1978 

OFFICIAL NTP
 

FILE COPY
 

National Cancer Institute 

CARCINOGENESIS DO NOT REMOVE FROM THIS OFFICE
 

Technical Report Series 

NO. 104 

BIOASSAY OF 

ANILAZINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 101-05-3 

NCI-CG-TR-104 

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

Public Health Service 

National Institutes of Health 
BIOASSAY OF
 

ANILAZINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1354
 
BIOASSAY OF
 

ANILAZINE
 

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
 

anilazine 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 environmental chemicals have the
 

capacity to produce cancer in animals. Negative results, in
 

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

than control animals, do not necessarily mean that the test
 

chemical is not a carcinogen, inasmuch as the experiments are
 

conducted under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals
 

under the conditions of the test and indicate that exposure to
 

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

of the risk to man from animal carcinogens requires a wider
 

analysis.
 

CONTRIBUTORS; This bioassay of anilazine was conducted by Gulf
 

South Research Institute, New Iberia, Louisiana, initially under
 

direct contract to NCI and currently under a subcontract to
 

Tracor Jitco, Inc., prime contractor for the NCI Carcinogenesis
 

Testing Program.
 

The experimental design for this bioassay is based on guidelines
 

for carcinogen bioassays in small animals that have been
 

established by NCI^. The doses for the chronic studies were
 

selected by Drs. E. E. Storrs2 and 0. G. Fitzhugh3*^, and the
 

principal investigator was Mr. R. J. Wheeler2. Chemicals were
 

analyzed by Mr. Wheeler and dosed feed mixtures by Mr. S. M.
 

Billedeau2. The results of these analyses were reviewed by Dr.
 

C. W. Jameson-^. Histologic examination of animal tissues was
 

performed by Drs. R. A. Ball2 and E. Bernal2, and the diagnoses
 

ill
 
included in this report represent the interpretation of these
 

pathologists.
 

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

Mason Research Institute^. Statistical analyses were performed
 

by Dr. J. R. Joiner^ and Ms. P. L. Yong^, using methods selected
 

for the bioassay program by Dr. J. J. Gart".
 

This report was prepared at Tracer Jitco-^ under the direction of
 

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

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

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

R. L. Schueler, pathologist; Dr. G. L. Miller, Mr. W. D.
 

Reichardt, and Ms. L. A. Waitz, bioscience writers; and Dr. E. W.
 

Gunberg, technical editor, assisted by Ms. Y. E. Presley and Ms.
 

P. J. Graboske.
 

The following scientists at NCI were responsible for evaluating
 

the bioassay experiment, interpreting the results, and reporting
 

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

Fielding Douglas, Dr. Dawn G. Goodman^, Dr. Richard A. Griesemer,
 

Dr. Morton H. Levitt, Dr. Harry A. Milman, Dr. Thomas Orme, Dr.
 

Robert A. Squire", Dr. Sherman Stinson, Dr. Jerrold M. Ward, and
 

Dr. Carrie E. Whitmire.
 

Icarcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

^Gulf South Research Institute, Atchafalaya Basin Laboratories,
 

P. 0. Box 1177, New Iberia, Louisiana.
 

-^Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

^4208 Dresden Street, Kensington, Maryland.
 

5

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

Rockville, Maryland.
 

iv
 
^Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Studies and Statistics, Division of Cancer
 

Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

'Now with Clement Associates, Inc., 1010 Wisconsin Avenue, N.W.,
 

Suite 660, Washington, D.C.
 

"Now with the Division of Comparative Medicine, Johns
 

Hopkins University, School of Medicine, Traylor Building,
 

Baltimore, Maryland.
 
SUMMARY
 

A bioassay of anilazine for possible carcinogenicity was
 

conducted by administering the test chemical in feed to Fischer
 

344 rats and B6C3F1 mice.
 

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

anilazine at one of two doses, either 500 or 1,000 ppm, for 103
 

weeks and then observed for 1-6 additional weeks. Matched
 

controls consisted of 25 untreated rats and 25 untreated mice of
 

each sex. All surviving rats were killed at 103-104 weeks; all
 

surviving mice were killed at 107-109 weeks.
 

Administration of the anilazine had no appreciable effect on body
 

weight in the rats and female mice; however, there was a
 

decreased gain in mean body weight in the dosed male mice.
 

Survival also was essentially unaffected. Survival in all groups
 

of dosed and control rats and mice was at least 80% at the end of
 

90 weeks on study, except for the male control mice; thus,
 

sufficient numbers of animals were at risk in most groups for
 

development of late-appearing tumors.
 

No tumors occurred in dosed rats or mice of either sex at
 

incidences that were significantly higher than those in
 

corresponding controls. Male and female rats and female mice may
 

have been able to tolerate higher doses.
 

It is concluded that under the conditions of this bioassay,
 

anilazine was not carcinogenic for either Fischer 344 rats or
 

B6C3F1 mice.
 

vii
 
TABLE OF CONTENTS
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals 5
 

D. Animal Maintenance 5
 

E. Subchronic Studies c.. 7
 

F. Chronic Studies 8
 

G. Clinical and Pathologic Examinations 11
 

H. Data Recording and Statistical Analyses 12
 

III. Results - Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 21
 

D. Statistical Analyses of Results (Rats).. 24
 

IV. Results - Mice 27
 

A. Body Weights and Clinical Signs (Mice) 27
 

B. Survival (Mice) 29
 

C. Pathology (Mice) 31
 

D. Statistical Analyses of Results (Mice) 31
 

V. Discussion 33
 

VI. Bibliography 35
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Administered Anilazine in the Diet 39
 

Table Al Summary of the Incidence of Neoplasms in Male Rats
 

Administered Anilazine in the Diet 41
 

Table A2 Summary of the Incidence of Neoplasms in Female Rats
 

Administered Anilazine in the Dietttt.. 45
 

ix
 
Page 

Appendix B Summary of the Incidence of Neoplasms in Mice 

Administered Anilazine in the Diet 49 

Table Bl Summary of the Incidence of Neoplasms in Male Mice 

Administered Anilazine in the Diet 51 

Table B2 Summary of the Incidence of Neoplasms in Female 

Mice Administered Anilazine in the Diet 55 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered Anilazine 

in the Diet 59 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered Anilazine 

in the Diet 61 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered Anilazine 

in the Diet 65 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered Anilazine 

in the Diet 69 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered Anilazine 

in the Diet 71 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered Anilazine 

in the Diet.. 74 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Administered Anilazine in the Diet 77 

Table El Analyses of the Incidence of Primary Tumors in 

Male Rats Administered Anilazine in the Diet 79 

Table E2 Analyses of the Incidence of Primary Tumors in 

Female Rats Administered Anilazine in the Diet 83 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered Anilazine in the Diet 89 
Table Fl Analyses of the Incidence of Primary Tumors in 

Male Mice Administered Anilazine in the Diet 91 

Table F2 Analyses of the Incidence of Primary Tumors in 

Female Mice Administered Anilazine in the Diet 95 

Appendix G Analysis of Formulated Diets for 

Concentrations of Anilazine 97 

TABLES 

Table 1 Anilazine Chronic Feeding Studies in Rats 9 

Table 2 Anilazine Chronic Feeding Studies in Mice 10 

FIGURES 

Figure 1 Growth Curves for Rats Administered Anilazine 

in the Diet 20 

Figure 2 Survival Curves for Rats Administered Anilazine 

in the Diet 22 

Figure 3 Growth Curves for Mice Administered Anilazine 

in the Diet 28 

Figure 4 Survival Curves for Mice Administered Anilazine 

in the Diet 30 

xi
 
I. INTRODUCTION
 

Cl
 

^..
 Cl.
 

