OFFICIAL NTP 

FILE COPY 

National Cancer Institute 

CARCINOGENESIS DO NOT REMOVE FROM THIS OFFICE 

Technical Report Series 

No. 107 

1978 

BIOASSAY OF 

5-NITRO-o-TOLUIDINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 99-55-8 

NCI-CG-TR-107 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 

National Institutes of Health 

:>x; 

BIOASSAY OF
 

5-NITRO-o-TOLUIDINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
 

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1357
 

REPORT ON THE BIOASSAY OF 5-NITRO-o-TOLUIDINE
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENESIS TESTING PROGRAM
 

DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

FOREWORD: This report presents the results of the bioassay of
 

5-nitro-o-toluidine conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer Institute
 

(NCI), National Institutes of Health, Bethesda, Maryland. This is
 

one of a series of experiments designed to determine whether selected
 

chemicals have the capacity to produce cancer in animals. Negative
 

results, in which the test animals do not have a significantly greater
 

incidence of cancer than control animals, do not necessarily mean the
 

test chemical is not a carcinogen because the experiments are conducted under a limited set of circumstances. Positive results demonstrate that the test chemical is carcinogenic for animals under the
 

conditions of the test and indicate a potential risk to man. The
 

actual determination of the risk to man from animal carcinogens requires a wider analysis.
 

CONTRIBUTORS: This bioassay of 5-nitro-o-toluidine was conducted by
 

Mason Research Institute, Worcester, Massachusetts, initially under
 

direct contract to the NCI and currently under a subcontract to Tracer
 

Jitco, Inc., prime contractor for the NCI Carcinogenesis Testing Program.
 

The experimental design was determined by the NCI Project Officers, Dr. J. H. Weisburger (1,2) and Dr. E. K. Weisburger (1). The
 

principal investigators for the contract were Dr. E. Smith (3) and
 

Dr. A. Handler (3). Animal treatment and observation were supervised
 

by Mr. G. Wade (3) and Ms. E. Zepp (3).
 

Histopathologic examinations were performed by Dr. R. W. Fleischman (3) and Dr. D. S. Wyand (3) at the Mason Research Institute, and
 

the diagnoses included in this report represent the interpretation of
 

these pathologists. Histopathology findings and reports were reviewed
 

by Dr. R. L. Schueler (4).
 

Compilation of individual animal survival, pathology, and summary
 

tables was performed by EG&G Mason Research Institute (5); the statistical analysis was performed by Mr. W. W. Belew (6), using methods
 

selected for the Carcinogenesis Testing Program by Dr. J. J. Gart (7).
 

This report was prepared at METREK, a Division of The MITRE Corporation (6) under the direction of the NCI. Those responsible for
 

111
 
this report at METREK are the project coordinator, 'Dr. L. W. Thomas
 

(6), task leader Dr. M. R. Kornreich (6), senior biologist Ms. P.
 

Walker (6), biochemist Dr. B. Fuller (6), chemist Dr. N. Zimmerman
 

(6), and technical editor Ms. P» A. Miller (6). The final report was
 

reviewed by members of the participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

the results, and reporting the findings: Dr. K. C. Chu (1), Dr. C.
 

Cueto, Jr. (1), Dr. J. F. Douglas (1), Dr. D. G. Goodman (1), Dr. R.
 

A. Griesemer (1), Dr. H. A. Milman (l), Dr. T. W. Orme (1), Dr. R. A.
 

Squire (1,8), Dr. J. M. Ward (1), and Dr. C. E. Whitmire (1).
 

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 No wit w h the Naylor Dana Institute for Disease Prevention, American Health Foundation, Hammon House Road, Valhalla, New York.
 

3.	 Mason Research Institute, 57 Union Street, Worcester, Massachusetts.
 

4.	 Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

5.	 EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

6.	 The MITRE Corporation, METREK Division, 1820 Dolley Madison
 

Boulevard, McLean, Virginia.
 

7.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

Cause and Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
 

8.	 No wit w h the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

iv
 
SUMMARY
 

A bioassay of 5-nitro-o-toluidine for possible carcinogenicity
 

was conducted using Fischer 344 rats and B6C3F1 mice. 5-Nitro-otoluidine was administered in the feed, at either of two concentrations, to groups of 50 male and 50 female animals of each species.
 

The time-weighted average high and low dietary concentrations of
 

5-nitro-o-toluidine were, respectively, 0.01 and 0.005 percent for
 

rats, and 0.23 and 0.12 percent for mice. After a 78-week period
 

of compound administration, observation of the rats continued for
 

an additional 30 to 31 weeks and observation of the mice continued
 

for up to an additional 20 weeks.
 

For each species, 50 animals of each sex were placed on test as
 

controls and fed only the basal diet.
 

There were no significant positive associations between the concentration of 5-nitro-o-toluidine administered and mortality in rats
 

or mice of either sex, and adequate numbers of animals in all groups
 

survived sufficiently long to be at risk from late-developing tumors.
 

No unusual tumors were observed in rats of either sex and no
 

convincing statistical evidence was provided for a significant positive association between compound administration and the incidence of
 

any neoplasm in rats.
 

Among mice there was a statistically significant positive association between administration of the chemical and the incidences of
 

hepatocellular carcinomas in both males and females. The combined
 

incidence of hemangiomas and hemangiosarcomas in male mice and the
 

incidence of hemangiosarcomas in female mice were not statistically
 

significant in relation to their respective control groups. However,
 

they were considered to be associated with compound administration
 

since they rarely occur in untreated B6C3F1 mice.
 

Under the conditions of this bioassay 5-nitro-o-toluidine was
 

carcinogenic in B6C3F1 mice, causing hepatocellular carcinomas in
 

both sexes, an increase in the combined incidence of hemangiomas and
 

hemangiosarcomas in male mice, and an increased incidence of hemangiosarcomas in female mice. The compound was not carcinogenic in
 

Fischer 344 rats.
 

v
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 3
 

A. Chemicals 3
 

B. Dietary Preparation 3
 

C. Animals 3
 

D. Animal Maintenance 5
 

E. Selection of Initial Concentrations 8
 

F. Experimental Design 9
 

G. Clinical and Histopathologic Examinations 12
 

H. Data Recording and Statistical Analyses 14
 

III. CHRONIC TESTING RESULTS: RATS 19
 

A. Body Weights and Clinical Observations 19
 

B. Survival 19
 

C. Pathology 22
 

D. Statistical Analyses of Results 23
 

IV. CHRONIC TESTING RESULTS: MICE 33
 

A. Body Weights and Clinical Observations 33
 

B. Survival 33
 

C. Pathology 36
 

D. Statistical Analyses of Results 37
 

V. DISCUSSION 44
 

VI. BIBLIOGRAPHY 46
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 5-NITRO-o-TOLUIDINE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 5-NITRO-o-TOLUIDINE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 5-NITRO-o-


TOLUIDINE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 5-NITRO-o-


TOLUIDINE D-l
 

vii
 
Figure Number
 

1
 

2
 

Table Number
 

Al
 

LIST OF ILLUSTRATIONS 

Page 

CHEMICAL STRUCTURE OF 5-NITRO-o-TOLUIDINE 4 

GROWTH CURVES FOR 5-NITRO-o-TOLUIDINE CHRONIC 

STUDY RATS 20 

SURVIVAL COMPARISONS OF 5-NITRO-o-TOLUIDINE 

CHRONIC STUDY RATS 21 

GROWTH CURVES FOR 5-NITRO-o-TOLUIDINE CHRONIC 

STUDY MICE 34 

SURVIVAL COMPARISONS OF 5-NITRO-o-TOLUIDINE 

CHRONIC STUDY MICE 35 

LIST OF TABLES 

Page 

DESIGN SUMMARY FOR FISCHER 344 RATS-­

5-NITRO-o-TOLUIDlNE FEEDING EXPERIMENT 10 

DESIGN SUMMARY FOR B6C3F1 MICE—5-NITRO-oTOLUIDINE FEEDING EXPERIMENT 11 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

5-NITRO-o-TOLUIDINE 24 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 5-NITRO-o-TOLUIDINE 28 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

5-NITRO-o-TOLUIDINE 38 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 5-NITRO-o-TOLUIDINE 40 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 5-NITRO-o-TOLUIDINE 

viil 

A-3 
LIST OF TABLES (Concluded) 

Table Number Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 5-NITRO-o-TOLUIDINE A-8 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 5-NITRO-o-TOLUIDINE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 5-NITRO-o-TOLUIDINE B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 5-NITRO-oTOLUIDINE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 5-NITROo-TOLUIDINE C-9 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 5-NITRO-oTOLUIDINE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 5-NITROo-TOLUIDINE D-7 

ix
 

I. INTRODUCTION
 

5-Nitro-o-toluidine (NCI No. C01843), an intermediate in the
 

synthesis of azo dyes, was selected for bioassay by the National Cancer Institute in an attempt to elucidate those chemicals which may be
 

responsible for the increased incidence of bladder cancer observed
 

among workers in the dye manufacturing industry (Wynder et al., 1963;
 

Anthony and Thomas, 1970). Aromatic nitro and amino compounds are
 

thought to contribute to the increased cancer risk in this industry
 

(Wynder et al., 1963).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 2-methyl-5-nitro-benzeneamine.
 

Other frequently used chemical names include 4-nitro-2-aminotoluene
 

and 6-methyl-3-nitroaniline. The dye industry commonly refers to
 

this compound as Fast Scarlet Base G or Colour Index (C.I.) Azoic
 

Diazo Component 12 (C.I. 37105).
 

According to the U.S. International Trade Commission (1977a),
 

1.25 x 10 pounds of Azoic Diazo Component 12, salt were produced by
 

three companies in 1975. Two of these companies and one other company also produced additional quantities of the free base listed as
 

either C.I. Azoic Diazo Component, base or as 5-nitro-o-toluidine.
 

Although quantities produced were considered proprietary, there is
 

reason to believe that production of the free base rivaled that of
 

*
 

The CAS registry number is 99-55-8,
 
the salt, since in 1973, 3.53 x 10 pounds of 5-nitro-o-toluidine were
 

produced as compared to 3.60 x 10 pounds of Azoic Diazo Component 12,
 

salt (U.S. International Trade Commission, 1975; as cited in Urso,
 

1977). Imports of 5-nitro-o-toluidine through principle U.S. customs
 

3
 districts amounted to 4.63 x 10" pounds in 1975 (U.S. International
 

Trade Commission, 1977b; as cited in Urso, 1977).
 

5-Nitro-o-toluidine finds application as an intermediate in the
 

synthesis of Pigment Red 17 (C.I. 12 390) and Pigment Red 22 (C.I.
 

12 315). Used in conjunction with certain C.I. coupling components,
 

5-nitro-o-toluidine also serves as a precursor for a wide assortment
 

of azo dyes of various red, yellow, orange, violet, and brown hues
 

(Society of Dyers and Colourists, 1971a, Society of Dyers and Colourists, 1971b, and Schlopfer, 1973; as cited in Urso, 1977).
 

Since 5-nitro-o-toluidine and its salts appear to be used on a
 

commercial scale exclusively by the dye and pigment industry, the
 

potential for exposure to the compound may be greatest for workers at
 

dye manufacturing facilities.
 

Methemoglobinemia is the major toxic effect observed following
 

excessive absorption of 5-nitro-o-toluidine (Hamblin, 1967). Symptoms of exposure include bluish lips and/or fingernails, headache,
 

nausea, and fatigue (Synalloy Corporation, 1977). In addition, upon
 

dermal contact the compound may irritate the skin, resulting in dermatitis (Synalloy Corporation, 1977).
 
II. MATERIALS AND METHODS 

A. Chemicals
 

5-Nitro-o-toluidine (Figure 1) was purchased from J. T. Baker
 

Chemical Company and chemical analysis was performed by Mason Research
 

Institute. The experimentally determined melting point of 104° to
 

106°C suggested a compound of high purity due to its narrow range and
 

close proximity to the literature value (Weast, 1977) of 107° to
 

108°C. Ultraviolet analysis of the purchased chemical showed X at
 

max
 

230.256 and 290. The literature (Sadtler Standard Spectra) listed a
 

\ only at 227, thereby indicating the presence of impurities,
 max
 

Throughout this report the term 5-nitro-o-toluidine is used to
 

represent this compound.
 

B. Dietary Preparation
 

The basal laboratory diet for both treated and control animals
 

consisted of Wayne Lab-Blox®(Allied Mills, Inc., Chicago, Illinois).
 

5-Nitro-o-toluidine was administered to the treated animals as a
 

component of the diet. The chemical was mixed with the feed using a
 

6 kg capacity Patterson-Kelley twin-shell stainless-steel V-blender.
 

After 20 minutes of blending, prepared diets were placed in double
 

plastic bags and stored in the dark at 4°C. Mixtures were used for
 

1 week only.
 

C. Animals
 

Two animals species, rats and mice, were used in the carcinogenicity bioassay. Fischer 344 rats and B6C3F1 mice were obtained through
 
FIGURE 1

 

CHEMICAL STRUCTURE OF 5-NITRO-o-TOLUIDINE 
contracts of the Division of Cancer Treatment, National Cancer
 

Institute. Rats for use in the treated groups were supplied by ARS/
 

Sprague-Dawley, Madison, Wisconsin. Rats to be used in the control
 

groups were obtained from Laboratory Supply Company, Inc., Indianapolis, Indiana. All mice were supplied by Charles River Breeding
 

Laboratories, Wilmington, Massachusetts. Upon arrival, a sample of
 

animals was examined for parasites and other signs of disease. Some
 

male rats from ARS/Sprague-Dawley were diagnosed as having pneumonia;
 

only disease-free animals were used for the chronic bioassay. The
 

remaining animals were quarantined for two weel^s prior to initiation
 

of test. Animals were assigned to groups and distributed among cages
 

so that average body weight per cage was approximately equal for a
 

given sex and species.
 

D. Animal Maintenance
 

All animals wene housed by species in rooms having a temperature
 

range of 23° to 34°C. Inpoming air was filtered through Tri-Dek
 

(R)
 15/40 denier Dacron filters (Tri-Dim Filter Corp., Hawthorne, New
 

Jersey) providing six changes of room air per hour. Fluorescent
 

lighting was provided on a 12-hour-daily cycle.
 

Rats were housed five per cage by sex. During quarantine and
 

for the first 11 months of the study, they were housed in stainless-


and galvanized-steel wire-mesh cages suspended above newspapers.
 

Newspapers were replaced daily and cages and racks washed weekly.
 
After the first 11 months and for the remainder of the study, rats
 

were kept in suspended polycarbonate cages equipped with disposable
 

nonwoven fiber filter sheets. Corncob bedding and clean cages were
 

provided twice weekly. Stainless steel racks were cleaned once every
 

two weeks and disposable filters were replaced at that time.
 

Mice were housed by sex in polycarbonate cages. During quarantine and periods of chemical administration, cages were fitted with
 

perforated stainless-steel lids. During the observation period
 

following chemical administration, stainless-steel wire bar lids were
 

used. Both types of lids were from Lab Products, Inc., Garfield, New
 

Jersey. Nonwoven fiber filter bonnets were used over cage lids. Animals were housed ten per cage for the first 16 months of study and
 

five per cage thereafter. Cages, lids, filters, and bedding were provided three times per week when cage populations were ten and twice
 

per week when cage populations were reduced to five. Ab-sorb-dri
 

hardwood chip bedding (Wilner Wood Products Company, Norway, Maine)
 

was used through the first 7 months of study, followed by SAN-I-CEL
 

corncob bedding (Paxton Processing Company, Paxton, Illinois) for the
 

next 12 months. After 19 months on study, animals were placed on a
 

second corncob bedding (Bed-o-Cobs , The Andersons Cob Division,
 

Maumee, Ohio). Reusable filters and pipe racks were sanitized every
 

2 weeks throughout the study.
 

