National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 160 

1979 

BIOASSAY OF 

2,4,5-TRIMETHYLANILINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 137-17-7 

NCI-CG-TR-160 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

2,4,5-TRIMETHYLANILINE
 

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
 

DREW Publication No. (NIH) 79-1716
 
i i

 
BIOASSAY OF
 

2,4,5-TRIMETHYLANILINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORD: This report presents the results of the bioassay of
 

2,4,wimethylaniline 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 that are carcinogenic in animals
 

requires a wider analysis.
 

CONTRIBUTORS: This bioassay of 2,4,5-trimethylaniline was
 

conducted by The NCI Frederick Cancer Research Center (FCRC) (11,
 

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, Mr. L.
 

Callahan for management of the computer branch, and Mr. R. Cypher
 

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

computer services. Necropsies were performed by Drs. B. Ulland,
 

R. Schueler, R. Ball, and R. Cardy. Histopathologic evaluations
 

were performed by Drs. M. D. Reuber and R. N. Empson, Jr., and
 

the diagnoses included in this report represent their
 

interpretation.
 

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

Mason Research Institute (3). The statistical analyses were
 

performed by Dr. J. R. Joiner (4) and Ms. P. L. Yong (4), using
 

methods selected for the bioassay program by Dr. J. J. Gart (5).
 

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 (1).
 

This report was prepared at Tracor Jitco (4) under the direction
 

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

Dr. C. R. Angel, Acting 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. Owen, Ms. M. S. King, and Mr. W. D. Reichardt,
 bioscience writers; and Dr. E. W. Gunberg, technical editor,
 

assisted by Ms. Y. E. Presley.
 

The following scientists at NCI were responsible for evaluating
 

the bioassay, 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. Ham ,
 

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

Rockville, Maryland.
 

(4) Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 Mary1and.
 

(5) 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 2,4,5-trimethylaniline for possible carcinogenicity 

was conducted by administering the test chemical in feed to F344 

rats and B6C3F1 mice. 

Groups of 50 rats and 50 mice of each sex were administered 

2,4,5-trimethylaniline at one of two doses, either 200 or 800 ppm 

for the rats and either 50 or 100 ppm for the mice, for 101 

weeks. Matched controls consisted of 20 untreated rats and 20 

untreated mice of each sex. All surviving animals were killed at 

the end of administration of the test chemical. 

Mean body weights of the dosed male and female rats were 

generally lower than those of corresponding controls; mean body 

weights of the dosed mice were only slightly lower in the males 

than in the corresponding controls and were unaffected or 

affected irregularly in the females. Survival was not affected 

significantly when the rats or mice were administered the test 

chemical and was 70% or greater in all dosed or control groups. 

Sufficient numbers of animals were at risk for the development of 

late-appearing tumors. 

In the rats, hepatocellular carcinomas or neoplastic nodules 

occurred at incidences that were dose related in both males and 

females (P less than or equal to 0.001), and in direct 

comparisons the incidences were significantly higher in the 

high-dose males, high-dose females, and low-dose females (P less 

than or equal to 0.004) than in corresponding controls (males: 

controls 1/19, low-dose 6/50, high-dose 20150; females: controls 

0/20, low-dose 12/49, high-dose 27/50). In addition, 

alveolar/branchiolar carcinomas or adenomas occurred in the 

female rats at incidences that were dose related (P = 0.003), and 

in a direct comparison the incidence was significantly higher in 

the high-dose group (P = 0.017) than in the corresponding control 

group (controls 0120, low-dose 3/43, high-dose 11/50). 

In the female mice, hepatocellular carcinomas occurred at 

incidences that were dose related (P less than or equal to 

0.0011, and in direct comparisons the incidences were 

significantly higher (P less than or equal to 0.001) in the lowand high-dose animals than in corresponding contro1s (controls 

0120, low-dose 18/49, high-dose 40150). Because historical 

records of this laboratory for control B6C3F1 male mice show a 

relatively high incidence of hepatocellular carcinomas, an 

increased incidence of these tumors in 2,4,5-trimethylaniline 
dosed male mice as compared with matched controls could not be 

clearly associated with administration of the test compound. 

It is concluded that under the conditions of this bioassay, 

2,4,5-trimethylaniline was carcinogenic for male and female F344 

rats and female B6C3F1 mice, inducing hepatocellular carcinomas or. 

neoplastic nodules in the rats of each sex, alveolar/bronchiolar 

carcinomas in the female rats, and hepatocellular carcinomas in 

the female mice. 

v i 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 5
 

E. Subchronic Studies 7
 

F. Chronic Studies 10
 

G. Clinical and Pathologic Examinations '12
 

H. Data Recording and Statistical Analyses 13
 

III. Results . Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (~ats) 19
 

C. Pathology (~ats) 22
 

D. Statistical Analyses of Results (~ats) 25
 

IV. Results . Mice .... 29
 

A. Body Weights and Clinical Signs ice) 29
 

B. Survival ice) 29
 

C. Pathology ice). 32
 

D. Statistical Analyses of Result? ice) 36
 

V. Discussion 39
 

VI. Bibliography 43
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Administered 2.4.5.Trimethylaniline
 

in the Diet 45
 

Table A1 Suranary of the Incidence of Neoplasms in
 

Male Rats Administered 2.4.5.Trimethylaniline
 

in the Diet 47
 

vii
 
Page 

Table A2 Summary of the Incidence of Neoplasms in 

Female Rats Administered 2,4,5-Trimethylaniline 

in the Diet 51 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Administered 2,4,5-Trimethylaniline in 

the Diet 55 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Administered 2,4,5-Trimethylaniline 

in the Diet 57 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Administered 2,4,5-Trimethylaniline 

in the Diet 61 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered 2,4,5­

Trimethylaniline in the Diet 65 

Table Cl Summary of the Incidence of Nonneoplastic Lesions 

in Male Rats Administered 2,4,5-Trimethylaniline 

in the Diet 67 

Table C2 Summary of the Incidence of Nonneoplastic Lesions 

in Female Rats Administered 2,4,5-Trimethylaniline 

in the Diet 72 

Appendix D Summary of the Incidence of Nonneoplastic Lesions 

in Mice Administered 2,4,5-Trimethylaniline 

in the Diet 77 

Table Dl Summary of the Incidence of Nonneoplastic Lesions 

in Male Mice Administered 2,4,5-Trimethylaniline 

in the Diet 79 

Table D2 Summary of the Incidence of Nonneoplastic Lesions 

in Female Mice Administered 2,4,5­

Trimethylaniline in the Diet 82 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Administered 2,4,5-Trimethylaniline 

in the Diet 85 

viii
 
Page 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Administered 2.4.5-Trimethylaniline 

in the Diet 87 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Administered 2.4.5-Trimethylaniline 

in the Diet 94 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered 2,4,5-Trimethylaniline 

in the Diet 101 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Administered 2.4.5-Trimethylaniline 

in the Diet 103 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Administered 2,4,5­

Trimethylaniline in the Diet 106 

TABLES 

Table 1 2.4.5-Trimethylaniline Subchronic Feeding 

Studies in Rats 8 

Table 2 2.4.5-Trimethylaniline Subchronic Feeding 

Studies in Mice 9 

Table 3 2.4.5-Trimethylaniline Chronic Feeding Studies 

in Rats and Mice 11 

FIGURES 

Figure 1

Figure 2

 Growth Curves for Rats Administered 

2.4.5-Trimethylaniline in the Diet

 Survival Curves for Rats Administered 

2.4.5-Trimethylaniline in the Diet

 20 

 21 

Figure 3 Growth Curves for Mice Administered 

2.4.5-Trimethylaniline in the Diet 30 

Figure 4 Survival Curves for Mice Administered 

2.4.5-Trimethylaniline in the Diet 3l 

ix
 

I. INTRODUCTION
 

2,4,5-Trimethylaniline (CAS 137­

17-7; NCI C02299), or pseudocumidine, is a component of a 

mixture of aromatic amines used 

in the synthesis of the red dye 

Ponceau 3R. This dye is produced 2, 4, 5-Trimethylaniline 

by diazotizing a mixture of amine 

intermediates, some of which have been identified as methyl-, 

dimethyl-, or trimethylanilines, and coupling them with 2-naphthol3,6-disulfonic acid. Ponceau 3R is therefore a complex mixture 

containing some 1-(2,4,5-trimethylpheny1azo)-2-naphthol-3,6­

disulfonic acid rice et al., 1961; Hansen et al., 1963). 

Ponceau 3R has been used as a color additive in foods since 1907.
 

It was certified as FD&C (Food Drug and Cosmetic) Red No. 1 from
 

1940 until 1960, at which time it was withdrawn from general use.
 

Provisional recertification as Ext. D&C (External Drug and
 

Cosmetic) Red No. 15 was granted shortly thereafter in 1961, but
 

revoked in 1968 (Hansen et al., 1963; Code of Federal Regulations,
 

1977).
 
The acute oral LD of 2,4,5-trimethylaniline in male Osborne-


Mendel rats when administered in an aqueous solution by stomach
 

tube was 1,585 mg/kg, which was intermediate in relation to six
 

anilines derived from Ponceau 3R (Lindstrom et al., 1969).
 

Administration of 2,4,5-trimethylaniline in the diet at 375 to
 

5,000 ppm, for 90 days resulted in increased liver and kidney
 

weights; at 750 to 5,000 ppm slight focal bile duct proliferation
 

was found (Lindstrom et al., 1969).
 

Ponceau 3R was found to produce liver tumors of the hepatocytes
 

and bile duct in rats (Hansen et al., 1963; Grice et al., 1961).
 

The azo linkage of some components of Ponceau 3R are cleaved in
 

vivo to give l-amino-2-naphthol-3,6-disulfonic acid and the
 

various aniline moieties, including 2,4,5-trimethylaniline;
 

however, since the naphthol derivative is a component of other
 

nontoxic food dyes, these carcinogenic effects have been attributed
 

to the anilines (Lindstrom et al., 1969). Earlier 2-year studies
 

conducted by the National Cancer Institute in male Charles River
 

rats and in male and female HaM/lCR mice suggested that
 

2,4,5trimethylaniline was carcinogenic in the mice and possibly
 

also in the rats (Homburger et al., 1972; Weisburger et al., in
 

press). 2,4,5-Trimethylaniline was selected for study in the
 

Careinogenesis Testing Program, using an expanded bioassay
 

protocol.
 
11. MATERIALS AND METHODS
 

A. Chemical
 

2,4,5-Trimethylaniline was obtained from Research Organic/
 

Inorganic Chemical as a fine, gray-white powder. Its melting
 

point was 64~ . Elemental analysis showed 78.6% carbon, 9,9%
 

hydrogen, and 10.2% nitrogen (theoretical: 80.0% C, 9.6% H, and
 

10.4% N). Analysis by gas-liquid chromatography indicated two
 

components, one of which was greater than 99.9%. Its infrared
 

spectrum was consistent with its chemical structure, and mass
 

spectral analysis gave a molecular ion at m/e 135 and a base peak
 

at m/e 120.
 

The test material was stored at 5 C until used.
 

