National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

NO. 153 

1979 

M 

BIOASSAY OF 

o-TOLUIDINE HYDROCHLORIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 636-21-5 

NCI-CG-TR-153 

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

Public Health Service 

National Institutes of Health 
BIOASSAY OF
 

o-TOLUIDINE HYDROCHLORIDE
 

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) 79-1709
 
ii

 
BIDASSAY OF
 

o-TOLUIDINE HYDROCHLORIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Careinogenesis 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
 

o-toluidine hydrochloride 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. A negative result, in
 

which the test animals do not have a greater incidence of cancer
 

than control animals, does not necessarily mean that the test
 

chemical is not a carcinogen, inasmuch as the experiments are
 

conducted under a limited set of circumstances. A positive
 

result demonstrates that the test chemical is carcinogenic for
 

animals under the conditions of the test and indicates that
 

exposure to the chemical is a potential risk to man. The actual
 

determination of the risk to man from chemicals found to be
 

carcinogenic in animals requires a wider analysis.
 

CONTRIBUTORS: This bioassay of o-toluidine hydrochloride was
 

conducted by the NCI Frederick Cancer Research Center (FCRC) (1),
 

Frederick, Maryland, operated for NCI (2) by Litton Bionetics,
 

Inc.
 

The manager of the bioassay at FCRC was Dr. B. Ulland, the
 

toxicologist was Dr. E. Gordon, and Drs. R. Cardy and D. Creasia
 

compiled the data. Ms. S. Toms was responsible for management of
 

data, Mr. D. Cameron for management of histopathology, and Mr. L.
 

Callahan for management of the facilities. Mr. A. Butler
 

performed the computer services. Histopathologic evaluations for
 

rats were performed by Dr. J. F. Hardisty (3), and the
 

histopathologic evaluations for mice were performed by Dr. C. E.
 

Gilmore (3). The diagnoses included in this report represent the
 

interpretations of Drs. Hardisty and Gilmore.
 

Animal pathology tables and survival tables were compiled at EG&G
 

Mason Research Institute (4). Statistical analyses were
 

performed by Dr. J. R. Joiner (5) and Ms. P. L. Yong (5), using
 

methods selected for the bioassay program by Dr. J. J. Gart (6).
 

iii
 
The chemicals used in this bioassay were analyzed at FCRC (1) by
 

Dr. W. Zielinsky. The chemical analyses and narrative were
 

reviewed and approved by Dr. W. Lijinsky.
 

This report was prepared at Tracer Jitco (5) under the direction
 

of NCI. Those responsible for the report at Tracer Jitco were
 

Dr. L. A. Campbell, Director of the Bioassay Program; Dr. S. S.
 

Olin, Deputy Director for Science; Dr. J. F. Robens,
 

toxicologist; Dr. R. L. Schueler, pathologist; Dr. G. L. Miller,
 

Ms. L. A. Waitz, Ms. M. S. rKing, and Mr. W. D. Reichardt,
 

bioscience writers; and Dr. E. W. Gunberg, technical editor,
 

assisted by Ms. Y. E. Presley and Ms. P. J. Graboske.
 

The following scientists at NCI were responsible for evaluating
 

the bioassay experiment, interpreting the results, and reporting
 

the findings: Dr. Kenneth C. Chu, Dr. Cipriano Cueto, Jr., Dr.
 

J. Fielding Douglas, Dr. Richard A. Griesemer, Dr. Thomas E.
 

Hamm, Dr. William V. Hartwell, Dr. Morton H. Levitt, Dr. Harry A.
 

Milman, Dr. Thomas W. Orme, Dr. A. R. Patel, Dr. Sherman F.
 

Stinson, Dr. Jerrold M. Ward, and Dr. Carrie E. Whitmire.
 

(1)	 Frederick Cancer Research Center, P.O. Box B, Frederick,
 

Maryland.
 

(2)	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes
 

of Health, Bethesda, Maryland.
 

(3)	 Experimental Pathology Laboratories, Inc., P.O. Box 474,
 

Herndon, Virginia.
 

(4)	 EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

(5)	 Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

(6)	 Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute,
 

National Institutes of Health, Bethesda, Maryland.
 

iv
 
SUMMARY
 

A bioassay of o-toluidine hydrochloride for possible carcinogenicity was conducted by administering the test chemical in feed
 

to F344 rats and B6C3F1 mice.
 

Groups of 50 rats of each sex and 50 mice of each sex were administered o-toluidine hydrochloride at one of several doses, either
 

3,000 or 6,000 ppm for the rats and either 1,000 or 3,000 ppm for
 

the mice, for 101 to 104 weeks. Matched controls consisted of 20
 

untreated rats of each sex and 20 untreated mice of each sex.
 

All surviving rats and mice were killed at the end of administration of the test chemical.
 

Mean body weights of dosed male and female rats and mice were
 

lower than those of corresponding matched controls and were dose
 

related. Mortalities of the male and female rats were dose
 

related and were relatively high at the end of the bioassay.
 

Mortalities of the male and female mice were not, however,
 

significantly affected by administration of the test chemical.
 

In rats, the administration of the test chemical induced several
 

types of sarcomas of the spleen and other organs in both males
 

and females, mesotheliomas of the abdominal cavity or scrotum in
 

males, and transitional-cell carcinomas of the urinary bladder in
 

females. Administration of the o-toluidine hydrochloride also
 

resulted in increased incidences of fibromas of the subcutaneous
 

tissue in the males and fibroadenomas or adenomas of the mammary
 

gland in the females.
 

In mice, hemangiosarcomas were induced at various sites in males,
 

and hepatocellular carcinomas or adenomas were induced in females.
 

Under the conditions of this bioassay, o-toluidine hydrochloride
 

was carcinogenic in both male and female F344 rats and B6C3F1
 

mice, producing a significant increased incidence of one or more
 

types of neoplasms.
 
vi

 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. AnimaIs 4
 

D. Animal Maintenance 5
 

E. Subchronic Studies 7
 

F. Chronic Studies 10­

G. Clinical and Pathologic Examinations 10
 

H. Data Recording and Statistical Analyses 14
 

III. Results - Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 19
 

C. Pathology (Rats) 22
 

D. Statistical Analyses of Results (Rats) 29
 

IV. Results -Mice 37
 

A. Body Weights and Clinical Signs (Mice) 37
 

B. Survival (Mice) 37
 

C. Pathology (Mice) 40
 

D. Statistical Analyses of Results (Mice) 45
 

V. Discussion 47
 

VI. Bibliography 51
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Administered o-Toluidine Hydrochloride
 

in the Diet 55
 

Table Al Summary of the Incidence of Neoplasms in Male Rats
 

Administered o-Toluidine Hydrochloride
 

in the Diet 57
 

vii
 
Table A2 Summary of the Incidence of Neoplasms in 

Female Rats Administered o-Toluidine Hydrochloride 

in the Diet 61 

Appendix B Summary of the Incidence of Neoplasms in Mice 

Administered o-Toluidine Hydrochloride in 

the Diet 65 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Administered o-Toluidine Hydrochloride 

in the Diet 67 

-Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Administered o-Toluidine Hydrochloride 

in the Diet 71 

Appendix C Summary of the Incidence of Nonneoplastic Lesions 

in Rats Administered o-Toluidine Hydrochloride 

in the Diet 75 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered 

o-Toluidine Hydrochloride in the Diet 77 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered 

o-Toluidine Hydrochloride in the Diet 82 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered o-Toluidine 

Hydrochloride in the Diet 87 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered 

o-Toluidine Hydrochloride in the Diet 89 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered 

o-Toluidine Hydrochloride in the Diet 92 

Appendix E Analyses of the Incidence of Primary 

Tumors in Rats Administered o-Toluidine 

Hydrochloride in the Diet 95 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Administered o-Toluidine 

Hydrochloride in the Diet 97 

viii
 
Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Administered o-Toluidine 

Hydrochloride in the Diet 109 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered o-Toluidine Hydrochloride 

in the Diet 119 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Administered o-Toluidine 

Hydrochloride in the Diet 121 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Administered o-Toluidine 

Hydrochloride in the Diet 127 

TABLES 

Table 1 o-Toluidine Hydrochloride Subchronic Feeding 

Studies in Rats and Mice 8 

Table 2 o-Toluidine Hydrochloride Chronic Feeding Studies 

in Rats 11 

Table 3 o-Toluidine Hydrochloride Chronic Feeding Studies 

in Mice 12 

FIGURES 

Figure 1 Growth Curves for Rats Administered 

o-Toluidine Hydrochloride in the Diet 20 

Figure 2 Survival Curves for Rats Administered 

o-Toluidine Hydrochloride in the Diet 21 

Figure 3 Growth Curves for Mice Administered 

o-Toluidine Hydrochloride in the Diet 38 

Figure 4 Survival Curves for Mice Administered 

o-Toluidine Hydrochloride in the Diet 39 

ix
 
X 
I. INTRODUCTION
 

o-Toluidine (CAS 636-21-5; NCI
 

• HCI
 

C02335) and its hydrochloride
 

salt are dye intermediates used
 

in the manufacture of a large
 

number of textile dyes which
 

o-Toluidine hydrochloride
 

include some of the azo,
 

triarylmethane, sulfur, and
 

indigoid compounds (Society of Dyers and Colourists, 1971). In
 

addition, there are numerous substituted o-toluidines that are
 

used as dye intermediates (Society of Dyers and Colourists,
 

1971). o-Toluidine also functions as a photographic dye
 

(Stanford Research Institute, 1976), as a reagent in a clinical
 

assay for glucose (Indriksons, 1975) and hemoglobin (Ferretti et
 

al., 1971), and as an antioxidant in the manufacture of rubber
 

(Stanford Research Institute, 1976).
 

This chemical has been in commercial use in the United States for
 

over 50 years (International Agency for Research on Cancer,
 

1974). The annual U. S. production volume is not reported;
 

however, 24,720 pounds were imported to this country in 1976
 

(United States International Trade Commission, 1977).
 
The oral LD of o-toluidine hydrochloride has been reported as 

2,951 rag/kg body weight when administered in water by stomach 

tube to male Osborne-Mendel rats (Lindstrom et al., 1969); that 

of o-toluidine has been reported as 900 mg/kg body weight when 

the undiluted chemical was administered by intubation to SpragueDawley rats (Jacobson, 1972) and as 515 mg/kg body weight when 

the chemical was administered in oil to mice of unspecified 

strain (Lunkin, 1967). Toxic properties of o-toluidine, in common with those of the analog aniline, include the ability to 

induce the formation of methemoglobin in man (Hamblin, 1963), 

rats (Lunkin, 1967), and mice (Nomura, 1977) and to cause 

hematuria in man (Hamblin, 1963). 

The primary urinary metabolic products identified when 

o-toluidine hydrochloride was administered subcutaneously to male 

CDF rats were the sulfate and glucuronide conjugates of 

4-amino-m-cre sol and N-acetyl-4-amino-m-cresol (Son et al., 1977). 

Preliminary investigations of o-toluidine sponsored by the 

National Cancer Institute indicated that this chemical was 

carcinogenic (Homburger et al., 1972; Russfield et al., 1973a). 

o-Toluidine hydrochloride was selected for testing in the 

Carcinogenesis Testing Program to confirm these earlier results 

under the conditions of a standard bioassay. 
II. MATERIALS AND METHOD^
 

A. Chemical
 

o-Toluidine hydrochloride (2-aminotoluene hydrochloride ; C?H ~NCl) 

was obtained as technical-grade nonformulated material from 

American Cyanamid Co. as a fine, grayish powder. Its purity was 

estimated by reversed-phase high-pressure liquid chromatography 

as greater than 99%, with the presence of one minor contaminant 

of less than 1%. Thin layer chromatography showed only one spot, 

with no impurities. Mass spectral analysis showed a molecular 

ion for the free amine at m/e 107, and a base peak at m/e 106. 

The melting point of the test material was 215 C (literature: 

215 C). Elemental analysis showed 57.8% carbon, 7.2% hydrogen, 

and 9.5% nitrogen (theoretical: 58.5% carbon, 7.0% hydrogen, and 

9.7% nitrogen). 

B. Dietary Preparation 

Test diets containing o-toluidine hydrochloride were prepared 

fresh every 1 to 1-1/2 weeks in 6- to 12-kg batches at the 

appropriate doses. A known weight of the chemical was first 
mixed with an equal weight of autoclaved Wayne® Sterilizable
 

Lab Meal (Allied Mills, Inc., Chicago, 111.), using a mortar and
 

pestle. The mixing was continued with second and third additions
 

of feed, and final mixing was performed with the remaining
 

quantity of feed for a minimum of 15 minutes in a Patterson-Kelly
 

twin-shell blender. The diets were stored at 5 C until used.
 

C. Animals
 

Male and female F344 (Fischer) rats and B6C3F1 mice were obtained
 

as 4-week-old weanlings, all within 3 days of the same age, from
 

the NCI Frederick Cancer Research Center animal farm (Frederick,
 

Md.) monitored by the Division of Cancer Treatment, NCI. The
 

animals were housed within the test facility for 2 weeks and then
 

were assigned four rats to a cage and five mice to a cage on a
 

weight basis for each cage of animals of a given species and
 

sex. For use in the chronic study, male rats were required to
 

weigh 90 to 105 g, averaging at least 100 g; female rats, 80 to
 

95 g, averaging at least 90 g; male mice, 18 to 22 g, averaging
 

at least 19.5 g; and female mice 17 to 21 g, averaging at least
 

18.5 g. Individual animals were identified by ear punch.
 
D. Animal Maintenance
 

The animals were housed in polycarbonate cages (Lab Products, 

Inc., Garfield, N. J.), 19 x 10-1/2 x 8 inches for the rats and 

11-1/2 x 7-1/2 x 5 inches for the mice. The cages were suspended 

from aluminum racks (Scientific Cages, Inc., Bryan, Tex.) and 

were covered by nonwoven polyester-fiber 12-mil-thick filter 

paper (Hoeltge, Inc., Cincinnati, Ohio). The bedding used was 

Absorb-dri® hardwood chips (Northeastern Products, Inc., 

Warrenburg, N. Y.). The feed supplied was presterilized Wayne® 

Sterilizable Lab Meal with 4% fat, provided ad libitum in 

suspended stainless steel hoppers and replenished at least three 

times per week. Water, acidified to pH 2.5, was supplied ad_ 

libitum from glass bottles. Sipper tubes (Lab Products, Inc.) 

were suspended through the tops of the cages. 

The contaminated bedding was disposed of through an enclosed 

vacuum line that led to a holding tank from which the bedding was 

fed periodically into an incinerator. The cages were sanitized 

twice per week and the feed hoppers twice per month at 82 to 

88 C in a tunnel-type cagewasher (Industrial Washing Corp., 

Mataway, N. J.), using the detergents, Clout® (Pharmacal 

Research Laboratories, Greenwich, Conn.) or Oxford D'Chlor 

(Oxford Chemicals, Atlanta, Ga.). The glass bottles were 
sanitized at 82 to 88 C in a tunnel-type bottle washer
 

(Consolidated Equipment Supply Co., Mercersburg, Pa.) three times
 

per week, using a Calgen Commercial Division detergent (St.
 

Louis, Mo.). The racks for the cages were sanitized at or above
 

82 C in a rack washer (Consolidated Equipment Supply Co.) once
 

per month, using the Calgen Commercial Division detergent, and
 

the filter paper was changed at the same time.
 

The air in the animal rooms was maintained at a temperature of 22
 

to 24 C and 45 to 55% relative humidity. Incoming air was
 

passed through a filter of 65% efficiency and a bag filter of 95%
 

efficiency at the intake and expelled without recirculation
 

through a "Z"-type roughing filter of 30% efficiency and a bag
 

system of 90 to 95% efficiency at the exhaust (American Air
 

Filters, Louisville, Ky.; Mine Safety Appliances, Pittsburgh,
 

Pa.). Room air was changed 15 times per hour. The air pressure
 

was maintained negative to a clean hallway and positive to a
 

return hallway. Fluorescent lighting was provided automatically
 

on a 12-hour-per-day cycle.
 