Water was available ad libitum for both species from 250 ml water
 

bottles equipped with rubber stoppers and stainless steel sipper tubes.
 
Glass water bottles were used for the first 8 months of study; polycarbonate bottles were used for the remainder of the bioassay. Bottles
 

were replaced twice weekly and, for rats only, water was supplied as
 

needed between changes.
 

Wayne Lab-Blox^ feed was used throughout the entire bioassay.
 

Pelleted feed was supplied during the quarantine period and the final
 

observation period. During chemical administration, all treated
 

animals were supplied with meal containing the appropriate concentrations of 5-nitro-o-toluidine. Control animals were supplied with
 

untreated meal. Alpine®aluminum feed cups (Curtin Matheson Scientific, Inc., Woburn, Massachusetts) equipped with stainless steel
 

baffles were used to dispense feed. Food was replenished daily and
 

the food assembly was replaced weekly. During the untreated observation period, rats were fed pellets on the cage floor. During this
 

same period, mice received pellets from a food hopper incorporated
 

into the cage lid.
 

Treated and control rats were housed in a room with other rats
 

ie
 

receiving diets containing hydrazobenzene (530-50-7); 2-aminoanthraquinone (117-79-3); 3-amino-9-ethylcarbazole hydrochloride; 4-nitroanthranilic acid (619-17-0); 6-nitrobenzimidazole (94-52-0); APC
 

(8003-03-0); 1-nitronaphthalene (86-57-7); 2,4-diaminoanisole sulfate
 

(615-05-4); and 2-methyl-l-nitroanthraquinone (129-15-7).
 

*
 

CAS registry numbers are given in parentheses.
 
Treated and control mice were housed in a room with other mice
 

receiving diets containing 2,5-toluenediamine sulfate (6369-59-1);
 

hydrazobenzene (530-50-7); 3-amino-9-ethylcarbazole hydrochloride;
 

6-nitrobenzimidazole (94-52-0); 3-nitro-p-acetophenetide (1777-84-0);
 

5-nitro-o-anisidine (99-59-2); acetylaminofluorene (53-96-3); acetone
 

(67-64-1); l-amino-2-methylanthraquinone (82-28-0); 3-amino-4-ethoxyacetanilide (17026-81-2); 1-nitronaphthalene (86-57-7); 2,4-diaminoanisole sulfate (615-05-4); APC (8003-03-0); 5-nitroacenaphthene
 

(602-87-9); 3-amino-4-ethoxyacetanilide (17026-81-2); 2-aminoanthraquinone (117-79-3); 2,4-dinitrotoluene (121-14-2); and N,N-dimethylp-nitrosoaniline (138-89-6).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

5-nitro-o-toluidine for administration to treated animals in the
 

chronic studies, Subchronic toxicity studies were conducted with both
 

rats and mice. In the initial phase of Subchronic toxicity testing,
 

5-nitro-o-toluidine was incorporated into the basal laboratory diet
 

and supplied to five of six rat groups (each consisting of five males
 

and five females) at concentrations of 0.009, 0.019, 0.037, 0.07, and
 

0.15 percent. The sixth group served as the control, receiving only
 

the basal laboratory diet. Each of these concentrations produced
 

severe weight depression and deaths within the first 3 weeks of study.
 

In the second phase of Subchronic toxicity testing, animals of each
 

species were distributed among five groups, each consisting of five
 

8
 
males and five females. 5-Nitro-o-toluidine was incorporated into
 

the basal laboratory diet and supplied to four groups of each species
 

in concentrations of 0.009, 0.019, 0.037, and 0.07 percent. The
 

dosed dietary preparations were administered for 4 weeks, followed by
 

a 2-week observation period during which animals were fed the basal
 

diet. No deaths occurred at these concentrations. Based upon weight
 

depression relative to untreated controls, the initial high and low
 

concentrations selected for rats in the chronic bioassay were 0.009
 

and 0.0045 percent, respectively. The initial high and low concentrations selected for mice in the chronic bioassay were 0.01 and 0.005
 

percent, respectively.
 

F. Experimental Design
 

The experimental design parameters for the chronic bioassay
 

(species, sex, group size, concentrations administered, duration of
 

treated and untreated observation periods, and time-weighted average
 

concentrations) are summarized in Tables 1 and 2.
 

At initiation of the study all the rats were approximately 6
 

weeks old. The initial dietary concentrations of 0.009 and 0.0045
 

percent were administered for the first 8 weeks. Throughout this
 

report those rats initially receiving the former concentration are
 

referred to as the high dose groups and those initially receiving the
 

latter concentration are referred to as the low dose groups. High and
 

low concentrations were increased in week 9 to 0.01 and 0.005 percent,
 

respectively. Dosed feed was administered to treated rats for a
 
MALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSE
 

FEMALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSE
 

a
 

TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

5-NITRO-o-TOLUIDINE FEEDING EXPERIMENT
 

5-NITRO-o 

INITIAL TOLUIDINE OBSERVATION PERIOD TIME-WEIGHTED 

GROUP CONCENTRATION TREATED UNTREATED AVERAGE 

SIZE (PERCENT) (WEEKS) (WEEKS) CONCENTRATION3 

50 0 0 108 0
 

50 0.0045 8 0.005
 

0.005 70
 

0 30
 

50 0.009 8 0.01
 

0.01 70
 

0 30
 

50 0 0 109 0
 

50 0.0045 8 0.005
 

0.005 70
 

0 30
 

50 0.009 8 0.01
 

0.01 70
 

0 31
 

2 (concentration X weeks received)
 

Z(weeks receiving chemical)
 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

5-NITRO-o-TOLUIDINE FEEDING EXPERIMENT
 

5-NITRO-o 

INITIAL TOLUIDINE OBSERVATION PERIOD TIME-WEIGHTED 

GROUP CONCENTRATION TREATED UNTREATED AVERAGE 

SIZE (PERCENT) (WEEKS) (WEEKS) CONCENTRATION3 

MALE 

CONTROL 50 0 0 95 0
 

LOW DOSE 49 0.005 18 0.12
 

0.15 60
 

0 19
 

HIGH DOSE 50 0.01 18 0.23
 

0.30 60
 

0 19
 

FEMALE
 

CONTROL 50 0 0 96 0
 

LOW DOSE 50 0.005 18 0.12
 

0.15 60
 

0 19
 

HIGH DOSE 50 0.01 18 0.23
 

0.30 60
 

0 20
 

am. . . . S (concentration X weeks received)
 

(weeks receiving chemical)
 

11
 
78-week period, during which untreated meal was available to controls.
 

The compound administration period was followed by an observation
 

period of 30 to 31 weeks.
 

At initiation of the study all mice were approximately 6 weeks
 

old. For the first 18 weeks dietary concentrations of 0.01 and 0.005
 

percent were administered. Throughout this report those mice initially receiving the former concentration are referred to as the high
 

dose groups and those initially receiving the latter concentration are
 

referred to as the low dose groups. Because of a lack of weight depression in the high and low dose mice, concentrations were increased
 

in week 19 to 0.3 and 0.15 percent, respectively, for the remainder
 

of the dosing period. After the 78-week period of compound administration, mice were observed for a period of up to 20 additional weeks.
 

Control mice were fed the basal laboratory diet for 95 to 96 weeks.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the experiment. Body weights were recorded twice weekly for the first
 

12 weeks of the study and at monthly intervals thereafter. Food
 

consumption, for two cages from each group, was monitored for seven
 

consecutive days once a month for the first nine months of the
 

bioassay and for three consecutive days each month thereafter. From
 

the first day, all animals were inspected twice daily for mortality.
 

The presence of tissue masses and lesions was determined by monthly
 

observation and palpation of each animal.
 

12
 
A necropsy was performed on each animal regardless of whether it
 

died, was killed when moribund, or was sacrificed at the end of the
 

bioassay. The animals were euthanized by carbon dioxide inhalation,
 

and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examination of major tissues, organs,
 

and gross lesions taken from sacrificed animals and, whenever possible,
 

from animals found dead.
 

Tissues were preserved in 10 percent buffered formalin, embedded
 

in paraffin, sectioned, and stained with hematoxylin and eosin prior
 

to microscopic examination. An occasional section was subjected to
 

special staining techniques for more definitive diagnosis.
 

Slides were prepared from the following tissues: skin, subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph
 

nodes, thymus, heart, salivary gland, liver, gallbladder (mice), pancreas, esophagus, stomach, small intestine, large intestine, kidney,
 

urinary bladder, pituitary, adrenal, thyroid, parathyroid, brain,
 

ear, testis, prostate, uterus, mammary gland, and ovary.
 

A few tissues were not examined for some animals, particularly
 

for those that died early. Also, some animals were missing, cannibalized, or judged to be in such an advanced state of autolysis as
 

to preclude histopathologic interpretation. Thus, the number of animals for which particular organs, tissues, or lesions were examined
 

microscopically varies and does not necessarily represent the number
 

of animals that were placed on experiment in each group.
 

13
 
H. Data Recording and Statistical Analyses
 

Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data System
 

(Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design, clinical observations, survival, body weight, and individual pathologic results,
 

as recommended by the International Union Against Cancer (Berenblum,
 

1969). Data tables were generated for verification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques described in this section. Those analyses of the experimental results
 

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

procedure of Kaplan and Meier (1958) and are presented in this report
 

in the form of graphs. Animals were statistically censored as of the
 

time that they died of other than natural causes or were found to be
 

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

on survival used the method of Cox (1972) when testing two groups for
 

equality and used Tarone's (1975) extensions of Cox's methods when
 

testing a dose-related trend. One-tailed P-values have been reported
 

for all tests except the departure from linearity test, which is only
 

reported when its two-tailed P-value is less than 0.05.
 

14
 
The incidence of neoplastic or nonneoplastic lesions has been
 

given as the ratio of the number of animals bearing such lesions at a
 

specific anatomic site (numerator) to the number of animals in which
 

that site was examined (denominator). In most instances, the denominators included only those animals for which that site was examined
 

histologically. However, when macroscopic examination was requited
 

to detect lesions prior to histologic sampling (e.g., skin or mammary
 

tumors), or when lesions could have appeared at multiple sites (e.g.,
 

lymphomas), the denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a significantly higher proportion of tumors than did the control animals.
 

As a part of these analyses, the one-tailed Fisher exact test (Cox,
 

1970, pp. 48-52) was used to compare the tumor incidence of a control
 

group to that of a group of treated animals at each dose level. When
 

results for a number of treated groups, k, are compared simultaneously
 

with those for a control group, a correction to ensure an overall
 

significance level of 0.05 may be made. The Bonferroni inequality
 

(Miller, 1966, pp. 6-10) requires that the P-value for any comparison
 

be less than or equal to 0.05/k. In cases where this correction was
 

used, it is discussed in the narrative section. It is not, however,
 

presented in the tables, where the Fisher exact P-values are shown.
 

15
 
The Cochran-Armitage test for linear trend in proportions, with
 

continuity correction (Armitage, 1971, pp. 362-365), was also used
 

when appropriate. Under the assumption of a linear trend, this test
 

determined if the slope of the dose-response curve is different from
 

zero at the one-tailed 0.05 level of significance. Unless otherwise
 

noted, the direction of the significant trend was a positive dose relationship. This method also provides a two-tailed test of departure
 

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

tumors. In this analysis, deaths that occurred before the first
 

tumor was observed were excluded by basing the statistical tests on
 

animals that survived at least 52 weeks, unless a tumor was found at
 

the anatomic site of interest before week 52. When such an early
 

tumor was found, comparisons were based exclusively on animals that
 

survived at least as long as the animal in which the first tumor was
 

found. Once this reduced set of data was obtained, the standard procedures for analyses of the incidence of tumors (Fisher exact tests,
 

Cochran-Armitage tests, etc.) were followed.
 

When appropriate, life-table methods were used to analyze the
 

incidence of tumors. Curves of the proportions surviving without an
 

observed tumor were computed as in Saffiotti et al. (1972). The week
 

during which animals died naturally or were sacrificed was entered as
 

the time point of tumor observation. Cox's methods of comparing
 

16
 
these curves were used for two groups; Tarone's extension to testing
 

for linear trend was used for three groups. The statistical tests for
 

the incidence of tumors which used life-table methods were one-tailed
 

and, unless otherwise noted, in the direction of a positive dose
 

relationship. Significant departures from linearity (P < 0.05, two-


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared to its control was calculated from
 

the exact interval on the odds ratio (Gart, 1971). The relative risk
 

is defined as p /p where p is the true binomial probability of the
 t c t
 

incidence of a specific type of tumor in a treated group of animals
 

and p is the true probability of the spontaneous incidence of the
 

c
 

same type of tumor in a control group. The hypothesis of equality
 

between the true proportion of a specific tumor in a treated group
 

and the proportion in a control group corresponds to a relative risk
 

of unity. Values in excess of unity represent the condition of a
 

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical analyses. The interpretation of the limits is that in approximately 95
 

percent of a large number of identical experiments, the true ratio
 

of the risk in a treated group of animals to that in a control group
 

would be within the interval calculated from the experiment. When
 

the lower limit of the confidence interval is greater than one, it
 

17
 
can be inferred that a statistically significant result (a P < 0.025
 

one-tailed test when the control incidence is not zero, P < 0.050
 

when the control incidence is zero) has occurred. When the lower
 

limit is less than unity but the upper limit is greater than unity,
 

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

of the induction of tumors by the test chemical which could not be
 

detected under the conditions of this test.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

Slight mean body weight depression was observed for high dose
 

male rats and for both high and low dose female rats when compared to
 

their respective controls (Figure 2).
 

Palpable subcutaneous masses occurred in two high dose females,
 

while one low dose female had a growth on its ear and a small crusted
 

lesion was observed on one control male. No other clinical abnormalities were observed.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 5-nitro-o-toluidine-dosed groups are shown in Figure 3. For male rats the Tarone test did not indicate a significant
 

positive association between dosage and mortality. The Cox tests did
 

not indicate a significant difference in survival between either dosed
 

group and the control group. For female rats the Tarone test did not
 

show a significant association between dosage and mortality.
 

Five male rats were sacrificed from each group in week 78.
 

Adequate numbers of males were at risk from late-developing tumors
 

with 56 percent (28/50) of the high dose, 54 percent (27/50) of the
 

low dose, and 66 percent (33/50) of the control rats alive on test
 

until the termination of the study. Five of the seven low dose rats
 

and three of the four high dose rats that died in weeks 2 through 4
 

had pneumonia.
 

19
 
750 750 

— 600 

— 450 

-300 

I-150 

TIME ON TEST (WEEKS) 

120 

750­ •750 

600 — -600 

tr 

2 

H 450­ X 

UJ 

g 

§300-

CQ 

-450 

-300 

150­ -150 

FEMALE RATS 

I 

15 

\ 

30 45 60 75 

TIME ON TEST (WEEKS) 

I 

90 105 120 

FIGURE 2 

GROWTH CURVES FOR 5-NITRO-o-TOLUIDINE

20 

 CHRONIC STUDY RATS 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIV

p


bo
 

I
 

CO
 

c
 

33 m
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33
 

O
 

O
 

33
 

1
 

o _,
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O
 

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C
 

D
 

33
 

H
 

o


b
 
Five female rats were sacrificed from each group in week 78.
 