B. Dietary Preparation
 

Test diets containing 2,4,5-trimethylaniline were prepared at
 

Frederick Cancer Research Center (FCRC) every 1 to 1-112 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
 
waynea Sterilizable Lab Meal containing 4% fat (Allied Mills,
 

Inc., Chicago, 111.1, 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-pell^@ twin-shell
 

blender. The diets were routinely stored at 7 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 FCRC (~rederick, ~d) , which is monitored by the Division
 

of Cancer Treatment, NCI. The animals were housed within the
 

test facility for 2 weeks and were then assigned four rats of the
 

same sex to a cage and five mice of the same sex to a cage. The
 

initial weights for male rats were 90 to 105 g, averaging at
 

least 100 g; for female rats, 80 to 95 g, averaging at least 90
 

g; for male mice, 18 to 22 g, averaging at least 19.5 g; and for
 

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 (~ab Products
 

Inc., Garfield, N. J.1, 19 x 10-112 x 8 inches for the rats and
 

11-112 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, 0hio). The bedding used was
 

~bsorb-dri@ hardwood chips (Northeastern Products, Inc.,
 

Warrenburg, N. Y) . The feed supplied was presterilized ~ayne@
 

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

suspended stainless steel hoppers and replenished as required, at
 

least three times per week. Water, acidified to pH 2.5, was
 

supplied £d_ libitum from glass bottles with sipper tubes
 

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
 

8 8 ~ in a tunne1-type cagewasher (industrial Washing Machine
 

Corp., Mataway, N. J.), using the detergents, clout *(~harmacal
 

Research Laboratories, Greenwich, Conn.) or Oxford DtChlor
 

(Oxford Chemicals, Atlanta, ~a.1. The glass bottles and sipper
 
tubes were sanitized at 82 to 88'~ 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 ~ in a rack washer (Consolidated
 

Equipment CO.) once per month, using the Calgen Commercial
 

Division detergent, and the filter paper was changed at the same
 

time.
 

The animal rooms were maintained at 22 to 24 ~ 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 2,4,5-trimethylaniline and their corresponding
 

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

of the following chemicals:
 
(CAS 128-37-0) butylated hydroxytoluene (BHT)
 

(CAS 88-96-0) phthalamide
 

Mice administered 2,4,5-trimethylaniline and their corresponding
 

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

of the following chemicals:
 

(cAS 156-62-7) calcium cyanamide
 

(GAS 999-81-5) (2-chloroethyl) trimethylammonium chloride (CCC)
 

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

(cAS 19010-66-3) lead dimethyldithiocarbamate
 

(cAS 86-30-6) N-nitrosodiphenylamine
 

(GAS 88-96-01 phthalamide
 

(CAS 120-62-7) piperonyl sulfoxide
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses (MTD's) of 2,4,5-trimethylaniline, 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 2,4,5-trimethylaniline at one of several doses
 

for 7 weeks, followed by 1 week of observation, and groups of
 

five control animals of each species and sex were administered
 

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

1 and 2 show doses fed, the survival of animals in each dosed
 

group at the end of the study, and the mean body weights of each
 
Table 1. 2,4,5-Trimethylaniline Subchronic 

Feeding Studies in Rats 

Male Female 

Mean Weight Mean Weight 

at Week 7 at Week 7 

Dose as % of as % of 

(ppm) Survival (a) Control Survival (a) Control 

RATS 

0 515 100 515 100 

1,000 515 93 515 515 

1,500 5/5 92 5/5 83 

2,000 5/5 86 5/5 84 

3,200(b) 5/5 67 5/5 73 

4,600 5/5 56 3/5 64 

(a)	 Number surviving/number in group.
 

(b)	 Small amount os f bile-duct hyperplasia and periportal edema
 

were observed in livers. Focal hyperplasia and hepatocellular
 

hypertrophy were occasionally noted.
 
Table 2. 2,4,5-Trimethylaniline Subchronic
 

Feeding Studies in Mice
 

Male	 Female
 

Mean Weight	 Mean Weight
 

at Week 7 at Week 7
 

Dose as % of as % of
 

(ppm) Survival (a) Control Survival (a) Control
 

MICE
 

First Study
 

0 4/5 100 5/5 100
 

1,000 5/5 86 5/5 87
 

1,500 5/5 83 5/5 82
 

2,000 5/5 80 2/5 90
 

3,200 5/5 71 5/5 79
 

4,600 5/5 72 5/5 74
 

Second Study
 

0 5/5 100 5/5 100
 

25 5/5 107 5/5 100
 

50 5/5 100 5/5 102
 

100 5/5 101 5/5 82
 

200 5/5 105 5/5 95
 

500 5/5 97 5/5 92
 

l,000(b) 5/5 82 5/5 84
 

(a)	 Number surviving/number in group.
 

(b)	 Very slight to moderate increase in splenic hematopoiesis in
 

one female and three males. A trace of Kupffer-cell
 

pigmentation in livers of four males and five females.
 
dosed group at week 7, expressed as percentages of mean body
 

weights of controls. At the end of the subchronic studies, all
 

animals were killed using C02 and necropsied. Histopathologic
 

findings are included as footnotes to tables 1 and 2.
 

Ten percent depression in body weight was one of the main
 

criteria used 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.
 

The low and high doses were set at 200 and 800 ppm for rats and
 

at 50 and 100 ppm for mice.
 

I?. Chronic Studies
 

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

feeding studies are shown in table 3.
 

10
 
Table 3. 2y4y5-Trimethylaniline Chronic Feeding
 

Studies in Rats and Mice
 

2,4,5-Trimethyl- Time on
 

Species, Sex, Initial No. aniline in Diet (b) Study
 

and Test Group Animals (a) (ppm) (weeks)
 

RATS
 

Male
 

Matched-Control 20 0 101
 

Low-Dose 50 200 101
 

High-Dose 50 800 101
 

Fema1e
 

Matched-Control 20 0 101
 

Low-Dose 50 200 101
 

High-Dose 50 800 101
 

MICE
 

Male
 

Matched-Control 20 0 101
 

Low-Dose 50 50 101
 

High-Dose 50 100 101
 

Female
 

Matched-Control 20 0 101
 

Low-Dose 50 50 101
 

High-Dose 50 100 101
 

(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.
 

11
 
G. Clinical and Patholoeic Examinations
 

All animals were observed twice daily. 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 those that survived to the end of the
 

bioassay were killed using CO and necropsied.
 

Gross and microscopic examinations of major tissues, major
 

organs, and all gross lesions were performed. The tissues were
 

preserved in 10% neutral buffered 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,
 

sublingual, and submaxillary), liver, pancreas, esophagus,
 

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

kidney, urinary bladder, pituitary, adrenal, thyroid,
 

parathyroid, testis, prostate, mammary gland, aterus, ovary,
 

brain (cerebrum and cerebellum), and all tissue masses.
 

Peripheral blood smears also were made for all animals, whenever
 

possible.
 

12
 
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.
 

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 inhahart 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 (~erenblum, 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
 

13
 
carcinogenicity are discussed in the statistical narrative
 

section.
 

Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically
 

censored as of the time that they died of other than natural
 

causes or were found to be missing; animals dying from natural
 

causes were not statistically censored. Statistical analyses for
 

a possible dose-related effect on survival used the method of Cox
 

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

extensions of Cox's 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 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
 

14
 
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
 

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 (~iller, 1966) requires that the
 

P value for any comparison be less than or equal to 0.051k. 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 (~rmitage, 19711, 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 relation15
 
ship. This method also provides a two-tailed test of departure
 

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

tests on animals that survived at least 52 weeks, unless a tumor
 

was found at the anatomic site of interest before week 52. When
 

such an early tumor was found, comparisons were based exclusively
 

on animals that survived at least as long as the animal in which
 

the first tumor was found. Once this reduced set of data was
 

obtained, the standard procedures for analyses of the incidence
 

of tumors isheher 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
 

16
 
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 art, 1971). The
 

relative risk is defined as p /p where p is the true
 

binomial probability of the incidence of a specific type of tumor
 

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.
 

17
 
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 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
 
111. RESULTS - RATS
 

A. Bodv Weights and Clinical Signs (Rats)
 

Mean body weights of low- and high-dose males and high-dose
 

females were lower than those of corresponding controls
 

throughout the bioassay (figure 1); mean body weights of low-d'ose
 

females were lower than those of corresponding controls only
 

after week 46. Other clinical signs were common to both control
 

and dosed groups of rats.
 

B. Survival (Rats)
 

Estimates of the probabilities of survival for male and female
 

rats administered 2,4,5-trimethylaniline in the diet at the doses
 

of this bioassay, together with those of the matched controls,
 

are shown in the Kaplan and Meier curves in figure 2. The result
 

of the Tarone test for dose-related trend in mortality is not
 

significant in either sex.
 

In male rats, 43/50 (86%) of the high-dose group, 37/50 (74%) of
 

t,he low-dose group, and 16/20 (80%) of the matched-control group
 

19
 
TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered 2,4,5-Trimethylaniline in the Diet 

20 
TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered 2,4,5-Trimethylaniline in the Diet 

21 
lived to the end of the bioassay. In females, 42/50 (84%) of
 

each dosed group and 14/20 (70%) of the matched-control group
 

lived to the end of the bioassay.
 

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

development of late-appearing tumors.
 

C. Pathology (~ats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A, tables A1 and A2; findings on nonneoplastic lesions
 

are summarized in Appendix C, tables Cl and C2.
 

Neoplasms were observed with greatest frequency in the livers and
 

lungs of dosed male and female rats.
 

Hepatocellular carcinomas generally were observed on gross
 

examination; histologically they were well-differentiated or
 

poorly differentiated, and a few were undifferentiated,
 

carcinomas. The cells in well-differentiated carcinomas had
 

vesicular nuclei and eosinophilic cytoplasm and grew in cords two
 

or several cells in thickness. Cells in poorly differentiated
 

carcinomas had basophilic or eosinophilic cytoplasms and grew in
 

22
 
sheets. Undifferentiated carcinoma cells varied greatly in size
 

and shape and were quite anaplastic. Bile-duct carcinomas were
 

well differentiated. Cells varied in size but usually tended to
 

be columnar and formed ducts. Neoplastic nodules were distinct,
 

small, early lesions in which the cells were clearly demarcated
 

from, and compressed the adjacent parenchymal cells, at least
 

focally. Cells had lightly staining eosinophilic cytoplasm and
 

occasional double nuclei. The incidences of the different
 

neoplasms of the liver in the rats were as follows:
 

Alveolar/bronchiolar carcinomas of the lung were well-


differentiated papillary adenocarcinomas or poorly differentiated
 

carcinomas. Cells in the well-differentiated carcinomas were
 

23
 
columnar and formed papillary growths as well as glands. Cells
 

in poorly differentiated carcinomas were small cells growing in
 

sheets. The incidences of alveolar/bronchiolar carcinomas and
 

adenomas in the rats were as follows:
 

Male Fema1e
 

Matched Low High Matched Low High
 

Control Dose Dose Control Dose Dose
 

Number of Tissues
 

Examined 20 49 50 20 43 50
 

Alveolar/Bronchiolar
 

Carcinoma 1(5%) 0 2(4%) 2(5%) 2(4%)
 

Alveolar/Bronchiolar
 

Adenoma 0 0 5(10%) 0 1(2%) 9(18%)
 

Number of Anima1s
 

with Tumors 1(5%) 7(14%) 0 3(7%) 11(22%)
 

There also were neoplasms of the hematopoietic system and vascular
 

system in some groups of rats; however, these occurred in both
 

control and dosed groups.
 

A variety of nonneoplastic lesions and disorders were encountered
 

with regularity in both control and dosed rats. Such lesions were
 

considered to be common in aged F344 rats.
 