Aniiazine
 

Anilazine (CAS 101-05-03; NCI C08684) is the internationally
 

accepted (ISO) generic name for the triazine fungicide
 

2,4-dichloro-6-(o-chloroanilino)-s-triazine. Anilazine was
 

originally synthesized and screened for herbicidal activity.
 

Although anilazine is virtually nonphytotoxic, it was found to have
 

broad-spectrum fungicidal effects, and was marketed in 1955 as an
 

agricultural fungicide (Spencer, 1973; Gysin, 1972).
 

Since that time, anilazine has been used to control fungal
 

infections of plant foliage and in some cases, of seeds. In 1975,
 

an estimated 200,000 pounds of anilazine were applied to vegetable
 
crops in the United States. Smaller amounts of anilazine were used
 

on lawns and turf (Ayers and Johnson, 1976).
 

In general, many heterocyclic nitrogen compounds are toxic to fungi
 

(Lukens, 1969). Anilazine is a nonspecific fungicide, functioning
 

as an alkylating agent. The triazine ring loses a chlorine atom,
 

and then reacts rapidly with amino and thiol groups by nucleophilic
 

substitution. The second chlorine atom on the triazine ring is
 

equally reactive initially, but loses reactivity following the
 

removal of the first chlorine (Lukens, 1969; Burchfield, 1967). In
 

this way it is conserved for a future reaction at a more specific
 

site. High concentrations are required for fungicidal activity,
 

presumably because the chemical must weaken the cell membrane of
 

the fungus before it can have any critical effects on cell
 

organelles (Lukens, 1969).
 

This compound was selected for testing because its use results in
 

its distribution in the environment and in food products. It is
 

structurally related to cyanuric acid, which was thought to be
 

carcinogenic at the time anilazine was considered for testing
 

(Pliss and Zabezhinsky, 1970). As an anilino compound, anilazine
 

is related to the monocyclic aromatic amines, such as o-toluidine,
 

which are also carcinogens (Homburger et al., 1972; Russfield et
 

al. , 1973).
 
II. MATERIALS AND METHODS
 

A. Chemical
 

Anilazine was obtained in several batches as technical-grade
 

Dyrene® from the Chemagro Agricultural Division of the Mobay
 

Chemical Corp., Kansas City, Missouri. The identity and purity
 

of Lot No. 4050279, which was used in the subchronic studies and
 

part of the chronic studies, was confirmed in analysis at Gulf
 

South Research Institute. The melting range was 151-154°C
 

(literature:159-160°C) (Farm Chemicals Handbook, 1977). Vapor
 

phase chromatography performed using two different stationery
 

phases of differing polarities gave a single peak in each case.
 

Elemental analyses (C, H, Cl, N) were correct for CgHsN^C^, the
 

molecular formula of anilazine. Nuclear magnetic resonance,
 

infrared, and ultraviolet spectra were consistent with the
 

structure. No analysis was performed on Lot No. 4050432 of
 

anilazine, which was used for the remainder of the chronic
 

studies.
 

B. Dietary Preparation
 

All diets were formulated every week using ground Wayne® Lab Blox
 

animal feed (Allied Mills, Inc., Chicago, 111.) to which was
 

added the required amount of anilazine. The test compound was
 

first dissolved in a small amount of acetone (Mallinckrodt Inc.,
 
St. Louis, Mo.) and this solution was then added to the feed.
 

Corn oil (LouAna®, Opelousas Refinery, Opelousas, La.) was also
 

added to the feed, primarily as a dust suppressant. The diets
 

were mixed mechanically in a Hobart blender for not less than 25
 

minutes to assure homogeneity and to allow for evaporation of the
 

acetone. Final diets, including those for the control groups of
 

animals, contained corn oil equal to 2% of the final weight of
 

feed.
 

The stability of anilazine in feed was tested by determining the
 

concentration of the compound in formulated diets at intervals
 

over a 7-day period. Diets containing 200 or 2,000 ppm anilazine
 

showed a significant decrease in concentration (57 and 70%
 

compound remaining, respectively) on standing at ambient
 

temperature in an open feeder for this period. Therefore, diet
 

mixtures used in the actual tests were stored at -20°C and were
 

kept for no longer than 1 week.
 

As a quality control test on the accuracy of preparation of the
 

diets, the concentration of anilazine was determined in randomly
 

selected batches of formulated diets at 8-week intervals during
 

the chronic studies. The results are summarized in Appendix G.
 

At each dietary concentration used in the chronic studies (500 or
 

1,000 ppm), the mean of the analytical concentrations for the
 

checked samples was within 1.6% of the theoretical concentration,
 
and the coefficient of variation was never more than 0.04. Thus,
 

the evidence indicates that the formulated diets were prepared
 

accurately.
 

C. Animals
 

Fischer 344 rats and B6C3F1 hybrid mice of each sex, obtained
 

through contracts of the Division of Cancer Treatment, NCI, were
 

used in these bioassays. The rats and mice were bred at and
 

supplied from the Frederick Cancer Research Center, Frederick,
 

Maryland. On arrival at the laboratory, the rats were
 

quarantined for 14 days and the mice for 30 days. Following
 

quarantine, animals were assigned to dosed or control groups.
 

The rats were 45 days of age and the mice were 73 days of age
 

when placed on study.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. The temperature range was 22-24°C, and the relative
 

humidity was maintained at 40-70%. The air in each room was
 

filtered through permanent air maze filters (Air Maze Incom
 

International, Cleveland, Ohio), and room air was changed 10-12
 

times per hour. Fluorescent lighting provided illumination 10
 

hours per day. Food and tap water were provided ad libitum.
 
Fresh feed was provided daily, and any feed remaining from the
 

previous day was discarded.
 

The rats were housed individually in hanging galvanized steel
 

mesh cages (Hoeltge, Cincinnati, Ohio), and the mice were housed
 

in polypropylene cages (Lab Products, Inc., Garfield, N.J.)
 

containing five animals per cage. Mouse cages were covered with
 

polyester filter bonnets (Lab Products, Inc.). The rat cages
 

were sanitized every 2 weeks. The mouse cages were sanitized two
 

times per week. Cages and racks were washed in an industrial
 

washer (Industrial Washing Machine Corp., Matawan, N.J.) at 82°C
 

with Acclaim® detergent (Economics Laboratory, Inc., St. Paul,
 

Minn.) and then rinsed. Absorbent Kimpak® cage liners (Kimberly
 

Clark Corp., Neenah, Wis.) under the rat cages were changed two
 

times per week. Absorb-dri® hardwood chip bedding (Lab Products,
 

Inc.) used in the mouse cages was provided two times per week.
 

Filter bonnets were sanitized each week. Feed jars and water
 

bottles were changed and sanitized two times per week. Sipper
 

tubes and stoppers were sanitized two times per week.
 

Filter bonnets, feed jars, water bottles, sipper tubes, and
 

stoppers were washed in a Vulcan Autosan washer (Louisville, Ky.)
 

at 82°C, using Acclaim® detergent, and then rinsed.
 

Cage racks for each species were rotated to a new position in the
 
room once per week; at the same time, each cage was moved to a
 

different row within the same column of a rack. Rats and mice
 

were housed in separate rooms. Control and dosed rats were
 

housed on the same rack, whereas cages for control and dosed mice
 

were placed in separate racks in the same room. Anilazine was
 

the only compound on test in each room.
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted with rats and mice to
 

estimate the maximum tolerated doses of anilazine, on the basis
 

of which two concentrations (hereinafter referred to as the "high
 

dose" and the "low dose"), were chosen for administration in the
 

chronic studies. In the subchronic studies, anilazine was added
 

to the animal feed at concentrations of 250, 500, 1,000, 2,000,
 

4,000, 8,000, and 16,000 ppm for 13 weeks for both rats and mice.
 