With 66 percent (33/50) of the high dose, 56 percent (28/50) of the
 

low dose, and 68 percent (34/50) of the control rats alive on test
 

until the termination of the study, adequate numbers of females were
 

at risk from late-developing tumors.
 

C. Pathology
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A (Tables Al and A2); findings on nonneoplastic lesions are
 

summarized in Appendix C (Tables Cl and C2).
 

Possible compound-related findings were limited to the occurrence
 

of hepatocellular carcinomas in treated male rats. None of the 47
 

controls or 41 low dose animals developed the lesion; however, 3/46
 

(7 percent) of the high dose males had hepatocellular carcinomas. In
 

contrast, 5/47 (11 percent) control males had neoplastic nodules in
 

the liver as did 1/41 (2 percent) in the low dose and 1/46 (2 percent)
 

in the high dose animals. The morphological features of the hepatocellular carcinomas and neoplastic nodules were similar to those
 

described in the literature (Squire and Levitt, 1975). Neoplastic
 

nodules were small and compressed the adjacent parenchyma in areas.
 

Neoplastic cells were large and had acidophilic cytoplasm and hyper-


chromatic nuclei. A few mitotic figures were present. Hepatocellular
 

carcinomas were composed of liver plates which were several cells in
 

thickness. Pleomorphism in the size of neoplastic hepatocytes was
 

noted. Cytoplasm of the cells was acidophilic or vacuolated and
 

22
 
nuclei were large with prominent nucleoli. Mitotic figures were not
 

numerous.
 

Certain other neoplastic lesions occurred more frequently in the
 

controls than in treated animals. However, their incidence is considered to be within the normal range of variation for Fischer 344 rats.
 

The variety of degenerative and inflammatory lesions was similar
 

in control and treated rats and incidences of these lesions were
 

within the normal range of variation for Fischer 344 rats. These
 

nonneoplastic lesions were, therefore, not considered to be compound-


related.
 

In this bioassay of 5-nitro-o-toluidine, the incidence of hepatocelluar carcinomas was higher in the high dose males than in the
 

low dose or control groups. However, the numbers of animals with
 

this lesion were too small to permit the determination that the
 

effect was compound-related.
 

Under the conditions of this study, there was no conclusive
 

evidence to indicate that the dietary administration of 5-nitro-otoluidine was carcinogenic to Fischer 344 rats.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

rats are summarized in Tables 3 and 4. The analysis is included for
 

every type of tumor in either sex where at least two such tumors were
 

observed in at least one of the control or 5-nitro-o-toluidine-dosed
 

23
 
___ 

___ 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH 5-NITRO-o-TOLUIDINE*
 

HIGH
 

DOSE
 

1/48(0.02)
 

P = 0.027(N)
 

0.140
 

0.003
 

1.027
 

108
 

2/48(0.04)
 

N.S.
 

0.392
 

0.039
 

2.259
 

100
 

3/46(0.07)
 

N.S.
 

Infinite
 

0.616
 

Infinite
 

108
 

TOPOGRAPHY :MORPHOLOGY
 

Skin and Subcutaneous Tissue: Fibroma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

N> Hematopoietic System: Leukemia or
 

Malignant Lymphoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

7/47(0.15)
 

P = 0.013(N)
 

98
 

5/47(0.11)
 

N.S.
 

_—
 

84
 

0/47(0.00)
 

P = 0.039
 

LOW
 

DOSE
 

2/48(0.04)
 

N.S.
 

0.280
 

0.030
 

1.378
 

107
 

6/48(0.13)
 

N.S.
 

1.175
 

0.321
 

4.548
 

85
 

0/41(0.00)
 

_.__
 
___ 

___ 

TOPOGRAPHY rMORPHOLOGY
 

Liver: Hepatocellular Carcinoma or
 

Neoplastic Nodule^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma
 

S3 

Ui P Values0 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocy toma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

CONTROL
 

5/47(0.11)
 

N.S.
 

108
 

8/47(0.17)
 

N.S.
 

108
 

7/47(0.15)
 

N.S.
 

108
 

LOW
 

DOSE
 

1/41(0.02)
 

N.S.
 

0.229
 

0.005
 

1.931
 

107
 

2/36(0.06)
 

N.S.
 

0.326
 

0.035
 

1.507
 

78
 

1/41(0.02)
 

P = 0.045(N)
 

0.164
 

0.004
 

1.194
 

107
 

HIGH
 

DOSE
 

4/46(0.09)
 

N.S.
 

0.817
 

0.172
 

3.557
 

108
 

8/43(0.19)
 

N.S.
 

1.093
 

0.392
 

3.040
 

82
 

3/45(0.07)
 

N.S.
 

0.448
 

0.079
 

1.825
 

83
 
___ 

___ 

TABLE

TOPOGRAPHY :MORPHOLOGY
 

Thyroid: C-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma^*
 S3 

P Values0 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pancreatic Islets: Islet-Cell Adenoma or
 

Islet-Cell Carcinomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

3 (CONTINUED) 

CONTROL
 

2/46(0.04)
 

N.S.
 

108
 

4/46(0.09)
 

N.S.
 

108
 

4/45(0.09)
 

N.S.
 

——
 

85
 

LOW
 

DOSE
 

0/39(0.00)
 

N.S.
 

0.000
 

0.000
 

3.960
 

2/39(0.05)
 

N.S.
 

0.590
 

0.056
 

3.867
 

107
 

2/41(0.05)
 

N.S.
 

0.549
 

0.052
 

3.606
 

107
 

HIGH
 

DOSE
 

2/41(0.05)
 

N.S.
 

1.122
 

0.085
 

14.845
 

101
 

3/41(0.07)
 

N.S.
 

0.842
 

0.130
 

4.671
 

101
 

5/44(0.11)
 

N.S.
 

1.278
 

0.295
 

6.041
 

88 
TABLE 3 (CONCLUDED)
 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Testis: Interstitial-Cell Tumor 

P Values0 

44/47(0.94) 

N.S. 

34/41(0.83) 

N.S. 

38/46(0.83) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.886 

0.785 

1.054 

0.882 

0.788 

1.048 

Weeks to First Observed Tumor 78 78 78 

Treated groups received time-weighted average doses of 0.005 or 0.01 percent in feed.
 

Number of tumor-bearing animals/number of animals examined at site (proportion).
 Q
 

The probability level for the Cochran-Armitage test is given beneath the incidence of tumors in
 

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 
_ _ _ 

___ 

___ 

TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 5-NITRO-o-TOLUIDINEe
 

HIGH
 

DOSE
 

4/50(0.08)
 

N.S.
 

0.768
 

0.162
 

3.356
 

93
 

10/45(0.22)
 

N.S.
 

0.568
 

0.266
 

1.145
 

87
 

0/49(0.00)
 

N.S.
 

0.000
 

0.000
 

1.034
 

TOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

NJ Weeks to First Observed Tumor
 00
 

Pituitary: Adenoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Cortical Adenoma or Cortical
 

Carcinoma*5
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

5/48(0.10)
 

N.S.
 

96
 

18/46(0.39)
 

N.S.
 

94
 

4/47(0.09)
 

P = 0.025(N)
 

96
 

LOW
 

DOSE
 

5/48(0.10)
 

N.S.
 

1.000
 

0.246
 

4.070
 

78
 

14/40(0.35)
 

N.S.
 

0.894
 

0.476
 

1.637
 

78
 

1/44(0.02)
 

N.S.
 

0.267
 

0.006
 

2.561
 

108
 
___ 

___ 

TOPOGRAPHY :MORPHOLOGY
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma
 

N5
 P Values0
 

Q
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Uterus: Endometrial Stromal Polyp
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED)
 

CONTROL
 

3/46(0.07)
 

N.S.
 

109
 

14/48(0.29)
 

N.S.
 

P = 0.025
 

76
 

15/47(0.32)
 

N.S.
 

78
 

LOW
 

DOSE
 

2/38(0.05)
 

N.S.
 

0.807
 

0.070
 

6.662
 

108
 

5/48(0.10)
 

P = 0.019(N)
 

0.357
 

0.109
 

0.957
 

98
 

13/44(0.30)
 

N.S.
 

0.926
 

0.460
 

1.833
 

74
 

HIGH
 

DOSE
 

1/47(0.02)
 

N.S.
 

0.326
 

0.006
 

3.887
 

98
 

12/50(0.24)
 

N.S.
 

0.823
 

0.389
 

1.715
 

78
 

14/48(0.29)
 

N.S.
 

0.914
 

0.463
 

1.795
 

92
 
TABLE 4 (CONCLUDED)
 

Treated groups received time-weighted average doses of 0.005 or 0.01 percent in feed.
 

Number of tumor-bearing animals/number of animals examined at site (proportion).
 £
 

The probability level for the Cochran-Armitage test is given beneath the incidence of tumors in
 

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 

e

The probability level of the test for departure from linear trend is given beneath the control
 

group when P < 0.05.
 

u>
 

o
 
groups and where such tumors were observed in at least 5 percent of
 

the group.
 

For male rats the Cochran-Armitage test indicated a significant
 

(P = 0.039) positive association between compound administration and
 

the incidence of hepatocellular carcinomas. The Fisher exact tests,
 

however, were not significant.
 

Significant Cochran-Armitage test results in a negative direction
 

were observed for fibroma of the skin or subcutaneous tissue in males
 

(P = 0.013) and for the combined incidence of cortical adenomas or
 

cortical carcinomas of the adrenal gland in females (P = 0.025), but
 

Fisher exact tests were not significant under the Bonferroni criterion.
 

In females the Fisher exact test showed a significantly (P =
 

0.019) lower incidence of mammary fibroadenomas in the low dose than
 

in the control. The Cochran-Armitage test and the high dose to control comparison, however, were not significant.
 

No statistical tests for other sites were significant under the
 

Bonferroni criterion. Based upon these results, there was no convincing evidence that 5-nitro-o-toluidine was a carcinogen in male or
 

female rats.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

risk have been estimated and entered in the tables based upon the
 

observed tumor incidence rates. In many of the intervals shown in
 

Tables 3 and 4, the value one is included; this indicates the absence
 

31
 
of statistically significant results. It should also be noted that
 

many of the confidence intervals have an upper limit greater than one,
 

indicating the theoretical possibility of tumor induction in rats by
 

5-nitro-o-toluidine that could not be established under the conditions
 

of this test.
 

32
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Mean body weight depression, as compared to controls, was observed in both sexes of treated mice. The effect was more pronounced
 

among females than males (Figure 4).
 

No differences in clinical abnormalities were observed between
 

treated and untreated mice of either sex.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 5-nitro-o-toluidine-dosed groups are shown in Figure 5. For male mice the Tarone test did not indicate a positive association between dosage and mortality. For female mice the Tarone
 

test did not show a significant association between dosage and
 

mortality.
 

Five male mice were sacrificed from the high dose group in week
 

79 and five from the control group in week 78. Adequate numbers of
 

males were at risk from late-developing tumors with 76 percent
 

(38/50) of the high dose, 82 percent (41/50) of the low dose, and 86
 

percent (43/50) of the controls alive on test until the termination
 

of the study.
 

Five female mice were sacrificed from the high dose group in
 

week 79 and five from the control group in week 78. With 72 percent
 

(36/50) of the high dose, 84 percent (42/50) of the low dose, and
 

72 percent (36/50) of the control mice alive on test until the
 

33
 
50 

TIME ON TEST (WEEKS) 

T 

15 30 60 75 120 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR 5-NITRO-o-TOLUIDINE CHRONIC STUDY MICE 

34 

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from late-developing tumors.
 

C. Pathology
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B (Tables Bl and B2); findings on nonneoplastic lesions are
 

summarized in Appendix D (Tables Dl and D2).
 

Hepatocellular carcinoma was observed in 29/45 (64 percent) high
 

dose male, 12/44 (27 percent) low dose male, and 12/50 (24 percent)
 

control male mice. In females, the incidence was 20/45 (44 percent)
 

high dose, 7/46 (15 percent) low dose, and 2/47 (4 percent) control
 

mice. The first mouse that died with hepatocelluar carcinoma was a
 

high dose male in week 79. This was in contrast to week 97 in high
 

dose females, week 94 for control males, and week 94 for control females. The hepatocellular carcinomas were similar to those described
 

by Reuber (1975). No hepatic tumor was judged to be benign, and
 

hepatocellular carcinomas in two control animals had metastasized to
 

the lungs. Hepatocellular carcinomas involved a part or an entire
 

lobe of the liver. The lobular architecture was not preserved and
 

sinusoids in areas were distended. Some pleomorphism in the size of
 

neoplastic hepatocytes was evident. Cytoplasm was acidophilic; in
 

some it was vacuolated, suggesting fatty infiltration. Nuclei were
 

hyperchromatic. Mitotic figures were numerous.
 

The incidence of other types of tumors was sporadic in the
 

treated groups of both sexes.
 

36
 
Low incidences of hemangiomas and hemangiosarcomas (all sites)
 

were observed in dosed mice. The incidences of male mice having
 

either hemangiomas or hemangiosarcomas were 1/50 (2 percent) controls,
 

0/47 low dose, and 4/48 (8 percent) high dose. Among female mice, no
 

hemangiomas were observed, but the incidences of hemangiosarcomas
 

(all sites) were 1/48 (2 percent) controls, 5/47 (11 percent) low
 

dose and 3/47 (6 percent) high dose. These tumors are rarely seen in
 

untreated B6C3F1 mice.
 

There were numerous inflammatory and degenerative lesions noted.
 

None were judged to be related to the administration of 5-nitro-otoluidine.
 

This histopathologic evaluation provides evidence for the carcinogenicity of 5-nitro-o-toluidine as hepatocellular carcinomas were
 

induced in both male and female B6C3F1 mice. In addition, there may
 

be an association between compound administration and the induction
 

of hemangiomas and hemangiosarcomas.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

mice are summarized in Tables 5 and 6. The analysis is included for
 

every type of tumor in either sex where at least two such tumors were
 

observed in at least one of the control or 5-nitro-o-toluidine-dosed
 

groups and where such tumors were observed in at least 5 percent of
 

the group.
 

High incidences of hepatocellular carcinomas were observed
 

in mice of both sexes. In both sexes the Cochran-Armitage tests
 

37
 
_ _ _ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 5-NITRO-o-TOLUIDINEc
 

TOPOGRAPHY :MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Carcinoma
 

P Val»esc
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CO Lung: Alveolar/Bronchiolar Adenoma or
 00
 Alveolar/Bronchiolar Carcinoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Malignant
 

Lymphoma"
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

5/50(0.10)
 

P = 0.014(N)
 

95
 

5/50(0.10)
 

N.S.
 

95
 

5/50(0.10)
 

N.S.
 

P = 0.042
 

74
 

LOW
 

DOSE
 

1/45(0.02)
 

N.S.
 

0.222
 

0.005
 

1.881
 

96
 

3/45(0.07)
 

N.S.
 

0.667
 

0.109
 

3.219
 

96
 

7/47(0.15)
 

N.S.
 

1.489
 

0.438
 

5.551
 

96
 

HIGH
 

DOSE
 

0/46(0.00)
 

P = 0.035(N)
 

0.000
 

0.000
 

0.860
 

2/46(0.04)
 

N.S.
 

0.435
 

0.043
 

2.506
 

97
 

0/48(0.00)
 

P = 0.031(N)
 

0.000
 

0.000
 

0.825
 
TABLE 5 (CONCLUDED)
 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Liver: Hepatocellular Carcinoma 12/50(0.24) 12/44(0.27) 29/45(0.64) 

P Values 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

P < 0.001 ___ N.S. 