Based on the histopathologic examination, the incidence of
 

24
 
neoplasms of the liver and the lung was increased in the male and
 

female rats administered 2,4,5-trimethylaniline.
 

D. Statistical Analyses of Results (~ats)
 

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.
 

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

dose-related trend in the incidence of hepatocellular carcinomas is
 

significant (P = 0.002). The result of the Fisher exact test shows
 

that the incidence of the tumors in the high-dose group is
 

significantly higher (P = 0.020) than that in the matched-control
 

group. When the incidence of either hepatocellular carcinomas or
 

neoplastic nodules in male rats is analyzed, increased significance
 

is observed in the Cochran-Armitage test for linear trend (P less
 

than 0.001) and in the Fisher exact test (P = 0.004) between the
 

high-dose and control groups. In females, the result of the
 

Cochran-Armitage test on the incidence of hepatocellular carcinomas
 

is significant (P less than 0.001). The Fisher exact comparison of
 

the incidences of tumors in the high-dose and matched-control
 

25
 
groups shows a P value of 0.039, which is above the 0.025 level
 

required for significance when the Bonferroni inequality criterion
 

is used for multiple comparison. However, when the incidence of
 

either hepatocellular carcinomas or neoplastic nodules in female
 

rats is analyzed, significant results of the Cochran-Armitage test
 

(P less than 0.001) and Fisher exact test (P less than or equal to
 

0.010) between each dosed group and the control group are
 

observed. The statistical conclusion is that the incidence of
 

liver tumors in rats of each sex is associated with the
 

administration of 2,4,5-trimethylaniline.
 

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

incidence of lung tumors is significant (P = 0.0031, and the result
 

of the Fisher exact test shows that the incidence of tumors in the
 

high-dose group is significantly higher (P = 0.017) than that in
 

the matched-control group. The statistical conclusion is that the
 

incidence of lung tumors in female rats is associated with the
 

administration of the test chemical.
 

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

incidence of lung tumors, the incidence of bile-duct carcinoma of
 

the liver, and the incidence of C-cell carcinoma of the thyroid are
 

significant, but the results of the Fisher exact test are not
 

significant.
 

26
 
Significant dose-related trends in the negative direction are
 

observed in the incidence of hematopoietic tumors and in the
 

incidence of mesotheliomas of the tunica vaginalis in male rats.
 

In females, the incidence of endometrial stromal polyps is higher
 

in the matched-control group than in the low-dose group.
 

In summary, the incidences of lung tumors in female rats and
 

hepatic tumors in rats of each sex were associated with the
 

administration of 2,4,5-trimethylaniline.
 

27
 
28

 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs ice)
 

Mean body weights of the dosed male mice were slightly lower than
 

those of the corresponding controls throughout the bioassay for
 

the high-dose group and after week 16 for the low-dose group
 

(figure 3) . Mean body weights of the high-dose females were
 

essentially the same as those of the corresponding controls
 

throughout the bioassay; mean body weights of the low-dose
 

females were slightly lower than those of the controls for the
 

first 30 weeks and slightly higher thereafter. Clinical signs
 

occurred at comparable incidences in control and dosed groups of
 

both male and female mice.
 

B. Survival ice)
 

Estimates of the probabilities of survival for male and female
 

mice administered 2,4,5-trimethylaniline in the diet at the doses
 

of this bioassay, together with those of the matched controls,
 

are shown in the Kaplan and Meier curves in figure 4. The result
 

29
 
TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Administered 2,4,5-Trimethylaniline i n the Diet 

30 
TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves for Mice Administered 2,4,5-Trimethylaniline in the Diet 
of the Tarone test for dose-related trend in mortality is not
 

significant in either sex.
 

In male mice, 38/50 (76%) of the high-dose group, 43/50 (86%) of
 

the low-dose group, and 16/20 (80%) of the matched-control group
 

lived to the end of the bioassay. In females, 45/50 (90%) of the
 

high-dose group, 39/50 (78%) of the low-dose group, and 17/20 (85%)
 

of the matched-control group lived to the end of the bioassay.
 

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

development of late-appearing tumors.
 

C. Patholoev (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.
 

Neoplasms were observed with greatest frequency in the liver,
 

hematopoietic system, and vascular system in mice administered
 

2,4,5-trimethylaniline.
 

The incidence of hepatocellular carcinomas was increased in dosed
 

32
 
male and female mice when compared with their respective controls.
 

These tumors generally were observed on gross examination; histologically they were well-differentiated or poorly differentiated,
 

and a few were undif ferentiated, carcinomas. The cells in well-


differentiated carcinomas had vesicular nuclei and eosinophilic
 

cytoplasm and grew in cords two or several cells in thickness.
 

Cells in poorly differentiated carcinomas had basophilic or
 

eosinophilic cytoplasms and grew in sheets. Undifferentiated
 

carcinoma cells varied greatly in size and shape and were quite
 

anaplastic. Carcinomas of the liver metastasized to the lungs in
 

one control male, two low-dose males, and one high-dose male. The
 

incidences of neoplasms of the liver and gallbladder in the mice
 

were as follows:
 

Male Female 

Matched Low High Matched Low High 

Control Dose Dose Control Dose Dose 

Number of Tissues
 

Examined 20 50 50 20 49 50
 

Hepatocellular
 

Carcinoma 5(25%) 26(52%) 27(54%) 18(36%) 40(80%)
 

Bile-Duct
 

Carcinoma 0 2(4%) 2(4%) 0 0
 

Hyperplastic
 

Nodu1e 1(5%) 3(6%) 7(14%) 4(8%) 13(26%)
 

Gallbladder
 

Carcinoma 0 1(2%) 0
 

Number of Anima1s
 

with Tumors 5(25%) 27(54%) 27(54%) 0 18(36%) 40(80%)
 

33
 
Neoplasms of the hematopoietic system were all diagnosed as
 

lymphomas. The incidences of these hematopoietic neoplasms were
 

not, however, higher in dosed groups than in corresponding
 

controls.
 

Incidences of neoplasms of the lung and of the vascular system
 

were slightly increased in female mice administered 2,4,5­

trimethylaniline when compared with controls. Carcinomas of the
 

lung were well-differentiated papillary adenocarcinomas or poorly
 

differentiated carcinomas. Cells in the well-differentiated
 

carcinomas were columnar and formed papillary growths as well as
 

glands. Cells in poorly differentiated carcinomas were small
 

cells growing in sheets. The incidences of alveolar/bronchiolar
 

adenomas and carcinomas were as follows:
 

Male Female
 

Matched Low High Matched Low High
 

Control Dose Dose Control Dose Dose
 

Number of Tissues
 

Examined 20 50 50 19 49 48
 

Alveolar/Bronchiolar
 

Carcinoma 4(20%) 7(14%) 1(2%) 0 4(8%) 6(12%)
 

Alveolar/Bronchiolar
 

Adenoma 0 2(4%) 0 1(2%) 0
 

Number of Animals
 

with Tumors 4(20%) 9(18%) 1(2%) 0 5(10%) 6(12%)
 

34
 
Hemangiomas and hemangiosarcomas in female mice receiving
 

2,4,5-trimethylaniline most often were seen in lymph nodes, but
 

they also were present in the bone, skeletal muscle, liver,
 

testis, and adipose tissue. Hemangiosarcomas were made up of
 

proliferating endothelial cells that formed blood-filled vascular
 

channels and generally were invasive. The incidences of the
 

different neoplasms of the vascular system were as follows:
 

Male Female
 

Matched Low High Matched Low High
 

Control Dose Dose Control Dose Dose
 

Number of Tissues
 

Examined 20 50 50 20 49 50
 

Hemangi os arcoma 2(10%) 3(6%) 6(12%) 1(5%) 11(22%) 7(14%)
 

Hemangi oma 0 0 0 0 1(2%) 0
 

Number of Anima1s
 

with Tumors 2(10%) 3(6%) 6(12%) 1(5%) 11(22%) 7(14%)
 

A variety of nonneoplastic lesions and disorders were encountered
 

with regularity in both control and dosed mice. Such lesions
 

were considered common in aged B6C3F1 mice.
 

Based on the histopathologic examination, incidences of neoplasms
 

of the liver were increased in male and female B6C3F1 mice
 

administered 2,4,5-trimethylaniline, and incidences of neoplasms
 

of the lung and of the vascular system were increased in females.
 

35
 
D. Statistical Analyses of Results ice)
 

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 result of the Cochran-Armitage test for the
 

incidence of hepatocellular carcinomas is significant (P =
 

0.039). The Fisher exact comparison of the incidences of tumors
 

in the matched-control group and the low-dose group shows a P
 

value of 0.035, which is above the 0.025 level required for
 

significance when the Bonferroni inequality criterion is used for
 

multiple comparison. The comparison between the control and
 

high-dose groups shows a P value of 0.025, which is at the
 

critical point when the Bonferroni inequality criterion is used
 

for multiple comparison. The historical records of this
 

laboratory for control B6C3F1 male mice having hepatocellular
 

carcinomas show an incidence of 137/422 (32%), with incidences in
 

individual control groups as high as 11/19 (58%) and 10120 (50%),
 

as compared with 4/20 (20%) in the control group in this
 

bioassay, 26/50 (52%) in the low-dose group, and 27/50 (54%) in
 

the high-dose group. In females, the result of the Cochran-


Annitage test for the incidence of hepatocellular carcinomas is
 

36
 
significant (P less than 0.001), and the results of the Fisher
 

exact test show that the incidence of these tumors in each dosed
 

group is significantly higher (P less than or equal to 0.001)
 

than that in the matched-control group. The statistical conclusion is that the incidence of hepatocellular carcinomas in
 

female mice is associated with the administration of 2,4,5­

tr imethylan ili ne.
 

In female mice, the result of the Cochran-Annitage test for the
 

incidence of carcinomas of the pituitary is significant (P =
 

0.0431, but the results of the Fisher exact test are not
 

significant. When the incidence of the combination of carcinomas
 

or adenomas of the pituitary in female mice is analyzed, the
 

results of the statistical test are not significant'.
 

In male mice, significant results in the negative direction are
 

observed in the incidence of lung tumors. In females, a
 

significant trend in the negative direction is observed in the
 

incidence of lymphomas.
 

37
 

V. DISCUSSION
 

Mean body weights of the dosed male and female rats were
 

generally lower than those of corresponding controls; mean body
 

weights of the dosed mice were only slightly lower in the males
 

than in the corresponding controls and were unaffected or
 

affected irregularly in the females. Survival was not affected
 

significantly when the rats or mice were administered the test
 

chemical and was 70% or greater in all dosed or control groups.
 

Sufficient numbers of animals were at risk for the development of
 

late-appearing tumors.
 

In the ratsy hepatocellular carcinomas or neoplastic nodules
 

occurred at incidences that were dose related in both males and
 

females (P less than or equal to O.OOl), and in direct
 

comparisons the incidences were significantly higher in the
 

high-dose males high-dose females and low-dose females (P less
 

than or equal to 0.004) than in corresponding controls (males:
 

controls 1/19 low-dose 6/50, high-dose 20150; females: controls
 

O/2Oy low-dose 12/49, high-dose 27/50). In addition, alveolar/
 

bronchiolar carcinomas or adenomas occurred in the female rats at
 

incidences that were dose related (P = 0.003), and in a direct
 

39
 
comparison the incidence was significantly higher in the high-


dose group (P = 0.017) than in the corresponding control group
 

(controls 0120, low-dose 3/43, high-dose 11/50).
 