Each dosed group, as well as the untreated controls, consisted of
 

10 male and 10 female animals. Diets were stored at -20°C and
 

fresh feed was provided each day. Animal weights were measured
 

each week. All animals were killed at the end of the 13-week
 

test period.
 

After 13 weeks on study, the mean weight gain in male rats was
 

unaffected at doses of up to 4,000 ppm, 87% of controls at 8,000
 

ppm, and 59% of controls at 16,000 ppm. In females, the mean
 
weight gains were unaffected at doses up to 8,000 ppm, and were
 

79% of controls at 16,000 ppm.
 

In both male and female mice, the mean body weight gains were
 

above controls at all doses up to 8,000 ppm. At 16,000 ppm,
 

weight gain in males was 76% of controls, and weight gain in
 

females was 94% of controls.
 

Gross and microscopic examination of tissues of the rats and mice
 

gave no evidence of any effect of anilazine. Analysis of rat and
 

mouse feces showed unreacted anilazine present in the feces,
 

especially at the higher doses. No unreacted anilazine was
 

detected in any of the rat and mouse tissues tested from the
 

group receiving 16,000 ppm, including the heart, lung, brain,
 

fat, muscle, and kidney, at a minimal detectable concentration of
 

0.1 micrograms per gram of tissue.
 

The low and high doses for the chronic studies in both rats and
 

mice were set at 500 and 1,000 ppm, respectively.
 

F. Chronic Studies
 

The test groups, doses administered, and times on study of the
 

chronic studies are shown in tables 1 and 2.
 
Table 1. Anilazine Chronic Feeding Studies in Rats
 

Sex and
 

Test
 

Group
 

Male
 

Matched-Control
 

Low-Dose
 

High-Dose
 

Female
 

Matched-Control
 

Low-Dose
 

High-Dose
 

Initial
 

No. of
 

Animals3
 

25
 

50
 

50
 

25
 

50
 

50
 

Anilazine
 

in Dietb
 

(ppm)
 

0
 

500
 

1,000
 

0
 

500
 

1,000
 

Time
 

Dosed
 

(weeks)
 

103
 

103
 

103
 

103
 

on Study
 

Observed
 

(weeks)
 

103-104
 

0-1
 

0-1
 

103-104
 

0-1
 

0-1
 

a

All rats were at 45 days of age when placed on study.
 

^Diets were provided ad libitum.
 
Table 2. Anilazine Chronic Feeding Studies in Mice
 

Sex and
 

Test
 

Group
 

Male
 

Matched-Control
 

Low-Dose
 

High-Dose
 

Female
 

Matched-Control
 

Low-Dose
 

High-Dose
 

Initial
 

No. of
 

Animals3
 

25
 

50
 

50
 

25
 

50
 

50
 

Anilazine
 

in Dietb
 

(ppm)
 

0
 

500
 

1,000
 

0
 

500
 

1,000
 

Time
 

Dosed
 

(weeks)
 

103
 

103
 

103
 

103
 

on Study
 

Observed
 

(weeks)
 

107-109
 

4-5
 

4-6
 

107-109
 

4-6
 

4-6
 

a

All mice were at 73 days of age when placed on study.
 

"Diets were provided ad libitum.
 

10
 
G. Clinical and Pathologic Examinations
 

All animals were observed twice per day for signs of toxicity,
 

weighed at regular intervals, and palpated for masses at each
 

weighing. Animals that were moribund at the time of clinical
 

examination and those that survived to the end of the bioassay
 

were sacrificed under pentobarbital anesthesia and necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

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

from killed animals and from animals found dead. The following
 

tissues were examined microscopically: skin, lungs and bronchi,
 

trachea, bone and bone marrow, spleen, lymph nodes, heart,
 

salivary gland, liver, gallbladder (mice), pancreas, stomach,
 

small intestine, large intestine, kidney, urinary bladder,
 

pituitary, adrenal, thyroid, parathyroid, mammary gland, prostate
 

or uterus, testis or ovary, and brain. Occasionally, additional
 

tissues were also examined microscopically. The different
 

tissues were preserved in 10% buffered formalin, embedded in
 

paraffin, sectioned, and stained with hematoxylin and eosin.
 

Special staining techniques were utilized when indicated for more
 

definitive diagnosis.
 

A few tissues from some animals were not examined, particularly
 

from those animals that died early. Also, some animals may have
 

11
 
been missing, cannibalized, or judged to be in such an advanced
 

state of autolysis as to preclude histopathologic evaluation.
 

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 (Linhart et al., 1974). The data elements include
 

descriptive information on the chemicals, animals, experimental
 

design, clinical observations, survival, body weight, and
 

individual pathologic results, as recommended by the
 

International Union Against Cancer (Berenblum, 1969). Data
 

tables were generated for verification of data transcription and
 

for statistical review.
 

These data were analyzed using the appropriate statistical
 

techniques described in this section. Those analyses of the
 

experimental results that bear on the possibility of
 

carcinogenicity are discussed in the statistical narrative
 

sections.
 

Probabilities of survival were estimated by the product-limit
 

12
 
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 as the ratio of the number of animals bearing such lesions
 

at a specific anatomic site (numerator) to the number of animals
 

in which that site is examined (denominator). In most instances,
 

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic examination was required to detect lesions prior to histologic sampling
 

(e.g., skin or mammary tumors), or when lesions could have
 

appeared at multiple sites (e.g., lymphomas), the denominators
 

consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

13
 
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 (Miller, 1966) requires that the
 

P value for any comparison be less than or equal to 0.05/k. In
 

cases where this correction was used, it is discussed in the narrative section. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

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

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

assumption of a linear trend, this test determines if the slope
 

of the dose-response curve is different from zero at the one-


tailed 0.05 level of significance. Unless otherwise noted, the
 

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

14
 
first tumor was observed were excluded by basing the statistical
 

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

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

such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were
 

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

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

methods of comparing these curves were used for two groups;
 

Tarone's extension to testing for linear trend was used for three
 

groups. The statistical tests for the incidence of tumors which
 

used life-table methods were one-tailed and, unless otherwise
 

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P < 0.05, two-tailed test)
 

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared to its control was calculated
 

15
 
from the exact interval on the odds ratio (Gart, 1971). The
 

relative risk is defined as Pt/Pc where pt is the true binomial
 

probability of the incidence of a specific type of tumor in a
 

dosed group of animals and pc is the true probability of the
 

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

a specific tumor in a dosed group and the proportion in a control
 

group corresponds to a relative risk of unity. Values in excess
 

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

approximately 95% of a large number of identical experiments, the
 

true ratio of the risk in a dosed group of animals to that in a
 

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P < 0.025 one-tailed test when the control
 

incidence is not zero, P < 0.050 when the control incidence is
 

zero) has occurred. When the lower limit is less than unity, but
 

the upper limit is greater than unity, the lower limit indicates
 

the absence of a significant result while the upper limit
 

16
 
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.
 

17
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of the dosed male and female rats were slightly
 

lower than those of the corresponding controls throughout the
 

bioassay (figure 1).
 

During the first 6 months of the bioassay, the appearance and
 

behavior of the dosed rats was generally comparable to that of
 

the control rats. During the second 6 months, adverse clinical
 

signs were noted at a low incidence among dosed rats. These
 

signs included loss of weight, rough hair coats, discolored
 

(dark) urine, loose stools, and pale mucous membranes. Adverse
 

clinical signs were noted with an increased frequency during the
 

second year of the bioassay, among all groups, dosed and control,
 

but predominantly in dosed groups. These signs included loss of
 

weight, rough hair coats, poor food consumption, loose stools,
 

hematuria, vaginal bleeding, tachypnea, dyspnea, discolored
 

(dark) urine, impaired equilibrium, pale mucous membranes,
 

mucous-like vaginal discharge, hyporeactivity, and tissue masses.
 