1.136 

0.521 

2.463 

P < 0.001 

2.685 

1.548 

4.775 

Weeks to First Observed Tumor 94 96 79 

Circulatory System:

Hemangioma" 

P Values0 

 Hemangio sarcoma or 

1/50(0.02) 

N.S. 

0/47(0.00) 

N.S. 

4/48(0.08) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.000 

0.000 

19.825 

4.167 

0.432 

200.683 

Weeks to First Observed Tumor 95 85 

'Treated groups received time-weighted average doses of 0.12 or 0.23 percent in feed.
 

Number of tumor-bearing animals/number of animals examined at site (proportion).
 

The probability level for the Cochran-Armitage test is given beneath the incidence of tumors in
 

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The probability level of the test for departure from linear trend is given beneath the control
 

group when P < 0.05.
 
___ 

___ 

TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH 5-NITRO-o-TOLUIDINE2
 

TOPOGRAPHY: MORPHOLOGY
 

Lung : Alveolar/Bronchiolar Adenoma or
 

Alveolar/Bronchiolar Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma°
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Circulatory System: Hemangio sarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW
 

CONTROL DOSE
 

2/46(0.04) 3/45(0.07)
 

N.S. N.S.
 

1.533
 

0.184
 

17.640
 

96 88
 

7/48(0.15) 5/47(0.11)
 

N.S. N.S.
 

0.729
 

0.196
 

2.475
 

83 97
 

1/48(0.02) 5/47(0.11)
 

N.S. N.S.
 

5.106
 

0.602
 

235.906
 

92 95
 

HIGH
 

DOSE
 

0/45(0.00)
 

N.S.
 

0.000
 

0.000
 

3.446
 

5/47(0.11)
 

N.S.
 

0.729
 

0.196
 

2.475
 

79
 

3/47(0.06)
 

N.S.
 

3.064
 

0.257
 

157.292
 

76
 
TABLE 6 (CONCLUDED)
 

LOW HIGH
 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE
 

Liver: Hepatocellular Carcinoma 2/47(0.04) 7/46(0.15) 20/45(0.44)
 

P Values P < 0.001 N.S. P < 0.001
 

Relative Risk (Control) 3.576 10.440
 

Lower Limit 0.727 2.776
 

Upper Limit 33.800 86.450
 

Weeks to First Observed Tumor 94 97 97
 

Pituitary: Adenoma NOS 5/43(0.12) 0/34(0.00) 0/39(0.00)
 

P Values0 P = O.OIO(N) P = 0.049(N) P = 0.035(N)
 

Relative Risk (Control) 0.000 0.000
 

Lower Limit 0.000 0.000
 

Upper Limit 0.989 0.867
 

Weeks to First Observed Tumor 95
 

Treated groups received time-weighted average doses of 0.12 or 0.23 percent in feed.
 

Number of tumor-bearing animals/number of animals examined at site (proportion).
 

"The probability level for the Cochran-Armitage test is given beneath the incidence of tumors in
 

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0 .05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 
indicated a significant (P < 0.001) positive association between compound administration and tumor incidence, supported by significant
 

(P < 0.001) results for the Fisher exact test comparing the tumor
 

incidence in the high dose treated group to that in the controls.
 

Based on these results, the administration of 5-nitro-o-toluidine was
 

associated with an elevated incidence of hepatocellular carcinomas in
 

both male and female mice.
 

Hemangiosarcomas and hemangiomas were noted in both male and female mice, but Fisher exact tests and Cochran-Armitage tests were not
 

significant. In historical data collected by this laboratory for the
 

NCI Carcinogenesis Testing Program, 5/350 (1.4 percent) untreated
 

male and 5/350 (1.4 percent) untreated female B6C3F1 mice had either
 

hemangiomas or hemangiosarcomas at all body sites. When a binomial
 

distribution with a probability of incidence of 5/350 was assumed,
 

the probability of observing 4 or more mice with such tumors out of
 

48 males (as in the high dose males) was P < 0.005, a significant
 

result. Similarly, the probability of observing 5 or more mice with
 

such tumors out of 47 females (as in the low dose females) was P <
 

0.001, a significant result.
 

For females the Cochran-Armitage test indicated a significant
 

(P = 0.010) negative association between dose and the incidence of
 

pituitary adenomas NOS. The Fisher exact test results, however, were
 

not significant under the Bonferroni criterion.
 

Similarly, a significant (P = 0.014) negative association was
 

observed for the incidence of alveolar/bronchiolar carcinomas in male
 

42
 
mice. Again, the Fisher exact test results were.not significant
 

under the Bonferroni criterion. No other statistical tests for any
 

site were significant under the Bonferroni criterion.
 

43
 
V. DISCUSSION
 

There were no significant positive associations between the concentration of 5-nitro-o-toluidine administered and mortality in rats
 

or mice of either sex, and adequate numbers of animals in all groups
 

survived sufficiently long to be at risk from late-developing tumors.
 

No unusual tumors were observed in rats of either sex and no
 

convincing statistical evidence was provided for a significant
 

positive association between compound administration and the incidence
 

of any neoplasm in rats.
 

In mice hepatocellular carcinomas were observed in 12/50 (24
 

percent), 12/44 (27 percent), and 29/45 (64 percent) of the control,
 

low dose, and high dose males, respectively, and in 2/47 (4 percent),
 

7/46 (15 percent), and 20/45 (44 percent) of the control, low dose,
 

and high dose females. There was a statistically significant positive
 

association between administration of the chemical and tumor incidence
 

in both males and females. The high dose to control Fisher exact comparisons supported the findings for both sexes.
 

The incidences of hemangiomas and hemangiosarcomas in dosed mice
 

were increased relative to controls. Among male mice, either hemangiomas or hemangiosarcomas were found in 1/50 (2 percent) control,
 

0/47 low dose, and 4/48 (8 percent) high dose animals. Among female
 

mice, no hemangiomas were observed, but the incidences of hemangiosarcomas were 1/48 (2 percent) in the control, 5/47 (11 percent) in
 

the low dose, and 3/47 (6 percent) in the high dose group. Although
 

44
 
the incidences in dosed groups were not statistically significant when
 

compared to the corresponding control groups, hemangiomas and hemangiosarcomas are rarely seen in untreated B6C3F1 mice. The incidences
 

in the high dose male mouse group and low dose female mouse group,
 

when considered in relation to historical data on untreated B6C3F1
 

mice at this laboratory (5/350 or 1.4 percent for each sex), are
 

sufficiently high to be considered to be related to administration of
 

the compound. When a binomial distribution and a spontaneous incidence rate corresponding to the appropriate historical control
 

incidence were assumed, the incidences of hemangiomas and hemangiosarcomas observed in this bioassay were significant.
 

There were no other tumors in mice for which a statistically
 

significant positive association was established between compound
 

administration and incidence.
 

Under the conditions of this bioassay 5-nitro-o-toluidine was
 

carcinogenic in B6C3F1 mice, causing hepatocellular carcinomas in
 

both sexes, causing an increase in the combined incidence of hemangiomas and hemangiosarcomas in male mice, and causing an increased
 

incidence of hemangiosarcomas in female mice. The compound was not
 

carcinogenic in Fischer 344 rats.
 

45
 
VI. BIBLIOGRAPHY
 

Anthony, H.M., and G.M. Thomas, "Tumors of the Urinary Bladder: An
 

Analysis of the Occupations of 1,030 Patients in Leeds, England."
 

Journal of the National Cancer Institute 45:879-895, 1970.
 

Armitage, P., Statistical Methods in Medical Research, Chapter 14.
 

J. Wiley & Sons, New York, 1971.
 

Berenblum, I., editor, Carcinogenicity Testing. International Union
 

Against Cancer, Technical Report Series, Vol. 2. International
 

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS) Ninth
 

Collective Index, Volumes 76-85, 1972-1976. American Chemical
 

Society, Washington, B.C., 1977.
 

Cox, D.R., Analysis of Binary Data, Chapters 4 and 5. Methuen and Co.,
 

Ltd., London, 1970.
 

Cox, D.R., "Regression Models and Life-Tables." Journal of the Royal
 

Statistical Society, Series "B" 34:187-220, 1972.
 

Gart, J.J., "The Comparison of Proportions: A Review of Significance
 

Tests, Confidence Limits, and Adjustments for Stratification."
 

International Statistical Institute Review 39:148-169, 1971.
 

Hamblin, D.O., "Aromatic Nitro and Amino Compounds." Industrial Hygiene and Toxicology, Second revised edition. Volume II: Toxicology. F.A. Patty, editor. pp. 2105-2169, 1967.
 

Kaplan, E.L., and P. Meier, "Nonparametric Estimation from Incomplete
 

Observations." Journal of the American Statistical Association
 

_5_3:457-481, 1958.
 

Linhart, M.S., J.A. Cooper, R.L. Martin, N.P. Page, and J.A. Peters,
 

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

Miller, R.G., Simultaneous Statistical Inference. McGraw-Hill Book
 

Co., New York, 1966.
 

Reuber, M.D., "Transplantation Studies." Pathology of Mouse Hepatic
 

Neoplasia, W.H. Butler and P.M. Newberne, editors. Elsevier
 

Scientific Publishing Company, Amsterdam, 1975.
 

46
 
Sadtler Standard Spectra. Sadtler Research Laboratories, Philadelphia, Pennsylvania. UV No. 3620.
 

Saffiotti, U., R. Montesano, A.R. Sellakumar, F. Cefis, and D.G.
 

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

by Different Numbers of Administration of Benzo (a) Pyrene and
 

Ferric Oxide." Cancer Research 32:1073-1079, 1972.
 

Schlopfer, L.A., "Naphthal Red and Brown Pigments." Pigment Handbook,
 

Volume 1. T.C. Patton, editor. Wiley-Interscience, New York,
 

1973. 

Society of Dyers and Colourists. Colour Index, 3rd edition, Volume 1. 

Yorkshire, England, 1971b. 

Society of Dyers and Colourists. Colour Index, 3rd edition, Volume 4. 

Yorkshire, England, 1971a. 

Squire, R.A. and M.H. Levitt, "Report of a Workshop on Classification
 

of Specific Hepatocellular Lesions in Rats." Cancer Research 35:
 

3214-3223, 1975.
 

Synalloy Corporation, Material Safety Data Sheet on Fast Scarlet G
 

Base. Synalloy Corporation, Blackman Uhler Chemical Division.
 

Spartanburg, South Carolina, 1977.
 

Tarone, R.E., "Tests for Trend in Life-Table Analysis." Biometrika
 

62^.679-682, 1975.
 

Urso, S., Research Analyst, Chemical-Environmental Program, Chemical
 

Industries Center, Stanford Research Institute, Menlo Park, California. Personal communication, 1977.
 

U.S.	 International Trade Commission, Imports of Benzenoid Chemicals
 

and Products, 1975. USITC Publication 806, U.S. Government
 

Printing Office, Washington, D.C., 1977b.
 

U.S.	 International Trade Commission, Synthetic Organic Chemicals, U.S.
 

Production and Sales, 1973. USITC Publication 718, U.S. Government Printing Office, Washington, D.C., 1975.
 

U.S.	 International Trade Commission, Synthetic Organic Chemicals, U.S.
 

Production and Sales, 1975. USITC Publication 804, U.S. Government Printing Office, Washington, D.C., 1977a.
 

Weast, R.C., editor, Handbook of Chemistry and Physics, 58th edition.
 

Chemical Rubber Company Press, Cleveland, Ohio, 1977.
 

47
 
Wynder, E.L., J. Onderdonk and N. Mantel, "An Epidemiological Investigation of Cancer of the Bladder." Cancer 16:1388-1407, 1963.
 

48
 
Review of the Bioassay of 5-Nitro-o-Toluidine* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

April 26, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academla, industry, organized labor, public interest
 

groups, State health officials, and quasi-public health and
 

research organizations. Members have been selected on the
 

basis of their experience in carcinogenesis or related
 

fields and, collectively, provide expertise in chemistry,
 biochemistry, biostatistics, toxicology, pathology, and
 

epidemiology. Representatives of various Governmental
 

agencies participate as ad hoc members. The Data Evaluation/
 

Risk Assessment Subgroup of the Clearinghouse is charged
 

with the responsibility of providing a peer review of
 

reports prepared on NCI-sponsored bioassays of chemicals
 

studied for carcinogenicity. It is in this context that the
 

below critique is given on the bioassay of 5-Nitro-o-
 Toluidine for carcinogenicity.
 

The primary reviewer agreed with the conclusion given
 

in the report, although his preference was to qualify
 

5-Nitro-o-Toluidine as an hepatocarcinogen. After a brief
 

description of the experimental design, he said that the
 

bioassay was valid within the experimental limitations of
 

the study. He noted, however, that the mice were exposed
 

at a concentration of about 100 times more than the rats.
 Since 5-Nitro-o-Toluidine is an azo dye intermediary, the
 

primary reviewer suggested that the report indicate
 

specifically that bladder tumors were not detected among
 

the treated animals.
 

The secondary reviewer agreed that 5-Nitro-o-Toluidine
 

was carcinogenic, based on the increased incidence of
 

hepatocellular cinomas in treated mice. He opined that
 

the bioassay was adequate on which to judge the carcino­ genicity of 5-Nitro-o-Toluidine. The secondary reviewer
 

49
 
noted that the statement in the report, regarding Anthony
 

and Thomas's work on bladder cancer in dye workers, may
 

be questionable and should be reexamined. He concluded
 

that 5-Nitro-o-Toluidine may be a potential human carcinogen.
 

A motion was approved unanimously that the report on
 

the bioassay of 5-Nitro-o-Toluidine be accepted as written.
 