In the mice, hepatocellular carcinomas occurred at incidences
 

that were dose related in both males and females (P less than or
 

equal to 0.039), and in direct comparisons the incidences were
 

significantly higher (P less than or equal to 0.025) in the high-


dose males, high-dose females, and low-dose females than in
 

corresponding controls (males: controls 5/20, lm-dose 26/50,
 

high-dose 27/50; females: controls 0120, low-dose 18/49,' high-


dose 40150). However, the comparison between the controls and
 

the high-dose males shows a P value of 0.025, which is at the
 

critical point when the Bonferroni inequality criterion is used
 

for multiple comparison. The historical records of this
 

laboratory for control B6C3Fl male mice having hepatocellular
 

carcinomas show an incidence of 1371422 (32%), with incidences of
 

individual control groups as high as 11/19 (58%) and 10120
 

(50%). Thus, in male mice these tumors cannot be clearly
 

associated with administration of 2,4,5-trimethylaniline.
 

In prior investigations of the carcinogenicity of 2,4,5-trimethylaniline administered in the diet in 2-year studies, hepatic and
 

pulmonary tumors occurred in increased incidences in male and
 

40
 
female HaMIICR mice, but marginal results were obtained using
 

male Sprague-Dawley rats (Homburger et al., 1972; Weisburger et
 

al* , in press). The low and high doses of test chemical used in
 

these studies were 3,000 and 6,000 ppm for 5 months and 1,500 and
 

3,000 ppm for the subsequent 13 months for the rats and 6,000 and
 

12,000 ppm for 18 months for the mice. The occurrence of hepatic
 

tumors in B6C3Fl mice administered 2,4,5-trimethylaniline in the
 

present bioassay is consistent with the occurrence of these
 

tumors in the HaMIICR mice of the earlier studies. The occurrence of hepatic tumors in male and female F344 rats and
 

pulmonary tumors in female F344 rats administered 2,4,5-trimethylaniline in the present bioassay differs, however, from the
 

marginal results obtained in the earlier studies using male
 

Sprague-Dawley rats. Although the doses of test chemical used in
 

the earlier studies were higher than those used in the present
 

bioassay, especially for the mice, the duration of administration
 

was shorter in the earlier studies. The induction of liver
 

adenomas or carcinomas in rats of various strains administered
 

Ponceau 3R in the feed rrice et al.? 1961; Hansen et al., 1963)
 

may be attributable to the in vivo cleavage of the dye to yield
 

its aniline moieties, among which 2,495-trimethylaniline is a
 

predominant component (Lindstrom et al., 1969).
 

It is concluded that under the conditions of this bioassay;
 

41
 
2,4,5-trimethylaniline was carcinogenic for male and female F344
 

rats and female B6C3Fl mice, inducing hepatocellular carcinomas or
 

neoplastic nodules in the rats of each sex, alveolar/bronchiolar
 

carcinomas in the female rats, and hepatocellular carcinomas in
 

female mice.
 

42
 
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 of the
 

Panel on Carcinogenicity £f_ the Cancer Research Cornmission-- of the
 

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

Code of Federal Regulations, 2J. CFR 81.30, 1977.
 

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

SOC. B 34:187-220, 1972.
 

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

London, 1970, pp. 48-52.
 

Gart, J. J., The comparison of proportions: a review of significance tests, confidence limits and adjustments for stratification. Rev. Int. Stat. Inst. 39:148-169, 1971.
 

Grice, H. C., Mannell, W. A., and Allmark, M. G., Liver tumors in
 

3 :509 520
 rats fed Ponceau 3R. Toxicol. Appl. Pharmacol. - - , 1961.
 

Hansen, W. H., Kent, J. D., Fitzhugh, 0. G., and Nelson, A. A.,
 

Chronic oral toxicity of Ponceau 3R. Toxicol. Appl. Pharmacol.
 

5:105-118, 1963.
 

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.
 

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

incomplete observations. J. Amer. 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.
 

43
 
Miller, R. G., Jr., Simultaneous Statistical Inference, McGraw-


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

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

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

induced by different numbers of admidstrations of benzo(a)
 

pyrene and ferric oxide. Cancer Res. 32} 1073-1081, 1972.
 

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

62:679-682, 1975.
 

Weisburger, E. K., Russfield, A. B., Hornburger, F., 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, National Cancer Institute, Bethesda,
 

Md. (in press).
 

44
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED 2,4,5-TRIMETHYLANILINE
 

45
 
46

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED 2,4,5-TRIMETHYLANILINE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 20 20 20
 

ANIMALS NECROPSIED 20 0 0
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 20 20 20
 

INTEGUMENTARY SYSTEM
 

XSKIN (20) (50) (50)
 

SQUANOUS CELL CARCINOMA 1 (2SO
 

XSUBCUT TISSUE (20) (50) (50)
 

SQUAMOUS CELL PAPILLOMA 1 (5%)
 

SQUAMOUS CELL CARCINOMA
 1 (2%)
 FIBROMA 1 (5%)
 

RESPIRATORY SYSTEM
 

#LUNG (20) (49) (50)
 

CARCINOMA, NOS, METASTATIC
 1 (2%)
 ALVEOLAR/BRONCHIOLAR ADENOMA 5 (10%)


ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (5%) 2 (4%)
 

HEMATOPOIETIC SYSTEM
 

XMULTIPLE ORGANS (20) (50) (50)
 

MALIGNANT LYMPHOMA, NOS 2 (4%)


LEUKEMIA,NOS (20%) 10 (20%) 3 (6X)
 

MONOCYTIC LEUKEMIA 1 (2%)
 

#LYMPH NODE (20) (50) (50)
 

MALIGNANT LYMPHOMA, NOS 1 (2%)
 

CIRCULATORY SYSTEM
 

XMAMMARY GLAND (20) (50) (50)
 

HEMANGIOSARCOMA 1 (25O
 

#SPLEEN (20) (49)
 

HEMANGIOSARCOMA 1 (2%)
 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

47
 
TABLE A1.MALE RATS: NEOPLASMS (CONTINUED)

 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

SLIVER ( 19) (50) (50)
 

LYMPHANGIOMA 1 (2%)
 

DIGESTIVE SYSTEM
 

#SALIVARY GLAND (20) (50) (49)
 

ADENOCARCINOMA, NOS 1 (5%)
 

OLIVER ( 19) (50) (50)
 

BILE DUCT CARCINOMA 4 (8*/.)
 

NEOPLASTIC NODULE 1 (5%) 3 (6%) 11 (22%)
 

HEPATOCELLULAR CARCINOMA 3 (6%) 11 (22%)
 

#PANCREAS (19) (50) (48)


ACINAR-CELL ADENOMA 1 (2%)
 

#STOMACH ( 19) (50) (50)


SQUAMOUS CELL PAPILLOMA 1 (2%)
 

#PEYERS PATCH (20) (50) (50
 

LEIOMYOSARCOMA 1 (2%)
 

#COLON ( 19) (50) (50)


ADENOMATOUS POLYP, NOS 1 (5%)
 

URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

#PITUITARY (20) (50) (50)


CARCINOWA,NOS 5 (25%) 6 (12%) 10 (20%)
 

ADENOMA, NOS 3 (15%) 11 (22%) 15 (30%)
 

#ADRENAL (20) (50) (50)


CORTICAL CARCINOMA 2 (4%)
 

PHEOCHROMOCYTOMA 2 (10%) 5 (10%) 5 (10%)
 

GANGLIONEUROWA 1 (2%)
 

#THYROID (20) (50 (50
 

FOLLICULAR-CELL ADENOMA 1 (5%)
 

tt NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)

 

FOLLICULAR-CELL CARCINOMA
 

C-CELL ADENOMA
 

C-CELL CARCINOMA
 

#PANCREATIC ISLETS
 

ISLET-CELL ADENOMA
 

ISLET-CELL CARCINOMA
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND
 

FIBROMA
 

LEIONYOSARCOMA
 

FIBROADENOMA
 

#TESTIS
 

INTERSTITIAL-CELL TUMOR
 

INTERSTITIAL-CELL TUMOR, MALIGNA
 

NERVOUS SYSTEM
 

#BRAIN
 

ASTROCYTOMA
 

OLIGODENDROGLIOMA
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

KPERITONEUM
 

MESOTHELIOMA, METASTATIC
 

XTUNICA VAGINALIS
 

MESOTHELIOMA, NOS
 

MATCHED
 

CONTROL
 

(19)
 

(20)
 

(20)
 

6 (30%)
 

8 (GO'/.)
 

(20)I
 

1 (5%)
 

1 (5%)
 

(20)
 

(20)
 

2 (10%)
 

LOW DOSE
 

1 (2%)
 

1 (2%)


1 (2%)
 

(50)
 

2 (4%)
 

1 (2%)
 

(501 )
 1 (2%)
 

1 (2%)
 

1 (2%)
 

(50) )
 

10 (20%)
 

32 (6450
 

(49)


1 (2%)
 

(20)


1 (2%)
 

(50)
 

HIGH DOSE
 

2 (4%)
 

4 (8%)
 

(48)
 

1 (2%)
 

(50)


1 (2%)
 

(49)
 

13 (27%)
 

32 (65%)
 

(50)
 

(20)
 

(50)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

49
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MESOTHELIOMA, MALIGNANT 1 (2%) 

ALL OTHER SYSTEMS 

HEAD 

ADENOCARCINOMA, NOS, METASTATIC 

ORBITAL REGION 

CARCINOMA,NOS 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 0 0
 

NATURAL DEATHS 10
 

MORIBUND SACRIFICE 3
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 16 37
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORSX 19 48 50
 

TOTAL PRIMARY TUMORS 39 99 127
 

TOTAL ANIMALS WITH BENIGN TUMORS 10 25 32
 

TOTAL BENIGN TUMORS 15 31 46
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 15 44 45
 

TOTAL MALIGNANT TUMORS 2 1 70
 

TOTAL ANIMALS WITH SECONDARY TUMORS# 1 2
 

TOTAL SECONDARY TUMORS 1 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 3 3 11
 

TOTAL UNCERTAIN TUMORS 3 3 11
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

50
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED 2,4,5-TRIMETHYLANILINE IN THE DIET 

MATCHED 

CONTROL LOW DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS MISSING 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

20 

20 

20 

50 

1 

49 

49 

INTEGUMENTARY SYSTEM
 

^SKIN (20) (49)
 

SQUAMOUS CELL CARCINOMA
 

HSUBCUT TISSUE (20) (49)
 

SQUAMOUS CELL CARCINOMA 1 (2K)
 

RESPIRATORY SYSTEM
 

#LUNG (20) (43)
 

CARCINOMA,NOS 1 (2%)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (2%)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 2 (5%)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (20) (49)
 

MALIGNANT LYNPHOMA, NOS 4 (8%)
 

LEUKEMIAPNOS 1 (5*)
 

#SPLEEN (20) (49)
 

MALIGNANT LYMPHOMA, NOS 1 (2%)
 

^INTESTINAL TRACT (20) (49)
 

MALIGNANT LYMPHOMA, NOS
 

SLIVER (20) (49)
 

MALIGNANT LYMPHO.MA, NOS
 

CIRCULATORY SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

2 (4X)
 

(50)
 

(50)
 

9 (18%)
 

2 (4%)
 

(50)
 

5 (10%)
 

1 (2%)
 

(49)
 