At week 84, a majority of low-dose females appeared hyperactive,
 

but stabilized. At week 103, when placed on control diet, the
 

high-dose males, low-dose females, and high-dose females appeared
 

hyperactive.
 

19
 
g 

UJ 

g 

Q 

O 

at 

B 

D

O

A

MALE RATS 

 MATCHED CONTROL 

 LOW DOSE 

 HIGH DOSE 

10 20 30 40 50 60 70 

TIME ON STUDY (WEEKS) 

90 100 110 

DO 

>

Q 

O 

CD 

Z

 250­

 200­

100<| 

FEMALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

20 30 40 50 60 70 

TIME ON STUDY (WEEKS) 

80 90 100 110 

Figure 1. Growth Curves for Rats Administered Anilazine in the Diet 
B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered anilazine in the
 

diet at the doses of this bioassay, together with those of the
 

controls, are shown in figure 2. The result of the Tarone test
 

for dose-related trend in mortality is not significant in either
 

sex.
 

In male rats, 37/50 (74%) of each dose group and 20/25 (80%) of
 

the control group were still alive at 2 years on study. In
 

females, 34/50 (68%) of the high-dose group, 37/50 (74%) of the
 

low-dose group, and 16/25 (64%) of the control group were alive
 

at 2 years on study.
 

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

development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

are summarized in Appendix C, tables Cl and C2.
 

A variety of neoplasms occurred in both the dosed and control
 

animals. An increased incidence of certain types of endocrine
 

neoplasms occurred in the dosed animals.
 

21
 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVA

*c

S j i "8 8 

fe

i I1 C; §3 83 

la C> O D > O C]

1 S

> -n m 

Js» S1 S

> GO

1?

LO

W DOSE

 

HIGH DOSE

 

i ?

! s 

' 3J 

i

! 5 3 H I. O CO C

r O

d s- s m m cu O E? 2 O

Ni Z 01.

| CO 

cn

H

0 60 7

 

STUDY (WEEKS 

C

O

<•

CD

^

CD

~ S­ — o

a>

N_

3* 

CD 

S" 

>
The finding of adenocarcinoma in the jejunum of 2/47 (4%) of the
 

high-dose male rats is of some interest, because of the relative
 

rarity of this neoplasm as a spontaneous entity. The primary
 

neoplasm in one animal was characterized by pleomorphic
 

epithelial cells which were invading the subjacent lamina
 

propria, submucosa, and muscularis externa. The less
 

differentiated neoplastic cells were compact cells whose nuclei
 

and cytoplasm stained deeply with hematoxylin. Differentiation
 

to goblet cells was frequently noted. Metastatic foci having
 

morphologic features closely resembling those of the primary
 

tumor were found in the lungs. Implants were also noted on the
 

serosa of the gastrointestinal tract.
 

The primary neoplasm in the jejunum of the second animal was a
 

broad-based tumor with increased mitoses in the epithelial cells,
 

moderate cellular pleomorphism, and increased cytoplasmic basophilia. In some areas the basement membrane was indistinct. In
 

one area there was morphologic evidence of invasion of tumor
 

cells in the submucosa, with encroachment upon the inner layer of
 

the muscularis externa.
 

Other neoplasms occurred at approximately the same incidences in
 

dosed and control animals in this bioassay, or were neoplasms
 

whose rates of occurrence were not appreciably above those in
 

control animals in this and other laboratories.
 

23
 
A variety of nonneoplastic lesions occurred in both dosed and
 

control animals. There were instances in which lesions occurred
 

only or with increased incidence in dosed animals; however, the
 

incidence, distribution, and severity of these lesions are
 

similar to those which are known to occur spontaneously in aged
 

Fischer 344 rats.
 

Based on the histopathologic examination, there was no clear
 

evidence for the carcinogenicity of anilazine in Fischer 344 rats
 

under the conditions of this bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

The results of the Cochran-Armitage test for dose-related trend
 

in incidences of tumors and those of the Fisher exact test
 

comparing the incidence of tumors in the dosed groups with those
 

in the corresponding control groups are not significant in either
 

sex.
 

In each of the 95% confidence intervals of relative risk, shown
 

in the tables, the value of one is included; this indicates the
 

24
 
absence of significant positive results. It should also be noted
 

that each of the intervals has an upper limit greater than one,
 

indicating the theoretical possibility of the induction of tumors
 

by anilazine, which could not be detected under the conditions of
 

this bioassay.
 

25
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

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

those of the corresponding controls, beginning at about week 30;
 

however, there was no relationship to dose. The mean body
 

weights of the dosed females were essentially unaffected (figure
 

3).
 

During the first 6 months of the bioassay, the appearance and
 

behavior of the dosed mice was generally comparable to that of
 

the control mice. During the next 6 months, adverse clinical
 

signs were noted at a fairly low incidence. These signs included
 

alopecia, hyperactivity, hyporeactivity, mucous in feces,
 

obesity, bloating (or abdominal distention), and discolored
 

(yellow) hair coats. Fighting was observed among all male mice,
 

but predominantly among low-dose males. This increased
 

aggression persisted until termination of the bioassay. A few
 

high-dose females and low- and high-dose males had single areas
 

of depigmentation of both skin and hair coats. Hyperemia of ears
 

and feet was also noted on a few animals in these same groups.
 

During the second year of the bioassay, adverse clinical signs
 

were noted at an increased frequency in all groups, but
 

predominantly in the dosed groups of both sexes. These signs
 

27
 
D D

DDDDD D O

D DD 

D DD 

 DO D D DDD D 

ODD D 

D D

1

"""""1 0 0 QOQQ Q QOAQAQAAA O OQQQ °OQO O D 

 AAAQA AAQQAO Q 

DDD ° 

35­

2 BBSS80 

I 30­ 8? 

111 ' s8 

I "1 B8 

Z 20­

UJ 

5 

15­

MALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

10 20 30 40 BO 60 70 80 90 100

TIME ON STUDY (WEEKS), 

_

5 

i­

<3 

Ul 

35H

30­

­ -°DC 

I

<n 1

20­

UJ 

2 15­

n=°M 

n " 

D

O

A

FEMALE MICE 

 MATCHED CONTROL 

 LOW DOSE 

 HIGH DOSE 

10 20 30 40 50 60 70

TIME ON STUDY (WEEKS) 

 80 90 100 110 

Figure 3. Growth Curves for Mice Administered Anilazine in the Diet 

28 

 110 
included tachypnea, obesity, mucous in feces, rough hair coats,
 

and abdominal distention.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered anilazine in the
 

diet at the doses of this bioassay, together with those of the
 

controls, are shown in figure 4.
 

In male mice, the result of the Tarone test for dose-related
 

trend in mortality is significant (P = 0.006), but in the
 

negative direction. There were 44/50 (88%) of the high-dose
 

group, 40/50 (80%) of the low-dose group, and 16/25 (64%) of the
 

control group alive at 2 years on study. In females, the result
 

of the Tarone test is not significant. An indicated departure
 

from linear trend is observed (P = 0.017), because the high-dose
 

female mice survived longer than the low-dose group. All 50 of
 

the high-dose female mice, 49/50 (98%) of the low-dose group, and
 

all 25 of the control group survived beyond week 52 on study.
 

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

development of late-appearing tumors.
 

29
 
_1

CC

 0.70 

 0.60­

6--, 

00 

<

CO 

O 

cc 

0­

°-4°­

 0.30 

D

O

A

MALE MICE 

 MATCHED CONTROL 

 LOW DOSE 

 HIGH DOSE 

10 20 30 40 50 60 70 

TIME ON STUDY (WEEKS) 

90 100 110 

<3 )­

> v> ­. 

