -nitrotol uene-exposed

groups at 13 and 26 weeks. PGST-positive foci are considered to be a preneoplastic chan

ge 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-acetylaminofluoren e (Stenbäck et al. , 1994) and estrogen (Ogawa et al. , 1995). With hepatocarcinogens , PGST-pos itive

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-we ek recovery period but the volume fraction increases. After 26 weeks of continu ous exposure to o -nitrotoluene, the number and volume fraction of foci wer e approximately 2-fold and 10-fold greater, respectively, compare d to those in the 13-week exposure group. In the stop-exposu

re groups (normal and altered gastrointestinal flora) the number of foci decreased to approximately half of that seen at 13 weeks, b ut the volume fraction of foci continued

### 56 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## to increase during the recovery period. This partial regression of foci of alteration, persistence of oval cell h yperplasia, and the presence of a small number of cholangiocellular neoplasms at 2 6

we eks suggests the potential for a strong carcinogenic response in the liver of rats administere d

o -nitr otoluene

over a longer period of time. In previous 2-year studies of dinitrotoluene, a che mical structurally related to o -nitrotoluene, there were increases in hepatocellular an d cholangiocarcinoma (Rickert et al. , 1987).

In contrast to o -nitrotoluene exposure, the only liver effect seen with o -toluidine hydrochlorid

e exposure was a minimal accumulation of hemosiderin pigment in the cytoplasm of the Kupffe r cells lining the hepatic sinusoids. This was considered to be secondary to the increased erythrocyte destru ction. Immunohistochemical staining of liver sections demonstrated an increase i n PGST-positive

foci in the o -toluidine hydrochloride group relative to controls after 26 weeks of continuous exposure. How ever, 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 -toluidin e hydrochloride there was no evidence of a carcinogenic effect in the liver of rats (NCI, 1979a).



## K IDNEY AND 

## U RINARY 

## B LADDER 

## Hyaline droplet accumulation was present in the renal tubule epithelium and appeare d 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 spont

aneous nephropathy of rats were slightly increased in incidence and severity i n the stop-exposure groups compared to the controls, suggesting incomplete resolution of renal toxicity. Previous studies have shown t hat 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 i ncrease in -globulin are typically associated with an increase in the severity o f 2μ 

## lesion s associated with spontaneous nephropathy; with chronic exposure, an increased incidence of renal tubule neo

plasms is sometimes observed (Baetcke et al. , 1991). The only renal effect of

o -to luidine

hydrochloride was an increase in hemosiderin pigment in tubule epithelium. Th e incidence of pigment accumulation increased with exposure duration and decreased in th e stop -exposure

study. This pigment accumulation was most likely secondary to the increase dO -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 57



## erythrocyte

de struction reflected by the spleen changes in these rats. In 2-year studies of o toluidine 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 uri nary bladder of rats continuously exposed to o -toluidine hydrochloride for 13 o r 26 weeks, t

here was a mild, diffuse hyperplasia of the transitional epithelium. There was n o evidence of treatment-rela

ted urinary calculus formation. Complete regression of the hyperplasia occurred in the stop-exposure group. In the 2-year study, continuous exposure to o -toluidin e hydroc hloride 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 -anisidin e (NCI, 1978d), and p -cr

esidine (NCI, 1979b) also caused hyperplasia and neoplasia of the urinary bladder epithelium in 2-year studies.