(50)
 

1 (25O
 

(50)
 

1 (25O
 

51
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)

 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

DIGESTIVE SYSTEM
 

#SALIVARY GLAND (20) (49) (49)
 

CARCINOSARCOMA 1 (2%)
 

SLIVER (20) (49 (50)
 

BILE DUCT CARCINOMA 1 (2%)
 

NEOPLASTIC NODULE 12 (24%) 20 (40%)
 

HEPATOCELI.ULAR CARCINOMA 9 (18%)
 

URINARY SYSTEM
 

#URINARY BLADDER ( 19) (49) (49)
 

LEIOMYOSARCOMA 1 (2%)
 

ENDOCRINE SYSTEM
 

#PITUITARY (20) (48) (49)
 

CARCINOMA,NOS 3 (15%) 12 (25%) 5 (10%)
 

ADENOMA, NOS 5 (25%) 10 (21%) 17 (35%)
 

#ADRENAL (20) (49) (49)
 

CORTICAL ADENOMA 2 (10%) 2 (4%) 1 (2%)
 

PHEOCHROMOCYTOMA 2 (4%)
 

KTHYROID (20) (46) (49)
 

FOLLICULAR-CELL CARCINOMA 1 (2%)
 

C-CELL ADENOMA 6 (13%) 5 (10%)
 

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

SPANCREATIC ISLETS (20) (49) (49)
 

ISLET-CELL CARCINOMA 1 (2%)
 

REPRODUCTIVE SYSTEM
 

%MAMMARY GLAND (20) (49) (50)
 

CARCINOMA,NOS 1 (2%)
 

SQUANOUS CELL CARCINOMA 1 (2%)
 

ADENOCARCINOMA, NOS 6 (12%) 4 (8%)
 

CYSTADENOMA, NOS 1 (2%)
 

CYSTADENOCARCINOMA, NOS 2 (%%I
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

52
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE 

FIBROMA 

FIBROADENOMA 

2 ( 10%) 

5 (25%) 

2 (4%)

8 (16%) 

#UTERUS 

ENDOMETRIAL STROMAL POLYP 

(20) 

6 (30%) 

(49) 

1 (2%) 

SOVARY 

GRANULOSA-CELL CARCINOMA 

(20) (49) 

NERVOUS SYSTEM
 

#BRAIN (20) (48)
 

ASTROCYTOMA
 

SPECIAL SENSE ORGANS
 

%MIDDLE EAR (20) (49)
 

SQUAMOUS CELL CARCINOMA
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 50
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 14 42
 

ANIMAL MISSING
 1
 

a INCLUDES AUTOLYZED ANIMALS
 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

1 (2%)
 

7 (14%)
 

(49)
 

7 (14%)
 

(49)
 

1 (2%)
 

(48)
 

1 (2%)
 

(50)
 

50
 

42
 

53
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)

 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS#
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 17 39 45 

 25 77 107 

 16 25 31 

 20 32 48 

5 24 29 

5 33 39 

 12 20 

 12 20 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

54
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED 2,4,5-TRIMETHYLANILINE
 

55
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED 2.4, &TRINETHYLANILINE IN THE DIET 

HATCHED 

CONTROL LOW DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

20 

20 

20 

20 

20 

20 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

#LUNG (20) (50)
 

%ILE DUCT CARCINOMA, METASTATIC 1 (2%)
 

HEPATOCELLULAR CARCINOMA, METAST 1 (5%) 2 (4%)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 2 (6%)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 4 (20%) 7 ( 16%)
 

TUBULAR-CELL ADENOCARCINOMA, MET
 

HEMATOPOIETIC SYSTEM
 

%MULTIPLE ORGANS (20) (50)
 

MALIGNANT LYMPHOMA, NOS 2 (105!) 4 (85!)
 

#SPLEEN (19) (50)
 

MALIGNANT LYMPHOMA, NOS 2 (45!)
 

#LYMPH NODE (20) (50)
 

TUBULAR-CELL ADENOCARCINOMA, MET
 

MALIGNANT LYMPHOMA, NOS 1 (5%) 1 (2%)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

ÃˆMESENTERI L. NODE (20) (20)
 

MALIGNANT LYMPHOMA, NOS 1 (2*/.)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (25!)
 

ÃˆPEYER PATCH (19) (45)
 

MALIGNANT LYMPHOMA, NOS 2 (4%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

X NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

20
 

20
 

20
 

(50)
 

1 (2%)
 

1 (2%)
 

(50)
 

5 ( 105!)
 

(49)
 

(50)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

(20)
 

(45)
 

57
 
TABLE 01. MALE MICE: NEOPLASMS (CONTINUED)

 

CIRCULATORY SYSTEM
 

*MULTIPLE ORGANS
 

HEMANGIOSARCOMA
 

#BONE NARROW
 

HEMANGIOSARCOMA
 

#SPLEEN
 

HEMANGIOSARCOMA
 

*ADIPOSE TISSUE
 

HEMANGIOSARCOMA
 

OLIVER
 

HEMANGIOSARCONA
 

UTESTIS
 

HEMANGIOSARCONA
 

DIGESTIVE SYSTEM
 

XINTESTINAL TRACT
 

ADENOCARCINOMA, NOS
 

#LIVER
 

BILE DUCT CARCINOMA
 

HEPATOCELLULAR CARCINOMA
 

^GALLBLADDER
 

CARCINOMA,NOS
 

URINARY SYSTEM
 

SKIDNEY
 

TUBULAR-CELL ADENOCARCINOMA
 

ENDOCRINE SYSTEM
 

#THYROID
 

FOLLICULAR-CELL CARCINOMA
 

MATCHED
 

CONTROL
 

(20)
 

(20)
 

(19)
 

2 (11%)
 

(20)
 

(20)
 

(20)
 

(20 ) 

(201
 

5 (25%)
 

(20)
 

(20)
 

(19 ) 

LOW DOSE 

(50)


2 (45O
 

(50)
 

(50
 

(50)
 

(50)
 

(50)
 

(50) 

(2K) 

(50)


2 (4%)
 

26 (52%)
 

(50)
 

(50)
 

(50 ) 

HIGH DOSE 

(50)


1 (25O
 

(50)
 

9 f & V ^
 

(49)
 

(50)
 

1 (2%)
 

(50)
 o t £• v ^
 

(50)
 

(50) 

(50)


2 Cby 5
 

27 (54%)
 

(50)
 

(50)
 

1 (23O
 

(48 ) 

» NUMBER OF ANIMALS UITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

58
 
TABLE B1. MALE MICE; NEOPLASMS (CONTINUED)

 

KPANCREATIC ISLETS
 

ISLET-CELL ADENOMA
 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

SHARDERIAN GLAND
 

ADENOCARCINOMA, NOS
 

PAPILLARY CYSTADENOCARCINOMA,NOS
 

MUSCULOSKELETAL SYSTEM
 

*INTERCOSTAL MUSCLE
 

HEPATOCELLULAR CARCINOMA, METAST
 

BODY CAVITIES
 

*MEDIASTINUM
 

HEPATOCELLULAR CARCINOMA, METAST
 

ALL OTHER SYSTEMS
 

DIAPHRAGM
 

HEPATOCELLULAR CARCINOMA, NETAST
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

(19) (49) (49) 

1 (5X) 

(20) (20) (20) 

1 

2 (4X) 

(20) (20) (20) 

1 (25O 

(20) (50)

1 (2%) 

(50) 

1 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

59
 
TABLE 11.MALE MICE: NEOPLASMS (CONTINUED)

 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 2 0 50
 

NATURAL DEATHS 4 7 4
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED 8
 

TERMINAL SACRIFICE 16 43 38
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY T~~ORSH 112 2 3

34 4 32
 

TOTAL PRIMARY TUMORS
 15 56 

TOTAL ANIMALS WITH BENIGN TUMORS 1 2

 

TOTAL BENIGN TUMORS 1 2

 

TOTAL ANIMALS WITH MALIGNANT TUMORS 1111 33 3 32
 

14 54

TOTAL MALIGNANT TUMORS 14 54 45
 

TOTAL ANIMALS WITH SECONDARY TUMORS# 11 22 2
 

TOTAL SECONDARY TUMORS 11 66 3
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

x PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

60
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED 2,4,5-TRIMETHYLANILINE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 20 50
 50
 

ANIMALS MISSING 1
 

ANIMALS NECROPSIED 20 49 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 20 49 50
 

INTEGUMENTARY SYSTEM
 

HSUBCUT TISSUE (20) (49) (50)
 

LIPOMA 1 (2%)
 

NEUROFIBROSARCOMA 1 (5%)
 

RESPIRATORY SYSTEM
 

#LUNG (19) (49) (481
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (2%)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 4 (8%) 6 (13%)
 

HEMATOPOIETIC SYSTEM
 

^MULTIPLE ORGANS (20) (49 (50)
 

MALIGNANT LYMPHOMA, NOS 3 (15%) 13 (27%) 3 (6%)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 2 (10%) 2 ('4%)
 

#SPLEEN (19) (49) (49)
 

MALIGNANT LYMPHOMA, NOS 1 (5X) 2 (4X) 2 (4X)
 

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

MALIGNANT LYMPHOMA, NOS 3 (65O
 

SLIVER (20) (49) (50)
 

MALIGNANT LYMPHOMA, NOS 1 (55O
 

#KIDNEY (20) (49) (49)
 

MALIGNANT LYMPHOMA, NOS 1 (5%)
 

#THYMUS ( 18) (46) (44)
 

MALIGNANT LYMPHOMA, NOS 1 (6%) 1 (2%) 1 (2%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

61
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED)

 

CIRCULATORY SYSTEM
 

*MULTIPLE ORGANS
 

HEMANGIOSARCOMA
 

*SKIN
 

HENANGIOSARCONA
 

#BONE MARROW
 

HEMANGI'OSARCOMA
 

#SPLEEN
 

HEMANGIOSARCOMA
 

#LYMPH NODE
 

HENANGIOSARCONA
 

SMESENTERIC L. NODE
 

HEMANGIOSARCOMA
 

*ADIPOSE TISSUE
 

HEMANGIOSARCONA
 

O N E
 

HEMANGIOSARCOMA
 

IVER
 

HEMANGIOSARCOMA
 

#OVARY
 

HENANGIOMA
 

DIGESTIVE SYSTEM
 

SLIVER
 

HEPATOCELLULAR CARCINOMA
 

#CECUM
 

LEIOMYOSARCOMA
 

URINARY SYSTEM
 

#KIDNEY
 

CARCINOMA,NOS
 

ft NUMBER OF ANIMALS WITH TISSUE x NUMBER OF ANIMALS NECROPSIED 

MATCHED
 

CONTROL
 

(20)
 

(20)
 

(20)
 

(19)
 

(20)
 

(20)
 

(20)
 

(20)


1 (5%)
 

(20)
 

(19)
 

(20)
 

(17)
 

(20)
 

LOW DOSE 

(49)


1 (2%)
 

(49)
 

(49)
 

7 (145O
 

(49)
 

1 (23O
 

(49)
 

(49 ) 

(49)


1 (2%)
 

(49)
 

(49)
 

1 (25O
 

(19)
 

(20)
 

18 (373O
 

(48)
 

1 (2X)
 

(20)
 

1 (2X)
 

HIGH DOSE 

(50)
 

(50)


1 (2%)
 

(49)
 

(49)
 

(49)


1 (2X)
 

(49)
 