Rats administered o-toluidine hydrochloride and their controls
 

were housed in the same room as rats on feeding studies of the
 

following chemicals:
 
(CAS 85-44-9) phthalic anhydride
 

(CAS 95-80-7) 2,4-diaminotoluene
 

Mice administered o-toluidine hydrochloride and their controls
 

were housed in the same room as mice on feeding studies of the
 

following chemicals:
 

(CAS 3165-93-3) 4-chloro-o-toluidine hydrochloride
 

(CAS 97-77-8) ethyl tuads
 

(CAS 128-04-1) sodium diethyldithiocarbamate
 

(CAS 128-37-0) butylated hydroxytoluene (BHT)
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses (MTD's) of o-toluidine hydrochloride, on the
 

basis of which two concentrations (referred to in this report as
 

"low" and "high" doses) were selected for administration in the
 

chronic studies. Groups of five rats and five mice of each sex
 

were fed diets containing one of several doses of o-toluidine
 

hydrochloride for 7 weeks, followed by 1 week on basal diet.
 

Groups of five control animals of each species and sex received
 

basal diet only. Each animal was weighed twice per week. Table
 

1 shows the doses fed and the mean body weights of dosed animals
 

at week 7, expressed as percentages of mean body weights of the
 

controls.
 
Table 1. o-Toluidine Hydrochloride Subchronic Feeding Studies
 

in Rats and Mice
 

Mean Weight at Week 7 as /I of Control
 

Dose
 

(ppm)
 

RATS
 

1,000
 

2,000
 

3,000
 

4,000
 

6,000
 

6,200
 

12,500
 

25,000
 

50,000
 

MICE
 

3,100
 

6,200
 

8,000
 

10,000
 

12,500
 

20,000
 

25,000
 

50,000
 

Male
 

93
 

87
 

88
 

88
 

88
 

91
 

74
 

45
 

51
 

89
 

86
 

85
 

83
 

79
 

74
 

74
 

63
 

Female
 

106
 

104
 

98
 

99
 

92
 

93
 

88
 

60
 

42
 

88
 

83
 

85
 

81
 

81
 

81
 

77
 

69
 
At the end of the subchronic studies, all animals were killed
 

using CO and necropsied. Small amounts of renal and splenic
 

pigmentation were observed in male and female rats receiving
 

12,500 ppm. The red pulp of the spleens in male and female mice
 

at 50,000 ppm had pigment deposition, apparently lipofuscin.
 

Similar pigmentation was observed in trace to small amounts in
 

the tubular epithelium of the kidneys, and there were trace
 

amounts of pigment seen in Kupffer's cells of the hepatic
 

sinusoids.
 

All dosed and control animals survived to the end of the
 

subchronic studies except male and female rats administered
 

50,000 ppm. In these groups four males and three females died by
 

week 3 on study.
 

Ten percent depression in body weight was taken as the major
 

criterion for the estimation of MTD's. The doses required to
 

produce this response were determined by the following
 

procedure: first, least squares regressions of mean body weights
 

versus days on study were used to estimate mean body weights of
 

each of the dosed groups at day 49. Next, probits of the percent
 

weights of the dosed groups at day 49 relative to weights of
 

corresponding control groups were plotted against the logarithms
 

of the doses, and least squares regressions fitted to the data
 
were used to estimate the doses required to induce 10% depression
 

in weight.
 

Based on the data thus obtained, the low and high doses for rats
 

were set at 3,000 and 6,000 ppm; and for mice at 1,000 and 3,000
 

ppm.
 

F. Chronic Studies
 

The test groups, doses administered, and durations of the chronic
 

feeding studies are shown in tables 2 and 3.
 

G. Clinical and Pathologic Examinations
 

All animals were checked twice daily for deaths. Observations
 

for sick, tumor-bearing, and moribund animals were recorded
 

daily. Clinical examination and palpation for masses were
 

performed each month, and the animals were weighed at least once
 

per month. Moribund animals and animals that survived to the end
 

of the bioassay were killed using CO. and necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

10
 
Table 2. o-Toluidine Hydrochloride Chronic Feeding Studies
 

in Rats
 

o-Toluidine 

Sex and Initial Hydrochloride Time 

Test No. of Doses (b) on 

Group Animals (a) (ppm) Study 

Male
 

Matched-Control 20 0 104
 

Low-Dose 50 3,000 104
 

High-Dose 50 6,000 101 (c)
 

Female
 

Matched-Control 20 0 104
 

Low-Dose 50 3,000 104
 

High -Dose 50 6,000 104
 

(a) All animals were 6 weeks of age when placed on study.
 

(b) Test and control diets were provided ad libitum 7 days per
 

week.
 

(c) Administration of the test chemical was terminated at the
 

time indicated due to deaths of all animals.
 

11
 
Table 3. o-Toluidine Hydrochloride Chronic Feeding Studies
 

in Mice
 

o-Toluidine 

Sex and Initial Hydrochloride Time 

Test No. of Doses (b) on 

Group Animals (a) (ppm) Study 

Male
 

Matched-Control 20 0 103
 

Low-Dose 50 1,000 103
 

High-Dose 50 3,000 102
 

Female
 

Matched-Control 20 0 103
 

Low-Dose 50 1,000 103
 

High-Dose 50 3,000 103
 

(a) All animals were 6 weeks of age when placed on study.
 

(b) Test and control diets were provided ad libitum 7 days per
 

week.
 

12
 
examination of major tissues, major organs, and all gross lesions.
 

The tissues were preserved in 10% formalin, embedded in paraffin,
 

sectioned, and stained with hematoxylin and eosin. The following
 

tissues were examined microscopically: skin, lungs and bronchi,
 

trachea, bone marrow (femur), spleen, lymph nodes (mesenteric and
 

submandibular), thymus, heart, salivary glands (parotid,
 

sub lingual, and submaxillary), liver, pancreas, esophagus,
 

stomach (glandular and nonglandular), small and large intestines,
 

kidney, urinary bladder, pituitary, adrenal, thyroid,
 

parathyroid, pancreatic islets, testis, prostate, mammary gland,
 

uterus, ovary, brain (cerebrum and cerebellum), and all tissue
 

masses. Peripheral blood smears also were made for all animals,
 

whenever possible.
 

Necropsies were also performed on all animals found dead, unless
 

precluded in whole or in part by autolysis or cannibalization.
 

Thus, the number of animals from which particular organs or
 

tissues were examined microscopically varies and does not
 

necessarily represent the number of animals that were placed on
 

study 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 appropriate statistical
 

techniques described in this section. Those analyses of the
 

experimental results that bear on the possibility of carcinqgenicity 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
 

14
 
(1972) for testing two groups for equality and Tarone's (1975)
 

extensions of Cox methods for testing for 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.
 

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 is examined (denominator). In most instances,
 

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic
 

examination was required 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) was used to compare the tumor incidence of
 

a control group with that of a group of dosed animals at each
 

dose level. When results for a number of dosed groups (k) are
 

15
 
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) 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.
 

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

continuity correction (Armitage, 1971), was also used. Under the
 

assumption of a linear trend, this test determines 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 is 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
 

16
 
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 an animal died naturally or was sacrificed
 

was entered as the time point of tumor observation. Cox's
 

methods of comparing 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 less than 0.05,
 

two-tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with 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
 

t c t
 

binomial probability of the incidence of a specific type of tumor
 

17
 
in a dosed group of animals and p is the true probability of
 

the spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

a specific tumor in a dosed 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
 

dosed 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% of a large number of identical experiments, the
 

true ratio of the risk in a dosed 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 can be inferred that a statistically
 

significant result (P less than 0.025 one-tailed test when the
 

control incidence is not zero, P less than 0.050 when the control
 

incidence is zero) has occurred. When the lower limit is less
 

than unity, but the upper limit is a 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. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of both dosed male and dosed female rats were
 

lower than those of corresponding matched controls and were dose
 

related throughout the bioassay (figure 1). No other clinical
 

signs were reported which could clearly be related to
 

administration of the test chemical. Some fluctuations in the
 

growth curves may be due to mortality; as the size of a group
 

diminishes, the mean body weight may be subject to variation.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered o-toluidine
 

hydrochloride in the diet at the doses of this bioassay, together
 

with those of the matched controls, are shown in figure 2. In
 

each sex, the result of the Tarone test for positive dose-related
 

trend in mortality is significant (P less than 0.001). An
 

indicated departure from linear trend is observed in
 

19
 
I 300 

, °AA 

o 

0 " °° ° 

O 

O 

CO 

g MALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

DD H D I 300 00 e> 0 0 0 

> o AA 

D 

O A CD 

Z 200 

< 

FEMALE RATS 

D MATCHED CONTROL 

I I O LOW DOSE 

A HIGH DOSE 

TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered o-Toluidine Hydrochloride in the Diet 

20 
n r. r. .-• .-..-.-. -.-. r; .•:.-..-..-. .• .~ r. .-..-..-. r.n * r:.~ r. n n~.r 

ttl 

\»* - ­1 

\ 

i 

A

V 

O 

^ 

A; 

A ••: 

DC 

D 

CO 

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6, 

4. 

A 

A: 6 A! 1 

m 

O 

cc A: 

a. 4.. ^

A, 

MALE RAT! 3 6

i 

^i 

0.20 [ [H MATCHED CC 

(3 LOW DOSE ^

A: 

A: ^ HIGH DOSE 

\

1 

45 60 75 

TIME ON STUDY (WEEKS) 

.-..-..-..-..-..-vs -..-.. -..-.. -..£>„„

1"£.~.-.A. 

t J>--, 1 

*•&-•*> 

\

, ^ 

\. 

0.70 • ^ 

> i 

A CC 

A 6 UO 4. t 

m A 

CD 

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O. 

]

FEMAL

 MATCHE

E RA

D CO 

T S \

c) LOW DOSE 

_/\ HIGH DOSE 

0 00 ­

TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered o-Toluidine Hydrochloride in the Diet 

21 
male rats (P = 0.019) due to the steep decrease in survival
 

observed in the dosed groups.
 

C. Pathology (Rats)
 

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.
 

Proliferative lesions and neoplasms involving mesenchymal tissue
 

in the spleen, other visceral organs, and the peritoneal lining
 

of the abdominal cavity and scrotum were observed only in the
 

dosed rats. Proliferative lesions involving the transitional-


cell epithelium of the urinary bladder and renal pelvis also were
 

observed only in the dosed rats. Other neoplasms which were
 

observed with increased frequency in the dosed rats when compared
 

with control rats were fibromas of the subcutaneous tissue and
 

fibroadenomas of the mammary gland.
 

Many of the dosed rats had benign neoplasms involving the fibrous
 

connective tissue of the subcutaneous tissue and mammary gland.
 

In the male rats these neoplasms were predominately fibrous and
 

were classified as fibromas. These neoplasms were characterized
 

22
 
by whorls and interlacing bundles of fibrous connective tissue.
 

Most of the neoplasms were relatively acellular and contained
 

abundant mature collagen. In the female rats, most were
 

classified as fibroadenomas. These neoplasms were characterized
 

by focal proliferations of well-differentiated fibrous connective
 

tissue surrounding proliferating mammary acinar and ductular
 

epithelium. There was much structural variation present in these
 

neoplasms. In some of the neoplasms, the connective tissue
 

stroma was predominant; in others, there was a marked epithelial
 

overgrowth. Several of the fibroadenomas in the female rats and
 

fibromas in the male rats occurred at multiple sites within the
 

subcutaneous tissue, and there were also a variety of malignant
 

mesenchymal neoplasms involving only the subcutaneous tissue of
 

the dosed male rats. The incidence of these neoplasms may be
 

summarized as follows:
 

No. Tissues Examined 

Histologically 

Control

20 

Males

Low

 Dose

50 

 High 

 Dose

49 

 Females 

 Control Dose

20 

Low

50

 Dose 

 High 

)OS{ 

 49 

SUBCUTANEOUS TISSUE 

Osteosarcoma
 

Myxosarcoma
 

Fibresarcoma
 

Sarcoma, NOS
 

Lipoma
 

Fibroma
 

MAMMARY GLAND
 

Adenocarc inoma
 

Adenoma, NOS
 

Fibroadenoma
 20 35

 

0

0

0

0

0 

2 

0

0

0

0

0

4 

0

0

0

0

0

0 

1

0

2

0

2 

0 

2

1

1 

4 

28 27 

0

0

0

0

0

0 

1

0 

0

0 

0

1 

6 

0

0

1 

0

0 

7 

0

0

0 

23
 
Proliferative lesions of the transitional-cell epithelium of the
 

urinary bladder occurred only in dosed rats. These lesions were
 

more frequent in the female rats than in the males. The incidence
 

of these lesions may be summarized as follows:
 

Males Females 

Low High Low High 

Control Dose Dose Control Dose Dose 

No. Tissues Examined 

Histologically 20 50 44 20 45 47 

URINARY BLADDER 

Squamous-cell 

Carcinoma 0 1 0 0 0 0 

Transitional-ce11 

Carcinoma 0 3 0 0 9 22 

Transitional-cell 

Papilloma 0 0 1 0 1 0 

Epithelial 

Hyperplasia 0 9 7 0 21 13 

Hyperplasia of the transitional-cell epithelium of the urinary 

bladder was characterized by a diffuse thickening of the 

transitional-cell epithelium of the bladder. In these lesions 

there was no evidence of invasion into the submucosa or wall of 

the urinary bladder. There appeared to be a dose-related 

progression, particularly in the female rats, from 

transitional-cell epithelial hyperplasia to transitional-cell 

carcinomas of the urinary bladder. The transitional-cell 

carcinomas varied considerably in appearance, but they were 

usually large, papillary neoplasms protruding into the lumen of 

24
 
the urinary bladder. Invasion of malignant epithelium into the
 

underlying stroma was frequently present. A few of the smaller
 

carcinomas were not papillary and were characterized by a focal
 

mucosal thickening of the atypical epithelial cells with nests of
 

malignant epithelium present in the submucosa. A few of the
 

dosed rats also had hyperplastic lesions involving the
 

transitional epithelium lining the renal pelvis. One low-dose
 

male rat and one high-dose female rat had a transitional-cell
 

carcinoma in the renal pelvis.
 

Neoplasms involving the spleen, other abdominal viscera, and the
 

peritoneal lining of the abdominal cavity and scrotum occurred
 

only in dosed rats. The spleen was the most frequent organ
 

involved and appears to be a specific target for the carcinogenic
 

activity of o-toluidine hydrochloride. The histologic appearance
 

of each type of neoplasm was similar regardless of the organ
 

involved. All of these neoplasms were of mesenchymal origin and
 

appeared to be closely related. All of the neoplasms were poorly
 

differentiated and difficult to classify. The specific classification of these sarcomas refers to the most differentiated cell
 

type observed histologically. These mesenchymal neoplasms
 

appeared to be highly malignant as evidenced by a high incidence
 

of invasion into adjacent organs and soft tissues and/or
 

metastasis to other sites.
 

25
 
The neoplasms diagnosed as sarcoma, NOS, were too undifferentiated to classify. They were composed of pleomorphic cells with
 

large, vesicular nuclei and abundant eosinophilic cytoplasm.
 