(/> 

u_ 

o 050_ 

CO 

C^D 

o 

CC 

o 

A 

FEMALE 

MATCHED 

LOW DOSE 

HIGH DOSE 

MICE 

CONTROL 

o nn40 50 60 70 

TIME ON STUDY (WEEKS) 

100 110 

Figure 4. Survival Curves for Mice Administered Anilazine in the Diet 

30 
C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B, tables Bl and B2; findings on nonneoplastic lesions
 

are summarized in Appendix D, tables Dl and D2.
 

A number of neoplastic and nonneoplastic lesions were observed in
 

mice of the control and dosed groups with approximately equal
 

frequency. One exception was the occurrence of diverse types of
 

sarcomas in the subcutaneous tissue in dosed mice, but not in
 

control mice. The incidence of these neoplasms was 5/49 (10%) in
 

the low-dose males, 0/50 in the high-dose males, and 1/50 (2%) in
 

the high-dose females. The absence of sarcomas in the high-dose
 

males and the low number in the high-dose females suggests that
 

they occurred spontaneously.
 

Based on the histopathologic examination, there was no evidence
 

for the carcinogenicity of anilazine in B6C3F1 mice under the
 

conditions of this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals in one group and with an incidence of at least
 

5% in one or more than one group.
 

31
 
The results of the Cochran-Arm!tage test for dose-related trend
 

in incidences of tumors and those of the Fisher exact test
 

comparing the incidences of tumors in the dosed groups with those
 

in the corresponding control groups are not significant in the
 

positive direction in either sex.
 

In male mice, the incidence of liver tumors in the control group
 

is significantly higher than that in the low-dose group.
 

In each of the 95% confidence intervals of relative risk, shown
 

in the tables, the value of one or less than one is included;
 

this indicates the absence of significant positive results. It
 

should also be noted that each of the intervals (except that for
 

the incidence of liver tumors in the low-dose male mice) has an
 

upper limit greater than one, indicating the theoretical
 

possibility of the induction of tumors by anilazine, which could
 

not be detected under the conditions of this bioassay.
 

32
 
V. DISCUSSION
 

The anilazine was generally nontoxic for the rats and mice under
 

the conditions of this bioassay. The chemical in the feed was
 

unstable at room temperature; however, the feed was changed each
 

day to compensate for the loss at ambient temperatures.
 

Administration of the chemical had only a slight effect on mean
 

body weights in male or female rats and female mice; however,
 

there was a more obvious decrease in mean body weight gain in the
 

dosed male mice. Survival also was essentially unaffected.
 

Except for the control male mice, survival in all groups of rats
 

and mice was at least 80% at the end of 90 weeks on study; thus,
 

sufficient numbers of animals were at risk in most groups for the
 

development of late-appearing tumors. Male and female rats and
 

female mice may have been able to tolerate higher doses.
 

No tumors occurred in dosed male or female rats or mice at
 

incidences that were statistically significantly higher than
 

those in corresponding controls. However, adenocarcinomas
 

occurred in the intestines of two high-dose male rats. Although
 

these lesions are generally rare in rats, their low incidence in
 

this study does not permit implication of a compound-related
 

effect.
 

The acute oral LD5Q for anilazine in rats (strain not specified)
 

33
 
has been reported as 2,700 mg/kg body weight; also, feeding of
 

the test chemical to rats (strain not specified) at doses of 50
 

to 5,000 ppm in the diet over a period of 2 years had no effect
 

on weight gain, survival, histological appearance of tissues, or
 

incidence of tumors (Lehman, 1965). A number of triazine
 

analogues related to anilazine have been reported to induce
 

tumors in rats or mice or both. These include 2,4,6-tris(laziridinyl)-s-triazine (Hendry et al., 1951; Shimkin, 1954; Roe
 

and Salaman, 1955; Walpole, 1958; Conklin et al., 1965),
 

2,4,6-trichloro-s-triazine (Pliss, 1966), 2-chloro-4,6-bis(ethylatnino)-s-triazine (Pliss and Zabezhinsky, 1970), 4,6-diamino-2­

(5-nitro-2-furyl)-s-triazine and its bisacetamide (Cohen et al. ,
 

1973), hexamethylmelamine (Cohen et al., 1973), azacytidine
 

(Stoner et al. , 1973), and hexahydro-1,3,5-trinitroso-s-triazine
 

(Urban and Danz, 1976). Prolonged contact with anilazine can
 

cause skin irritation in humans (Kambe et al. , 1970; Gosselin et
 

al., 1976).
 

It is concluded that under the conditions of this bioassay,
 

anilazine was not carcinogenic for either Fischer 344 rats or
 

B6C3F1 mice.
 

34
 
VI. BIBLIOGRAPHY
 

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

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

Ayers, J. H. and Johnson, 0. H., Fungicides. In: Chemical
 

Economics Handbook, Stanford Research Institute, Menlo Park,
 

Calif., 1976, sec. 573.5003 E-F, 573.5007 G-H, and 573.5007
 

U-Y.
 

Berenblum, I., ed., Carcinogenicity Testing; A Report of the
 

Panel on Carcinogenicity of the Cancer Research Commission
 

of_ the UICC, Vol. "I. International Union Against Cancer,
 

Geneva, 1969.
 

Burchfield, H. P., Chemical and physical interactions. In:
 

Fungicides - An Advanced Treatise, Vol _1_, Torgeson, D. C. ,
 

ed., AcademicTress, New York, 1967, pp. 502-506.
 

Cohen, S. M. , Erturk, E. , von Esch, A. M. , Crovetti, A. J. , and
 

Bryan, G. T., Carcinogenicity of 5-nitrofurans,
 

5-nitroimidazoles, 4-nitrobenzenes, and related compounds.
 

J. Natl Cancer Inst. 51(2):403-417, 1973.
 

Conklin, J. W. , Upton, A. C., and Christenberry, K. W., Further
 

observations on late somatic effects of radiomimetic
 

chemicals and x-rays in mice. Cancer Res. 25:20-28, 1965.
 

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 & Co., Ltd., London,
 

1970, pp. 48-52.
 

Farm Chemicals Handbook, Meister Publishing Co., Willoughby,
 

Ohio, 1977, p. D106.
 

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.
 

Gosselin, R. E., Hodge, H. C. , Smith, R. P., and Gleason, M. N. ,
 

Clinical Toxicology of Commercial Products, Fourth Edition,
 

The Williams & Wilkins Co., Baltimore, Md., 1976, p. 202.
 

35
 
Gysin, H. , The chemical structure and biological relationship of
 

s-triazines. In: Herbicides, Fungicides, Formulation
 

Chemistry, Vol 5_, Tahori, A. S., ed., Gordon and Breach
 

Science Publishers, New York, 1972, pp. 1-3, 5-8, 27, 241,
 

and 246.
 

Hendry J. A., Homer, R. J. , and Rose, F. L. , and Walpole, A. L. ,
 

Cytotoxic agents: III, derivates of ethyleneimine. Brit. J.
 

Pharmacol. 6:357-410, 1951.
 

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

J. H., Carcinogenicity of simple aromatic amine derivatives
 

in mice and rats. Toxicol. Appl. Pharmacol. 22:280-281,
 

1972.
 

Kambe, Y., Matsushima, S., Matsumura, T., Kuroume, T., and
 

Suzuki, S., Studies on patch tests on contact dermatitis
 

caused by pesticides. In: Fourth International Congress of
 

Rural Medicine, Kuroiwa, H., ed. , Japan Association of Rural
 

Medicine, Tokyo, 1970, pp. 55-57.
 