(50)
 

(50)


3 (6%)
 

(50)
 

1 (2%)
 

(19)
 

(20)
 

40 (80%)
 

(47)
 

(20)
 

 EXAMINED MICROSCOPICALLY 

62
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ENDOCRINE SYSTEM
 

#PITUITARY (19) (48) (50)


CARCINOMA,NOS 4 (8X)
 

ADENOMA, NOS 1 (5X) 1 (2X) 1 (2X)
 

(ADRENAL (20) (49) (49)
 

CORTICAL CARCINOMA 1 (25O 1 (2X)
 

#THYROID (20) (45) (49)
 

FOLLICULAR-CELL ADENOMA 1 (5X)
 

REPRODUCTIVE SYSTEM
 

XMAMMARY GLAND (20) (49) (50)
 

CARCINOMA,NOS 1 (2X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

XHARDERIAN GLAND (20) (49) (50)
 

ADENOCARCINOMA, NOS
 1 (25O
 PAPILLARY ADENOCARCINOMA 2 (43O
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

%MULTIPLE ORGANS (20) (+9) (50)
 

LEIOMYOSARCOMA
 1 (5%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

63
 
TABLE 12. FEMALE MICE: NEOPLASMS (CONTINUED)

 

SITE UNKNOWN
 

LEIOMYOSARCOMA
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATURAL DEATHS


MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIMAL MISSING


3 INCLUDES AUTOLY2ED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS#
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 20 50 50 

3 10 5 

 17 39 45 

1 

 12 41 45 

 15 59 7 1 

2 4 1 

2 4 1 

 12 41 45 

 13 55 70 

X PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

64
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED 2,4,5-TRIMETHYLANILINE
 

65
 
66

 
TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED 2,4, 6-TRIMETHYLANILINE

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

(50)
 

(50)
 

2 (5%)
 

(50)
 

11 (22%)
 

(50)
 

1 (2%)
 

(49)
 

2 (45J)
 

(49)
 

1 (2X)
 

(50)
 

25 (50%)
 

ANIMALS
 

ANIMALS
 

ANIMALS
 

INITIALLY IN STUDY
 

NECROPSIED
 

EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

%SKIN
 

ABSCESS, NOS
 

KSUBCUT TISSUE
 

EPIDERMAL INCLUSION CYST
 

RESPIRATORY SYSTEM
 

#TRACHEA
 

LYMPHOCYTIC INFLAMMATORY INFILTR
 

• LUNG
 

INFLAMMATION, SUPPURATIVE
 

HYPERPLASIA, ALVEOLAR EPITHELIUM
 

HEMATOPOIETIC SYSTEM
 

#BONE MARROW
 

HYPERPLASIA, NOS
 

#SPLEEN
 

CONGESTION, ACUTE
 

HENOSIDEROSIS
 

HYPERPLASIA, NOS
 

HENATOPOIESIS
 

#SPLENIC SINUSOIDS
 

HYPERPLASIA, NOS
 

#LYMPH NODE
 

HYPERPLASIA. NOS
 

MATCHED
 

CONTROL
 

20
 

20
 

20
 

(20)


1 (55O
 

(20)
 

1 (55O
 

(20)
 

1 (5%)
 

(20)
 

1 (5%)
 

(20)
 

(20)
 

1 (55O
 

(20)
 

(20)
 

 IN THE DIET 

LOW DOSE
 

50
 

50
 

50
 

(50)
 

(50)
 

(50)
 

(49)
 

4 (8%)
 

(50)
 

(49)
 

1 (25O
 

1 (25O
 

(49)
 

(50)
 

14 (28%)
 

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

67
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

CIRCULATORY SYSTEM
 

#SPLEEN (20) (49) (49)
 

THROMBOSISp NOS 1 (5%)
 

#LYMPH NODE (20) (50) (50)

LYPlPHANGIECTASIS 1 (2%) 

#MANDIBULAR L. NODE (20) (50) (50) 

LYNPHANGIECTASIS 1 (2%) 

#HEART (20) (50) (48) 

FIBROSIS, FOCAL 1 (2%) 

PERIARTERITIS 1 (555) 2 (4%) 1 (25O 

#HEART/ATRIUM (20) (50) (48)

 

THROMBOSIS, NOS
 1 (255) 

#HEARTIVENTRICLE (20) (50) (48) 

THRONBOSIS, NOS 1 (255) 

#MYOCARDIUM (20) (50) (48) 

FIBROSIS 1 (5%) 7 (14%) 9 (19%) 

FIBROSISp FOCAL 13 (65%) 10 (20%) 25 (52%) 

#ENDOCARDIUM OF LEFT (20) (50) (48)

 

HYP'ERPLASIA~ NOS 1 (5%)

 

#PANCREAS (19) (50) (48) 

PERIARTERITIS 1 (5%) 3 (6%) 

XMESENTERY (20) (50) (50)

 

PERIARTERITIS
 1 (5%) 1 (2%) 5 (10%) 

DIGESTIVE SYSTEM
 

#LIVER (19) (50) (50)

METAMORPHOSIS FATTY 2 (4%) 4 (8%) 

HEHOSIDEROSIS 1 (2%) 

HYPERPLASIAp NOS 1 (2%) 20 (40%) 

HYPERPLASIA, FOCAL 1 (5X) 1 (2%) 5 (10%) 

HYPERPLASIAI DIFFUSE 1 (2%) 1 (2%) 

#LIVER/CENTRILOBULAR (19) (50) (50)

 

NECROSIS* NOS 2 (4%)

 

« NUMBER OF ANIMALS WITH TISSUE EXARINED PIICROSCOPICALLY
 * NUHBE~ OF ANIMALS NECROPSIED
 

68
 
TAKE C1. MALE RATS: NtmNEOtLASTlC LESIONS 

MATHE0 

CONTROL LOW DOSE HIGH DOSE 

METAMORPHOSIS FATTY	 1 (2%) 

#PANCREAS ( 19) (50) (481 

INFLAPlHATION, CHRONIC 1 (2%) INFLANPIATION, CHRONIC FOCAL 1 (5%) 1 (2%) 3 (6%) 

#STONACH (19) (50) (50) 

INFLAf'UlATION, FOCAL 1 (2%) 

URINARY SYSTEN
 

#KIDNEY	 (20) (50) (50) INFLAMWATION, INTERSTITIAL 2 (4%)

 INFLAMflATION, CHRONIC FOCAL 1 (5%) 15 (30%) 15 (30%)

INFLAMMATIONt CHRONIC DIFFUSE 2 (4%)

 HYPERPLASIA, TUBULAR CELL 1 (2%)

 HYPERPLASIA, DIFFUSE 1 (2%)
 

ENDOCRINE SYSTEM
 

#PITUITARY
 (20) (50 ) (50) CYST* NOS 2 (10%)

 

HYPERPLASIA, FOCAL 1 (2X)

 

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

NODULE 1 (2%) 

HYPERPLASIA, NOS 1 (2%) 

#ADRENAL MEDULLA (20) (50) (50) 

HYPERPLASIA, NO5 5 (16%) 

HYPERPLASSA, FOCAL 1 (5%) 1 (2%) 

•THYROID	 (26) (56) (51) 

HYPERPLASIA, C-CELL 8 (16%) 18 (36%) 

REPRODUCTIVE SYSTEM.
 

HMANMARY GLAND (20) (50) (50) DILATATION/DUCTS 1 (5%) 5 (10%) ATROPHY, NO5 1 (2%) HYPERTROPHY, NOS 1 (5%) 1 (2%) HYPERPLASSA> NOS 18 (36%) 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED PlICRO5COPICALLY
 * NUPIBER OF ANIMALS NECROPSIED
 

69
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

HYPERPLASIA, DIFFUSE 1 (2%)
 

#PROSTATE (11) (49) (48)
 

INFLAMWATION, FOCAL 1 (2%)
 

INFLAPlWATIONt SUPPURATIVE 1 (9%) 3 (6x1 1 (2%)


INFLAMNATION, ACUTE FOCAL 1 (2%)
 

INFLAMMATION ACTIVE CHRONIC 1 (2%)


FIBROSIS 1 (2%)
 

ATROPHY, NOS I8 (37%) 21 (44%)


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

HYPERPLASIA, FOCAL 6 (12%)
 

HYPERPLASIA, PAPILLARY 1 (2%)
 

HYPERPLASIA, CYSTIC 1 (9%)
 

%SEMINAL VESICLE (20) (50) (50)
 

INFLAMMATION, SUPPURATIVE 1 (2%)
 

#TESTIS (20) (50) (49)
 

ATROPHY, NOS 41 (82%) 37 (76%)
 

HYPERPLASIA, INTERSTITIAL CELL 1 (5%) 9 (18%) 2 (4%)
 

%EPIDIDYMIS (20) (50) (50)
 

INFLAMMATION, CHRONIC 1 (2%)
 

NERVOUS SYSTEM
 

#BRAIN (20) (49) (50)
 

HYDROCEPHALUS, NOS 1 (2%)
 

SPECIAL SENSE ORGANS
 

NONE
 

NUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED NICROSCOPICAL'LY
 x NUMBER OF ANIMALS NECROPSIED
 

70
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ALL OTHER SYSTEMS 

SITE UNKNOWN 

ABSCESS, NOS 1 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 1 1
 

# NUMBER OF ANIMALS WITH TISSUE EXANINED PlICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

71
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NOMNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED 2,4,8-TRMETHYLANILINE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIMALS NISSING 1
 

ANINALS NECROPSIED 20 49 58
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 20 49 20
 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

UTRACHEA (20) (49) (49)
 

LYMPHOCYTIC INFLAMMATORY INFILTR 1 (5%)
 

#LUNG/BRONCHUS (20) (20) (20)
 

LYMPHOCYTIC INFLAMMATORY INFILTR 1 (2%)
 

#LUNG/BRONCHIOLE (20) (20) (20)
 

LYMPHOCYTIC INFLAMMATORY INFILTR 1 (2%)
 

HUNG (20) (43) (50)
 

HYPERPLASIA, ALVEOLAR EPITHELIUM 1 C2JS) 7 (14%)
 

HEMATOPOIETIC SYSTEM
 

#BONE NARROW (20) (49) (50)
 

HYPERPLASIA, NOS 1 (5%) 1 (2%) 2 (4%)
 

HYPERPLASIA, HENATOPOIETIC 1 (5%)
 

#SPLEEN (20) (49) (49)
 

HEMOSIDEROSIS 3 (15%) 27 (55%) 12 (24%)
 

HYPERPLASIAS NOS 1 (2%)
 

HYPERPLASIA, HEMATOPOIETIC 1 (5%)
 

HYPERPLASIA, LYMPHOID 1 (5x1
 

HEMATOPOIESIS 2 (4%)
 

#LYMPH NODE (201 (49) (50)
 

HYPERPLASIA, NOS 1 (5%) 29 (59%) 29 (58%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

72
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

#MANDIBULAR L. NODE (20) (49) (50)
 

INFLAMMATION, , NECROTIZING 1 (5%)
 

HYPERPLASIA, LYMPHOID 2 (10%)
 

CIRCULATORY SYSTEM
 

#MANDIBULAR L. NODE (20) (49) (50)
 

LYMPHANGIECTASIS 1 (55O
 

#MESENTERIC L. NODE (20) (49) (50)
 

LYMPHANGIECTASIS 1 (5?O
 

#MYOCARDIUM (20) (49) (48)
 