Bizarre nuclear shapes and multinucleated cells were not
 

uncommon. Mitotic figures were scattered throughout these
 

neoplasms. The incidences of these lesions may be summarized as
 

follows:
 

Males Females 

Low High Low High 

Control Dose Dose Control Dose Dose 

No. Tissues Examined 

Histologically 20 49 42 20 49 49 

SPLEEN
 

Sarcoma, NOS
 

Osteosarcoma
 

Angiosarcoma
 

F ib roma
 

Angioma
 

Lipoma
 

No. Animals Necropsied 20 50 49 20 50 49
 

MULTIPLE ORGANS
 

Osteosarcoma
 

20 

11 

Fibrosarcoma
 

Angiosarcoma
 

Mesothelioma, NOS
 

Mesothelioma,
 

Malignant
 

Sarcoma, NOS
 

Fibroma
 

3

1 

9 

6

0

0 

1

1 

7

4

0

0 

0

0

0

0

0

0 

3

1 

0 

2 

0

1 

1

0

7 

10 

1

0 

0

0

0

0

0

0 

18 

0

1 

0 

0

1

1 

0 

0

0

0

0 

5 

1

0 

3

8

1

1 

0

0

0

0 

0

2

0 

0

1 

0 

0

0

0 

3 

0 

4

3

1 

0

0

0 

26
 
Males Females
 

Low High Low High
 

Control Dose Dose Control Dose Dose
 

No. Animals Necropsied 20 50 49 20 50
 49
 

BODY CAVITIES
 

Tunica Vaginalis
 

Mesothelioma,
 

Malignant 0 1 0 6 0 0 0
 

Mesentery
 

Mesothelioma, NO S 0 2 0 0 0 0
 

Sarcoma, NO S 0 0 0 0 0 1
 

Abdominal Cavity
 

Sarcoma, NO S 0 0 0 0 0 1
 

The fibrosarcomas were composed primarily of pleomorphic spindle-


shaped cells. The histologic appearance of these neoplasms was
 

quite variable. In some areas, these neoplasms were highly cellular with little evidence of collagen being produced. In these
 

areas, the cells had large nuclei with distinct nuleoli and a
 

high mitotic activity. Occasional multinucleated cells were
 

observed. In other areas, the neoplasm was relatively acellular
 

and contained abundant mature collagen. These neoplasms were
 

highly invasive and completely destroyed the spleen in several
 

animals, with invasion into the liver and other visceral organs.
 

In a few animals, fibrous neoplasms occurred that were acellular
 

and composed of interlacing bundles and whorls of mature
 

collagen. These neoplasms were growing primarily by expansion on
 

the serosal surfaces of the abdominal viscera and were classified
 

as fibromas. Within the neoplasms diagnosed as sarcoma, NOS,
 

fibrosarcoma, and fibroma, areas of osseous metaplasia were
 

27
 
sometimes observed. In these areas, the osseous tissue was
 

present as mature cancellous bone and did not appear to be part
 

of the proliferating tissue.
 

The osteosarcomas were composed of pleomorphic spindle-shaped
 

cells which were differentiating into osteoblasts that were
 

producing varying amounts of osteoid. In most areas, the
 

individual neoplastic cells were surrounded by acellular
 

eosinophilic material with little progression to bone. In other
 

areas of these neoplasms, the osteoid was differentiating into
 

mature cancellous bone. These neoplasms appeared to be forming
 

only in soft tissues, and no skeletal involvement was observed.
 

The angiosarcomas of the spleen usually replaced much of the
 

normal splenic architecture. There was great variation in the
 

histologic appearances of these neoplasms. In some areas, the
 

neoplasms were highly vascular and consisted of obvious vascular
 

spaces lined by pleomorphic endothelial cells. In other areas,
 

the stroma of the neoplasm was prominent and vascular spaces were
 

difficult to observe. Many of these vascular neoplasms contained
 

large blood-filled cysts that were associated with areas of
 

hemorrhage and necrosis.
 

Mesotheliomas were characterized as papillary proliferations of
 

28
 
fibrous connective tissue covered by plump mesothelial cells.
 

Foci of osseous metaplasia were sometimes observed in the fibrous
 

stroma of the malignant mesotheliomas.
 

A variety of other neoplastic and nonneoplastic lesions were
 

observed with approximately equal frequency in the control and
 

dosed rats. The nature, distribution, and incidence of these
 

neoplasms are similar to those known to occur in aged F344 rats.
 

Under the conditions of this study, o-toluidine hydrochloride is
 

carcinogenic when administered in the diet to F344 male and
 

female rats, inducing fibromas of the subcutaneous connective
 

tissue, transitional-cell carcinomas of the urinary bladder, and
 

several types of sarcomas involving the spleen and peritoneum.
 

There was also an increased incidence of fibroadenomas of the
 

mammary gland in dosed rats when compared with control rats.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals of one group and at an incidence of at least 5%
 

in one or more than one group.
 

29
 
In male rats, the result of the Cochran-Armitage test for
 

positive dose-related trend in the incidence of fibromas of the
 

subcutaneous tissue in the integumentary system is significant
 

(P = 0.001). An indicated departure from linear trend is
 

observed due to the steep increase of incidences of tumors in the
 

dosed groups. The Fisher exact test shows that the incidence in
 

each dosed group is significantly higher than that in the matched
 

controls (P less than 0.001). The statistical conclusion is that
 

the incidence of fibromas of the subcutaneous tissue in the
 

integumentary system of male rats is associated with the
 

administration of o-toluidine hydrochloride. The results of the
 

statistical tests for the incidence of this tumor in female rats
 

are not significant.
 

In male rats, the results of the Cochran-Armitage test for the
 

incidences of sarcomas of multiple organs and of fibrosarcomas of
 

multiple organs are significant (P = 0.003 and P less than 0.001,
 

respectively). The results of the Fisher exact test show that
 

the incidences in the high-dose group are significantly higher
 

(sarcomas: P = 0.016; fibrosarcomas: P less than 0.001) than
 

those in the controls. When the incidences of male rats with
 

sarcomas, fibrosarcomas, angiosarcomas, or osteosarcomas of
 

multiple organs are combined, the results of the Cochran-Armitage
 

test and those of the Fisher exact test are significant (P less
 

30
 
than or equal to 0.003). The statistical conclusion is that the
 

incidences of these tumors of multiple organs in male rats are
 

associated with the administration of o-toluidine hydrochloride.
 

In female rats, the results of the Cochran-Armitage test of the
 

incidence of animals with osteosarcomas of multiple organs and
 

also the incidence of animals with sarcomas, fibrosarcomas,
 

angiosarcomas, and osteosarcomas of multiple organs are
 

significant (P less than 0.001). Departures from linear trend
 

are observed (osteosarcoma: P = 0.008; combined incidence: P =
 

0.042) due to the steep increases in incidences of tumors in the
 

high-dose groups. The results of the Fisher exact test show that
 

the incidences of either of these tumors in the high-dose groups
 

are significantly higher (P less than or equal to 0.001) than
 

those of the controls. The statistical conclusion is that these
 

tumors of multiple organs in female rats are associated with
 

o-toluidine hydrochloride.
 

In male rats, the incidence of fibromas of the spleen in the
 

low-dose group is significantly higher (P = 0.024) than that in
 

the control group; the incidence in the high-dose group is not
 

significant. The result of the Cochran-Armitage for the incidence of this tumor is not significant.
 

31
 
In females, the result of the Cochran-Armitage test for the
 

incidence of angiosarcomas of the spleen is significant (P =
 

0.045). The Fisher exact test comparing the incidences of this
 

tumor in the high-dose group with the control group indicates a P
 

value of 0.036, which is above the 0.025 level required for
 

significance when the Bonferroni inequality criterion is used for
 

multiple comparison. However, when incidences of female rats
 

with sarcomas, angiosarcomas, or osteosarcomas in the spleen are
 

combined for analysis, the results of the Cochran-Armitage test
 

are significant (P = 0.018). The results of the Fisher exact
 

test comparing the combined incidence in the low-dose and control
 

groups indicate a P value of 0.036, which is above the 0.025
 

level required for significance when the Bonferroni inequality
 

criterion is used for multiple comparison, but the Fisher exact
 

test comparing the combined incidence in the high-dose and
 

control groups is significant (P = 0.010). The data in this
 

paragraph indicate that the incidence of those tumors at multiple
 

sites is significant.
 

In female rats, the results of the Cochran-Armitage test for the
 

incidence of transitional-cell carcinomas of the urinary bladder
 

or the combined incidence of transitional-cell carcinomas and
 

papillomas of the urinary bladder are significant (P less than
 

0.001). The results of the Fisher exact test comparing the
 

32
 
incidences of the dosed groups with that of the control group are
 

all significant (P less than 0.001 in the high-dose group for
 

both cases, P = 0.028 in the low-dose group for the incidence of
 

carcinomas, and P = 0.018 in the low-dose group for the combined
 

incidence). The statistical conclusion is that the incidence of
 

transitional-cell tumors of the urinary bladder in female rats is
 

dose-related to the administration of o-toluidine hydrochloride.
 

The results of the statistical tests for transitional-cell tumors
 

of the urinary bladder in male rats are not significant.
 

In female rats, the results of the Cochran-Armitage test for the
 

incidence of fibroadenomas of the mammary gland and the combined
 

incidence of fibroadenomas and adenomas of the mammary gland are
 

significant (P less than 0.001 and P = 0.001, respectively). The
 

Fisher exact test shows that the incidences in the high-dose
 

groups are significantly higher than those in their respective
 

controls (fibroadenomas: P = 0.002; adenomas and fibroadenomas:
 

P = 0.006). The statistical conclusion is that the incidence of
 

these mammary gland tumors in female rats is associated with the
 

administration of o-toluidine hydrochloride.
 

In male rats, the results of the Fisher exact test on the
 

incidence of mesotheliomas of multiple organs or of the tunica
 

vaginalis show a P value of 0.001 when the incidence in the
 

33
 
low-dose group is compared with that in the control group and a P
 

value of 0.036 when the incidence in the high-dose group is
 

compared with that in the control group. The latter is above the
 

0.025 level required for significance when the Bonferroni
 

inequality criterion is used for multiple comparison. The
 

historical records of this laboratory show an incidence of
 

mesotheliomas in male F344 rats of only 1/285 (0.4%). Using this
 

rate as a parameter (Fears, 1977) and assuming a binomial
 

distribution, the probability of the chance occurrence of nine or
 

more animals bearing these tumors in the high-dose group of 49
 

animals is less than 0.001.
 

Several significant incidences in the negative direction are
 

observed in each sex. This may have been because the dosed
 

animals did not survive as long as the control animals,
 

particulary after week 60 in male rats and after week 75 in
 

female rats.
 

In summary, the following tumors were related to the
 

administration of o-toluidine hydrochloride:
 

• Sarcomas of multiple organs in each sex,
 

• Fibromas of the subcutaneous tissue in male rats,
 

34
 
•	 Sarcomas of the spleen in female rats,
 

•	 Transitional-cell carcinomas of the urinary bladder in
 

female rats,
 

•	 Fibroadenomas or adenomas of the mammary gland in female
 

rats, and
 

•	 Mesotheliomas in multiple organs or the tunica vaginalis in
 

male rats.
 

35
 
36

 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of both dosed male and dosed female mice were
 

lower than those of corresponding controls, particularly after
 

week 18, and were dose related (figure 3). No other clinical
 

signs could clearly be related to administration of the test
 

chemical. Some fluctuations in the growth curves may be due to
 

mortality; as the size of a group diminishes, the mean body
 

weight may be subject to variation.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered o-toluidine
 

hydrochloride in the diet at the doses of this bioassay, together
 

with those of the matched controls, are shown in figure 4. The
 

result of the Tarone test for dose-related trend in mortality is
 

not significant in either sex.
 

37
 
00 8 

A A A 

D 

O D 

a a 

OO 

AAAA 

O 

A 

D 

O 

A 

8

A 

a 

D

O

A

MALE MICE 

 MATCHED CONTROL 

 LOW DOSE 

 HIGH DOSE 

40 SO 60 70 

TIME ON STUDY (WEEKS) 

5 40 

O 

>.

Q 

O 

m 

 30 

A

D 

A 

0 0 A fl 

8 FEMALE MICE 

D

O

A

 MATCHED CONTROL 

 LOW DOSE 

 HIGH DOSE 

0 10 20 30 40 50 60 70 80 90 100

TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Administered o-Toluidine Hydrochloride in the Diet 

 110 

38 
PROBABILITY O

F SURVIVAL PROBABILITY OF SURVIVAL 

p p o p p 

S § § 

8 S S S S 

1

1

11 re

1 P*

• 11 Crt

 <

01­

I

r r-

3 

r>B

! i s m« c

i 

FEMALE MIC 

11111

) LOW DOSE 

|

u\1

O 

w . 

23

; m 

fa

11 c

1 r

1

s'

111

1 U1 m

C 

t o

O

M •*.

LO 

en 

\ o-Toluidin

0 7 

JDY (WEEKS) 

cV

J

0 7 JDY (WEEKS) 

<" re<X•

1 ^

 

1n"

9i

J 

o §• 

0

j

CO

0J

 

51

re

0J

••>•

5' g?^

 

r*

3-

[ c

 >

S "

re" F* 

0

?3 • 
In male mice, 34/50 (68%) of the high-dose group, 43/50 (86%) of
 

the low-dose group, and 15/20 (75%) of the control group lived to
 

the end of the study. In females, 43/50 (86%) of the high-dose
 

group, 39/50 (78%) of the low-dose group, and 19/20 (95%) of the
 

control group lived to the end of the study.
 

Sufficient numbers of mice of each sex were at risk for the
 

development of late-appearing tumors.
 

C. Pathology (Mice)
 

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.
 

The most common neoplasms encountered were those involving the
 

liver (hepatocellular carcinoma and hepatocellular adenoma).
 

Incidence was slightly higher in the low-dose males than in the
 

control males and considerably higher in the dosed females than
 

in the control females. Distribution was as follows:
 

40
 
Control Low-Dose High-Dose
 

Male Female Male Female Male Female
 

Hepatocellular
 

Carcinoma 4(21%) 0 16(32%) 2(4%) 11(22%) 7(14%)
 

Hepatocellular
 

Adenoma 1(5%) 0 3(6%) 2(4%) 3(6%) 6(12%)
 

One hepatocellular carcinoma from a control male mouse and
 

another from a low-dose male mouse metastasized to the lungs.
 

The hepatocellular carcinomas had a wide morphologic spectrum
 

varying from relatively well to very poorly differentiated. Some
 

were small, unencapsulated, invasive nodules usually with increased basophilic staining; others were large masses involving
 

large portions of the liver. Some appeared as solid masses while
 

others formed cords several cell layers thick and were separated
 

by spaces that, in sections, contained only a small amount of
 

blood. All the tumors had increased mitotic figures.
 

The tumors classified as hepatocellular adenomas were single
 

nodules of normal to slightly basophilic-staining hepatic cells;
 

there was slight to moderate compression of the surrounding
 

hepatic parenchyma, and the hepatic architecture within the
 

tumors was distorted.
 

41
 
Some mice of each sex in both the dosed and control groups had
 

malignant lymphoma. In some instances, the tumor was confined to
 

one organ or location; in others, the tumor was widespread,
 

involving most organs and tissues in both the thoracic and
 

abdominal cavities. The incidence did not appear unusual for
 

this strain of mouse, and there was no apparent dose relation to
 

the incidence of the tumor.
 

There was an unusually high incidence of vascular tumors
 

(hemangiosarcoma, hemangioma) in high-dose male mice.
 

Distribution was as follows:
 

Control Low-Dose High-Dose
 

Male Female Male Female Male Female
 

Hemangiosarcoma 1 1 1 1 1 0 2
 

Hemangioma 1 0 1 0 2 1
 

A single control male had a hemangioma in the spleen and a
 

hemangiosarcoma in the liver. The hemangiosarcoma in the one
 

control female was located in the inguinal subcutis. One of the
 

two tumors in the low-dose males was in a mesenteric lymph node;
 

the other, in the spleen. The single tumor in a low-dose female
 

was in the periuterine fat.
 

42
 
All vascular tumors that occurred in high-dose male mice were in
 

the area of the abdominal genital organs; most were described
 

grossly as involving the periepididymal fat. Two of those in
 

high-dose females were in periuterine fat; the third was in the
 

liver.
 

Exact location of some of these tumors could not be determined
 

either by gross description or histologic examination. They
 

rarely involved the genital organs directly, but appeared to be
 

localized in the perigenital fat. No evidence of metastases was
 

found.
 