Members present were:
 

Michael Shimkin (Acting Chairman), University of California
 

at San Diego
 

Joseph Highland, Environmental Defense Fund
 

George Roush, Jr., Monsanto Company
 

Louise Strong, University of Texas Health Sciences Center
 

John Weisburger, American Health Foundation
 

(David Clayson, Eppley Institute for Cancer Research,
 submitted a written review)
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

<tU.S. GOVERNMEN T PRINTIN G OFFICE : 1978-260-899/3148
 

50
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 5-NITRO-o-TOLUIDINE
 

TABLE A1

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH 5-NITRO-o-TOLUIDINE 

CONTROL (UN1R) LOU DOSE HIGH DOSE
 

01-0055 01-0053 01-0054
 

ANIMALS INITIALLY IN STDDY 50 50 50
 

ANIMALS MISSING 2 1
 

ANIMALS NECROPSIED HI 48 48
 

JNIMALS EXAMINED HISTOPATHOLOGICALLY** 47 46 47
 

INTEGDMENTAR Y SYSTEM 

*SKIN (47) (48) (48) 

SQUANOHS CEIL PAPILLOMA 1 (2%) 1 (2X) 1 (2%) 

BASAL-CELL CARCINOM A 1 (2%) 

SEBACEOUS ADENOCASCINOMA 1 <2X) 

FIBROMA 1 (2%) 

LIPOMA 1 (2*) 

LEIOMYOSA8COMA 1 (2*) 

*SDBCDT TISSUE (47) (48) («8) 

BASAL-CELL CARCINOM A 1 (2X) 

FIBROMA 7 (15X) 1 (2*) 1 (2X) 

RESPIRATOR Y SYSTEM 

tLUNG (47) (46) (47) 

ALVEOLAR/BHONCHIOLAR ADENOMA 1 (2*) 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2X) 1 (2%) 

CORTICAL CARCINOMA, METASTATIC 1 (2*) 

HEMATOPOIETIC SYSTEM
 

*MDLTIPLE ORGANS (47) C--3) (48) 

MALIGNANT LYMPHOMA, NOS 1 (2%) 

LEUKEMIA, NOS 1 (2%) 

ONDIFFERENTIATED LEDKEMIA 1 (2X) MYELOMONOCYTIC LEUKEMIA 1 (2X) 5 (10X) 1 (2%) 

tBONE MAHROH (46) (41) (45)

 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2S) 

tSPLEEN (47) (41) (46) 

____ MOCINgUS_AP|NOCARCINOjlAJ HETASTA 

* NUMBER OF ANIMALS KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
TABLE Al (CONTINUED)
 

CONTROL (DNTR) LOU DOSE HIGH DOSE
 01-0055 01-0053 01-0054
 

HALIG.LYMPHOMA, LYBPHOCYTIC TYPE 1 (2X)
 

•MEDIASTINAL L.NODE (42) (36) (14)
 

MUCINOOS ADENOCARCINOBA, METASTA 1(2%)
 

•PEYERS PATCH (47) (40) (45)
 

MALIG.LYHPHOMA, HISTIOCYTIC TYPE 1 (2X)
 

CIFCDLATOHY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

*ORAL CAVITY (47) (48) (48)
 FIBROSARCOMA 1 (2%)
 

•LIVER	 (47) (41) (46)
 

NEOPLASTIC NODULE 5 (mj 1 (2X) 1 (2X)
 HEPATOCELLULAR CARCINOMA 3 (7X)
 

•PANCREAS	 (45) (41) (44)
 

MUCINOUS ADENOCARCINOMA, METASTA 1 (2X)
 

•STOMACH	 (47) (40) (46)
 MUCINOUS ADENOCAECINOMA, METASTA 1 (2X)
 

•ILIUM	 (47) (40) (45)
 

LEIOHYOSASCOMA 1 (2X)
 

•COLON	 (46) (40) (43)
 

MDCINOUS ADENOCAECINOMA 1 (2«) 1 (2X)
 

URINARY SYSTEM
 

•KIDNEY	 (47) (41) (46)
 

LIPOBA 1 (2%)
 

ENDOCRINE SYSTEM
 

•PITUITARY	 (47) (36) («3)
 

* NDMB5R OF ANINALS SITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-4
 
TABLE A1 (CONTINUED) 

CONTROL (UNTH) 

01-0055 

LOW DOSE 

01-0053 

ADENOMA , NOS 8 (17*) 2 (6X) 

•ADRENA L 

CORTICAL CARCINOMA 

PHEOCHROHOCYTOMA 

(47) 

7 (15*) 

(41) 

1 (2%) 

tTHYROID 

ADENOCARCINOMA, NOS 

FOLLICIJLAR-CELL ADENOMA 

C-CELL ADENOM A 

C-CELL CARCINOMA 

(46) 

1

2

 (UK ) 

 (4*) 

(39) 

2 (5*) 

tPARATHYROI D 

ADINOMA , NOS 

(21) 

1 (It) 

(24) 

tPANCREATIC ISLETS 

ISLET-CELL ADENOMA 

ISLET-CELL CA2CINOM A 

(45) 

3

1

 (7*) 

12%} 

(41) 

2 (5*) 

REPRODUCTIVE SYSTEM 

*MAH11ARY GLAH D (47) (48) 

ADENOMA, NOS 1 (2«) 

FIBROADENOMA 1 (2%} 

*PRIPDTIAL GLAN D (47) (48) 

SQUAMOUS CELL CARCINOM A 1 (2*) 

ADENOMA , NOS 1 12%} 

*SEHINAL VESICLE (47) (48) 

MUCINOUS ADENOCARCINOMA, METAST A 1 (2%) 

tTESTIS (47) (41) 

INTERSTITIAL-CELL TUMOR 44 (94*) 34 (83%) 

NERVOOS SYSTEM 

tCEREBELLDM (45) (40) 

GLIOMA, NOS 

SPECIAL SENSE ORGAN S 

*EAR CANAL (47) (48) 

SOJAMOnS_CELL_CARCINOM4 

* NOMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOMBER OF ANIMALS NECROPSIED
 

A-5
 

HIGH DOSE 

01-0054 

8 (19X) 

(45) 

1 (2*) 

3 (7») 

(41) 

1 (2%} 

1 (2%) 

1 (2%) 

2 (5%) 

(23) 

(44) 

5 (1U) 

(48) 

1 (2%) 

(48) 

(48) 

(46) 

38 (83*) 

(45) 

1 (2*) 

(48)
 
TABLE Al (CONTINUED) 

CONTROL (trNTH)

01-0055

 LOB DOSE

 01-0053

 HIGH DOSE 

 01-0051 

MUSCULOSKELETAL SYSTEM 

*STERNUM

MUCINOUS ADENOCARCINOMA, METASTA

 (H7)

 1 (2X) 

 (t8) (<t8) 

BODY CAVITIES 

*BODY CAVITIES

MESOTHELIOMA, NCS

MESOTHELIOMA, BALISNANT

 (47)

 2 (1)i) 

 (18)

 1 (2*)

 (t8) 

1 (2X) 

BLL OTHER SYSTE8S 

NONE 

ANIMAL DISPOSITION SDMMASY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS) 

MORIBUND SJCEIPICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TEBHINAL SACRIFICE 

ANIMAL MISSING 

50 

2 

8 

5 

33 

2 

50 

9 

8 

5 

27 

1 

50 

11 

6 

5 

28 

9 INCLUDES AUTOLYZJD ANIHALS 

NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

NUMBER OF ANIMALS NECROPSIED 

A-6
 
TABLE Al (CONCLUDED)
 

CONTROL(DNTR) LOW DOSE HIGH DOSE
 

01-0055 01-0053 01-0051
 

TUMOR SUMMARY
 

TOTAL ANIMALS KITH PRIMARY TUMORS* 17 37 42
 TOTAL PRIMARY TUMORS 96 59 76
 

TOTAL ANIMALS WITH BENIGN TUMORS «7 3U KO
 TOTAL BENISN TUMORS 76 17 59
 

TOTAL ANIMALS WITH MALIGNANT TDMORS 11 10 15
 TOTAL MALIGNANT TDMOES 15 10 15
 

TOTAL ANIMALS WITH SECONDARY TDMORSt 1 1
 TOTAL SECONDARY TDMORS 6 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN O R MALIGNANT 5 2 2
 TOTAL UNCERTAIN TUMORS 5 2 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PBIMARY OR MBTASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: METASTATIC TDM08S OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-7
 
TABLE A2
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED WITH 5-NITRO-o-TOLUIDINE
 

CONTROL (ONTR) LOW DOSE HIGH DOSE
 

02-0055 02-0053 02-0054
 

ANIMALS INITIALLY III STUDY 50 50 50
 

ANIMALS MISSING 2
 

ANIMALS NECROPSIED 48 48 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 47 47 50
 

INTEGUMENTARY SYSTEM
 

*SKIN (48) (48) (50)
 

SQOAMODS CELL PAPILLOMA 1 (2«)
 

BASAL-CELL CARCINOMA 1 (2X)
 

FIBROMA 1 (2*)
 

FIBROSARCOMA 1 (2X) 2 (4%)
 

*SOBCUT TISSUE (48) (48) (50)
 

FIBROMA 1 (2X)
 

RESPIRATORY SYSTEM
 

*LDNG (47) (47) (50)
 

SQDAMOUS CELL CARCINOMA 1 (2X)
 

ALVEOLAH/BRONCHIOLAR ADENOMA 1 (2*) 2 (4«)
 

FIB80SARCOMA 1 (2X)
 

FIBROSARCOSA, METASTATIC 1 (251)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (48) (48) (50)
 MALIGNANT LYMPHOMA, NOS 2 (4X) 1 (2K)
 MYELOHONOCYTIC LEDKEMIA 3 (6%) 4 (8%) 3 (6X)
 

#SPLESN (47) (44) (49)
 

MYELOMONOCYTIC LEUKEMIA 1 (2*)
 

tTHYMUS (39) (25) (37)
 

THYMOMA 1 (3«)
 

CIRCULATORY SYSTEM
 

tHEART (47) (44) (50)

 

FIBROSARCOMA. METASTATIC,
 

* NUMBE R OF ANIMALS «ITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-8 
TABLE A2 (CONTINUED) 

HIGH DOSE
 

02-0054
 

(50)
 

1 (2X)
 

1 (2X)
 

(48)
 

(15)
 

1 (2*)
 

10 (22%)
 

(19)
 

1 (2X)
 

1 (2X)
 

(47).
 

1 (2X)
 

1 (2X)
 

(50)
 

3 <6X)
 

12 (24X)
 

(50)
 

(48)
 

DISESTIVE SYSTEM
 

fLIVBR
 

NEOPLASTIC NODDLE
 

HEPATOCELLOLAR CARCINOMA
 

PIBROSARCOSA, METASTATIC
 

URINARY SYSTEM
 

tKIDNEY
 

SQOAMOUS CELL CARCINOMA, METASTA
 

ENDOCRINE SYSTEM
 

#PITniTARY
 

CiBCINOHA.NOS
 

ADENOMA, NOS
 

#&DRENAL
 

CORTICAL ADENOMA
 

CORTICAL CARCINOMA
 

PHEOCHROHOCYTOMA
 

LIPOMA
 

GANGLIONEDROHA
 

*THYROID
 

C-CELL ADENOMA
 

C-CELL CARCINOMA
 

REPRODUCTIVE SYSTEM
 

"•MAMMARY GLAND
 

ADENOMA, NOS
 

ADENOCARCINOMA, NOS
 

PAPILLARY CYSTADENOMA, NOS
 

FIBROADENOMA
 

*VAGIN»
 

SARCOHA, NOS
 

tDTERUS
 

AMNOCARCIHOMA. NOS
 

t NUMBER OF ANIMAL S WITH TISSUE

* NUMBER OF ANIMALS NECROPSIED 

CONTROL (ONTR)
 

02-0055
 

(K7)
 

(17)
 

(»6)
 

2 (4«)
 

18 (39X)
 

<«7)
 

3 (6X)
 

1 (2X)
 

1 <2X)
 

(«6)
 

3 (7X)
 

(18)
 

1 (2X)
 

1 (2X)
 1« (29X)
 

C*8)
 

1 (2X)
 

(47)
 

_2 SH&L- _.
 

 EXAMINED MICROSCOPICALLY 

A-9 

LOU DOSE
 

02-0053
 

(«3)
 

1 (2%)
 

1 (2X)
 

(11)
 

1 (2X)
 

(10)
 

14 (35X)
 

(11)
 

1 (2X)
 

1 (2X)
 

(38)
 

1 (3X)
 

1 (3X)
 

(18)
 

1 (2X)
 

5 (10X)
 

(48)
 

(11)
 
TABLE A2 (CONTINUED) 

LEIOMYOSABCOMA


ENDOMETRIAL STROMAL POLYP


#UTERUS/!NDOMETRIDM


CARCINOMA, NOS


*07ARY


GRANULOSA-CELL CARCINOMA


NERVOUS SYSTEM
 

• BRAIN


ASTROCYTOMA


SPECIAL SENSE ORGANS
 

NONE


HDSCOLOSKELBTAL SYSIIM
 

*MUSCLE OF NECK


FIBROSARCOHA, HETASTATIC


BODY CSVITIHS
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

&NIMBL DISPOSITION SOHMARY
 

ANIMALS INITIALLY IN STODY
 NATURAL DBATH3


MORIBUND SACRIFICE


SCHEDULED SACRIFICE


ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIMAL MISSING


CONTROL (UNTR)


02-0055


 15 (32*)


 (17)


 (46)


 1 (2*)
 

(47)


 1 (2*)
 

(48)


 50
 3


 6
 

5
 

 34


 2
 

 LOW DOSE HIGH DOSE
 

 02-0053 02-0051
 

1 (2%)
 

 13 (30*) 11 (29*)
 

 (44)	 (48)
 

2 (4*)
 

 (44) (48)
 

 (»4) (48)
 

,
 

 (48)	 (50)
 

1 (2*)
 

 50 50
 11 4
 

6 8
 

5 5
 

 28 33
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-IO
 
TABLE A2 (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

02-0055 02-0053 02-0054
 

TOHOR SUMMARY
 

TOTAL ANIMALS WITH PRIBABY TDMOBS*
 TOTAL PBIMABY TDBORS


TOTAL ANIMALS KITH BENIGN TUMORS
 TOTAL BENIGN TDMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS
 TOTAL MALIGNANT TDMORS


TOTAL ANIMALS WITH SECONDARY TBMORSf
 TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TOSORS UNCERTAIN­ BENIGN OR MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR BETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

 42 33 35
 70 51 57
 

 38 30 28
 53 39 11
 

 11 11 13
 17 11 16
 

1 1
 

1 1
 

1
 

1
 

* PRIMARY TDBORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: METASTATIC TUMORS OR TOMOES INVASIVE INTO AN ADJACENT ORGAN
 

A-1
 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 5-NITRO-o-TOLUIDINE
 

TABLE Bl

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH 5-NITRO-o-TOLUIDINE 

CONTROL (ONTR)

05-0070

 LOH DOSE

 05-0053

 HIGH DOSE 

 05-0054 

ANIMALS INITIALLY IN STDDY 50

ANIMALS MISSING

ANIHALS NECROPSIED 50

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 50

 50

 1

 47

 16

 50 

1 

 18 

 18 

INTEGOMENTAEY SYSTEM 

*SOECDT TISSDB

SARCOMA, NOS

HEMANGIOSABCOMA

 (50) (47)

 1 (2X) 

 (18) 

3 (6X) 

RESPISATORY SYSTEM 

ILUNG

HEPATOCELLOLAR CARCINOMA, METAST

ALVEOLAH/BRONCHIOLAR ADENOMA

ALVEOLAB/BBONCHIOLAR CARCINOMA

 (50)

 1 (2*) 

5 (10*)

 (45)

 2 (4X)

 1 (2X) 

 (46) 

2 (4*) 

HEMATOPOIETIC SYSTEM 

*MaLTIPLE ORGANS

MALIGNANT LYMPHOMA, NOS

MALIG.LYMPHOMA, HISTIOCYTIC TYPE

 (50)

 2 (4X)

 1 (2%) 

 (47)

 U (9X) 

 (48) 

*LYMPH NODE

MALIG.LYMPHOMA, HISTIOCYTIC TYPE

 (45)

 2 (4X) 

 (34) (36) 

IMES1NTERIC L. NODE

MALIGNANT LYMPHOMA, NOS

 (45) (34)

 2 (6K) 

 (36) 

tPEYBRS PATCH

MALIGNANT LYMPHOMA, NOS

 (49) (43)

 1 (2X) 

 (44) 

CIRCULATORY SYSTEM 

NOJIE 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D

* NUMBE R OP ANIMAL S SECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

B-3 
TABLE Bl (CONTINUED) 

 HIGH DOSE 

 05-005K 

 (15) 

 29 (6411) 

 (15) 

 (15) 

1 (2X) 

 (10)
 

 (11) 

 (15)
 

1 (2*)
 

 (18)
 