FIBROSIS 5 (25X)
 

FIBROSIS, FOCAL 4 (205O 6 C13%)
 

*PULMONARY ARTERY (20) (49) (50)
 

CALCIFICATION, FOCAL 1 (25O
 

XMESENTERY (20) (49) (50)


PERIARTERITIS 1 (2%)
 

DIGESTIVE SYSTEM
 

OLIVER (20) (49) (50)
 

METAMORPHOSIS FATTY 2 (4%) 3 (6%)
 

HEPATOCYTOMEGALY 1 (2%)
 

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

HYPERPLASIA, FOCAL 14 (70%) 24 (49%) 24 (48%)
 

HYPERPLASIA, DIFFUSE 1 (2%)
 

#BILE DUCT (20) (49) (50)
 

CYST, NOS 1 (2%)
 

#PANCREAS (20) (20) (20)
 

INFLAMMATION, CHRONIC FOCAL 1 (2X) 2 (4X)
 

#GASTRIC MUCOSA (20) (49) (50)
 

HYPERPLASIA, NOS 1 (25O
 

#GASTRIC SUBMUCOSA (20) (49) (50)
 

INFLAMMATION, NOS 1 (2%)
 

» NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

73
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

KOLON (19 ) (48 ) (49)

 

NEMATODIASIS 2 (454)

 

URINARY SYSTEM
 

#KIDNEY (20) (49) (49 ) 

HYDRONEPHROSIS 1 (5%) 

INFLAMMATION, INTERSTITIAL 1 (5%) 

PYELONEPHRITIS SUPPURATIVE 1 (5%) 

INFLAMMATION, CHRONIC FOCAL 1 (2%) 6 (12%) 

#URINARY BLADDER (19) (49 ) (49 )

 

ULCER, NOS 1 (5%)

 

ENDOCRINE SYSTEM
 

•PITUITARY (20) (48) (49 ) CYST, NOS 1 (554) 1 (254) 

HEMORRHAGIC CYST 1 (2*) 

HYPERPLASIA. NOS 3 (655) 13 (2754) 

HYPERPLASIA, FOCAL 1 (2%) 

WADRENAL CORTEX (20) (49) (49)

 

NODULE 2 (454)

 

#ADRENAL MEDULLA (20) (49) (49) 

HYPERPLASIA, NOS 1 (254) 1 (2%) 

#THYROID (20 ) (46) (49) 

HYPERPLASIA, C-CELL 2 (10% ) 9 (20%) 4 (8%) 

REPRODUCTIVE SYSTEM
 

%MAMMARY GLAND (20) (49 ) (50) DILATATION, NOS 1 (254) 

DILATATION/DUCTS 7 (3554) 1 (2J4) CYST, NOS 1 (254) 

HYPERPLASIA, NOS 1 (554) 19 (3954) 8 (1654) HYPERPLASIA, DIFFUSE
 

HYPERPLASIA, CYSTIC 1 (254) 

#UTERUS/ENDOMETRIUM (20 ) (49 ) (49 ) 

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

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

74
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

INFLAMMATION, SUPPURATIVE 1 (2%)
 

HYPERPLASIA, NOS 1 (2%)
 

#OVARY (20) (49) (49)
 

FOLLICULAR CYST, NOS 1 (5%)
 

HYPERPLASIA, STROMAL 3 (6%) 3 (6%)
 

NERVOUS SYSTEM
 

ttBRAIN (20) (48) (48)
 

HEMORRHAGE 1 (25O
 

INFLAMMATION, FOCAL 1 (2X)
 

INFLAMMATION, CHRONIC 1 (55S)
 

INFLAMMATION, CHRONIC FOCAL 1 (55O
 

tMEDULLA OBLONGATA (20) (48) (48)
 

ABSCESS, NOS 1 (2?O
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

MMUSCLE HIPITHIGH (20) (49) (50)
 

INFLAMMATION, NECROTIZING 1 (2%)
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

ANIMAL MISSING/NO NECROPSY
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

75
 
76

 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE ADMINISTERED 2,4,5-TRIMETHYLANILINE
 

77
 
78

 
TABLE Dl.

 

SUMMARY OF THE INCIDEIUCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED 2# 4# 6-TRIMETHYLANILINE IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIHALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NSUBCUT TISSUE
 

ABSCESS, NOS
 

RESPIRATORY SYSTEM
 

ttLUNG


HEMOSIDEROSIS
 

HYPERPLASIA, ALVEOLAR EPITHELIUM


HEMATOPOIETIC SYSTEM
 

#BONE MARROW
 

HYPERPLASIA, NOS
 

#SPLEEN
 

HYPERPLASIA, NOS
 

#LYPlPH NODE
 

HYPERPLASIA, NOS
 

#THYMUÂ
 

HYPERPLASIA, NOÂ
 

ZIRCULATORY SYSTEM
 

NCARDIOVASCULAR SYSTE
 

PERIARTERITIS
 

DIGESTIVE SYSTEM
 

#LIVER
 

NECROSIS, FOCAL
 

MATGHED
 

COUTROL
 

20
 

20
 

20
 

(20)
 

 (20)
 

 1 (5JJ)
 

(20)
 

( 19)
 

1 (5X)
 

(20)
 

2 (102)
 

(20)
 

(20)
 

(20)
 

LOW DOSE
 

0
 

0
 

20
 

(50)
 

(50)
 

1 (22)
 

(50)
 

(50)
 

3 (6*)
 

(50)
 

7 (14X)
 

(41)
 

(50)
 

(50)
 

HIGH DOSE
 

0
 

0
 

20
 

(50>
 

(50)
 

(50)


1 (2fc)
 

(49)
 

16 (335O
 

(50)
 

14 (28X)
 

(43)
 

1 (2%)
 

(50)


1 (25O
 

(50)
 

1 (2%)
 

ft NUflBER OF ANIMALS WITH TISSUE EXAPIINED MICROSCOPICALLY
 x NUNBER OF ANINALS NECROPSIED
 

79
 
TABLE Dl. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE

INFARCT, NOS

METAMORPHOSIS FATTY

HYPERPLASTIC NODULE

HYPERPLASIA, NOS

HYPERPLASIA, FOCAL 

 1 (5%) 

 1 (5%) 

 1 (5%) 

3 

1 

(6%) 

(2%) 

#LIVER/CENTRILOBULAR

NECROSIS, DIFFUSE 

HYPERPLASIA, NOS 

 (20) (50) 

11 (22%) 

#BILE DUCT (20) (20) 

LYMPHOCYTIC INFLANflATORY INFILTR 

#PANCREAS

INFLANMATIONr CHRONIC 

 (19) (19) 

1 (2JO 

ftGASTRIC MUCOSA (20) (50) 

NECROSIS, FOCAL 

URINARY SYSTEM 

#KIDNEY (20) (50

INFLAMMATION, CHRONIC FOCAL

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEN
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED NICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

 HIGH DOSE 

 1 (2%)
 

7 ( 14%) 5 (10%) 1 (2%) 

(50) 

1 (2%) 8 (16%) 

(20) 

1 (2%) 

(19) 

(50) 

1 (2%) 

1
 

1
 

80 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

ADIPOSE TISSUE 

INFARCT, NOS

i 

SPECIAL HORPHOLOGY SUHHARY 

1 

NO LESION REPORTED 5 2

AUTO/NECROPSY/HISTO PERF 1 

# NUNBER OF ANINALS WITH TISSUE EXANINED NICROSCOPICALLY 

* NUHBER OF ANIMALS NECROPSIED 

2 

81
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED 2#4#5-TRIMETHYLANILINE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIMALS NISSING 1
 

ANIMALS NECROPSIED 20 49 50
 

ANIMALS EXANINED HISTOPATHOLOGICALLY 20 49 50
 

INTEGUNENTARY SYSTEM
 

NSUBCUT TISSUE (20) (49) (50)
 

INFLAMMATION VESICULAR GRANULOMA 1 (5%)
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN (19) (49) (49)


ATROPHY, NOS 1 12'/.)


HYPERPLASIA, NOS 2 ( 1 U) 8 (165O 14 (29%)
 

HYPERPLASIA, LYNPHOID 2 (45O
 

#LYNPH NODE (20) (49) (49)
 

HYPERPLASIA? NOS 3 (15>O 4 (8%) 12 (24%)
 

ttTHYMUS (18) (46) (44)


HYPERPLASIA, NOS 1 (2X)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVER (20) (49) (50)
 

INFLANMATION? NECROTIZING 1
 

NECROSIS? FOCAL 1 (5%) 3 (6S)
 

# NUMBER OF ANIMALS UITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANINALS NECROPSIED
 

82
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

METAMORPHOSIS FATTY
 2 (4%) HYPERPLASTIC NODULE 4 (8%) 13 (26%) 

#LIVER/CENTRILOBULAR C20) (49) (50 )

 

HYPERPLASIA, NOS 1 (23O

 

#PANCREAS (20) (49 ) (50 ) 

DILATATION/DUCTS 4 

1 

HYPERPLASIA, NOS 1 (2%) 

URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

#THYROID (20) (45) (49)
 

HYPERPLASIA, C-CELL 1 (2%)
 

REPRODUCTIVE SYSTEM
 

#UTERUS/ENDOMETRIUN (20) (48) (49 ) 

INFLAMMATION, FOCAL 

HYPERPLASIA, NOS 12 (60X) 23 (48X) 36 (73*X) 

HYPERPLASIA, CYSTIC
 2 (103O 1 (2%) 

#OVARY ( 19) (47) (49)

 

CYST, NOS 2 (11%) 4 (9%)

 

CORPUS LUTEUM CYST 1 (2%) MULTIPLE CYSTS 1 (2%) 4 (.&'/.) 

HEMORRHAGIC CYST 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

83
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 5
 

ANIMAL MISSING/NO NECROPSY 1
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

84
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

RATS ADMINISTERED 2,4,5-TRIMETHYLANILINE
 

85
 
86

 
Table El. Analyses of the Incidence of Primary Tumors in
 

Male Rats Administered 2,4,5-Trimethylaniline in the Diet (a)
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar Carcinoma
 

or Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Lymphoma or
 

Leukemia (b)
 

P Values (c,d)
 

Relative Risk (Â£
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/20 (5)
 

P = 0.009
 

101
 

4/20 (20)
 

P = 0.006 (N)
 

72
 

Low
 

Dose
 

0149 (0)
 

N.S.
 

0.000
 

0.000
 

7.624
 

—
 

14/50 (28)
 

N.S.
 

1.400
 

0.520
 

5.303
 

74
 

High
 

Dose
 

7/50 (14)
 

N.S.
 

2.800
 

0.403
 

123.407
 

98
 

3/50 (6)
 

N.S.
 

0.300
 

0.049
 

1.642
 

95
 
Table El. Analyses of the Incidence of Primary Tumors in
 

Male Rats Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Liver: Bile-Duct Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lover Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tnmor
 

Matched Low High 

Control Dose Dose 

0/19 (0) 0/50 (0) 4/50 (8) 

P « 0.015 — N.S. 

Infinite 

— 0.368 

— Infinite 

— — 100 

0/19 (0) 3/50 (6) 11/50 (2 

P = 0.002 N.S. P = 0.02 

Infinite Infinite 

0.238 1.320 

Infinite Infinite 

101 97 
Table El. Analyses of the Incidence of Primary Tumors in
 

Male Rats Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Liver: Hepatocellular Carcinoma
 

or Neoplastic Nodule (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Carcinoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/19 (5)
 

P less than
 

0.001
 

101
 

5/20 (25)
 

N.S.
 