Hemangiosarcomas were characterized by poorly encapsulated or
 

nonencapsulated masses of basophilic spindle-shaped cells with no
 

particular histologic pattern. There were variably sized
 

vascular spaces separating the neoplastic cells within the tumor
 

mass. Mitotic figures were common in the tumor cells.
 

Hemangiomas were moderately well encapsulated masses usually
 

composed of a number of nearly equal-sized vascular spaces that
 

were lined with plump endothelial cells. Some of the spaces were
 

separated by sclerotic connective tissue.
 

43
 
Some of the vascular tumors contained large blood-filled cysts.
 

Two high-dose male mice had lesions classified as hematocysts in
 

the periepididymal fat. These were single blood-filled cysts
 

surrounded by connective and granulation tissue with no evidence
 

of neoplasia. The hematocysts may be related to the vascular
 

tumors, but evidence for such a relation is not evident on the
 

tissue sections.
 

In addition to the proliferative and neoplastic lesions just
 

described, there was a scattering of other tumors that were of
 

single occurrence or very low incidence. All were tumors that
 

may be expected in this strain of mouse and, therefore, are not
 

considered to be related to administration of the test chemical.
 

In addition to the proliferative lesions, there were a few
 

inflammatory and degenerative changes in some mice in each of the
 

groups. Incidences of these lesions appeared to be approximately
 

equal in control and dosed groups; none were considered to be
 

related to the test chemical.
 

In conclusion, o-toluidine hydrochloride is considered to be
 

carcinogenic in male and female B6C3F1 mice. This is based on
 

the increased incidences of hepatocellular neoplasms in female
 

mice and hemangiosarcomas in the periepididymal fat in high-dose
 

44
 
males and the unusual, although low, incidence of hemangiosarcomas
 

in the periuterine fat of females exposed to the compound.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals of one group and at an incidence of at least 5%
 

in one or more than one group.
 

In male mice, the results of the Cochran-Armitage test for the
 

incidence of hemangiosarcomas of all sites or the combined
 

incidence of hemangiomas and hemangiosarcomas of all sites are
 

significant (P = 0.004 and P = 0.002, respectively), but the
 

results of the Fisher exact test are not significant. The
 

historical records at this laboratory show an incidence of
 

hemangiosarcomas of 13/323 (4%) in the male control mice. Using
 

this rate as a parameter (Fears, 1977) and assuming a binomial
 

distribution, the probability level of obtaining 10 or more such
 

tumors, given a total of 50 animals, is less than 0.001. The
 

incidence of hemangioma and hemangiosarcoma combined is 17/323
 

(5%). Using this rate as a parameter and assuming a binomial
 

45
 
distribution, the probability level of obtaining 12 or more such
 

tumors, given a total of 50 animals, is less than 0.001
 

In female mice, when the animals with either hepatocellular
 

carcinomas or adenomas are combined for analysis, the result of
 

the Cochran-Armitage test for positive dose-related trend is
 

significant (P = 0.001). The Fisher exact test shows that the
 

incidence in the high-dose group is significantly higher (P =
 

0.007) than that in the control group. The statistical
 

conclusion is that the incidence of tumors of the liver in female
 

mice is associated with the administration of o-tuluidine hydrochloride. The current historical-control groups of female mice
 

at this laboratory have incidences of hepatocellular tumors
 

ranging from 0/20 to 2/19 (11%) with an overall incidence of
 

14/324 (4.3%). The results of the statistical tests on the
 

incidences of hepatocellular tumors in male mice are not
 

significant.
 

Several significant results in the negative direction are
 

observed in each sex in which the incidence in the control group
 

exceeds those in the dosed groups. They are: alveolar/bronchiolar tumors in male mice, lipoma of the mesentery in male
 

mice, and follicular-cell adenoma in female mice.
 

46
 
V. DISCUSSION
 

For rats and mice at the doses used in this bioassay there were
 

dose-related depressions in the amount of body weight gained in
 

both species and a dose-related increase in mortality in the
 

rats. Mortality in the male and female rats was dose related and
 

was relatively high at the end of the bioassay. Mortality in the
 

mice was unaffected by the administration of the test chemical.
 

Other clinical signs occurred only at low frequencies and were
 

common to dosed and control groups of animals.
 

In preliminary investigations on the carcinogenicity of
 

o-toluidine hydrochloride administered in the diet in 2-year
 

studies, urinary bladder cancers and subcutaneous fibromas or
 

fibrosarcomas were induced at significant incidences in male
 

Sprague-Dawley rats, and vascular tumors were induced at significant incidences in male and female Ha/ICR mice (Homburger et
 

al., 1972; Russfield et al., 1973a; Russfield et al., 1973b). A
 

report in press specifies the doses used in the preceding studies
 

as 8,000 and 16,000 ppm for 3 months and 4,000 and 8,000 ppm for
 

the subsequent 15 months for the rats, 16,000 and 32,000 ppm for
 

3 months and 8,000 and 16,000 ppm for the subsequent 15 months
 

for the mice (Weisburger et al., in press). In concurrent
 

47
 
studies, the m-toluidine and p-toluidine isomers only induced
 

liver tumors in the mice. The induction of bladder tumors in
 

rats administered aromatic azo compounds in the diet has been
 

attributed to toluidines generated as primary decompositon
 

products of the test compounds (Ekman and Strombeck, 1947,
 

1949). o-Toluidine has been reported to be nonmutagenic in the
 

Salmonella/microsome test (McCann et al., 1975; Ferretti et al.,
 

1977).
 

In the present study, the administration of the test chemical to
 

rats induced several types of sarcomas of the spleen and other
 

organs in both males and females, mesotheliomas of the abdominal
 

cavity or scrotum in males, and transitional-cell carcinomas of
 

the urinary bladder in females. Administration of the
 

o-toluidine hydrochloride also resulted in increased incidences
 

of fibromas of the subcutaneous tissue in the males and
 

fibroadenomas or adenomas of the mammary gland in the females.
 

In mice, hemangiosarcomas were induced at various sites in males,
 

and hepatocellular carcinomas or adenomas were induced in females.
 

The findings are consistent with those previously published but
 

showed, in addition, the induction of mesotheliomas in male rats,
 

fibroadenomas and adenomas of the mammary gland in female rats,
 

48
 
several types of sarcomas in both male and female rats, and
 

hepatocellular carcinomas or adenomas in female mice.
 

Under the conditions of this bioassay, o-toluidine hydrochloride
 

was carcinogenic in both male and female F344 rats and B6C3F1
 

mice, producing a significant increased incidence of one or more
 

types of neoplasms.
 

49
 
50

 
VI. BIBLIOGRAPHY
 

Armitage, P., Statistical Methods in Medical Research, John Wiley
 

& Sons, Inc., New York, 1971, pp. 362-365.
 

Berenblum, I., ed., Carcinogenicity Testing; A Report £f the
 

Panel on Carcinogenicity of the Cancer Research Commission of the
 

UICC, Vol. 2_. International Union Against Cancer, Geneva, 1969.
 

Cox, D.R., Regression models and life tables. £. R. Statist.
 

Soc. B 34:187-220, 1972.
 

Cox, D. R., Analysis of Binary Data, Methuen & Co., Ltd. London,
 

1970, pp. 48-52.
 

Ekman, B. and Strbmbeck, J. P., The effect of some splitproducts
 

of 2,3'-azotoluene on the urinary bladder in the rat and their
 

excretion on various diets. Acta Path. Microbiol. Scand. 26;
 

447-467, 1949.
 

Ekman, B. and Strombeck, J. P. , Demonstration of tumorigenic
 

decomposition products of 2,3-azotoluene. Acta Physiol. Scand.
 

14:43-50, 1947.
 

Fears, T. R., Tarone, R. E. and Chu, K. C., False-Positive and
 

False-Negative Rates for Carcinogenicity Screens. Cancer Res.
 

37:1941-1945, 1977.
 

Ferretti, J. J., Lu, W., and Liu, M. B., Mutagenicity of
 

benzidine and related compounds employed in the detection of
 

hemoglobin. Am. J. Clin. Pathol. 6X6) :526-527, 1977.
 

Gart, J. J., The comparison of proportions: a review of
 

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Stat. Inst. 3£: 148-169, 1971.
 

Hamblin, D. 0., Aromatic nitro and amino compounds. In:
 

Industrial Hygiene and Toxicology, Vol. 2^, Patty, F. A., ed.,
 

Interscience Publishers, New York, 1963, p. 2123.
 

Homburger, F., Friedell, G. H., Weisburger, E. K., and
 

Weisburger, J. H., Carcinogenicity of simple aromatic amine
 

derivatives in mice and rats. Toxicol. Appl. Pharmacol. 22(2);
 

280, 1972.
 

51
 
Indriksons, A., Hazards of o-toluidine. Clin. Chem. 21(9):1345,
 

1975.
 

International Agency for Research on Cancer, Some aromatic
 

amines, hydrazine and related substances, N-nitroso compounds and
 

miscellaneous alkylating agents. IARC Monographs on the
 

Evaluation of Carcinogenic Risk of_ Chemicals to^ Man, Vol. 4>,
 

International Agency for Research on Cancer, Lyon, France, 1974,
 

pp. 31-39.
 

Jacobson, K. H., Acute oral toxicity of mono- and di-alkyl
 

ring-substituted derivatives of aniline. Toxicol. Appl.
 

Pharmacol. 2^:153-154, 1972.
 

Kaplan, E. L. and Meier, P., Nonparametric estimation from
 

incomplete observations. J. Am. Statist. Assoc. 53^457-481, 1958.
 

Lindstrom, H. V., Bowie, W. C., Wallace, W. C., Nelson, A. A.,
 

and Fitzhugh, 0. G., The toxicity and metabolism of mesidine and
 

pseudocumidine in rats. J. Pharmacol. Exptl Therap. 167(2);
 

223-234, 1969. ~
 

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

Peters, J. A., Carcinogenesis bioassay data system. Comp. and
 

Biomed. Res. 7^:230-248, 1974.
 

Lunkin, V. N., Information for the hygienic establishment of the
 

maximum allowable concentration of para- and ortho- toluidines in
 

inland waters. Cited in: IARC Monographs on the Evaluation £f
 

Carcinogenic Risk £f Chemicals to_ Man, Vol. 4_, International
 

Agency for Research on Cancer, Lyon, France, 1974.
 

McCann, J., Choi, E., Yamasaki, E., and Ames, B.N., Detection of
 

carcinogens as mutagens in the SaImone1la/microsome test: assay
 

of 300 chemicals. Proc. Nat. Acad. Sci., U.S.A. 72(12);
 

5135-5139, 1975.
 

Miller, R., G., Jr., Simultaneous Statistical Inference,
 

McGraw-Hill Book Co., New York, 1966, pp. 6-10.
 

Nomura, A., Studies on sulfhemoglobin formation by various
 

drugs. Nippon Yakurigaku Zasshi 73(4);423-435, 1977.
 

Russfield, A. B., Homburger, F., Weisburger, E. K., and
 

Weisburger, J. H., Further studies on carcinogenicity of
 

environmental chemicals including simple aromatic amines.
 

Toxicol. Appl. Pharmacol. 25:446, 1973a.
 

52
 
Russfield, A. B., Boger, E., Homburger, F., Weisburger, E. K.,
 

and Weisburger, J. H., Effect of structure of seven methyl
 

anilines on toxicity and on incidence of subcutaneous and liver
 

tumors in Charles River rats. Fed. Proc. 3>2(3):3470, 1973b.
 

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

Kaufman, D. G., Respiratory tract carcinogenesis in hamsters
 

induced by different numbers of administrations of benzo(a)
 

pyrene and ferric oxide. Cancer Res. _32:1073-1081, 1972.
 

Society of Dyers and Colourists, The Colour Index, Dean House,
 

Yorkshire, England, 1971, pp. 4856-4857.
 

Son, 0. S., Weiss, L., Fiala, E. S., and Weisburger, E. K.,
 

Metabolism of the carcinogen o-toluidine. Proc. Am. Assoc.
 

Cancer Res. ^123, 1977.
 

Stanford Research Institute, o-Toluidine, Stage II, Chemical
 

Dossier, Stanford Research Institute, Menlo Park, Calif., April,
 

1976.
 

Tarone, R. E., Tests for trend in life table analysis.
 

Biometrika 62:679-682, 1975.
 

United States International Trade Commission, Synthetic Organic
 

Chemicals - United States Production and Sales,1976,USITC
 

Publication 833, U.S. Government Printing Office, Washington,
 

D.C., 1977.
 

Weisburger, E. K., Russfield, A. B., Weisburger, J. H., Boger,
 

E., Van Dongen, C. G., and Chu, K. C., Testing of 21
 

environmental aromatic amines or derivatives for long-term
 

toxicity or carcinogenicity (in press), National Cancer
 

Institute, Bethesda, Md.
 

53
 
54

 
APPENDIX A 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

RATS ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

55 
56

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALL Y IN STUDY 20 50 50
 

ANIMALS MISSING 1
 

ANIMALS NECROPSIED 20 50 49
 

ANIMAL S EXAMINED HISTOPATHOLOGICAL L Y 20 50 49
 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50) (49)
 

SQUAMOUS CELL CARCINOMA 2 (4%)
 

*SUBCUT TISSUE (20) (50) (49)
 

SARCOMA , NOS 1 (2%)
 

FIBROMA 28 (56%) 27 (55%)
 

FIBROSARCOMA 1 (2%) 2 (4%)
 

MYXOSARCOMA 2 (4%)
 

LIPOMA ft (8%) 2 (4%)
 

OSTEOSARCOMA 1 (2%)
 

RESPIRATORY SYSTEM
 

#LUNG (20) (50) (48)
 

ALVEOLAR/BRONCHIOLA R CARCINOMA 2 (4%) 2 (4%)
 

SARCOMA, NOS, METASTATIC 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

^MULTIPLE ORGANS (20) (50) (49)
 

MALIGNAN T LYMPHOMA, NOS 1 (5%)
 

MALIG. LYMPHOMA, UNDI FFER-TYPE 3 (15%) 2 (4%)
 

tSPLEEN (20) (49) (42)
 

SARCOMA, NOS 1 (2%) 3 (7%)
 

FIBROM A 10 (20%) 2 ( 5 % )
 

LIPOMA 1 ( 2 % )
 

ANGIOM A 1 ( 2 % )
 

ANGIOSARCOMA 7 (14%)
 

OSTEOSARCOMA 1 ( 2 % 1
 

#RENA L LYMPH NODE (20) (50) (47)
 

INTERSTITIAL-CELL TUMOR, METASTA 1 (5%)
 

# NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBE R OF ANIMAL S NECROPSIED
 

57
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

CIRCULATOR Y SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVE R (20) (50)
 

NEOPLASTIC NODULE 1 (5%)
 

HEPATOCELLULA R CARCINOMA 1 ( 2 % )
 

FIBROSARCOM A 1 (2%)
 

#PANCREAS (20) (47)
 

ACINAR-CEL L CARCINOM A
 

tLARG E INTESTINE (20) (47)
 

ADENOMATOUS POLYP, NOS 1 (2%)
 

URINAR Y SYSTEM
 

f KIDNEY (20) (50)
 

TUBULAR-CEL L ADENOCARCINOMA 1 (2%)
 

#KIDNEY/PELVIS (20) (50)
 

TRANSITIONAL-CELL CARCINOMA 1 (2%)
 

#URINAR Y BLADDER (20) (50)
 

SQUAMOUS CEL L CARCINOM A 1 (2%)
 

TRANSITIONAL-CEL L PAPILLOM A
 

TRANSITIONAL-CELL CARCINOM A 3 ( 6 % )
 

ANGIOMA
 

ANGIOSARCOMA 1 (2%)
 

ENDOCRIN E SYSTEM
 

#PITUITARY (20) (50)
 