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

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

1958.
 

Lehman, A. J. , Dyrene. In: Summaries of Pesticide Toxicity, The
 

Association of Food and Drug Officals of the United States,
 

Topeka, Kan., 1965, pp. 98-99.
 

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.
 

Lukens, R. J. , Heterocyclic nitrogen compounds. In: Fungicides ­

An Advanced Treatise, Vol _2, Academic Press, New York, 1969,
 

pp. 396, 411 -419, and 4~36I437.
 

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

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

Pliss, G. B., Blastomogenic action of cyanurchloride. Vopr.
 

Onkol. 12(4):78-82. 1966.
 

Pliss, G. B. and Zabezhinsky, M. A., On carcinogenic properties
 

of symmetrical triazine derivatives. Vopr. Onkol. 16(1);82,
 

1970.
 

36
 
Roe, F. J. C. and Salaman, 'I. H. , Further studies on incomplete
 

carcinogenusis: triethytene raelamine (T.E.M.),
 

1,2-benzanthracene and /3-propiolactone, as initiators o£
 

skin tumours formation in the mouse. brit. Jj^ Cancer
 

9(1):177-203, 1955.
 

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

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

environmental chemicals including simple aromatic amines.
 

Toxicpl. Appl. 1'harnacol. _25:446-447, 1973.
 

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

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

induced by different numbers of administrations of benzo (a)
 

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

Shirakin, M. B., Pulmonary-tumors induction in mice with chemical
 

agents used in the clinical management of lymphomas. Cancer
 

2:410-413, 1954.
 

Spencer, E. Y. , Guide _t_o the Chemicals Used in Crop Protection,
 

University of Western Ontario, London, Ontario, 1973, p. 17.
 

Stoner, G. U. , Shimkin, M. ^., Kniazeff, A. J., \veisburger, J.
 

11., Weisburger, E. K., and Gori, C. B. , Test for
 

carcinogenicity of food additives and chemotherapeutic
 

agents by the pulmonary tumor response in strain A mice.
 

Cancer Res. J33:3069-3085, 1973.
 

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

_62(3):679-682, 1975.
 

Urban, H. and Uanz, M. , Tumorinduzierende Wirkung von Trinitrosotriraethylen-triamin in Verbtndung -nit Oimethylsulfoxid.
 

Arch. Geschwulstforsch. 46(8):657-602, 1976.
 

Walpole, A. L. , Carcinogenic action of alkylating agents. Ann.
 

N-Y.Acad.Sci. 68(3):750-761, 1958.
 

37
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED ANILAZINE IN THE DIET
 

39
 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED ANILAZINE IN THE DIET 

LOW DOSE HIGH DOSE 

ANIMAL S INITIALL Y IN STUD Y 25 50 50 

ANIMAL S NECROPSIE E 25 50 50 

ANIMAL S EXAMINE E HISTOPATHOLOGICALL Y 25 50 50 

INTEGUMENTAR Y SYSTE M 

*SKIN (25) (50) (50) 

FIBROSARCOM A i (2%) 

*SU3CUT ^ISSUE (25) (50) (50)
 

FIBROMA 1 (2%)
 

5ESP T RAT03 Y SYSTE M 

#TUK 5 (25) (49 ) (49) 

ALVEOLAR/BSONCHIOLAR ADENOMA 2 (8%) 2 (4%)
 

ALVEOLAF/ERONCHTOLAR CARCINOMA 1 (2%)
 

HFMATOPOIETIC SYSTEM
 

"MULTIPLE ORGANS (25) (50) (50)
 

(JNDIFFERENTIATED LEUKEMIA 2 (3%) 1 (2%) 1(2%)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tJEJ'JW'1 (22) (47) (47)
 

ADENOCA?CT NCMA, NOS 2 (4%)
 

URINAK Y SYSTEM
 

NONE
 

t rUCBE B O F ANIMAL S WIT H TISSU E EXAMINE D MICROSCOPICALL Y 

* T1U»3ES OF ANIMAL S NECPOPSTE D 

41 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE CONTROL 

ENDOCRIN E SYSTEM 

(25) (44) (46) 

CARCINOMA , NOS 2 (4«) 

ADENOMA , NO S 6 (24% ) 16 (36% ) 15 (33X) 

#ADPENA L (23) (49) (49) 

PHEOCHSOMOCYTOMA 1 (2%) 1 (2X) 

tADFENAL MEDULLA (23) (49) (49) 

NEUROBLASTCMA 1 (2%) 

#THYROID (23) (38 ) (47) 

CAPILLAR Y ADENOM A 2 (5%) 

C-CEIL ADENOM A 1 (4%) 4 (11% ) 1 (2*) 

*PAPATHYP.OI D (16) (29) (33) 

ADENOMA , NO S 1 (3%) 

• PANCREATI C ISLETS (24 ) (47 ) (50) 

ISLET-CELL ADENOM A 1 (4%) 4 (9%) 5 (10%) 

°EPFQDUC m I V E SYSTE M 

*MAMMAR Y GLAN D (25) (50) (50) 

"IBFOJU 1 (2% ) 1 (2%) 

•TESTIS (24 ) (49) (49) 

TITEPSTITIAL-CEL L TUMO R 20 (83%) 43 (88% ) 44 (9C% ) 

NERVOU S SYS T E M 

NONE 

SPECIAL SENSE ORGAN S 

NON E 

M'JSC'.1I,OSKELE?AL SYSTEM 

*SKM!T. (25) (50 ) (50) 

_____ 03?_ EOS A ROOM A _________________________________________ 

# !1Uri3~E OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R O F ANIMAL S NECROPST.E D 

42 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE CONTROL 

BOD Y CAVITIE S 

*PEVITONEU M (25) (50) (50) 

"I2SQTHELIOMA , NOS 1 (2% )

 

A L I OTHE F SYSTEM S 

*I"UI TIPLE 0"GAN F (25) (50) (50) 

CAFCINO M A,NO S 1 (4%) 

ADENOCAPCINOMA , NOS 1 (2% ) 

ADENOCAPCTNOMA , NOS, METASTAT1C 1 (2X ) 

SAPCOKA , NOS 1 (H%) 

^ESOTHELIOMA , NOS 1 (2%) 

SI^E UNKNOW N 

PARAGANGLIOMA . NO S 1 

ANIMA L DISPOSITIO N SUMMAR Y 

ANI1ALS INITIALL Y IN STUD Y 2 5 50 50 

NATURA L CEATH3) 1 2 3 

MOP.IB'IND SACRIFICE 5 11 1U 

SCHEDULE D SACPIFTCE 

ACCIDENTALL Y KILLE D 1 

^EP^INA L SACRIFICE 18 37 33 

ANIMA L MISSING 

* INCLUDE S AUTOLYZE D ANIMAL S 

* NUMBEP OF ANIMALS WITH TISSUE EXAMINED MICEOSCOPICALLY
 

* NUMBER OF ANIHALS NECROPSIED
 

43
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE CONTROL 

TUMO R SUMMAR Y 

TOTA L ANIMAL S WIT H PPIMAR Y TUMOPS * 2 4 50 50

 T OTA L PRIMAR Y TUMOR S 3 4 77 79

 

TOTAL ANIMAL S WI^ H BENIG N TUMOR S 2 3 49 50

 

TOTAL BENIG N TtlMOP S 3 0 72 70 

TO^A L ANIMAL S KIT H MALIGNAN T TUMOR S 3 4 7 

TOTAL MALIGNAN T TUMORS 4 U 1 

"OTAL ANIMAL S WIT H SECONDAR Y TUMORS * 1

 