## S PLEEN 

## In creased spleen weights and incidences of hematopoiesis and hemosiderin accumulation wer e observe d in rats exposed to o -nitrotoluene. These findings are consistent with results fro m previous studies where o -nitrotoluene has been shown t o increase methemoglobin levels and cause

a mil d regenerative anemia (NTP, 1992). In the present study, there was little difference in th e severity of these effects between the 13- and 26-week continuous-exposure groups. The spleen toxi city seen with o -toluidine hydrochloride exposure was more severe than that caused b y
o -nitrotoluene. S

pleens appeared grossly enlarged and discolored, and spleen weights were twoto threefold

greater than those of the controls. These findings corresponded to the increase d incidences of congestio

n, 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, hemosi derin accumulation, fibrosis, and increased hematopoiesis) have been attributed to the denaturation of hemoglobin, Heinz bod y formation, and increased erythrocyte destruction in the spleen. o -Toluidine hydrochloride binds with hemog

lobin (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-relate d increase in the incidence of sarcomas of the spleen (NCI, 1979a). This increased incidence o f 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## A LTERED 

## G ASTROINTESTINAL 

## F LORA 

## D IET 

## There w as little evidence that the attempt to reduce gastrointestinal bacteria affected th e carcinogeni

c potential or the pattern and severity of o -nitrotoluene toxicity in male rats. Th e incidence of mesothelioma and the severity and pattern of toxic lesions in the liver (increase d weight, cytoplasmic vacuolization, oval cell hyperplasia, PGST-positive foci), kidney (hyalin e droplet accumulation), testes (degeneration), and spleen (hematopoiesis, hemosiderin, capsula r fibro sis) 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 o f cholangiocarcinomas, which were seen in 3 of 40 rats administered o -nitrotoluene in the normal diet. Howeve

r, the altered gastrointestinal flora did not prevent increased liver weight or hepatic toxicity, reflected by the p

resence of cytoplasmic vacuolization, PGST-positive foci, and oval cell hyperplasi a. Oval cell hyperplasia is considered to be a morphologic change preceding an d

assoc iated

with the development of cholangial neoplasms. Based on these findings, the shor t duration o f the study, and the small number of these liver neoplasms seen in the norma l gastrointestinal flora group, it is unlikely that the attempt to alter gastrointestinal flora prevented the development of cholangiocarcinoma.

Previou s 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 unsched uled DNA synthesis in primary hepatocyte cultures from male rats (Doolittle et al. , 1983). This effect of o -nitrotoluene was not seen i n similarly treated, bacteria-free rats. Gardner and Renwick (1978) demonstrated a reduction in the bacteria lly mediated reduction of nitrobenzoic acids in rats receiving a mixture of antibiotic s similar to that used in the current studies, but with the bacteriostat bacitracin rather than th e antifunga l agent nystatin. Although a significant alteration and reduction of the gastrointestina l flora was observed with daily administration of antibiotics in this study, this was only demonstrated for aer obes, and it was subsequently pointed out during a review of an earlier draft of thi s document that the obligate anaerobes may have been less affected by the antibiotics used. Thi s would have resulted in an insufficient alteration of flora to affect the hydrolytic and reductiv e activity of the intest

inal bacteria on the glucuronidated forms of the o -nitrotoluene secreted in the bile. Also, although bacte

rial 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 wer

e higher than those observed after one week of antibiotic treatment (Appendix C),O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 59



## and this increa

se in resistant bacteria may have been sufficient to adequately reduce the chemical conjugates in the intestine.

In an effor

t to further determine the efficacy of the antibiotic mixture used to reduc e gastro intestinal

flora, the pilot study described in Appendix C was repeated, and cultures wer e plated a nd enumerated under conditions appropriate for determining both aerobic and anaerobic bacte ria. The results of the second pilot study extended the findings of the initial study b y dem onstrating

a shift in the types of aerobic bacteria present, with a decline in enteric Gram

negative 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 obliga te anaerobic bacteria. Therefore, no conclusions concerning the involvement o f 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 devel opment of a high incidence of mesotheliomas and a small number o f cholangiocarcinomas in male rats after o nly 13 or 26 weeks of exposure. The increased incidence of mesothelioma indicat

es the relative greater carcinogenic potential or decrease in tumor latency for o -nitrotoluene compared to an equivalent dose and exposure time fo r o -toluidine hydrochloride. While the chem

icals are similar in structure, basic differences exist in the way o -nitrotoluene and