ADENOMA, NOS 4 (20%) 7 (14%)
 

#ADRENA L (20) (50)
 

PHEOCHROMOCYTOMA 3 (6%)
 

tTHYROID (20) (50)
 

FOLLICULAR-CEL L ADENOMA 1 (2%)
 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMALS NECROPSIED
 

HIGH DOSE 

(49)
 

2 (4%)
 

1 ( 2 % )
 

(41 )
 

1 (2%)
 

(46)
 

(49)
 

(49)
 

(44)
 

1 (2%)
 

1 (2%)
 

(44)
 

3 (7%)
 

(49)
 

2 (4%)
 

(45)
 

58
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

C-CELL ADENOMA 1 (5%) 2 (4%) 2 (4%)
 

C-CELL CARCINOMA 2 (10%)
 

tPANCREATIC ISLETS (20) (47) (41)
 

ISLET-CELL ADENOMA 1 (2%)
 

REPRODUCTIVE SYSTEM
 

XMAMMAR Y GLAND (20) (50) (49)
 

FIBROADENOMA 7 (14%) 1 (2%)
 

*PREPUTIAL GLAND (20) (50) (49)
 

ADENOMA, NOS 1 (2%)
 

#TESTIS (20) (50) (49)
 

INTERSTITIAL-CELL TUMOR 16 (80%) 41 (52%) 33 (67%)
 

INTERSTITIAL-CELL TUMOR, MALIGN A 1 (5%)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETA L SYSTEM
 

NONE
 

BODY CAVITIES
 

*TUNICA VAGINALI S (20) (50) (49)
 

MESOTHELIOMA , NOS 2 (4%)
 

MESOTHELIOMA , MALIGNAN T 10 (20%) 6 (12%)
 

A LL OTHER SYSTEMS
 

^MULTIPLE ORGANS (20) (50) (49)
 

SARCOMA , NOS 3 ( 6 % ) 11 (22%)
 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBE R OF ANIMAL S NECROPSIED
 

59
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

FIBROM A
 

FIBROSARCOM A
 

MESOTHELIOMA , NOS
 

MESOTHELIOMA , MALIGNAN T
 

ANGIOSARCOM A
 

OSTEOSARCOMA
 

ANIMA L DISPOSITION SUMMAR Y
 

ANIMALS INITIALLY IN STUDY
 

NATURA L DEATHS
 

MORIBUN D SACRIFIC E
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINA L SACRIFIC E
 

ANIMA L MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y
 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS*


TOTAL PRIMAR Y TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMAL S WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORSt


TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNAN T


TOTAL UNCERTAIN TUMORS


TOTAL ANIMAL S WITH TUMORS UNCERTAINPRIMAR Y OR METASTATIC
 

TOTAL UNCERTAI N TUMORS
 

20
 

1 8
 

 20


 29


 18


 21


 6


 7


 1


 1


 1


 1


1 (2%)
 

8 (16%) 20 ( 4 1 % )
 

1 (2%)
 

^ (8%) 3 ( 6 % )
 

1 ( 2 % ) 1 (2%)
 

3 (6%) 5 t 1 0% )
 

50 50
 

32
 

5
 

1 3
 

 50 48
 

 167 134
 

 43 42
 

 108 75
 

 42 47
 

 56 57
 

1
 

1
 

3 2
 

3 2
 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

t SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

60
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALL Y IN STUDY 20 50 50
 

ANIMAL S MISSING 1
 

ANIMAL S NECROPSIED 20 50 49
 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY 20 49 49
 

INTEGUMENTAR Y SYSTEM
 

*SUBCUT TISSUE (20) (50) (49)
 

FIBROMA 4 (8%) 2 (4%)
 

RESPIRATOR Y SYSTEM
 

#LUNG (20) (48) (49)
 

CARCINOMA , NOS, METASTATIC 1 (2%)
 

ALVEOLAR/BRONCHIOLA R CARCINOM A 1 (5%)
 

C-CELL CARCINOMA , METASTATIC 1 (5%)
 

PHEOCHROMOCYTOMA, METASTATIC 1 (2%)
 

ANGIOSARCOMA , METASTATIC 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

^MULTIPLE ORGANS (20) (50) (49)
 

MALIGNAN T LYMPHOMA , NOS 1 (5%)
 

MALIG.LYMPHOMA , UNO IFFER-TYPE 3 ( 6 %)
 

#SPLEEN (20) (49) (49)
 

SARCOMA , NOS 1 (2%) 3 (6%)
 

FIBROM A 4 (8%) i 6 (12%)
 

ANGIOSARCOMA 7 (14%) 9 (18%)
 

OSTEOSARCOMA 1 ( 2 % ) 1 (2%)
 

CIRCULATOR Y SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVE R (20) (49) (49)
 

NEOPLASTIC NODULE 2 (4%)
 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECROPSIED
 

61
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE
 

#STOMACH (20) (48)
 

SQUAMOUS CELL PAPILLOMA 1 (2%)
 

tLARG E INTESTINE (19) (44)
 

ANGI05ARCOMA 1 (2%)
 

URINAR Y SYSTEM
 

#KIDNEY/PELVIS (20) (49)
 

TRANSITIONAL-CELL CARCINOMA
 

tURINAR Y BLADDER (20) (45)
 

TRANSITIONAL-CEL L PAPILLOMA 1 (2%)
 

TRANSITIONAL-CEL L CARCINOMA 9 (20%)
 

ENDOCRINE SYSTEM 

#PITUITARY 

CARCINOMA , NOS 

ADENOMA, NOS 

(20) 

5 (25%) 

(47) 

1 (2%) 

12 (26%) 

#ADRENA L 

PHEOCHROMOCYTOMA 

PHEOCHROMOCYTOMA, MALIGNANT 

(20) (49) 

4 (8%) 

#THYROID 

FOLLICULAR-CELL ADENOMA 

C-CELL ADENOMA 

C-CELL CARCINOM A 

(20) 

1 (5%) 

(47) 

1 (2%) 

3 (6%) 

3 ( 6 «) 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND (20) (50)
 

ADENOMA , NOS 1 (5%)
 

ADENOCARCINOMA , NOS
 

FIBROADENOM A 6 (30%) 20 (40%)
 

*CLITORAL GLAND (20) (50)
 

ADENOMA, NOS 1 (2%)
 

tUTERUS (20) (49)
 

ENDOMETRIA L STROMAL POLYP 5 (25%) 4 (8%)
 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBE R OF ANIMAL S NECROPSIED
 

HIGH DOSE
 

(49)
 

1 (2%)
 

(42)
 

(49)
 

1 (2%)
 

(47)
 

22 (47X)
 

(46)
 

2 (4%)
 

(49)
 

1 (2%)
 

1 (2%)
 

(46)
 

1 (2%)
 

(49)
 

1 ( 2 % )
 

35 (71%)
 

(49)
 

1 (2%)
 

(48)
 

1 (2«>
 

62
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANGIOSARCOMA 1 (2%)
 

NERVOUS SYSTEM
 

#BRAIN (20) (48) (49)
 

CARCINOMA, NOS, INVASIVE 1 (2%)
 

GLIOMA, NOS 1 (2%)
 

SPECIAL SENSE ORGANS
 

*ZYMBAL'S GLAND (20) (50) (49)
 

SQUAMOUS CELL CARCINOMA 1 (2%)
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

^ABDOMINAL CAVITY (20) (50) (49)
 

SARCOMA, NOS 1 (2%)
 

^MESENTERY (20) (50) (49)
 

SARCOMA, NOS 1 (2%)
 

A LL OTHER SYSTEMS
 

^MULTIPLE ORGANS (20) (50) (49)
 

SARCOMA, NOS 1 (2%) 2 (4«)
 

FIBROSARCOMA 1 (2%)
 

ANGIOSARCOMA 1 (2%) 1 (2%)
 

OSTEOSARCOMA 1 8 (37%)
 

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

63
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

ANIMA L DISPOSITION SUMMARY
 

ANIMAL S INITIALL Y IN STUDY
 

NATURA L DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALL Y KILLE D
 

TERMINA L SACRIFICE
 

ANIMA L MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS*
 

TOTAL PRIMAR Y TUMORS
 

TOTAL ANIMAL S WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMAL S WITH MALIGNANT TUMORS
 

TOTAL MALIGNAN T TUMORS
 

TOTAL ANIMAL S WITH SECONDARY TUMORS*


TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNAN T


TOTAL UNCERTAI N TUMORS


TOTAL ANIMAL S WITH TUMORS UNCERTAINPRIMAR Y OR METASTATIC
 

TOTAL UNCERTAI N TUMORS
 

MATCHED
 

CONTROL


20


2


2


16


1 3


20


1 3


1 7


3


3


 1


 1


 LOW DOSE HIGH DOSE 

 50 50 

 18 35 

4 3 

1 

 28 1 0 

1 

 43 48 

 89 1 1 1 

 35 38 

 55 50 

 26 45 

 32 61 

3 1 

3 1 

2 

2 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIV E INTO AN ADJACENT ORGAN
 

64
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADIMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET
 

65
 
66

 
TABLE B1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE
 

ANIMALS INITIALL Y IN STUDY 20 50
 

ANIMAL S MISSING 1
 

ANIMAL S NECROPSIED 1 9 50
 

ANIMALS EXAMINED H ISTOPATHOIOGICAL LY 1 9 50
 

INTEGUMENTAR Y SYSTEM
 

*SUBCUT TISSUE (19) (50)
 

SARCOMA, NOS 1 (2%)
 

RESPIRATOR Y SYSTEM
 

#LUNG (19) (50)
 

HEPATOCELLULA R CARCINOMA , METAST 1 (5%) 1 (2%)
 

ALVEOLAR/BRONCHIOLA R ADENOMA 2 (11%) 1 (2%)
 

ALVEOLAR/BRONCHIOLA R CARCINOMA 3 (16%) 4 (8%)
 

HEMATOPOIETIC SYSTEM 

^MULTIPLE ORGANS 

MALIGNAN T LYMPHOMA, NOS 

MALIG . LYMPHOMA, HISTIOCYTIC TYPE 

(19) 

1 (5%) 

( 50) 

6 (12%) 

1 (2%) 

#SPLEEN 

HEMANGIOM A 

HEMANGIOSARCOM A 

MALIGNAN T LYMPHOMA, NOS 

(19) 

1 (5%) 

(49) 

1 (2%) 

#ABDOMINAL LYMPH NODE 

HEMANGIOSARCOM A 

(18) (48) 

#MESENTERIC L. NODE 

HEMANGIOM A 

MALIGNAN T LYMPHOMA , NOS 

(18) (48) 

1 

1 

(2%) 

(2%) 

tSMALL INTESTINE 

MALIGNAN T LYMPHOMA , NOS 

(17) (47) 

1 (2%) 

CIRCULATOR Y SYSTEM
 

NONE
 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECROPSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

(50)
 

2 (4%)
 

1 ( 2 % )
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

(48)
 

1 (2%)
 

(48)
 

(48)
 

2 (4%)
 

67
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIVE SYSTEM
 

#LIVE R (19) (50) (50)
 

ADENOMA , NOS 1 (5%)
 

HEPATOCELLULA R ADENOMA 1 (5%) 3 (6%) 3 (6%)
 

HEPATOCELLULAR CARCINOMA 4 (21%) 16 (32%) 11 (22%)
 

HEMANGIOSARCOM A 1 (5%)
 

tESOPHAGUS (17) (50) (50)
 

SQUAMOUS CEL L CARCINOMA 1 (2%)
 

#SMALL INTESTINE (17) (47) (48)
 

ADENOCARCINOMA , NOS 1 (6%)
 

ttCECUM (15) (49) (45)
 

SARCOMA , NOS 1 (2%)
 

URINAR Y SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

tPITUITAR Y (18) (46) (46)
 

ADENOCARCINOMA , NOS 1 (2%)
 

#ADRENA L (19) (50) (49 )
 

CORTICA L ADENOMA 2 (4%)
 

tTHYROID (19) (49) (48)
 

FOLLICULAR-CEL L ADENOMA 1 (5%) 1 (2%) 1 (2%)
 

FOLLICULAR-CELL CARCINOMA 1 (5%) 1 (2%) 1 (2%)
 

REPRODUCTIV E SYSTEM
 

NONE
 

NERVOU S SYSTEM
 

NONE
 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECROPSIED
 

68
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE ORGANS 

*EYE/LACRIMAL GLAND

ADENOMA , NOS

 (19)

 1 (5%)

 (50) (50) 

2 (4X) 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

XABDOMINAL CAVITY

SARCOMA, NOS

LIPOMA

HEMANGIOM A

HEMANGIOSARCOM A

 (19)

 1 (5%) 

 (50) (50) 

2 (4%) 

2 (4%) 

9 (18%) 

*MESENTERY

LIPOMA

 (19)

 2 (11%) 

 (50) (50) 

A LL OTHER SYSTEMS 

NONE 

ANIMA L DISPOSITION SUMMARY 

ANIMAL S INITIALLY IN STUDY 

NATURA L DEATH3 

MORIBUN D SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALL Y KILLE D 

TERMINA L SACRIFICE 

ANIMA L MISSING 

20 

15 

1 

50 50 

1 3 

1 

a INCLUDES AUTOLYZED ANIMAL S 

# NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY * NUMBE R OF ANIMAL S NECROPSIED 

69
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMAR Y TUMORS*


TOTAL PRIMAR Y TUMORS


TOTAL ANIMAL S WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNAN T TUMORS


TOTAL ANIMAL S WITH SECONDARY TUMORS*


TOTAL SECONDARY TUMORS


TOTAL ANIMAL S WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMAR Y OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 17 28 30 

 21 38 44 

8 6 1 5 

9 6 14 

 11 24 24 

 12 32 30 

1 1 

1 1 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

70
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALLY IN STUDY

ANIMALS MISSING

ANIMAL S NECROPSIED

ANIMAL S EXAMINED HISTOPATHOLOGICALLY

 20

 20

 20

 50

 1 

 49

 49

 50 

 50 

 50 

INTEGUMENTARY SYSTEM 

*SKIN

SARCOMA, NOS

 (20) (49) (50) 

1 (2%) 

*SUBCUT TISSUE

LIPOMA

HEMANGIOSARCOM A

NEUROFIBROSARCOM A

 (20)

 1 (5») 

1 (5K) 

 (49)

 1 (2%) 

 (50) 

RESPIRATORY SYSTEM 

#LUNG

ALVEOLAR/BRONCHIOLA R ADENOMA

ALVEOLAR/BRONCHIOLA R CARCINOMA

NEUROFIBROSARCOMA , METASTATIC

 (20)

 1 (5%)

 (48)

 1 (2%) 

1 (23;)

 1 (2«) 

 (48) 

2 (4%) 

HEMATOPOIETIC SYSTEM 

^MULTIPLE ORGANS

MALIGNAN T LYMPHOMA, NOS

MALIS.LYMPHOMA, HISTIOCYTIC TYPE 

 (20) 

1 (5V.) 