^OTA L SECONDAR Y TUMOP S 1 

TO^AI ANIMAL S WIT H TUMOR S UNCERTAIN ­

BENIG N O R MALIGNAN T 1 2 

TOTA L UNCERTAI N TUMOR S 1 2

 

TOTAL ANIMALS WITH TUMORS UNCERTAINFRIMAP-Y 013 METASTATIC
 

"'OTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONE&RY TUMORS
 

* SECONCAPY TUMOFS: METASTATIC TUMORS OR TUMORS INVASIV E INTO AN ADJACENT ORGA N
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED ANILAZINE IN THE DIET

 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S

ANIMAL S

ANIMAL S

 INITIALL Y IN STUDY

 NECROPSIE D

 EXAMINE D HI STOPATHOLOGICALL Y

 25

 25

 25

 50

 50

 50

 50 

 50 

 50 

INTEGUMENTAR Y SYSTE M 

*SKIN

BASAL-CEL L CARCINOM A

 .(25) (50) (50) 

1 (2%) 

*SUBCUT TISSUE

SARCOMA , NOS

 (25)

 1 (4S) 

 (50) (50) 

RESPIRATOR Y SYSTEM 

#LUN G

ALVEOLAR/BRONCHIOLA R ADENOM A

 (24) (49 )

 2 (4«)

 (50) 

1 (2») 

HEMA'f'OPOIETIC SYSTEM 

"MULTIPL E ORGAN S

UNDTFFEFEHTIATE D LEUKEMI A

 (25)

 3 (12% )

 (50)

 3 (6% )

 (50) 

1 (2X ) 

"MUSCLE OP TRUN K

MALIO.LYMPHOMA , LYMPHOCYTIC TYPE

 (25) (50) (50) 

1 (2%) 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIV E SYSTE M 

#LIVE F

NEOPLASTIC NODUL E

H^PATOCELLULA P CARCINOM A

 '

 (25) (48)

 1 (2% ) 

 (49) 

1 (2X ) 

tJEJUNU M (24) (50) (46 ) 

* NPMBE8 OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

45 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE CONTROL 

URINAR Y SYSTE M 

NON E 

ENDOCRIN E SYSTE M 

#PI"UITAR Y 

CARCINOMA , NO S 

ADENOMA , NO S 

(22 ) 

1 (5*) 

11 (50% ) 

(43) 

3 (7% ) 

22 (51S) 

#THYBOI D 

PAPILLAR Y ADENOM A 

C-CELL ADENOM A 

(214) (35) 

1 (3% ) 

*PA?!CREA m TC ISLETS 

TSLET-CEL L ADENOM A 

(24) 

1 (4S) 

(49) 

2 (4% ) 

REPRODUCTIV E SYSTE M 

*F!AMMAP. Y GLAN E (25) (50) 

ADENOCARCINOMA , NO S 

FIBROSftPCOM A 

FIBROABENOM A 1 (4S) 3 (6X) 

ttn^ERU S (22) (46) 

ADENOMA , BOS 1 (2%) 

ENDOMETPIA L STROMA L POLY P 6 (27*) 13 (28%) 

ENDOMETRIA L STROMA L SARCOM A 1 (2%) 

NEFVOtJ S SYSTE M 

NON 2 

SPECIA L SFNS E ORGAN S 

NON E 

HUSCHLOSK2LETAL
 

NONE
 

# '1UMBE? OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

(46) 

1 (2*) 

32 (70*) 

(46) 

1 (2% ) 

3 (7% ) 

(50) 

1 (2*) 

(50) 

1 (2*) 

1 (2*) 

7 (14% ) 

(43) 

11 (26% ) 

46
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 

LOW DOSE HIGH DOSE
 CONTROL
 

BOEY CAVITIES
 

ALL OTHER SYSTEMS 

"•MULTIPLE OPGANS 

SARCOMA, NOS 

CARCINOSARCOMA 

(25) (50) (50) 

1 (2X> 

1 (2S) 

"HORACTC CAVITY 

PARAGANGLIOMA, NOS 

ADIPOSE TISSUE 

LIFOMA 

ANIMAL DISPOSITION SUMMAR Y
 

ANIMALS INITIALLY IN STUDY 25 50 50
 

NATURAL CEATH9 2 5
 

MORIBUND SACRIFICE 12 15
 

**SCHEDULED SACRIFICE 1
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 16 35 30
 

ANIMA L MISSING
 

3LI"£LS2!S _AU^OLYZED_ANIMILS,
 

t NUMBER CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 **
 

Animal is in fact an early terminal sacrifice, but will
 

appear as a scheduled sacrifice due to system interpretation.
 

47
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 LOW DOSE HIGH DOSE
 CONTROL
 

TUFOF SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 22 12 43
 

"T^AL PRIMARY TUMORS 25 53 66
 

TOTAL ANIMALS WITH BENIGN TUMORS 17 36 40
 

"OTAL BENIGN TUMOBS 20 4U 56
 

TO^AL ANIMALS WITH MALIGNANT TUMORS 5 7 9
 

TOTAL MALIGNANT TUMORS 5 7 10
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TJMORS
 

"•OTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 2
 

^OTAL UNCERTAIN TUMORS 2
 

TO^AL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY CR METASTATIC
 

•"OTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONEARY TUMORS
 

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

48
 
APPENDIX B
 

SUMMARY OF Till: INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED ANILAZINE IN THE DIET
 
TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED ANILAZINE IN THE DIET

 

MATCHED LOW DOSE HIGH DOSE 

CONTROL 

ANIMAL S INITIALL Y I N STUD Y 25 50 50 

ANIMAL S NECEOPSIE D 23 49 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALL Y 23 49 50 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (23) (49) (50)
 

SARCOMA, NOS 3 (6%)
 

'IBRO?ARCOMA 1 (2%)
 

FIBROUS HISTIOCYTOMA, MALIGNANT 1 (2%)
 

HEM ANGIOSARCOMA 1 (2%)
 

*ESPTRATOFY SYSTEM
 

#! UN G (23) (48) (50) 

CARCINOMA , NO? , METASTATI C 1 (2% ) 

KEPATOCELLULA R CARCINOMA , METAST 1 (2S) 

AL VEOI AR/BRONCH'OLA R ADENOM A 2 (9%) 5 (1055) 4 (8%) 

ALVEOI.AP/ERCNCHIOLA P CARCINOM A 2 (9% ) 2 (4% ) 2 (>4%) 

"ISROUS HISTTOCYTriMA , METASTATI C 1 (2% ) 

"EMATOPOIETIC SYSTEM
 

*"U!TIPI,E ORGAN S (23) (49 ) (50) 

"AilGNAN T LYMPHOMA , NO S 2 (4X ) 

M ALIG. LYMPHOMA , LY1PHOCYTIC TYPE 1 (2%) 

NAIIG.LYMPHO f A, HISTIOCYTIC TYP E 2 (9%) 1 (2X ) 

4SPLEEN (23) (49) (50)
 

HEMANGI01A 1 (2%)
 

HEMANGIOSARCOf.A 1 (4%) 1 (2%) 1 (2<)
 

#IY«?H NODE (18) (45) (40)
 

ALVEOLAR/BHOKCHIOLAR CA, METASTA 1 (655)
 

"ALIGVANT LYMPHOMA, NOS 1 (2%)
 

I. ATOPY SYSTEM
 

* NIMBE? OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECPOPSIED
 

51
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

LOW DOSE HIGH DOSE 

LtUIN I KUL 

DIGESTIV E SYSTEM 

#IIVEP. (23) (49) (50) 

HEPA^OCELLULA F ADENOM A 1 (2%) 

HEPATOCELLULA E CARCINOM A 9 (39S) 5 (1C% ) 12 (24X) 

HEMANGIOSA5COM A 1 (2% ) 