DIGESTIVE SYSTEM 

ILIVEE

HEPATOCELLtlLAR CARCINOHA

H^HANGIOMA

UBINAB Y SYSTEM 

*KIDNEY

TDBOLAE-CELL ADENOCARCINOMA

*KIDNEY/CORTEX

TUBOLAR-CBLL ADENOMA

ENDOCEINE SYSTEM
 

#THYEOID


ADBHOCARCINOMA, NOS


• PANCREATIC ISLETS

ISLET-CELL ADENOM A

REPRODUCTIVE SYSTEM 

*TESTIS


HEMANGIOMA


NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*HARDEPIAN GLAND


ADENOMA, NOS


MDSCULOSKELETAL SYSTEM
 

NOJIJ
 

CONTROL (DNTE)

05-0070 LOS DOSE 05-0053

 (50) (11)

 12 (21)1) 12 (21%)

 1 (2X) 

 (19) (15)

 1 (2X) 

 (19) (15)

 (10) (38)

 1 (3X) 

 (16) (11)

 1 (2X) 

 (50) (15)

 (50) (17)

 1 (2X) 1 (2X) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS HECEOPSIED
 

B-4
 
TABLE Bl (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 05-0070 05-0053 05-005U
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

*MULTIPLE ORGANS (50) (47) (48)
 

NEOHOFIBEOSAECOHA 1 (2*)
 

ANIHAL DISPOSITION SUMMARY
 

ANIHALS INITIALLY IN STUDY 50 50 50
 

NATOS4L DEATHS 2 6 5
 

HOBIBONO SACRIFICE 1 1
 

SCHEDULED SACRIFICE 5 5
 ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 43 41 38
 

ANIMAL MISSING 1 1
 

3 INCLUDES AOTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TOMORS* 23 20 31
 

TOTAL PRIMARY TOMORS 27 25 36
 

TOTAL ANIMALS WITH BENIGN TUMORS 3 3 4
 

TOTAL BENIGN TDMORS 3 3 4
 

TOTAL ANIMALS WITH MALIGNANT TOMORS 22 19 30
 TOTAL MALIGNANT TOMOSS 2U 22 32
 

TOTAL ANIMALS WITH SECONDARY TUMORS! 1
 TOTAL SECONDARY TOMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENISN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR MSTASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMOBS: ALL TUMORS EXCEPT SECONDARY TOMORS
 * SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-5
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH 5-NITRO-o-TOLUIDINE 

CONTROL (UNTR) LOS DOSE HIGH DOSE 

06-0070 06-0053 06-0054 

ANIMAL S INITIALLY IN STUDY 50 50 50 

ANIMAL S HISSING 1 

ANIMAL S NECHOPSIED 48 47 117 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY** 47 (17 1*7 

INTEGUMENTAR Y SYSTEM 

*SDBCDT TISSUE (48) (17) (1(7) 

HEMANGIOSAHCOM A 2 (4X) 1 (2%) 

RESPIRATORY SYSTEM
 

*LONG (46) (45) (45)
 HFPATOCELIOLAR CARCINOMA, METAST 1 (2%)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 3 (7X)
 

ALVEOLA8/BHONCHIOLAR CARCINOMA 2 (4*)
 

OSTEOSAHCOMA, METASTATIC 1 12%)
 

HBMATOPOIETIC SYSTEM
 

*MDLTIPLE ORGANS (48) (47) (47)
 

MALIGNANT LYMPHOMA, NOS 2 (4X) 4 (9X) 4 <9X)
 MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2XJ 1 (2*)
 LYMPHOCYTIC LEUKEMIA 1 (2X)
 ERYTHROCYTIC LEUKEMIA 1 (2X)
 

#SPLEEN (47) (45) (45)
 

HEMANGIOSAHCOMA 1 (2X)
 MALIGNANT LYHPHOMA, NOS 1 (2X)
 

fMESENTERIC L. NODE (36) (31) (U2)
 

MALIG.LYMPHOM1, HISTIOCYTIC TYPE 1 (3X)
 

tPEYERS PATCH (45) (46) l«2)
 MALIGNANT LYMPHOMJ, NOS 1 (2X)
 

CIRCULATOBY SYSTEM
 

fHEABT/VESTRICLE (44) (46) (45)
 

HEMANSIOSARCOMA LJ2S1.
 

* NUMBER OF ANIMALS SITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOMBER OF ANIMALS NECROPSIED
 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

B-6
 
TABLE B2 (CONTINUED) 

CONTBOL(ONTR) LOW DOSE HIGH DOSE 

06-0070 06-0053 06-0054 

DIGESTIVE SYSTEM 

tLIVEB (47) (16) (45)
 

HEPATOCELLOLAB CARCINOMA 2 (1*) 7 (15X) 20 (44X)
 

DHINBEY SYSTEM
 

IKIDNEY (45) (46) (45)
 

HEMANGIOSABCONA 1 (2X)
 

ENDOCRINE SYSTEM
 

tPITUITARY (43) (34) (39)
 

ADENOMA, NOS 5 (12S)
 

*ADREN1L (47) (44) (42)
 

CORTICAL ADENOMA 1 (2X)
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND (48) (47) (47)
 

ADENOCARCINOMA, NOS 1 (2X)
 

tDTEROS (43) (44) (41)
 

LEIOMYOMA 1 (2X)
 

tOVARY/OVIDUCT (43) (44) (41)
 

PAPILLARY ADENOMA 1 (2X)
 

*OVARY (45) (45) (41)
 

TOBDLAR ADENOMA 1 (2%)
 

NSBVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*HASDE8IAS GL&ND (K8) (47) (47)
 

ADENOMix_fi2S
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

B-7
 
TABLE B2 (CONTINUED) 

CONTROL(ONTR) LOW DOSE HIGH DOSE 06-0070 06-0053 06-0054 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY
 

HEMANGIOSARCOHA 2 (4*) 

A LL OTHER SYSTEMS 

OMENTD M 

HEMftNGIOS&RCOMA
 

ANIMAL DISPOSITION SDMMABY
 

AKIMALS INITIALLY IN STUDY 50 50 50
 NATURAL DE4TH8 6
 

MORIBUND SACRIFICE 3
 SCHEDULED SACRIFICE 5
 

ACCIDENTALLY KILLED
 TERMINAL SACEIFICE 36 12 36
 ANIMAL MISSING 1
 

NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 NUMBER OF ANIMALS NECROPSIED
 

B-8
 
TABLE B2 (CONCLUDED)
 

CONTROL(ONTR) LOB DOSE HIGH DOSE
 06-0070 06-0053 06-005U
 

TOUCH SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TDBOFS
 TOTAL BENIGN TDMORS


TOTAL ANIMALS HITH MALIGNANT TUMORS
 TOTAL MALIGNANT TUMORS


TOTAL ANIMALS KITH SECONDARY TUMORS*
 TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCEFTAINBENIGN OS MALISNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS DNCERTAINPHIBARY OR METASTATIC
 

TOTAL UNCERTAIN TOBOBS
 

 18
 22


 8


 9
 

 12
 13


 2
 

2
 

 19 25
 21 29
 

3 1
 

3 1
 

 17 21
 18 28
 

* PRIMARY TUMORS: ALL TDMORS EXCEPT SECONDARY TDMORS
 * SECONDARY TUMORS: BETASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-9
 

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 5-NITRO-o-TOLUIDINE
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 5-NITRO-o-TOLUIDINE 

CONTBOL(ONTR) LOH DOSE 

01-0055 01-0053 

ANIMALS INITIALLY IN STDDY 50 50 

ANIMAL S MISSING 2 1 ANIMAL S NECROPSIED 17 18 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY ** 17 16 

INTEGUMENTARY SYSTEM
 

*SKIN (17) (18)
 

EPIDERMAL INCLUSION CYST 1 (2%)
 INFLAMMATION, NECROTIZING 1 (2X)
 INFLAMMATION, ACUTE FOCAL 1 (2*1
 FIBSOSIS, FOCAL 1 (2X)
 HYPEBPLASIA, PSEDDOEPITHELIOMATO 1 (2X)
 

HYPERPLASIA, BASAL CELL 1 (2X)
 

ACANTHOSIS 1 (2X)
 

RESPIRATORY SYSTEM
 

»TRACHEA (16) (11)
 

INFLAMMATION, NOS 1 (2X)
 INFLAMMATION, ACOTE/CHRONIC 19 (13X)
 

INFLAMMATION, CHRONIC
 

METAPLASIA, SQOAMODS 1 (2X)
 

tLUNG/BRONCHUS (17) (16)
 ERONCHIECTASIS 1 <9X)
 

INFLAMMATION, ACUTE FOCAL 1 (2X)
 

ABSCESS, NOS 1 12%)
 

INFLAMMATION, ACDTE/CHRONIC
 

METAPLASIA, SQUAMOOS 1 (2X)
 

*LUNG/BRONCHIOLE (17) (16)
 

HYPEBPLASIA, LYMPHOID 5
 

fLUNG (17) (16)
 

INFLAMMATION, FOCAL 3 (7X)
 

INFLAMMATION, INTERSTITIAL 2 (IX)
 

ABSCESS, NOS 1 (2S)
 

^INFLAMMATION, _ACDTEyCHRONIC
 

* NUMBE R OF ANIMALS SITH TISSOE EXAMINE D MICROSCOPICALLY 

* NDMBER OF ANIMALS NECHOPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3
 

HIGH DOSE
 

01-005U
 

50 

18 

17 

(18)
 

(16)
 

22 (18*)
 

(17)
 

3 (6X)
 

5 (11X)
 

(17)
 

(17)
 

2 (IX)
 

1 (2X)
 
TABLE Cl (CONTINUED) 

CONTROL (ONTR) LOW DOSB HIGH DOSE
 01-0055 01-0053 01-0054
 

PNEUMONIA, CHRONIC HUEINE 4 (9*) 3 (6X)
 

INFLAMMATION, CHRONIC 1 (2*) 1 (2%)
 

HYPERPLASIA, ADENOMATOOS 1 (2*)
 

HEMATOPOIETIC SYSTEM
 

*BONE HARROW (46) (41) (45)
 

MYELOFIBROSIS 1 (2X)
 

*SPLEEN (47) (41) (46)
 

FIBROSIS, FOCAL 1 (2X)
 

INFARCT HEHORRHAGIC 1 (2»)
 

ATROPHY, NOS 1 (2%) 1 (2»)
 

HYPEBPLASIA, LYHPHOID 1 (2%)
 HEMATOPOIESIS 1 (2%)
 

tTRACHEAL LYMPH NODE (42) (36) (44)
 

CONSESTION, NOS 1 (3X) 1 (2X)
 

tPANCREATIC L.NODE (42) (36) (44)
 

INFLAMMATION, ACOTE/CHRONIC 1 (2S)
 

»ILEOCOLIC LYMPH NODE (42) (36) (44)
 

LYMPHADENOPATHY 1 (2X)
 

CISCOLATORY SYSTEM
 

tMYOCARDIUM (47) (41) (46)
 

INFLAMMATION, INTERSTITIAL 1 (2«) 5 (11X)
 INFLAMMATION, ACDTE/CHRONIC 1 (2X) 3 (7*)
 

FIBROSIS 15 (37X) 2 (Ml!)
 

FIBROSIS, FOCAL 5 (12X) 8 (17X)
 

FIBROSIS, DIFFUSE 1 (2%) 6 (13X)
 

DEGENERATION, NOS 10 (21X)
 

*AOETIC TUNICA MEDIA (47) (48) (48)
 

MINERALIZATION 1 (2X)
 

*CORONAPY ARTERY (47) (48) (48)
 

fllNEBALIZATION . 1 (2X)
 INFLAMMATION, ACDTB/CHRONIC 1 (2%)
 

*POLMONABY ARTERY (47) (48) (48)
 

* NDMBER OF ANIMALS »ITH TISSOE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-4
 
TABLE Cl (CONTINUED) 

*MEDIASTINAL ARTERY 

INFLAMMATION , ACDTE/CHRONIC 

DIGESTIVE SYSTEM
 

tLIVER
 

CONGESTION, PASSIVE
 

CHOLANGIOPIBROSIS
 

DEGENERATION, HYALINE
 

DEGENERATION, EOSINOPHILIC
 

NECROSIS, FOCAL
 

NECROSIS, HEMORRHAGIC
 

METAMORPHOSIS FATTY
 

BASOPHILIC CYTO CHANGE
 

FOCAL CELLULAR CHANGE
 

HYPERPLASIA, NODDLAH
 

HYPERPLASIA, FOCAL
 

fLIVER/CENTRILOBOLAR
 

METAMORPHOSIS FATTY
 

*BILE DOCT
 

FIBROSIS
 

HYPERPLASIA, NOS
 

HYPERPLASIA, FOCAL
 

tPANCREAS
 

INFLAMMATION, FOCAL
 

INFLAMMATION, ACOTE/CHRONIC
 

PERIARTEHI1IS
 

tPANCSEATIC ACINDS
 

ATROPHY, NOS
 

ATROPHY, FOCAL
 

HYPEBPLASIA, NOS
 

fSTOSACH
 

PERIARTERITIS
 

#GASTRIC MDCOSA
 

NECROSIS, FOCAL
 

*COLON
 

CONTROL(ONTR) 

01-0055 

(47)
 

1 (2X)
 

1 (2*)
 

3 (6X)
 

12 (26%)
 

(1(7)
 

4 <9X)
 

(45)
 

1 (2%)
 

(45)
 

3 (7X)
 

1 <2X)
 

1 (2X)
 

(47)
 

(46)
 

* NOMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOMBER OF ANIMALS NECEOPSIED
 

C-5
 

LOW DOSE 

01-0053 

(148)
 

(11)
 

1 (2X)
 

5 (12*)
 

2 <5X)
 

1 (10X)
 

16 (39X)
 

1 (2*)
 

(11)
 

5 (12X)
 

(18)
 

1 <2X)
 

1 (2X)
 

(11)
 

2 (5X)
 

3 <7X)
 

(41)
 

(40)
 

(40)
 

<3X)
 

(40)
 

HIGH DOSE 

01-0051 

(KB)
 

1 (2X)
 

(46)
 

<2X)
 

3 (7X)
 

1 (2X)
 

1 (2X)
 

7 (15X)
 

20 (43X)
 

1 (2X)
 

1 (2X)
 

(46)
 

5 (11X)
 

(18)
 

1 (2X)
 

3 (6X)
 

(44)
 

4 (9X)
 

4 (9X)
 

1 (2X)
 

(44)
 

(46)
 

1 (2X)
 

(46)
 

(43)
 

1 12S1.
 