(49) 

7 (14%) 

(50) 

4 (8%) 

1 (2%) 

tSPLEEN

MALIG . LYMPHOMA, HISTIOCYTIC TYPE 

 (20) (49) (48) 

1 (2H) 

#LIVE R

MALIGNAN T LYMPHOMA, NOS 

 (20) (49) 

1 (2%) 

(50) 

#SMALL INTESTINE

MALIGNAN T LYMPHOMA, NOS

MALIG.LYMPHOMA , HISTIOCYTIC TYPE

 (20) 

2 (10X) 

1 (5%) 

(47) 

2 (4«) 

(50) 

3 ( 6 %) 

CIRCULATORY SYSTEM 

NONE 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINED

* NUMBE R OF ANIMAL S NECROPSIED 

 MICROSCOPICALLY 

71
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

DIGESTIVE SYSTEM
 

#LIVE R (20) (49) (50)
 

HEPATOCELLULAR ADENOMA 2 (4%) 6 (12%)
 

HEPATOCELLULA R CARCINOM A 2 (4%) 7 (14%)
 

HEMANGIOSARCOMA 1 ( Z %)
 

URINAR Y SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

#THYROID (20) (44) (47)
 

FOLLICULAR-CEL L ADENOMA 3 (15%)
 

REPRODUCTIV E SYSTEM
 

*MAMMAR Y GLAND (20) (49) (50)
 

ADENOCARCINOMA, NOS 1 (2%) 1 (2%)
 

tUTERUS (20) (46) (48)
 

LEIOMYOSARCOM A 2 (4%)
 

NERVOU S SYSTEM
 

#BRAIN (20) (47) (48)
 

GLIOMA , NOS 1 (5%)
 

SPECIAL SENSE ORGANS
 

*EYE/LACRIMA L GLAND (20) (49) (50)
 

ADENOMA , NOS 1(2%) 1(2% )
 

MUSCULOSKELETA L SYSTEM
 

NONE
 

# NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

72
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY (20) (49) (50)
 

NEOPLASM, NOS 1 ( 2 % )
 

HEMANGIOMA 1 ( 2 % 1
 

HEMANGIOSARCOMA 1 (2«) 1 (2%)
 

ALL OTHER SYSTEMS
 

^MULTIPLE ORGANS (20) (49) (50)
 

NEUROFIBROSARCOMA 1 (2%)
 

ADIPOSE TISSUE
 

LIPOMA 1
 

ANIMAL DISPOSITION SUMMARY
 

ANIMAL S INITIALL Y IN STUDY 20 50 50
 

NATURAL DEATHS 1 8 7
 

MORIBUN D SACRIFICE 1
 

SCHEDULED SACRIFIC E
 

ACCIDENTALLY KILLE D 1
 

TERMINA L SACRIFICE 19 39 43
 

ANIMA L MISSING 1
 

3 INCLUDES AUTOLYZED ANIMAL S
 

f NUMBE R OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECROPSIED
 

73
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

TUMOR SUMMAR Y
 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS* 8 1 9 26
 

TOTAL PRIMAR Y TUMORS 1 1 23 32
 

TOTAL ANIMAL S WITH BENIGN TUMORS 3 4 9
 

TOTAL BENIGN TUMORS ft 4 9
 

TOTAL ANIMAL S WITH MALIGNANT TUMORS 6 1 7 1 7
 

TOTAL MALIGNANT TUMORS 7 1 9 22
 

TOTAL ANIMAL S WITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNAN T 1
 

TOTAL UNCERTAI N TUMORS 1
 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAI N TUMORS
 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

74
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET
 

75
 
76

 
TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMAL S INITIALLY IN STUDY 20 50 50
 

ANIMALS MISSING 1
 

ANIMAL S NECROPSIED 20 50 49
 

ANIMALS EXAMINED H ISTOPATHOLOGICAL L Y 20 50 49
 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50) (49)
 

EPIDERMA L INCLUSION CYST 1 (2%) 1 (2%)
 

RESPIRATOR Y SYSTEM
 

fTRACHEA (20) (50) (48)
 

INFLAMMATION, ACUTE 1 (2%)
 

#LUNG (20) (50) (48)
 

HEMORRHAG E 1 (2%)
 

INFLAMMATION , INTERSTITIAL 1 (5%)
 

BRONCHOPNEUMONIA, ACUTE 1 (2%)
 

INFLAMMATION , CHRONIC 4 (20%) 1 (2%)
 

GRANULOMA , NOS 2 (4%)
 

PERIVASCULITIS 4 (8%)
 

HYPERPLASIA, ALVEOLA R EPITHELIUM 3 (15%) 1 ( 2 % ) 2 (4%)
 

*LUNG/ALVEOLI (20) (50) (48)
 

HISTIOCYTOSIS 1 (5%)
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN (20) (49) (42)
 

FIBROSIS 1 3 (27%) 5 (12%)
 

METAMORPHOSIS FATTY 6 (12%) 2 (5%)
 

LYMPHOID DEPLETION 2 (4%) 7 (17%)
 

HYPERPLASIA, MESOTHELIAL 1 ( 2 % )
 

METAPLASIA, OSSEOUS 2 (4%)
 

HEMATOPOIESIS 1 (5%) 3 (6%)
 

•SPLENIC	 CAPSULE (20) (49) (42)
 

HEMORRHAGI C CYST 1 (2%)
 

t NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

77
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

HYPERPLASIA , MESOTHELIA L 18 (37%) 5 (12%)
 

METAPLASIA , OSSEOUS 1 (2%)
 

ttMANDIBULAR L. NODE C20) (50) (47)
 

LYMPHANGIECTASIS 2 (4%)
 

ftMESENTERIC L. NODE (20) (50) (47)
 

LYMPHANGIECTASIS 1 (2%)
 

CIRCULATOR Y SYSTEM
 

#HEART C20) ( 50) (48)
 

PERIARTERITI S 1 ( 2%)
 

tMYOCARDIU M (20) (50) (48)
 

INFLAMMATION , CHRONIC 3 (15%)
 

FIBROSIS 1 7 (34%) 8 (17%)
 

*CORONAR Y ARTER Y (20) (50) (49)
 

MEDIA L CALCIFICATIO N 1 (5%) 1 (2%)
 

*PULMONAR Y ARTER Y (20) (50) (49)
 

MEDIA L CALCIFICATIO N 6 (30%) 5 (10%) 1 (2%)
 

DIGESTIVE SYSTEM
 

#LIVE R (20) (50) (49)
 

FIBROSIS 1 (2%)
 

CHOLANGIOFIBROSIS 9 (18%)
 

NECROSIS, NOS 2 ( 1 0%) 1 2 (24%) 1 7 (35%)
 

METAMORPHOSI S FATTY 1 (5%) 2 (4%) 2 (4%)
 

CYTOPLASMIC VACUOL IZAT I ON 7 (35%)
 

FOCAL CELLULA R CHANGE 2 (4%) 3 (6%)
 

HEPATOCYTOMEGAL Y 1 (5%) 1 ( 2 % )
 

ANGIECTASIS 1 (2%)
 

HEMATOPOIESIS 2 (4%)
 

tBIL E DUCT (20) (50) (49)
 

HYPERPLASIA , NOS 19 (95%) 1 1 (22%) 6 (12%)
 

#PANCREA S (20) (47) (41 )
 

STEATITIS 1 (2%)
 

PERIARTERITI S 1 (5%)
 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMAL S NECROPSIED
 

78
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE
 

#PANCREATIC ACINUS (20) (47)
 

ATROPHY, NOS 7 (35%) 2 ( 4 % )
 

#STOMACH (20) (50)
 

ULCER , NOS 1 (2%)
 

INFLAMMATION , ACUTE
 

INFLAMMATION, CHRONIC
 

*SMALL INTESTINE (20) (50)
 

HYPERPLASIA , LYMPHOID 1 (5%)
 

fLARGE INTESTINE (20) (47)
 

NEMATODIASIS 1 (5%) 2 (4%)
 

URINAR Y SYSTEM 

#KIDNEY 

HYDRONEPHROSIS 

PYELONEPHRITIS, NOS 

INFLAMMATION, CHRONIC 

NEPHROSIS, NOS 

HEMOSIDEROSIS 

(20) 

18 (90%) 

( 50 ) 

1 ( 2 % ) 

1 (2%) 

35 (70%) 

#KIDNEY/MEDULLA 

HYPERPLASIA , NOS 

(20) (50) 

1 (2%) 

*KIDNEY/TUBULE 

HEMOSIDEROSIS 

(20) (50) 

#KIDNEY/PELVIS 

HYPERPLASIA, EPITHELIAL 

(20) (50) 

1 (2%) 

#URINARY BLADDER 

HYPERPLASIA , EPITHELIA L 

(20) (50) 

9 (18%) 

ENDOCRINE SYSTEM
 

tPITUITARY (20) (50)
 

CYST, NOS 1 (5%) 1 (2%)
 

ANGIECTASIS 2 (4%)
 

tADRENA L (20) (50)
 

LYMPHOCYTIC INFLAMMATOR Y INFILTR 1 (2%)
 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMAL S NECROPSIED
 

HIGH DOSE
 

(41 )
 

1 ( 2 % )
 

(47)
 

1 ( 2 % )
 

2 (4%)
 

1 (2%)
 

(45)
 

(46)
 

1 (2%)
 

(49 )
 

34 (69%)
 

1 ( 2 % )
 

1 ( 2 % )
 

(49)
 

(49 )
 

3 (6%)
 

(49)
 

1 ( 2 % )
 

(44)
 

7 (16%)
 

(44)
 

1 (2%)
 

1 (2%)
 

(49 )
 

79
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

#ADRENAL CORTEX (20) (50) (49)
 

NECROSIS, NOS 1 (2%)
 

LIPOIDOSIS 1 (2?J)
 

HYPERPLASIA , NODULAR 1 (2X) 1 (2?«)
 

•ADRENAL	 MEDULL A (20) (50) (49)
 

HYPERPLASIA, NOS 2 (45O 1 (25{)
 

#THYROID (20) (50) (45)
 

CYSTIC FOLLICLES 2 (45S)
 

HYPERPLASIA , C-CELL 5 (25*)
 

HYPERPLASIA , FOLLICULAR-CEL L 1 (5%)
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND (20) (50) (49)
 

DILATATION/DUCTS 1 (25i)
 

•PROSTATE	 (19) (49) (45)
 

INFLAMMATION, ACUTE 1 (5«) 2 (4%) 1 (2%)
 

tTESTIS (20) (50) (49)
 

ATROPHY, NOS 1 (55{)
 

HYPERPLASIA, INTERSTITIAL CELL 3 (6%)
 

NERVOUS SYSTEM
 

*BRAIN (20) (50) (48)
 

HEMORRHAGE 1 (2%)
 

INFLAMMATION , NOS 1 (254)
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

^MESENTERY (20) (50) (49)
 

ABSCESS, NOS 1
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

80
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW QOSE HIGH DOSE 

PERIARTERITI S 1 (Z») 

A LL OTHER SYSTEMS
 

*MULTIPLE ORGANS (20) (50) (49)
 

HEMOSIDEROSIS 1 (2%)
 

SPECIAL MORPHOLOGY SUMMAR Y
 

ANIMA L MISSING/NO NECROPSY
 

f NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBE R OF ANIMAL S NECROPSIED
 

81
 

1 
TABLE C2. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

ANIMAL S INITIALL Y IN STUDY


ANIMAL S MISSING
 

ANIMAL S NECROPSIED


ANIMAL S EXAMINED H ISTOPATHOLOGICAL L Y


INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATOR Y SYSTEM
 

#LUNG


PNEUMONIA , ASPIRATION
 

INFLAMMATION , ACUTE
 

INFLAMMATION , CHRONIC
 

BRONCHOPNEUMONIA , CHRONIC


PERIVASCULITI S


HYPERPLASIA , ALVEOLA R EPITHELIUM
 

tLUNG/ALVEOL I


HISTIOCYTOSIS


HEMATOPOIETIC SYSTEM
 

ttSPLEEN


FIBROSIS
 

METAMORPHOSIS FATTY
 

HEMOSIDEROSIS


HEMATOPOIESIS


tSPLENIC CAPSULE


FIBROSIS
 

HYPERPLASIA , MESOTHELIAL
 

#LYMPH NODE


HYPERPLASIA , LYMPHOID
 

ttMANDIBULAR L. NODE


LYMPHANGIECTASI S
 

MATCHED
 

CONTROL
 

 20
 

 20
 

 20
 

 (20)
 

2 (1054)
 

3 (15%)
 

 (20)
 

1 (5%)
 

 (20)
 

4 (20%)
 

1 (5%)
 

 (20)
 

 (20)
 

 (20)
 

LOW DOSE
 

50
 

50
 

49
 

(48)
 

2 (4%)
 

4 (8%)
 

(48)
 

1 (2%)
 

(49)
 

6 (12%)
 

1 ( 2 % )
 

7 (14%)
 

2 (4%)
 

(49)
 

5 (10%)
 

32 (65%)
 

(49)
 

1 ( 2 % )
 

(49)
 

1 ( 2 % )
 

HIGH DOSE
 

50
 

1
 

49
 

49
 

(49)
 

1 (2%)
 

1 (2%)
 

2 (4%)
 

(49)
 

1 (2%)
 

(49)
 

3 (6%)
 

3 ( 6 % )
 

1 (2%)
 

(49)
 

12 (24%)
 

(48)
 

1 ( 2 % )
 

(48)
 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y
 

* NUMBE R OF ANIMAL S NECROPSIED
 

82
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGHI )OSE
 

HYPERPLASIA , LYMPHOID 1 f2 '/. ) 1 (2%)
 

CIRCULATOR Y SYSTEM
 

tHEAR T (20) (48) (49)
 

PERIARTERITI S 1 (2%)
 

#MYOCARDIU M (20) (48) (49)
 

FIBROSIS 1 ( 2 % ) 1 (2%)
 

*CORONARY ARTER Y (20) (50) ^ (49)
 

PERIVASCULITI S 3 M5%)
 

*PULMONARY ARTER Y (20) (50) (49)
 

MEDIA L CALCIFICATION 2 (4%) 1 (2%)
 

DIGESTIVE SYSTEM
 

SLIVER (20) (49) (49)
 

NECROSIS, NOS 1 (2%) 1 5 (31%)
 

INFARCT, NOS 1 (2%)
 

METAMORPHOSIS FATTY 2 (4%)
 

CYTOPLASMIC V ACUO L I ZAT I ON 3 ( 6 % )
 

FOCAL CELLULA R CHANGE 4 (20%) 22 (45%) 5 (10%)
 

HEPATOCYTOMEGALY 1 (5%) 3 (6%) 1 (2%)
 

HEMATOPOIESIS 1 (2%)
 

tLIVER/CENTRILOBULA R (20) (49) (49)
 

CYTOPLASMIC VACUOL IZATION 1 (2%)
 

tBILE DUCT (20) (49) (49)
 

HYPERPLASIA , NOS 1 (2%) 5 (10%)
 

#PANCREATIC ACINUS (20) (48) (46)
 

ATROPHY, NOS 2 (10%) 2 (4%)
 

#STOMACH (20) (48) (49)
 

ULCER , NOS 1 ( 2 % )
 

HYPERKERATOSIS 1 (2%)
 

tLARG E INTESTINE (19) (44) (42)
 

NEMATODIASIS 1 (2%)
 

HYPERPLASIA , LYMPHOID 1 (5%)
 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMAL S NECROPSIED
 

83
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

URINAR Y SYSTEM
 

tKIDNEY
 

MINERALIZATION
 

HYDRONEPHROSIS
 

PYELONEPHRITIS, NOS
 

INFLAMMATION , CHRONIC
 

NEPHROSIS, NOS
 

#KIDNEY/TUBULE
 

NECROSIS, NOS
 

f KIDNEY/PELVIS
 

MINERALIZATIO N
 

HYPERPLASIA , EPITHELIAL
 

#URINAR Y BLADDER
 

HYPERPLASIA , EPITHELIAL
 

METAPLASIA, SQUAMOUS
 

ENDOCRINE SYSTEM
 

f PITUITARY
 

CYST, NOS
 

ANGIECTASIS
 

SADRENA L CORTEX
 

LIPOIDOSIS
 

HYPERPLASIA , NODULAR
 

ANGIECTASIS
 

#THYROID
 

HYPERPLASIA , C-CEl,L
 

HYPERPLASIA , FOL L ICUL AR-CE L L
 

REPRODUCTIV E SYSTEM
 

*MAMMAR Y GLAND
 

DILATATION/DUCTS
 

*VAGINA
 

POLYP
 

# NUMBER OF ANIMAL S WITH TISSUE


* NUMBE R OF ANIMAL S NECROPSIED
 

MATCHED
 

CONTROL
 

(20)
 

8 (40%)
 

(20)
 

(20)
 