o -toluid ine

hydrochloride are metabolized, e.g., methyl group oxidation ( o -nitrotoluene) verse

s aromatic ring oxidation (

o -toluidine hydrochloride). It may be that the metabolite responsible for the i ncreased incidence of mesotheliomas may be a relatively minor metabolite of o -toluidin e hydrochlo ride and a major one for o -nitrotoluene. Although metabolites of both compounds are

excr eted 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 urinar y metabolites of each chemical. These findings, plus the presence of testicular degeneration , hepatoto xicity,

and cholangiocarcinoma only in o -nitrotoluene groups and the relatively greate r spleen toxicit

y seen in the o -toluidine hydrochloride groups, indicate that the important toxic and carcinogeni

c effects of o -nitrotoluene may not be attributed primarily to the intestinal trac t reduction of the nitro group to the amine.

### 60 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### 61 

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### 74 O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



## Z EIGER 

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esis (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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### A-1 

# A PPENDIX 

# 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 1

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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 NoneO -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 NoneO -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### B-1 

# A PPENDIX 

# 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 Hydrochloride 1

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** 4O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### C-1 

# A PPENDIX 

# 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



# E FFICACY AND 

# A CUTE 

# T OXICITY OF 

# A NTIBIOTIC 

# M IXTURE 

# (P ILOT 

# S TUDIES 

# )
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 acut e 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, th e 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). W eighed 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
ne gative. Rats were weighed and then randomly distributed into five groups. Four groups contained five rats each and were d esignated 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 Fo rmula Diet (Zeigler Brothers, Inc., Gardners, PA) in meal form were available ad libitum

.
Rat s designated for fecal sample collection were given a single daily gavage dose of the antibiotic mixture (20 m g tetracycline hydrochloride, 40 mg neom ycin 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) witho

ut 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. 2O -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 an d transported to Battelle's Microbiology Department on ice. The samples were diluted with sterile phosphate bu ffered saline (1:100 on a weight:weight basis) and mixed for 4 minutes with a Model 400 Stomacher Lab Blender. Three serial dilutio ns 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 we

re 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.

I n 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. Twent y-one male F344 rats were received from Taconic Laboratories (Germantown, NY). The rats wer e approximately 4 to 6 weeks old at r eceipt and were quarantined for 12 days. Blood samples were collected from five rats and the sera were analyzed for antib ody 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 d

osing on Day 7, cecal contents were collected from control and dosed rats. The samples fro m individual rats were diluted with s terile 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 plat es (trypticase soy agar plus 5% sheep blood) and anaerobic counts were determined with Brucella agar plate

s ( Br ucella agar plus 5% sheep blood, hemin, and Vitamin K1). The plates were incubated at 35 to 37 C for 24 t o 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 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 m ean 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).

Fol lowing the 7-day antibiotic administration period in the preliminary pilot study, total intestinal microflora wer e reduced in two of the four f ecal 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 flo or 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., Klebsiel

la 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.
Oblig ate anaerobes were less affected by antibiotic treatment than aerobes. More diverse colony types of Gram

- negative anaerobes (i.e., Bacteroides sp., Prevotel

la 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 anaerobe

s (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 result

s 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 effect iveness of the antibiotic mixture in the 26-week studies, cecal samples were collected at the 1 3-week interim evaluation from five rats in each of the following groups: altered flora control, normal flor a control, alt

ered 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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44 

### C-5

TABLE C1 Survival and Body Weights of Male F344/N Rats Following 7 Days

of Antibiotic Administration 1 Body Weight

Group Survival 2 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 Administration 1
Gastrointestinal Flora Counts Percent Day 1 2 Day 7 Reduction 3
Fecal Sample 4
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 -N ITROTOLUENE / O -T OLUIDINE H YDROCHLORIDE , NTP T OXICITY R EPORT N UMBER 44



### TABLE C3 Total Aerobic and Anaerobic Plate Counts of Male F344/N Rats Following 7 Days

of Antibiotic Administration 1

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