TABLE Cl (CONTINUED)
 

HIGH DOSE
 

01-0054
 

1 (2*)
 

(46)
 

35 (76X)
 

1 (2*)
 

(46)
 

(43)
 

(43)
 

2 (5*)
 

5 (12X)
 

(45)
 

2 (4X)
 

(45)
 

1 (2X)
 

2 (4X)
 

(45)
 

1 (2X)
 

1 (2X)
 

(41)


1 (2X)
 

1 (2X)
 

3 (7X)
 

PARASITISM
 

URINARY SYSTEM
 

tKIDNEY
 

CYST, NOS
 

GLOMIRULONEPHRITIS, NOS
 

GLOMEROLONEPHRITIS, FOCAL
 

GLOMEROLON5PHRITIS, SUBACDTE
 

INFLAMMATION, CHRONIC
 

tKIDNEY/TUBOLE
 

HEMOSIDEROSIS
 

IUEINARY BLADDER
 

HYPERPLASIS, EPITHELIAL
 

ENDOCRINE SYSTEM
 

JPITDITARY
 

HEMORRHAGE
 

HYPERPLASIA, FOCAL
 

*ADRENAL
 

METAMORPHOSIS FATTY
 

HYPERPLASIA, FOCAL
 

ANGIUCTASIS
 

»ADRENAL COHTBX
 

METAMORPHOSIS FATTY
 

HYPERPLASIA, FOCAL
 

•ADRENAL	 MEDULLA
 

CYST, NOS
 

HEMORRHAGIC CYST
 

HYPERPLASIA, NODULAR
 

HYPERPLASIA, FOCAL
 

tTHYROID
 

HYPERPLASIA, PAPILLARY
 

HYPERPLASIA, CYSTIC
 

HYPERPLASIA, C-CELL
 

METJiELA.S.,I.iJ,_SJ20AJ!0_nS
 

CONTROL (ONTR)
 

01-0055
 

(47)
 

1 (2X)
 

39 (83X)
 

(47)
 

(47)
 

1 (2X)
 

(47)
 

3 (6X)
 

(47)
 

1 <2X)
 

(47)
 

(47)
 

3 (6X)
 

(46)
 

4 (9X)
 

LOW DOSE
 

01-0053
 

3 (8X)
 

(41)
 

33	 (80X)
 

1 (2X)
 

(41)
 

2 (5X)
 

(40)
 

(36)
 

1 (3X)
 

(41)
 

3 (7X)
 

(41)
 

1 (2X)
 

(41)
 

1 (2X)
 

(39)
 

1 (3X)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-6
 
TABLE Cl (CONTINUED)
 

CONTROL (UNTR) LOW DOSE 01-0055 01-0053 

tPARATHYROID (21) (21) 

HYPERPLASIA, NOS 1 (IX)

 

tPANCBEATIC ISLETS (»5) (11) 

HYPEBPLASIi, FOCAL 1 (2X) 

REPRODUCTIVE SYSTEM 

*MAMMAR Y GLAN D («7) (18)

 

GALACTOCELE 1 (2X) 

HYPFBPLASIA, NOS 1 (2%)

 

HYPERPLASIA, FOCAL 

*PREPDTIAL GLAND (17) (18)

 

INFLAMMATION, ACUTE 1 (2%)

 

#PBOSTATE (16) (11) 

INFLAMMATION, ACDTE 7 (15X) 7 (17X) 

INFLAMMATION, ACUTE FOCAL 3 (7X) 3 (7X) 

ABSCESS, NOS 

INFLAMMATION , ACUTE/CHRONIC 2 (UX) 3 (7X) 

INFLAMMATION , CHBONIC FOCAL 1 (2X) 

ATROPHY, NOS 

*SEMINAL VESICLE (17) (18)

 

ATROPHY, NOS 

ITBSTIS (") (11)

DESENERATION, NOS 1 (2X) 31 (76X) 

HYPEBPLASIA, INTERSTITIAL CELL 1 (2X) 

mSTIS/TOBULE (17) (11)

 

DEGENERATION. DOS 1 (2X)

 

NERVOUS SYSTEM 

tBRAIN (15) (10)

 

CONGESTION, NOS 

SPECIAL SENSE ORGANS
 

__NOJE_
 

* NOMBER OF ANIMALS »ITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECSOPSIED
 

HIGH DOSE 01-0051 

(23) 

(11) 

(18) 

2 (UX) 

1 (2X) 

(18) 

(11)

5 (11X) 

10 (23X) 

1 (2X) 

2 (5X) 

1 (2X) 

(18) 

1 (2X) 

(16) 

31 (67*) 

(16) 

(15) 

1 (2X) 

C-7
 
TABLE Cl (CONCLUDED)
 

CONTROL(OKIE) LOB DOSE HIGH DOSE
 01-0055 01-0053 01-0051
 

MOSCCLOSKELETAL SYSTEM 

*STERNOM (47) (48) (18)
 

CSTEOSCLEROSIS 1 (2X)
 

BODY CAVITIES
 

*HESENTERY (17) (18) (48)
 

STEATITIS 1 (2*)
 

ALL OTHER SYSTEMS
 

*MOLTIPLE ORGANS (17) (18) (18)
 

POSTMORTEM CHANGE 3 (6X)
 

ADIPOSE TISSOE
 

INFLAMMATION, CHRONIC 1
 

SPECIAL MOHEHOLOGY SOMMAEY
 

ANIMAL MISSING/NO NECROPSY 2 1
 AOTO/NECHOPSY/NO HISTO 2 1
 ADTOLYSIS/NO NECROPSY 1 1 2
 

» NUMBER OF ANIMALS WITH TISSOE IXAMINED MICROSCOPICALLY
 * NOHBER OF ANIMALS NECROPSIED
 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WitH 5-NITRO-o-TOLUIDINE 

CONTROL (DNTB) LOW DOSE HIGH DOSE
 02-0055 02-0053 02-0054
 

ANIMALS INITIALLY IN STUDY 50 50 50
 ANIMALS HISSING 2
 ANIMALS NECHOPSIED 48 48 50
 ANIMALS EXAMINED HIS10PATHOLOGICALLY ** 17 47 50
 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

#TRACHEA (47) (45) (48)
 INFLAMMATION, ACUTE/CHRONIC 20 (44)1) 19 (40X)
 INFLAMMATION, CHRONIC 1 (2*)
 

METAPLASIA, SQDAMOOS 1 (2X)
 

#LUNG/BRONCHOS (47) (47) (50)
 BRONCHIECTASIS 5 (11*) 2 (4%)
 POLYP, INFLAMMATORY 1 (2%)
 

tLUNG/BRONCHIOLE (47) (47) (50)
 

HYPERPLASIA, PAPILLARY 1 (2*)
 

*LONG (47) (47) (50)
 CONGESTION, NOS 2 (4*)
 EDEMA, NOS 2 (4*) 1 (2%)
 INFLAMMATION, FOCAL 1 (2%) 3 (6%)
 INFLAMMATION, INTERSTITIAL 1 (2%) 1 (2X)
 ABSCESS, NOS 3 (6X)
 PNEUMONIA, CHRONIC MOHINE 2 (4S)
 

HYPERPLASIA, ALVEOLAR EPITHELIUM 1 (2*)
 

HEMATOPOIETIC SYSTEM
 

&SPLEEN (47) (44) (49) 

CONGESTION, NOS 1 (2X) 

HEMATOPOIESIS <2X) 1 (2X) 

tSUEMANDIBULAR L.NODE (40) (41) (46) 

HYPERPLASIA.NOS 2_I4S1 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS SECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-9 
TABLE C2 (CONTINUED)
 

" •
 

*THYMOS
 

CYST, NOS
 

CIBCULATOEY SYSIEH
 

tHEART
 

PERIARTEEITIS
 

*MYOCARDIOM
 

INFLAMMATION, INTERSTITIAL
 

INFLAHMATION, ACUTE/CHRONIC
 FIBROSIS
 

FIBROSIS, FOCAL
 FIBEOSIS, DIFFOSE
 

DEGENERATION, NOS
 

tENEOCAEDIOM
 

INFLAMMATION, FOCAL
 INFLAMMATION, ACUTE/CHBONIC
 

tCAHDIAC VALVE
 

INFLAMMATION, ACUTE/CHRONIC
 

*»ORTA
 INFLAMHATION, ACUTE/CHRONIC
 

*COHONAEY ARTERY
 

INFLAMMATION, ACUTE/CHRONIC
 

*POLMONARY ASTEEY
 MINERALIZATION
 

DIGESTIVE SYSTEM
 

*PAEOTID GLAND
 

HYPEEPLASIA, FOCAL
 

ISUEMAXILLARY GLAND
 INFLAMMATION, ACUTE/CHRONIC
 

tLIVER
 __ BEGENEBATION^NQS . .. .
 

COBTHOL(ONTH)
 

02-0055
 

(39)
 

(17)
 

(U7)
 

7 (15*)
 

(«7)
 

(47)
 

(US)
 

(»8)
 

(48)
 

(16)
 

(16)
 

(17)
 

1 JC251
 

LOU DOSE HIGH DOSE
 02-0053 02-0054
 

(25)	 (37)
 

1 <3X)
 

(»1) (50)
 

1 (2X)
 

(11) (50)
 

1 (2X)
 

6 (12X)
 

2 (5X) 6 (12X)
 

3 (7*) 2 (4X)
 

3 (7«)
 

(11) (50)
 

1 (2X)
 

1 (2*)
 

(11) (50)
 

1 <2X)
 

(18)	 (50)
 

1 (2X)
 

(48)	 (50)
 

1 (2X)
 

(48)	 (50)
 

1 (2X) 3 (6X)
 

(13)	 (48)
 

1 (2X)
 

(43)	 (48)
 

1 (2X)
 

(43) (50)
 

I	 NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NOMBES OF ANIMALS NECEOPSIED
 

c-io
 
TABLE C2 (CONTINUED)
 

CONTROL (DNTB)

02-0055 LOW DOSE 02-0053 

NECROSIS, FOCAL NECROSIS, DIFFUSE NtCHOSIS, HEMORBHAGIC METAMORPHOSIS FATTY 2 (411)

2 

1 

1 

8 

(5%) 

(2X> 

<2X) 

(19X) 

BASOPHILIC CYTO CHANGE 30 (70S) 

FOCAL CELLULAR CHANGE 25 (53X) 

ANGIECTASIS 

tLIVER/CENTRILOBOLAR (47) (43) 

METAMORPHOSIS FATTY 1 (2X) 

*BILE DOCT (48) (48) 

HYPERPLASIA, NOS 2 (4X) 

tPANCBEAS (46) (44) 

INFLAMMATION, FOCAL 

INFLAMMATION, ACDTE/CHRONIC 6 (14X) 

ATROPHY, FOCAL 

HYPERPLASIA, NOS 

2 

1 

(5X) 

(2%) 

fPANCREATIC ACINDS (44) 

ATBOPHY, NOS 8 (17X) 

tSTOBACH (46) (44) 

OLCER, FOCAL 1 (2X) 

•COLON (45) 

SEBATODIASIS 6 (15X) 

HYPEBPLASIA, LYHFHOID 

URINARY SYSTEH
 

IKIDNEY (U7) (41)
 

GLOBEEOLONEPHEITIS, NOS 15 (37X)
 

GLOMEROLONEPHRITIS, FOCAL 3 (7X)
 

INFLAHHATION, CHRONIC 29 (62X)
 

POS5PHORTEM CHANGE 1 (2X)
 

ENDOCRINE SYSIIM
 

tPITDITARY (46) (40)
 

CYST, NOS
 BIPERPLASIA. FOCAL, 2 <5%J_
 

* NUMBER OF ANIMALS SITH TISSUE EXAMINED MICROSCOPICALLY
 

* NDHBER OF ANIMALS NECBOPSIED
 

C-l
 

HIGH DOSE
 02-0054
 

7 (14%)
 

43 (86*)
 

1 (2X)
 

(50)
 

(50)
 

3 (6X)
 

(47)
 

5 (11%)
 

3 (6*)
 

(47)
 

(46)
 

(45)
 

7 (16X)
 

2 (4%)
 

(48)
 

25	 (52X)
 

2 (4X)
 

(45)
 

1 (2X)
 
TABLE C2 (CONTINUED) 

CONTBOL(UNTR) l LOU DOSE
 02-0055 02-0053
 

•ADRENAL	 (47)
 HEMORRHAGIC CYST 1 (2X)
 

•ADRENAL CORTEX (47) (44)
 CYST, NOS
 

DEGENERATION, NOS 3 (6X)
 METAMORPHOSIS FATTY 1 (2X) 5 (11X)
 HYPERPLASIA, NODULAR 2 (4X)
 HYPERPLASIA, FOCAL 3 (6X) 1 (2X)
 ANGIECTASIS 5 (11X)
 

•ADRENAL MEDULLA	 (47) (44)
 THROMBOSIS, NOS 1 (2X)
 HYPEBPLASIA, FOCAL 1 (2X)
 

•THYROID	 (46) (38)
 INFLAMMATION, ACUTE/CHRONIC 1 (3X)
 HYPERPLASIA, C-CELL 4 (9X) 1 (3X)
 

•PANCREATIC ISLETS (46) (44)
 HYPEBPLASIA, NOS 1 (2X)
 HYPERPLASIA, FOCAL
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND (48) (48)
 

DILATATION/DUCTS 3 (6X)
 GALACTOCEtE 7 (15X) 10 (21X)
 INFLAMMATION, ACUTE
 

HYPSRPLASIA, NOS 4 (8X) 18 (38X)
 HYPERPLASIA, FOCAL 1 (2X)
 

*NAHMARY DUCT (48) (48)
 FIBHOSIS
 

•UTERUS	 (47) (44)
 HYDEOMETRA 2 (4X)
 

• UTIEDS/ENDOMETRIUM (47) (44)
 INFLAMMATION, VESICULAR
 

INFLAMMATION, ACUTE 9 (19X) 24 (55X)
 INFLAMMATION, ACUTE VESICULAR
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NDMBEH OF ANIMALS NECROPSIED 

C-I2 

HIGH DOSE
 

02-0054
 

<«.
 

(49)
 

4 (8X)
 

4 (8X)
 

(49)
 

(47)
 

1 (2X)
 

(47)
 

1 (2X)
 

(50)
 

20 (40X)
 

1 (2X)
 

22 (44X)
 

(50)
 

(48)
 

(48)
 

1 <2X)
 

24	 (SOX)
 

1 (2X)
 
TABLE C2 (CONTINUED) 

CONTROL (BUTE) LOS DOSE HIGH DOSE
 

02-0055 02-0053 02-0054
 

HYPEBPLASIA, HOS 2 (5X) 5 (10X)
 

HYPEHPLASIA, CYSTIC 3 (6X) 6 (11*) 6 (13X)
 

HYPEBPLASIA, STROMAL 1 (2X)
 

*OVARY/OVIDOCT (44) (48)
 

INFLAMMATION, ACOTE 3 (6*) 1 (2*) 3 (6X)
 

INFLAMMATION ACTIVE CHRONIC 1 (21)
 

INFLAMMATION, ACOTE/CHRONIC 1 (2%)
 

INFLAMMATION, CHBONIC 1 <2X)
 

fOVAEY/PAROVARIAN (47) (44) (48)
 

ABSCESS, NOS 1 (2X)
 

#OVARY (16) (41) (18)
 CYST, NOS 1 (2X)
 

ABSCESS, NOS 1 (2X)
 INFLAMMATION, CHRONIC 1 (2X)
 

*0 VARY/FOLLICLE (16) (44, (48)
 

HYPERPLASIA, NOS 1 (2X)
 

NERVOUS SYSTEM
 

tBRAIN (47) (K8)
 

NECROSIS, HEBORBHAGIC 1 (2*)
 

SPECIAL SENSE ORGANS
 

*EYE/CRYSTALLINE LENS (U8) (50)
 

CATARACT 1 (2X)
 

MOSCULOSKELETAL SYSTEM
 

*BONE (U8) (K8) (50)
 

CSTEOSCLEROSIS
 

BODY CAVITIES
 

*PLEURA (48) (48) (50)
 

INFLAMMATION, CHRONIC 1 (2X)
 

*EPICARDIUM (18) (48) (50)
 

INFLAMMATION. CHBONIC
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED 8ICROSCOPICALLT
 * NUMBER OF ANIMALS NECROPSIED
 

C-I3
 
TABLE C2 (CONCLUDED) 

CONTROL (ONTR) LOU DOSE HIGH DOSE 

02-0055 02-0053 02-005<t 

*MESENTERY (48) (50)
 

STf&TITIS 1 (2*)
 

NECROSIS, PAT 1 (2%)
 

ALL OTHEE SYSTEMS
 

*MULTIPLE ORGANS (K8) (18) (50)
 