1 (5%)
 

(20)
 

(20)
 

(20)
 

1 (5%)
 

3 (15%)
 

1 (5%)
 

(20)
 

7 (35%)
 

(20)
 

2 (10%)
 

(20)
 

LOW DOSE
 

(49)
 

2 (4%)
 

1 (2%)
 

7 (14%)
 

(49)
 

(49)
 

3 ( 6 % )
 

(45)
 

21 (47%)
 

(47)
 

6 (13%)
 

4 (9%)
 

(49)
 

9 (18%)
 

(47)
 

7 (15%)
 

1 ( 2 % )
 

(50)
 

2 (4%)
 

( 50)
 

1 (2%)
 

 EXAMINED MICROSCOPICALLY
 

HIGH DOSE
 

(49)
 

1 (2%)
 

4 (8%)
 

1 (2%)
 

(49)
 

2 (4%)
 

(49)
 

4 (8%)
 

(47)
 

13 (28%)
 

1 (2%)
 

(46)
 

1 (2%)
 

1 (2%)
 

(49)
 

3 (6%)
 

(46)
 

2 (4%)
 

1 ( 2 % )
 

(49)
 

2 ( 4 % )
 

(49)
 

84
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

#UTERUS/ENDOMETRIUM


CYST, NOS


#OVAR Y


CYST, NOS


NERVOUS SYSTEM
 

#BRAIN


HEMORRHAG E


SPECIAL SENSE ORGANS
 

NONE
 

MUSCUIOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY


HEMORRHAGI C CYST


^MESENTERY


HEMORRHAGI C CYST


A LL OTHER SYSTEMS
 

^MULTIPLE ORGANS


HYPERPLASIA , LYMPHOID


HEMATOPOIESIS


SPECIAL MORPHOLOGY SUMMARY
 

ANIMA L MISSING/NO NECROPSY


AUTO/NECROPSY/NO HISTO


MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

 (20) (49) (48)
 

1	 (5%)
 

 (20)	 (49) (49)
 

1 (2%)
 

 (20)	 (48) (49)
 

1 (5%)
 

 (20)	 (50) (49)
 

1 (2%)
 

 (20)	 (50) (49)
 

1 (2%)
 

 (20) (50)	 (49)
 

1	 (5%)
 

1 (2%)
 

1
 

1
 

tt NUMBER OF ANIMALS WITH TISSUE	 EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

85
 
86

 
APPENDIX D 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MICE ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

87 
oo 

CO 
TABLE D1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIMALS MISSING 1
 

ANIMALS NECROPSIED 19 50 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 19 50 50
 

INTEGUMENTARY SYSTEM
 

NONE
 s
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN (19) (49) (50)
 

HYPERPLASIA, LYMPHOID 1 (5%) 1 (2K)
 

HEMATOPOIESIS 1 (5X) 6 (12%) 11 (225O
 

*MESENTERIC L. NODE (18) (48) (48)
 

HYPERPLASIA, LYMPHOID 1 (6%) 1 (2%)
 

CIRCULATOR Y SYSTEM
 

•MYOCARDIUM	 (19) (50) (50)
 

INFLAMMATION, FOCAL 1 (2%)
 

DIGESTIVE SYSTEM
 

#LIVE R 

INFLAMMATION , FOCAL 

NECROSIS, FOCAL 

MEGALOCYTOSIS 

HYPERPLASIA , NODULAR 

ANGIECTASIS 

(19) (50) 

1 (2?0 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2X) 

*LIVER/HEPATOCYTES 

CYTOPLASMIC VACUOLIZATION 

(19) 

2 (1 

(50) 

2 (4X) 

(50) 

1 (2X) 

# NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMAL S NECROPSIED
 

89
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

#PANCREAS
 

INFLAMMATION , FOCAL
 

PERIARTERITI S
 

ATROPHY, NOS
 

#SMALL INTESTINE
 

DIVERTICULU M
 

#LARG E INTESTINE
 

NEMATODIASIS
 

URINAR Y SYSTEM
 

#KIDNEY
 

INFLAMMATION , CHRONIC
 

HYPERPLASIA , TUBULA R CEL L
 

ENDOCRIN E SYSTEM
 

#PANCREATI C ISLETS
 

HYPERPLASIA , NOS
 

REPRODUCTIV E SYSTEM
 

SPROSTATE
 

INFLAMMATION , NOS
 

NERVOUS SYSTEM
 

fBRAIN
 

MINERALIZATIO N
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETA L SYSTEM
 

NONE
 

# NUMBE R OF ANIMAL S WIT H TISSUE


* NUMBE R OF ANIMAL S NECROPSIE D
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

(18)

(17)

(15)

 (47)

1 (2%) 

1 ( 2 X ) 

 (47)

1 ( 2%) 

 (49)

 (46) 

1 ( 2 '/. ) 

 (48) 

 (45) 

1 (2%) 

(18)

1(6%)

1 (6%) 

 (50) (50) 

 1(2%) 

(18)

3 (17%) 

 (47) (46) 

(17) (45) ' (42) 

1 (2%) 

(19)

6 (32%)

 (48)

 10 (21%)

 (50) 

 11 (22%) 

 EXAMINE D MICROSCOPICALL Y 

90
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

"ABDOMINAL CAVITY

HEMATOMA , NOS

HEMORRHAGI C CYST

 (19) (50) (50) 

1 (2%) 

2 

*MESENTERY

ABSCESS, NOS

 (19) (50)

 1 (2%) 

 (50) 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 1 12

ANIMA L MISSING/NO NECROPSY 1 

AUTO/NECROPSY/HISTO PERF 1

ft NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

x NUMBE R OF ANIMAL S NECROPSIED 

 11 

1 

91
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALLY IN STUDY 20 50 50 

ANIMAL S MISSING 1 

ANIMALS NECROPSIED 20 49 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 20 49 50 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

*LUNG (20) (48) (48)
 

INFLAMMATIO N PROL I FERATI VE 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN (20) (49) (48)
 

HYPERPLASIA , LYMPHOID 2 (45O 1 (2%)
 

HEMATOPOIESIS 9 (45«) 4 (8%) 6 ( 1.3X)
 

#LYMPH NODE (20) (49) (48)
 

HYPERPLASIA , LYMPHOID 1 (2%)
 

tMESENTERIC L. NODE (20) (49) (48)
 

CYST, NOS 1 (5%)
 

HYPERPLASIA, LYMPHOID 2 (4X)
 

CIRCULATOR Y SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVE R (20) (49) (50)
 

CYST, NOS 1 (2%)
 

NECROSIS, FOCAL 1 (2%)
 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 « NUMBE R OF ANIMAL S NECROPSIED
 

92
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

BASOPHILIC CYTO CHANGE
 

ANGIECTASIS
 

HEMATOPOIESIS
 

tSMALL INTESTINE
 

EPIDERMA L INCLUSION CYST
 

URINAR Y SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

ttPITUITARY
 

ANGIECTASIS
 

#THYROID
 

CYSTIC FOLLICLES
 

REPRODUCTIV E SYSTEM
 

#UTERUS/ENDOMETRIUM
 

CYST, NOS
 

#OVARY
 

CYST, NOS
 

NERVOUS SYSTEM
 

#BRAIN
 

MINERALIZATIO N
 

#CEREBELLU M
 

HEMORRHAG E
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETA L SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE
 * NUMBER OF ANIMALS NECROPSIED
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

1 (2%) 

3 (6%) 

1 (2%) 

(20) (47) (50) 

1 (2%) 

(20) (44) (45) 

1 (5%) 

(20) (44) (47) 

1 (2%) 2 (4%) 

(20) (46) (45) 

10 (50%) 14 (30%) 18 (38%) 

(20) (46) (47) 

1 (5%) 7 (15%) 6 (13%) 

(20) (47) (48) 

5 (25%) 12 (26%) 11 (23%) 

(20) (47) (48) 

1 (2%) 

 EXAMINED MICROSCOPICALLY 

93
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

*ABDOMINAL CAVITY

ABSCESS, NOS

*MESENTERY

CYST, NOS

 (20)

 (20)

 (49)

 (49)

 (50) 

1 (2%) 

 (50) 

1 (2%) 

A LL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

N O LESION REPORTED 2 

ANIMA L MISSING/NO NECROPSY 1 

AUTO/NECROPSY/HISTO PERF 2 

# NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

8 9 

94
 
APPENDIX E

 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

IN RATS ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

95 
96

 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

Topography: Morphology


Integumentary System: Fibroma of the
 

Subcutaneous Tissue (b)


P Values (c,d)


Departure from Linear Trend (e)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Integumentary System: Lipoma of
 

the Subcutaneous Tissue (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

 Control
 

 0/20 (0)
 

P = 0.001
 

P = 0.004
 

 0/20 (0)
 

 N.S.
 

Low
 

Dose
 

28/50 (56)
 

P less than 0.001
 

Infinite
 

3.873
 

Infinite
 

82
 

4/50 (8)
 

N.S.
 

Infinite
 

0.386
 

Infinite
 

High
 

Dose
 

27/49 (55)
 

P less than 0.001
 

Infinite
 

3.803
 

Infinite
 

62
 

2/49 (4)
 

N.S.
 

Infinite
 

0.125
 

Infinite
 

Weeks to First Observed Tumor 80 82
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Low
 

Topography: Morphology Control Dose
 

Integumentary System: Sarcoma, NOS,
 

Fibrosarcoma, Myxosarcoma, or
 

Osteosarcoma (b) 0/20 (0) 4/50 (8)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) Infinite
 

Lower Limit 0.386
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — 66
 

oo
 

Hematopoietic System:
 

Lymphoma (b) 4/20 (20) 2/50 (4)
 

P Values (c,d) P = 0.002 (N) N.S.
 

Relative Risk (f) 0.200
 

Lower Limit 0.020
 

Upper Limit 1.297
 

Weeks to First Observed Tumor 101 71
 

High
 

Dose
 

3/49 (6)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

72
 

0/49 (0)
 

P = 0.006 (N)
 

0.000
 

0.000
 

0.435
 
(continued) 

Topography:

Table El. Analyses of the Incidence of Primary Tumors in

Administered o-Toluidine Hydrochloride in the Diet

 Morphology Control 

Low 

Dose 

 Male

 (a) 

 Rats 

High 

Dose 

Spleen: Sarcoma, NOS (b) 0/20 (0) 1/49 (2) 3/42 (7) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.023 

Infinite 

Infinite 

0.298 

Infinite 

Weeks to First Observed Tumor — 104 72 

\o Spleen: Fibroma (b) 0/20 (0) 10/49 (20) 2/42 (5) 

P Values (c,d) N.S. P = 0.024 N.S. 

Departure from Linear Trend (e) P = 0.004 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

1.266 

Infinite 

Infinite 

0.146 

Infinite 

Weeks to First Observed Tumor 96 82 
(continued) 

Topography:

Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered o-Toluidine Hydrochloride in the Diet (a) 

 Morphology Control 

Low 

Dose 

High 

Dose 

Spleen: Angiosarcoma (b) 0/20 (0) 7/49 (14) 0/42 (0) 

P Values (c,d) N.S. N.S. — 

Departure from Linear Trend (e) P = 0.003 

Relative Risk ( f) 

Lower Limit 

Upper Limit 

Infinite 

0.826 

Infinite 

—• 

o 

o 

Weeks to First Observed Tumor — 83 — 

Spleen: Sarcoma, NOS, Angiosarcoma, or 

Osteosarcoma (b) 0/20 (0) 8/49 (16) 4/42 (10) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.972 

Infinite 

Infinite 

0.460 

Infinite 

Weeks to First Observed Tumor 83 72 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography: Morphology


Liver: Neoplastic Nodule or
 

Hepatocellular Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Urinary Bladder: Transitional-cell
 

Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Low High 

 Control Dose Dose 

 1/20 (5) 1/50 (2) 3/49 (6) 

 N.S. N.S. N.S. 

0.400 1.224 

0.005 0.108 

30.802 62.958 

 104 99 72 

 0/20 (0) 3/50 (6) 0/44 (0) 

 N.S. N.S. — 

__ 

Infinite 

0.250 

Infinite — 

— 

99 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography: Morphology


Urinary Bladder: Transitional-cell
 

Carcinoma or Papilloma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Adrenal: Pheochromocytoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Low High 

 Control Dose Dose 

 0/20 (0) 3/50 (6) 1/44 (2) 

 N.S. N.S. N.S. 

Infinite Infinite 

0.250 0.025 

Infinite Infinite 

— 99 72 

 0/20 (0) 3/50 (6) 2/49 (4) 

 N.S. N.S. N.S. 

Infinite Infinite 

0.250 0.125 

Infinite Infinite 

80 82 
Table El. Analyses df the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography; Morphology
 

Pituitary: Adenoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

i_*
 

o	 Thyroid: C-cell Carcinoma (b)
 

P Values (c,d)
 

Departure from Linear Trend (e)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Low 

Control Dose 

4/20 (20) 7/50 (14) 

N.S. N.S. 

0.700 

0.207 

2.994 

104 79 

2/20 (10) 0/50 (0) 

P = 0.028 (N) N.S. 

P = 0.048 

0.000 

0.000 

1.345 

104 

High
 

Dose
 

3/44 (7)
 

N.S.
 

0.341
 

0.056
 

1.857
 

0/45 (0)
 

N.S.
 

0.000
 

0.000
 

1.491
 

79 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered o-Toluidine Hydrochloride in the Diet (a) 

(continued) 

Low High 

Topography: Morphology Control Dose Dose 

Thyroid: C-cell Carcinoma 

or Adenoma (b) 3/20 (15) 2/50 (4) 2/45 (4) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 0.267 0.296 

Lower Limit 0.024 0.027 

Upper Limit 2.190 2.424 

Weeks to First Observed Tumor 104 99 78 

Mammary Gland: Fibroadenoma (b) 0/20 (0) 7/50 (14) 1/49 (2) 

P Values (c,d) N.S. N.S. N.S. 

Departure from Linear Trend (e) P = 0.008 

Relative Risk (f) Infinite Infinite 

Lower Limit 0.809 0.023 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor 74 93 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Low
 

Topography: Morphology Control Dose
 

Testis: Interstitial-Cell
 

Tumor (b) 17/20 (85) 41/50 (82)
 

P Values (c,d) P = 0.045(N) N.S.
 

Relative Risk (f) 0.965
 

Lower Limit 0.802
 

Upper Limit 1.310
 

Weeks to First Observed Tumor 104 70
 

o
 

Ul
 

Multiple Organs or Tunica Vaginal is:
 

Mesothelioma (b) 0/20 (0) 17/50 (34)
 

P Values N.S. P = 0.001
 

Departure from Linear Trend (e) P = 0.002
 

Relative Risk (f) Infinite
 

Lower Limit 2.256
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 66
 

High
 

Dose
 

33/49 (67)
 

N.S.
 

0.792
 

0.649
 

1.139
 

9/49 (18)
 

P = 0.036
 

Infinite
 

1.119
 

Infinite
 

72
 

72 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Low High
 

Topography: Morphology Control Dose Pose
 

Multiple Organs: Sarcoma, NOS (b) 0/20 (0) 3/50 (6) 11/49 (22)
 

P Values (c,d) P = 0.003 N.S. P = 0.016
 

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.250 1.413
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 84 61
 

Multiple Organs: Fibrosarcoma (b) 0/20 (0) 8/50 (16) 20/49 (41)
 

o
 

OS
 

P Values (c,d) P less than 0.001 N.S. P less than 0.001
 

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.952 2.750
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 70 67
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography: Morphology


Multiple Organs: Osteosarcoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Multiple Organs: Sarcoma, NOS,
 

Fibrosarcoma, Angiosarcoma, or
 

Osteosarcoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Control
 

 0/20 (0)
 

 N.S.
 

—
 

 0/20 (0)
 

P less than 0.001
 

Low
 

Dose
 

3/50 (6)
 

N.S.
 