101
 

Low
 

Dose
 

6/50 (12)
 

N.S.
 

2.280
 

0.311
 

102.629
 

92
 

6/50 (12)
 

N.S.
 

0.480
 

0.143
 

1.807
 

101
 

High
 

Dose
 

20/50 (40)
 

P = 0.004
 

7.600
 

1.394
 

304.933
 

97
 

10/50 (20)
 

N.S.
 

0.800
 

0.296
 

2.689
 

98
 
Table El. Analyses of the Incidence of Primary Tumors in
 

Male Rats Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Pituitary: Carcinoma, NOS, or
 

Adenoma, NOS (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
 

Matched Low High 

Control Dose Dose 

8/20 (40) 17/50 (34) 25/50 (50) 

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

0.85Q 1.250 

0.435 0.689 

1.957 2.700 

101 74 101 

2/20 (10) 5/50 (10) 5/50 (10) 

N.S. N.s. N.S. 

1.000 1.000 

0.184 0.184 

10.007 10.007 

101 92 °2 
Table El. Analyses of the Incidence of Primary Tumors in
 

Male Rats Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Thyroid: C-cell Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to Frrst Observed Tumor
 

Thyroid: C-cell Carcinoma or
 

Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

0/20 (0)
 

P - 0.047
 

—
 

0/20 (0)
 

N.S.
 

101
 

Low
 

Dose
 

1/50 (2)
 

N.S.
 

Infinite
 

0.022
 

Infinite
 

101
 

2/50 (4)
 

N.S.
 

Infinite
 

0.123
 

Infinite
 

101
 

High
 

Dose
 

4/50 (8)
 

N.S.
 

Infinite
 

0.386
 

Infinite
 

101
 

5/50 (10
 

N.S.
 

Infinite
 

0.525
 

Infinite
 

101
 
Table El. Analyses of the Incidence of Primary Tumors in
 

Male Rats Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Pancreatic Islets: Islet-cell
 

Carcinoma or Adenoma (b)
 

P Values (c,d)
 

Relative Risk (Â£
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Testis: Interstitial-cell Tumor (b)
 

P Values (c,d)
 

Relative Risk (Â£
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tunor
 

Matched
 

Control
 

0/19 (0)
 

N.S.
 

—
 

14/20 (70)
 

N.S.
 

97
 

Low High 

Dose Dose 

3/50 (6) 1/48 (2) 

N.S. N.S. 

Infinite Infinite 

0.238 0.022 

Infinite Infinite 

101 101 

41/50 (82) 40/49 (8 

N.S. N.S. 

1.171 1.166 

0.871 0.865 

1.704 1.699 

86 92 
Table El. Analyses of the Incidence of Primary Tumors in
 

Male Rats Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

(a) Dosed groups received 200 or 800 ppm.
 

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

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

the Cochran-Armitage test when P is 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 is less than 0.05; otherwise, not significant
 

(N.S.) is indicated.
 

(dl A negative trend (N) indicates a lower incidence in a dosed group than in the
 

control group.
 

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

0.05 for any comparison.
 

(f) The 95 percent 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 2,4,5-Trimethylaniline in the Diet (a)
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Hematopoietic System: Lymphoma or
 

Leukemia (b)
 

P Values (c,d)
 

Relative Risk (Â£
 

Lower Limit
 

Upper Limit
 

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

Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Liver: Hepatocellular Carcinoma
 

or Neoplastic Nodule (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Carcinoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (Â£
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

Control Dose Dose 

0120 (0) 12/49 (24) 27/50 (54) 

P less than P =.0.010 P less than 

0.001 0.001 

Infinite InÂinite 

1.561 3.725 

InÂinite Infinite 

— 101 89 

3/20 (15) 12/48 (25) 5/49 (10) 

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

1.667 0.680 

0.524 0.150 

8.505 4.092 

81 90 101 
Table E2. Analyses of the Incidence of Primary Tuaors in Female Rats
 

Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Pituitary: Carcinoma, NOS, or
 

Adenoma, MOS (b)
 

P Values (c,d)
 

Relative Risk (Â£
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Cortical Adenoma (b)
 

P Values (c,d)
 

Relative Risk (Â£
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

Control Dose Dose 

8/20 (40) 22/48 (46) 22/49 (45) 

N.S. N.S. S. 

1.146 1.122 

0.618 0.605 

2.516 2.471 

81 90 90 

2/20 (10) 2/49 (4) 1/49 (2) 

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

0.408 0.204 

0.032 0.004 

5.381 3.754 

101 101 101 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Thyroid: C-cell Carcinoma
 

or Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

Control Dose Dose 

1/20 (5) 7/46 (15) 7/49 (14) 

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

3.043 2.857 

0.439 0.411 

133.816 125.833 

101 74 74 

2/20 (10) 2/49 (4) 1/50 (2) 

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

0.408 0.200 

0.032 0.004 

5.381 3.681 

92 90 101 
VO
 

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

Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Mammary Gland: Adenocarcinoma,
 

NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low 

Control Dose 

0/20 (0) 6/49 (12) 

N.S. N.S. 

Infinite 

0.680 

Infinite 

— 93 

5/20 (25) 8/49 (16) 

N.S. N.S. 

0.653 

0.222 

2.293 

96 90 

High
 

Dose
 

4/50 (8)
 

N.S.
 

Infinite
 

0.386
 

Infinite
 

101
 

7/50 (14)
 

N.S.
 

0.560
 

0.180
 

2.029
 

89
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued) 

Topography: Morphology 

Ma

C

tched 

ontrol 

Low 

Dose 

High 

Dose 

Uterus: Endometrial Stromal 

Polyp (b) 

7/49 (14) 

6/20 (30) 1/49 ( 

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

Departure from Linear Trend (e) P = 0.001 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.068 

0.002 

0.516 

0.476 

0.163 

1.537 

Weeks to First Observed Tumor 77 101 94 

o
 

o
 

(a) Dosed groups received 200 or 800 ppm.
 

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

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

the Cochran-Armitage test when P is 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 is 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 the
 

control group.
 

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

0.05 for any comparison.
 

(f) The 95 percent confidence interval of the relative risk between each dosed
 

group and the control group.
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

MICE ADMINISTERED 2,4,5-TRIMETHYLANILINE
 

101
 
102

 
Table Fl. Analyses of the Incidence of Primary Tumors in

 

Male Mice Administered 2,4,5-Trimethylaniline in the Diet (a) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Lung: Alveolar/Bronchiolar Carcinoma (b) 4/20 (20) 7/50 (14) 1/50 (2) 

P Values (c,d) P = 0.010(N) N.S. P = 0.02KN) 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.700 

0.207 

2.994 

0.100 

0.002 

0.944 

Weeks to First Observed Tumor 95 79 83 

Lung: Alveolar/Bronchiolar Carcinoma 

or Adenoma (b) 4/20 (20) 9/50 (18) 1/50 (2) 

P Values (c,d) P = 0.009(N) N.S. P = 0.02KN) 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.900 

0.294 

3.660 

0.100 

0.002 

0.944 

Weeks to First Observed Tumor 95 79 83 
Table Fl. Analyses of the Incidence of Primary Tumors in
 

Male Mice Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 
Table Fl. Analyses of the Incidence of Primary Tumors in
 

Male Mice Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

(a) Dosed groups received 50 or 100 ppm.
 o
 

Ln (b) Number of tumor-bearing animalslnumber of animals examined at site (percent).
 

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

the Cochran-Armitage test when P is 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 is 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 the
 

control group.
 

(el The probability level for departure from linear trend is given when P is less than
 

0.05 for any comparison.
 

(f) The 95 percent 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 2,4,5-Trimethylaniline in the Diet (a) 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered 2,4,5-TrimethyIaniline in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Hematopoietic System: Lymphoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

All Sites: Hemangiosarcoma or
 

Hemangioma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

9/20 (45)
 

P = 0.035(N)
 

91
 

1/20 (5)
 

N.S.
 

101
 

Low
 

Dose
 

16/49 (33)
 

N.S.
 

0.726
 

0.383
 

1.588
 

69
 

11/49 (22)
 

N.S.
 

4.490
 

0.737
 

188.359
 

70
 

High
 

Dose
 

11/50 (22)
 

N.S.
 

0.489
 

0.232
 

1.152
 

69
 

7/50 (14)
 

N.S.
 

2.800
 

0.403
 

123.407
 

101
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered 2,4,5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morpholog Morphology


Liver: Hepatocellula Hepatocellular Carcinom Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Pituitary: Carcinoma, NOS (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

Control
 

0120 (0)
 

P less than
 

0.001
 

—
 

0119 (0)
 

P = 0.043
 

—
 

Low
 

Dose
 

18/49 (37)
 

P = 0.001
 

Infinite
 

2.451
 

Infinite
 

101
 

0/48 (0)
 

High
 

Dose
 

40150 (80)
 

P less than
 

0.001
 

Infinite
 

5.727
 

Infinite
 

96
 

4/50 (8)
 

N.S.
 

Infinite
 

0.368
 

Infinite
 

101
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered 2.4.5-Trimethylaniline in the Diet (a)
 

(continued)
 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Pituitary: Carcinoma, NOS, 

or Adenoma, NOS (b) 1/19(5) 1/48(2) 5/50(10) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.396 

0.005 

30.454 

1.900 

0.238 

87.985 

Weeks to First Observed Tumor 95 101 101 

(a) Dosed groups received 50 or 100 ppm. 

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

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

the Cochran-Armitage test when P is less than 0.05; otherwise, not significant 

(N.S.) is indicated. Beneath the incidence of tumors in a dosed grou is the 

probability level for the Fisher exact test for the comparison of that dosed group 

with the matched-control group when P is 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 the 

control group. 

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

0.05 for any comparison. 

(f) The 95 percent confidence interval of the relative risk between each dosed 

group and the control group. 
HO

 
Review of the Bioassay of 2.4,s-Trimethylaniline* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

December 13, 1978
 

The Clearinghouse on Environmental Carcinogens was established
 

in May, 1976, in compliance with MEW 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 on the Institute's bioassay program to identify and 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 Governsaental 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 2,4,5-Trimethylaniline for carcinogenicity.
 

The primary reviewer for the report on the bioassay of 2,4,5­

Trimethylaniline said that the compound was carcinogenic in both sexes
 

of treated rats and in treated female mice. Since the study was well
 

designed and conducted, he concluded that 2,4,5-Trimethylaniline may
 

pose a carcinogenic risk to humans.
 

The secondary reviewer said that the results were sufficiently
 

significant as to obviate the experimental shortcomings. It was moved
 

that the report on the bioassay of 2,4,5-Trimethylaniline be accepted
 

as written. The motion was seconded and approved without objection.
 

Clearinghouse Member- dresent:
 

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

Joaeph Highland, Environmental Defense Fund
 

William Lijinsky, Frederick Cancer Research Center
 

Henry Pitot, University of Wisconsin Medical Center
 

Verne A. Ray, Pfizer Medical Research Laboratory
 

Verald K. Rove, Dow Chemical USA
 

Michael Shimkin, University of California at San Diego
 

111
 
Louise Strong, University of Texas Health Sciences Center
 

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.
 

U.S . GOVERNMEN T PRINTIN G OFFICE : 1979-281 - 217/304 5
 

112
 

DHEW Publication No. (NIH) 79-1716