POSTMORTEM CHANGE 3 (6«) 3 (6*)
 

SPECIAL MORPHOLOGY SDBMARY
 

ANIMAL HISSING/NO HECFOPSY
 

SUTO/N15CROPSY/NO HISTO
 

ADTOLYSIS/NO NECROPSY
 

* NDHBER OF ANIMALS SITH TISSDE FXAMINED MICROSCOPICALLY
 

* NOMBER OP ANIMALS NECROPSIED
 

C-14
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 5-NITRO-o-TOLUIDlNE
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 5-NITRO-o-TOLUIDINE 

c:ONTROL(UNTR) LOS DOSE HIGH EOSE
 05-0070 05-0053 05-00511
 

ANIMALS INITIALLY IN STUDY 50 50 50
 ANIMALS MISSING 1 1
 ANIMALS NECBOPSIED 50 in us
 ANIMALS EXAMINED HISZOPATHOLOGIC ALLY ** 50 16 48
 

INTEGOMENTABY SYSTEM
 

*SKIN (50) (17) (K8)
 

ABSCESS, NOS 2 (IX)
 

*SUBCOT TISSUE (50) (t7) («8)
 ABSCESS, NOS 1 (2*)
 NECROSIS, FAT 1 (2%)
 

NECROSIS, HEMOHRHAGIC 1 (2%)
 

RESPIRATORY SYSTEM
 

#LDNG/BRONCHIOLE (50) (U5) (K6)
 

INFLAMMATION, NOS 1 (2X)
 PERI7ASCULITIS 1 (2%)
 

tLONG (50) (15) (K6)
 

HEMORRHAGE 2 (IX)
 HYPEHPLASIA, ALVEOLAR EPITHELIUM 2 (l»X)
 

HEBATOPOIETIC SYSTEM
 

#SPLEEN (50) (H3) (H5)
 HYPERPLASIA, NOS 1 (2%)
 

HYPERPLASIA. LYBPHOID 1 (2X)
 HEMATOPOIESIS 2 (4X)
 ERYTHROPOIESIS 3 (7S) 1 (2X)
 

ISPLENIC FOLLICLES (50) (13) (15)
 HYPERPLASIA, NOS 2 (IX)
 

•LYMPH NODE (15) (31) (36)
 _ _ HXPEHPLASIAf NOS 1 (3X)
 

* NUMBER OF ANIMAL S illTH TISSUE EXAMINE D MICROSCOPICALLY * NUMBER OF ANIMAL S NECROPSIED 

** EXCLUDES PARTIALLY ATJTOLYZED ANIMALS 

D-3 
IMESENTERIC L. NODE
 

HYPEBPLASIA, RETICOLUM CELL
 

CIBCOLATORY SYSTEM
 

#AORTIC VALVE
 

INFLAMMATION, ACDTE/CHRONIC
 

DIGESTIVP SYSTEM
 

tSALIVASY GLAND
 

PESIVASCDLITIS
 

*LIVER
 

HEPATITIS, TOXIC
 

NECROSIS, NOS
 

NECROSIS, FOCAL
 

METAMORPHOSIS FATTY
 

H^PATOCYTOMEGALY
 

MEGALOCYTOSIS
 

DEPLETION
 

HYPEEPLASIA, NODOLAR
 

HYPEBPLASTIC NODULE
 

HYPEBPLASIA, FOCAL
 

HYPEEPLASIA, DIFFUSE
 

HEMATOPOIESIS
 

tLIVER/CENTRILOBULAR
 

NECEOSIS, NOS
 

*LIVEP/KUPFFER CELL
 

HYPERPLASIA, NOS
 

*BILE DUCT
 

CYST, NOS
 

HYPEBPLASIA, NOS
 

*PANCREAS
 

INFLAMMATION, FOCAL
 

INFLAMMATION, INTERSTITIAL
 

#3ASTRIC MDCOSA
 

INFLSMMATjait FOCli
 

TABLE Dl (CONTINUED)
 

CONTBOL(UNTB) 

05-0070 

(45)
 

1 (2%)
 

1 (2%)
 

(49)
 

1 12%)
 

(50)
 

1	 <2X>
 

2	 («%)
 2	 (4X)
 

1	 (2*)
 

2	 (4X)
 

1 (2%)
 

1	 (2X)
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

(46)
 

1 (2%)
 

(49)
 

J I2*l_ _
 

LOW DOSE HIGH DOSE
 05-0053 05-0054
 

(30)	 (36)
 

(45)	 (45)
 

(42)	 (45)
 

(44)	 (45)
 

1 (2X)
 

1 (2%)
 

1 (2X)
 

1 (2%)
 

1	 (2X)
 

2	 (4X)
 

(44)	 (45)
 

(44)	 (45)
 

(47)	 (48)
 

1	 (2X)
 

1 (2X)
 

(44)	 (41)
 

1 (2X) 1 <2X)
 

(42)	 (44)
 

#	 NDMBER OF ANIMALS BITH TISSDE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECEOPSIED
 

D-4
 
fPEYERS PATCH


HYPE8PLASIA, DOS


*COLON


GRANULOHA, NOS


UBINAFY SYSTEM
 

IKIDNEY
 HYDBONEPHROSIS


GLOMEBOIONEPHRITIS, NOS


INFLAMMATION, IN1ERSTITIAL


*ORINAEY BLADDEB


HYPEBPLASIA, EPITHELIAL


ENDOCRINE SYSTEM
 

NOS!
 

BEPBODOCTIVE SYSTEM
 

*PRBPDTIAL GLAND


ABSCESS, NOS


NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCDLOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY


TABLED! (CONTINUED) 

CONTROL (UNTS)
 05-0070


 (49)


 1 (2X)


 (46)


 1 (2%)
 

(49)


 3 (6X)
 

(47)


 1 (2«)
 

(50)


 (50)


 LOU DOSE HIGH DOSE
 05-0053 05-0054
 

 (13)	 (44)
 

1 (2») 1 (2X)
 

 (39) (43)
 

 (45)	 (45)
 

1 (2X)
 

1 (2*)
 

 (41) (42)
 

 (47)	 (48)
 

1 (2X)
 

 (47) (48)
 

t	 NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 *	 NUMBER OF ANIMALS NECROPSIED
 

D-5
 
TABLE Dl (CONCLUDED)
 

CONTROL (ONTE) LOU DOSE HIGH DOSS

 

05-0070 05-0053 05-0054

 

*MESENTEBY (50) (147) (48)
 CONGESTION, NOS 1 (2%)
 

HEMORRHAGE 1 (2*)
 

AIL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SOMHABY
 

SO LESION REPORTED 12 19 10
 ANIMAL HISSING/NO NECROPSY 1 1
 

AOTO/NECROPSY/HISTO PEHF 1 2
 

AOTO/NECROPSY/NO HISTO 1
 

AtJTOLYSIS/NO NECROPSY 1
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NOMBER OF ANIMALS NECROPSIED
 

D-6
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 5-NITRO-o-TOLUIDINE

 

HIGH DOSE
 06-0054
 

50
 

47
 

47
 

(45)
 

(45)
 

(44)
 

(45)
 

1 (2X)
 

1 (2X)
 2 (4X)
 

(45)
 

(42)
 

(42)
 

ANIMALS INITIALLY III STDDY


ANIMALS HISSING
 

ANIHALS NBCBOPSIED


»NIHALS EXAMINED HISTOPATHOLOGICALLY**


INTEGUMENTARY SYSTBB
 

NONE
 

RESPIRATORY SYSTEM
 

*LUNG/BRONCH10LE
 

INFLAMMATION, NOS
 

*LDNG
 

INFLAMMATION, INTERSTITIAL
 

HEMATOPOIETIC SYSTEM
 

*BONE MARROW
 

MYELOFIBROSIS
 

ISPLEEN
 

ANGIECTASIS
 HYPEBPLASIA, LYMPHOID
 HEHATOPOIESIS
 ERYTHHOPOIESIS
 

tSPLENIC FOLLICLES
 HYPERPLASIA, NOS
 

*LYMPH NODE
 INFLAMMATION, NOS
 

HYPERPLASIA, NOS
 HYPERPLASIA, PLASMA CELL
 

IABDOMINAL LYMPH NODE
 PLJSMACYTOSIS
 

CONTROL (D NTP)
 

06-0070
 

 50
 

 48
 

 47
 

(46)
 

1 (2X)
 

(46)
 

1 (2X)
 

(46)
 

1 (2X)
 

(47)
 

1 (2X)
 

(47)
 

3 (6X)
 

(36)
 

1 (3X)
 

1 <3X)
 

1 (3X)
 

(36)
 

1 (3X1
 

LOW DOSE 

06-0053 

50
 

1
 

47
 

47
 

(45)
 

(45)
 

(45)
 

(45)
 

1 (2X)
 

2 (4X)
 

(45)
 

(31)
 

(31)
 

I NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NDMBER OF ANIMALS SECROPSIED
 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

D-7
 
TABLE D2 (CONTINUED)
 

CONTROL (UNTR) LOS DOSE HIGH DOSE
 06-0070 06-0053 06-0054
 

CIRCULATORY SYSTEM
 

IHEART/ATFIUM (14) (16) (45)
 

THROHBOSIS, NOS 1 (2%)
 

IHYOCARDIUM (44) (46) (45)
 

INFLAMMATION, ACUTE FOCAL 1 (2*)
 

FIBROSIS. FOCAL 1(2%)
 

#ENDOCARDIUM (44) (46) (45)
 

INFLAMMATION, CHRONIC 1 <2X)
 

#AORTIC VALVE (44) (46) (45)
 

INFLAMMATION, NOS 1 <2X)
 

DIGESTIVE SYSTHH
 

tSALIVAEY GLAND (45) (45) (42)
 

PERIVASCULITIS 3 (7!t)
 

PESIVASCOLAH CUFFING 1(2%)
 

OLIVER (47) (46) (45)
 

MULTIPLE CYSTS 1 (2*)
 

INFLAMMATION, ACOTE FOCAL 1 (2)6)
 

INFLAMMATION, ACUTE/CHRONIC 1 (2*)
 

NECROSIS, FOCAL 2 (4*)
 

NECROSIS, COAGOLATIVE 1(2%)
 

METAMORPHOSIS FATTY 1 (2%) 2 (4X)
 

HYPERPLASTIC NODULE 1 (2Jf) 2 (4X)
 

HYPERPLASIA, FOCAL 1 (2%) 1 (2X)
 

*LIVF.R/CAUDATE LOBE (47) (46) (45)
 

HEMORRHAGE 1(2%)
 

*BILE DUCT (48) (47) (47)
 

INFLAMMATION, ACUTE/CHRONIC 4 (8!8)
 

tPANCREAS (43) (44) (41)
 

H8M08RHAGIC CYST 1(2%)
 

INFLAMMATION, NOS 1 (2%)
 

INFLAMMATION, INTERSTITIAL 1 (2*)
 

PERIARTERITIS 1 (2«)
 

tPANCREATIC ACINUS (43) (44) (41) 

ATROPHYx_NflS 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-8
 
TABLE D2 (CONTINUED) 

COHTROL(USTR) LOU DOSE HIGH DOSE
 06-0070 06-0053 06-0054
 

#STOMACH (45) (<t2)
 

ULCER, FOCAL 1 (2X)
 

*PEYERS PATCH (15) (42)
 

HYPEHPLASIA, NOS 1 (2X)
 

URINARY SYSTEM
 

fKIDHEY (45) (46) (45)
 

HYDROSEPHROSIS 1 <2X)
 GLOHEROLONEPHRITIS, HOS 3 <7X)
 GLOMEHULONEPHRITIS, FOCAL 2 (4X)
 INFLAMMATION, INTERSTITIAL 1 (2X)
 GLOMEBOLONBPHRITIS, MEMBRANOUS 2 (4X)
 PYELONGPHEITIS, ACHTE/CHRONIC 1 (2X)
 GLOMEEULONEPHRITIS, CHRONIC 1 (2X)
 

IDEINAEY BLADDEE (45) (45) (43)
 

INFLAMMATION, CHEOSIC FOCAL 1 <2X)
 PERIAETERITIS 1 (2X)
 

ENDOCEIHE SYSTEM
 

IAD8ENAL CORTEX (47) (44) (42)
 

HEMORRHAGE 1
 

tTHYROID (41) (32) (29)
 

HYPERPLASIA, FOLIICDLiR-CELL 1 (2XJ
 

EEPEODUCTIVE SYSTEM
 

tUTERUS (43) (44) (41)
 

DILATATION, NOS 2 (5X)
 

HYD80METEA 3 (7X) 1 (2X)
 INFLAMMATION, ACUTE 1 (2X)
 ABSCESS, NOS 2 (5X)
 

tUTEEUS/ENDOMETEIOM (43) (44) (41)
 

INFLAMMATION, NOS 2 (5X)
 INFLAMMATION, SUPPUEATIVE 2 (5X) <2X)
 

6__U431
 

* SOMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NDMBER OF ANIMALS NECROPSIED
 

D-9
 
TABLE D2 (CONTINUED) 

CONTROL (ONTB) LOW DOSE HIGH DOSE 

06-0070 06-0053 06-005(4 

INFLAMMATION, ACUTE FOCAL 1 (2%)
 INFLAMMATION, ACUTE/CHBONIC 3 (7X)
 HYPERPLASIA, NOS 1 (2*)
 HYPERPLASIA, CYSTIC 20 (47*) 18 (41*) 15 (37*)
 METAPLASIA, SQUAMOUS 1 (2*)
 

#OVABY/OVIDOCT (13) (44) (41)
 INFLAMMATION, SOPPUEATIVE 4 (9%)
 ABSCESS, NOS 1 (2*)
 

#OVAEY (15) (45) CYST, NOS 1 (2*) 4 (10*) 

FOLLICD1AE CYST, NOS 1 (2%) INFLAMMATION, SOPPUSATIVE 6 (13*) 

INFLAMMATION, CHBONIC 1 <2*) 

ABSCESS, CHRONIC 1 (2*) 

PEEIABTERIfIS 1 (2*) 

NEEVOO S SYSTEM 

#BBAIR/I1ENINGES (46)
 

INFLAMMATION, ACUTE/CHRONIC (2X) 

INFLAMHATION, CHKONIC FOCAL (2%)

 

SPECIAL SENSE OEGANS
 

NONE
 

MUSCOLOSKELITAL SYSTIH
 

NONE
 

BODY CAVITIES
 

*A.BDOMINAL CAVITY 08) (17) (17) 

HEMATOMA, NOS 1 (2«) 

ALL OTHEE SYSIHMS 

*MOLTIPLE ORGANS (18) (17) (17) 

# NUMBER OF ANIHALS BITH TISSDE EXAMINED MICBOSCOPICA1LY
 

* NUMBEB OF ANIMALS NECEOPSIED
 

D-IO
 
TABLE D2 (CONCLUDED) 

CONTROL (UNTP) LO» DOSE HIGH DOSE 

06-0070 06-0053 06-0051* 

SPECIAL MORPHOLOGY SUMMAR Y 

HO LESION BEPOETED 10 7 ANIMAL MISSING/NO NECROPSY 1 

AUTO/NECROPSY/HISTO PERF 1 2 

ADTO/NECROPSY/NO HISTO 1 AUTOLYS IS/NO NECBOPSY 2 2 3 

* NOSBER OF ANIMAL S SITH TISSUE EXAMINE D MICROSCOPICALLY 

* HOMBSE OF ANIMAL S NECROPSIED 

D-ll 

DHEW Publication No. (NIH) 78-1357