Infinite
 

0.250
 

Infinite
 

92
 

15/50 (30)
 

P = 0.003
 

Infinite
 

1.964
 

Infinite
 

70
 

High
 

Dose
 

5/49 (10)
 

N.S.
 

Infinite
 

0.536
 

Infinite
 

82
 

37/49 (76)
 

P less than 0.001
 

Infinite
 

5.361
 

Infinite
 

61
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

(a) Dosed groups received 3,000 or 6,000 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors in a dosed group is the probability level for the Fisher exact test for
 

the comparison of that dosed group with the matched-control group when P less than 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
 

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

Low High
 

Topography: Morphology Control Dose Dose
 

Integumentary System: Fibroma of the
 

Subcutaneous Tissue (b) 0/20 (0) 4/50 (8) 2/49 (4)
 

P Values (c,d) M.S. N.S. N.S.
 

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.386 0.125
 

Upper Limit Infinite Infinite
 

o Weeks to First Observed Tumor — 99
 VO
 

Hematopoietic System: Lymphoma (b) 1/20 (5) 3/50 (6) 0/49 (0)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) 1 . 200 0.000
 

Lower Limit 0.106 0.000
 

Upper Limit 61.724 7.624
 

Weeks to First Observed Tumor 101 96
 

87 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued) 

Topography: Morphology Control 

Low 

Dose 

High 

Dose 

Spleen : Sarcoma, NOS (b) 0/20 (0) 1/49 (2) 3/49 (6) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.023 

Infinite 

Infinite 

0.255 

Infinite 

Weeks to First Observed Tumor 104 81 

Spleen: Fibroma (b) 0/20 (0) 4/49 (8) 6/49 (12)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.394 0.680
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 104 87
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography: Morphology


Spleen: Angiosarcoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Spleen: Sarcoma, NOS,
 

Angiosarcoma, or Osteosarcoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Control
 

 0/20 (0)
 

P = 0.045
 

—
 

 0/20 (0)
 

P = 0.018
 

Low
 

Dose
 

7/49 (14)
 

N.S.
 

Infinite
 

0.826
 

Infinite
 

103
 

9/49 (18)
 

P = 0.036
 

Infinite
 

1.119
 

Infinite
 

92
 

High
 

Dose
 

9/49 (18)
 

P = 0.036
 

Infinite
 

1.119
 

Infinite
 

65
 

12/49 (24)
 

P = 0.010
 

Infinite
 

1.561
 

Infinite
 

65
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

(continued) 

Topography;

Administered o-Toluidine Hydrochlor

 Morphology Control 

ide in the Diet (a) 

Low 

Dose 

High 

Dose 

Urinary Bladder:

Carcinoma (b) 

 Transitional-cell 

0/20 (0) 9/45 (20) 22/47 (47) 

P Values (c,d) P less than0.001 P = 0.028 P less than 0 .001 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

1.219 

Infinite 

Infinite 

3.182 

Infinite 

Weeks to First Observed Tumor 103 65 

Urinary Bladder: Transitional-cell
 

Carcinoma or Papilloma (b) 0/20 (0) 10/45 (22) 22/47 (47)
 

P Values (c,d) P less than 0.001 P = 0.018 P less than 0.001
 

Relative Risk (f) Infinite Infinite
 

Lower Limit 1.379 3.182
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 103 65
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Low
 

Topography: Morphology Control Dose
 

Pituitary: Adenoma, NOS (b) 5/20 (25) 12/47 (26)
 

P Values (c,d) P = 0.009 (N) N.S.
 

Relative Risk (f) 1.021
 

Lower Limit 0.400
 

Upper Limit 3.310
 

Weeks to First Observed Tumor 104 88
 

M Adrenal: Pheochromocytoma (b) 0/20 (0) 4/49 (8)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) Infinite
 

Lower Limit 0.394
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 104
 

High
 

Dose
 

2/46 (4)
 

P = 0.023 (N)
 

0.174
 

0.018
 

0.975
 

89
 

2/49 (4)
 

N.S.
 

Infinite
 

0.125
 

Infinite
 

104
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued) 

Topography: Morphology Control 

Low 

Dose 

High 

Dose 

Thyroid: C-cell Carcinoma (b) 1/20 (5) 3/47 (6) 0/46 (0) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.277 

0.112 

65.563 

0.000 

0-. 000 

8.111 

Weeks to First Observed Tumor 104 104 — 

Thyroid: C-cell Carcinoma or
 

Adenoma (b) 1/20 (5) 6/47 (13) 1/46 (2)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) 2.553 0.435
 

Lower Limit 0.346 0.006
 

Upper Limit 114.692 33.420
 

Weeks to First Observed Tumor 104 75 104
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued) 

Low High 

Topography: Morphology Control Dose Dose 

Mammary Gland: Fibroadenoma (b) 6/20 (30) 20/50 (40) 35/49 (71) 

P Values (c,d) P less than 0.001 N.S. P = 0.002 

Relative Risk (f) 1.333 2.381 

Lower Limit 0.631 1.240 

Upper Limit 3.541 5.608 

Weeks to First Observed Tumor 97 94 72 

Mammary Gland: Fibroadenoma or
 

Adenoma, NOS (b) 7/20 (35) 20/50 (40) 35/49 (71)
 

P Values (c,d) P = 0.001 N.S. P = 0.006
 

Relative Risk (f) 1.143 2.041
 

Lower Limit 0.575 1.130
 

Upper Limit 2.760 4.361
 

Weeks to First Observed Tumor 97 94 72
 
Table E2. Analyses of the Incidence of Primary

(continued) 

Administered o-Toluidine Hydrochloride

Topography: Morphology Control 

Uterus: Endometrial Stromal 

Polyp (b) 5/20 (25) 

P Values (c,d) P = 0.004 (N) 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 104 

Multiple Organs: Osteosarcoma (b) 0/20 (0)
 

P Values (c,d) P less than 0.001
 

Departure from Linear Trend (e) P = 0.008
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 

 Tumors in Female Rats 

 in the Diet (a) 

Low High 

Dose Dose 

4/49 (8)	 1/48 (2)
 

N.S.	 P = 0.007 (N)
 

0.327	 0.083
 

0.074	 0.002
 

1.385	 0.690
 

88 104
 

0/50 (0)	 18/49 (37)
 

P = 0.001
 —
 

Infinite
 

2.451
 —
 Infinite
 

84 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats

 

Administered o-Toluidine Hydrochloride in the Diet (a) 

(continued) 

Low High 

Topography: Morphology Control Dose Dose 

Multiple Organs: Sarcoma, NOS, 

Fibrosarcoma, Angiosarcoma, 

or Osteosarcoma (b) 0/20 (0) 3/50 (6) 21/49 (43) 

P Values (c,d) P less than 0.001 N.S. P less than 0.001 

Departure from Linear Trend (e) P = 0.042 

Relative Risk ( f ) Infinite Infinite 

Lower Limit 0.250 2.899 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor 76 76 

(a) Dosed groups received 3,000 or 6,000 ppm. 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the CochranArmitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath 

the incidence of tumors in a dosed group is the probability level for the Fisher exact test for 

the comparison of that dosed group with the matched-control group when P less than 0.05; 

otherwise, not significant (N.S.) is indicated. 

(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group. 

(e) The probability level for departure from linear trend is given when P less than 0.05 for any 

comparison. 

( f) The 95% confidence interval of the relative risk between each dosed group and the control group. 
118

 
APPENDIX F 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

IN MICE ADMINISTERED o-TOLUIDINE HYDROCHLORIDE IN THE DIET 

119 
120

 
Table Fl. Analyses of the Incidence of Primary Tumors in Male

Administered o-Toluidine Hydrochloride in the Diet (a) 

 Mice 

Topography: Morphology Control

Low 

 Dose 

High 

Dose 

Lung: Alveolar/Bronchiolar 

Carcinoma (b) 3/19 (16) 4/50 (8) 1/50 (2) 

P Values (c,d) P = 0.032 (N) N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.507 

0.097 

3.234 

0.127 

0.003 

1.487 

Weeks to First Observed Tumor 103 103 102 

N> 

Lung: Alveolar/Bronchiolar 

Carcinoma or Adenoma (b) 5/19 (26) 5/50 (10) 3/50 (6) 

P Values (c,d) P = 0.033 (N) N.S. P = 0.032 (N) 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.380 

0.102 

1.500 

0.228 

0.040 

1.071 

Weeks to First Observed Tumor 103 54 102 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Low
 

Topography: Morphology


Hematopoietic System: Lymphoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


M All Sites: Hemangiosarcoma (b)
 K3
 

P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Control
 

 1/19(5)
 

 N.S.
 

 101
 

 1/19 (5)
 

P = 0.004
 

99
 

Dose
 

9/50 (18)
 

N.S.
 

3.420
 

0.536
 

146.437
 

101
 

1/50 (2)
 

N.S.
 

0.380
 

0.005
 

29.260
 

103
 

High
 

Dose
 

4/50 (8)
 

N.S.
 

1.520
 

0.167
 

73.309
 

50
 

10/50 (20)
 

N.S.
 

3.800
 

0.613
 

160.949
 

94
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography: Morphology


All Sites: Hemangioma or
 

Heraangiosarcoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


NJ
 

CO Liver: Hepatocellular Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Control
 

 1/19 (5)
 

P = 0.002
 

 99
 

 4/19 (21)
 

 N.S.
 

94
 

Low
 

Dose
 

2/50 (4)
 

N.S.
 

0.760
 

0.043
 

43.961
 

103
 

16/50 (32)
 

N.S.
 

1.520
 

0.587
 

5.639
 

70
 

High
 

Dose
 

12/50 (24)
 

N.S.
 

4.560
 

0.767
 

190.018
 

102
 

11/50 (22)
 

N.S.
 

1.045
 

0.367
 

4.095
 

92 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography: Morphology


Liver: Hepatocellular Carcinoma
 

or Adenoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Liver: Hepatocellular Adenoma
 

or Adenoma, NOS (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Low High 

 Control Dose Dose 

 5/19 (26) 19/50 (38) 14/50 (28) 

 N.S. N.S. N.S. 

1.444 1.064 

0.635 0.438 

4.371 3.368 

 94 70 92 

 2/19 (11) 3/50 (6) 3/50 (6) 

 N.S. N.S. N.S. 

0.570 0.570 

0.073 0.073 

6.511 6.511 

103 103 94 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Topography: Morphology


Thyroid: Follicular-cell Adenoma
 

or Carcinoma (b) ­

P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


NJ
 

Ul Mesentery: Lipoma (b)


P Values (c,d)


Departure from Linear Trend (e)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Control
 

 2/19 (11)
 

 N.S.
 

 103
 

 2/19 (11)
 

P = 0.041 (N)
 

 P = 0.011
 

103
 

Low
 

Dose
 

2/49 (4)
 

N.S.
 

0.388
 

0.031
 

5.108
 

103
 

0/50 (0)
 

N.S.
 

0.000
 

0.000
 

1.278
 

High
 

Dose
 

2/49 (4)
 

N.S.
 

0.388
 

0.031
 

5.108
 

0/50 (0)
 

N.S.
 

0.000
 

0.000
 

1.278
 

102 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

(a) Dosed groups received 1,000 or 3,000 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors in a dosed group is the probability level for the Fisher exact test for
 

the comparison of that dosed group with the matched-control group when P less than 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
 

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

Low
 

Topography: Morphology Control Dose
 

Hematopoietic System: Lymphoma (b) 4/20 (20) 10/49 (20)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) 1.020
 

Lower Limit 0.346
 

Upper Limit 4.068
 

Weeks to First Observed Tumor 103 80
 

N3
 

All Sites: Hemangiosarcoma
 

or Hemangioma (b) 1/20 (5) 1/49 (2)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) 0.408
 

Lower Limit 0.005
 

Upper Limit 31.413
 

Weeks to First Observed Tumor 103 103
 

High
 

Dose
 

9/50 (18)
 

N.S.
 

0.900
 

0.294
 

3.660
 

88
 

3/50 (6)
 

N.S.
 

1.200
 

0.106
 

61.724
 

102
 
Table F2. Analyses of the Incidence of Primary

Administered o-Toluidine Hydrochloride

(continued) 

Topography: Morphology Control 

Liver: Hepatocellular Carcinoma (b) 0/20 (0) 

P Values (c,d) P = 0.015 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

NJ Liver: Hepatocellular Carcinoma 00 

or Adenoma (b) 0/20 (0) 

P Values (c,d) P = 0.001 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

 Tumors in Female Mice 

 in the Diet (a) 

Low 

Dose 

High 

Dose 

2/49 (4) 7/50 (14) 

N.S. N.S. 

Infinite 

0.125 

Infinite 

Infinite 

0.809 

Infinite 

103 102 

4/49 (8) 13/50 (26) 

N.S. P = 0.007 

Infinite Infinite 

0.394 1.674 

Infinite Infinite 

103 102 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered o-Toluidine Hydrochloride in the Diet (a)
 

(continued)
 

Low High
 

Topography: Morphology Control Dose Dose
 

Thyroid: Follicular-cell 

Adenoma (b) 3/20 (15) 0/44 (0) 0/47 (0) 

P Values (c,d) P = 0.014 (N) P = 0.027 (N) P = 0.024 (N) 

Departure from Linear Trend (e) P = 0.005 

Relative Risk (f) 0.000 0.000
 

Lower Limit 0.000 0.000
 

Upper Limit 0.747 0.700
 

NJ Weeks to First Observed Tumor 103 

(a) Dosed groups received 1,000 or 3,000 ppm. 

(b) Number of tumor-bearing animaIs/number of animals examined at site (percent). 

(c) Beneath the incidence of tumors in the control group is the probability level for the 

Cochran-Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. 

Beneath the incidence of tumors in a dosed group is the probability level for the Fisher exact 

test for the comparison of that dosed group with the matched-control group when P less than 

0.05; otherwise, not significant (N.S.) is indicated. 

(d) A negative trend (N) indicates a lower incidence in => dosed group than in a control group. 

(e) The probability level for departure from linear trend is given when P less than 0.05 for any 

comparison. 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group. 
130

 
Review of the Bioassay of o-Toluidine Hydrochloride* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

October 25, 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 academia, industry,
 

organized labor, public interest groups, and State health officials.
 

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 o-Toluidine Hydrochloride for
 

carcinogenicity.
 

The primary reviewer for the report of the bioassay of O-Toluidine
 

hydrochloride said that, under the conditions of test, the compound
 

was carcinogenic in both sexes of treated rats and mice. After briefly
 

describing the experimental design, he noted an increased incidence of
 

urinary bladder epithelial and spleen capsule mesothelial hyperplasia
 

in both sexes of treated rats. He suggested that this finding was
 

worthy of special mention in the report. The primary reviewer pointed
 

out that it was necessary to use historical control mice for the
 

statistical analysis of hemangiosarcomas due to the small number of
 

matched controls. Based on the results of the bioassay, he said that
 

o-Toluidine hydrochloride would have to be considered to pose some human
 

cancer risk.
 

The secondary reviewer said that the bioassay of o-Toluidine
 

hydrochloride was acceptable and that the results clearly indicated
 

that the compound was carcinogenic. He also concluded that the
 

compound must be considered to be a potential human carcinogen.
 

It was moved that the report on the bioassay of o-Toluidine
 

hydrochloride be accepted as written. The motion was seconded and
 

approved unanimously.
 

131
 
Clearinghouse Members present:
 

Arnold L. Brown (Chairman), University of Wisconsin Medical School
 

Joseph Highland, Environmental Defense Fund
 

William Lijinsky, Frederick Cancer Research Center
 

Henry Pitot, University of Wisconsin Medical Center
 

Verne A. Ray, Pfizer Medical Research Laboratory
 

Kenneth Wilcox, Michigan State Health Department
 

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.
 

132
 

. S . GOVERNMEN T PRINTIN G OFFICE : 1978-281-217/3338
 

DHEW Publication No. (NIH) 79-1709