*EILE DUCT (23) (49 ) (50) 

CARCINOMA,NO S 1 (2% ) 

ICOLON (20) (43) (47 ) 

LEIOMYOM A 1 (2X ) 

URIN«R Y SYSTE M 

#KTDNE Y (23) (49) (50) 

ALVEOLAR/BRONCHIOLA P CA, METAST A 1 (2%) 1 (2*) 

ENDOCRIN E SYSTEM 

• THYROI D (20 ) (43) (39) FOLLICt.'LAP-CELL ADENOM A 1 (3X) 

PEPRHDUC7IVE SYSTEM
 

JtTESTIS (23) (49 ) (50) 

SEMINOMA/EYSGERHIKOM A 1 (25) 

NERV^'I S SYSTSM 

NON E 

SPECTAL SENSE ORGANS
 

*HARDERIAN GLAND (23) (49) (50)
 

ADENOMA , NOS 2 (4%)
 

MUSC"LOSKELETAL SYSTEM
 

VONS
 

» i:UM3ER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECPOPSIED
 

52
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

 LOW DOSE HIGH DOSE CONTROL

BOD Y CAVITIE S 

*PEPTTONEU 1

ALVEOLAR/ESONCHIOLA R CA , METAST A

 (23)

 1 (4% ) 

 (49 ) (50) 

*PLE'I5A

ALVEOLAP./BFONCHTOLA P

ALVEOLAP/EROHCHIOLA R

 CA,

 CA,

 INVASI V

 METAST A

 (23)

 1 (4%)

 (49)

 1 (2X) 

 (50) 

1 (2X) 

A LL OTHER SYSTEM S 

NON E 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALL Y III STUDY 25 50 50 

NATURA L DEATHS 6 8 2 

MORIBUN D SACRIFICE 5 9 7 

**SCHEDULED SACRIFICE 5 7 

ACCIDENTALL Y KILLED 1 

TERMINA L SACRIFICE 8 33 34 

ANIMA L MISSING 

1_SI£LHDES_AUTQLYZED_ANJMALS 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECPOPSIED
 

Animals are in fact early terminal sacrifices, but will
 

appear as scheduled sacrifices due to system interpretation.
 

53 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

p/IATCHED LOW DOSE HIGH DOSE cJONTROL 

TUPO P SUMMAR Y 

TOTAL ANIMAL S WIT H PRIMAR Y TUMORS * 15 20 25 

TOTA L PRIMAR Y TUMOR S 16 25 27 

TOTAL ANIMAL S WITH BENIG N TUMOR S 2 8 6 

TOTAL BENIG N TUMORS 2 8 7

 

TOTAL ANIMAL S WITH MALIGNAN T TUMOBS 11* 16 20 

TOTAL MALIGNAN T TUMOR S 14 17 20 

TOTAL ANIFAL S WIT H SECONDAR Y TUMCRS t 1 3 2 

TOTAL SECONDAR Y TUMORS 3 U 3 

TOTAL ANIMAL S HITH TUMORS UNCERTAIN ­ BENIGN O R "ALIGNAN T

 

TOTAL UNCERTAI N TUMORS 

TOTAL ANIMAL S WITH TUMORS UNCEHTAIN­ PRTMAR Y OR METASTATIC

 

TOTAL UNCERTAI N TUHORS 

* PRIMAR Y TUMORS : ALL TUMORS EXCEFT SECONDARY TUMORS * SECONDAR Y TUMOPS : METASTATIC TUBCRS OR TUMORS INVASIV E INTO AN ADJACENT ORGA N 

54 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED ANILAZINE IN THE DIET 

MATCHED 

CONTROL LOWD >OSE 

ANIMALS INITIALLY IN STUDY 25 50 ' 

ANIMALS NECROPSIED 25 47 

ANIMALS EXAMINED HI STOPATHOLOGICALLY 25 47 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (25) (47)
 

SARCOMA, NOS
 

PESPISATORY SYSTEM
 

#!UVG (23) (47)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (2%)
 

ALVECLAR/BRONCHIOLAR CARCINOMA
 

HEMATOPOIETIC SYSTEM
 

* MULTIPLE ORGANS (25) (47)
 

MALIGNANT LYMPHOMA, NOS 1 (4%) 4 (9%)
 

f ALIG.LYMPHOKA, LYMPHOCYTIC TYPE 1 (4%) 2 (4%)
 

KALIG. LYMPHOMA, HISTIOCYTIC TYPE 1 (4%) 1 (2%)
 

MYELOMONOCYTIC LEUKEMIA
 

HONOCYTIC LEUKEMIA 1 (2%)
 

tSPLEEN (25) (47)
 

FEMANGIOMA 1 (2%)
 

tLIVES (24) (45)
 

MALIGNANT LYMPHOMA, NOS 1 (2«)
 

V ALIG. LYMPHOKA, LYMPHOCYTIC TYPE
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIV E SYSTE M 

tLIVSR (24) (45) 

___ _ HePATOCELLULAR_CAECINOB A 

# NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALL Y * IinMBER OF ANIMAL S NECEOPSIED 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

1 (2»)
 

(50)
 

1 (2X)
 

1 (2%)
 

(50)
 

8 (16%)
 

1 (2%)
 

(49)
 

(48)
 

1 (2%)
 

(48) 

55 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE CONTROL 

URINARY SYSTEM
 

NONE
 

ENDOCRIN E SYSTEM 

tFI^UITA^Y (23) (43) (44 ) 

CHROMOPHOB E ADENOM A 1 (US) 3 (7%) 1 (2%) 

*THYROID (25) 144) (48) 

FOLLTCULAR-CEL L ADENOM A 1 (2%)

 

^PRODUCTIVE SYSTEM 

*MAMMAR Y GLAN D (25) (47) (50) 

CARCINOMA , NOS 1 (2%) 

ADENOMA , NOS 1 (2X) 

ADENOCARCINOMA , .NOS 1 (28) 

#UTERUS (25) (43) (48) 

FIBROM A 1 (2%) 

ENDOMETRIA L STPOMA L POLYP 1 (2%) 1 (2%) 

*OVAS Y (25) (46 ) (49) 

CYSTADENOMA , NO S 1 (2% ) 1 (2X) 

^EF.ATOMA , BENIG N 1 (2% ) 

NERVOU S SYSTE M 

'10NE 

SPFCTA L SENS E ORGAN S 

+HASDERIA N GLAN D (25) (47) (50) 

ADENOMA , NO S 1 (2S) 

"USCULOSKELETA L SYSTE M 

NON3 

* NUMBE" . O F ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R O F ANTMAL S NECPOPSIE D 

56 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

BOD Y CAVITIES 

*ABD1MTNA L CAVITY (25) (47) (50) 

T.TPOM A 2 (4% ) 

*PE"ITONEU M (25) (47) (50) 

T.IPOH A 1 (2% ) 

ALL OTHER SYSTEMS 

NON E 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALLY IN STUD Y 25 50 50 

NATURA L DEATHS 6 3 

MORIBUN D SACRIFICE 3 11 6 

**SCHEDULED SACRIFICE 5 5 8 

ACCIDENTALL Y KILLE D 1 • "ERMINAL SACRIFICE 17 27 33 

ANIMA L MISSING 

3 INCLUDES AUTOLYZE D ANIMAL S 

SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUMOFS * 22 18 

TOTAL PRIMAR Y TUMORS 25 19 

TO"AL ANIMAL S WIT H BENIGN TONORS 14

 

TOTAL BENIGN TUMORS T*

 

TOTAL ANIMAL S WIT H MALIGNAN T TO PORS 11 14 

TOTAL MALIGNAN T TUMORS 11 14 

ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TO^AL ANIMALS WITH TUMORS UNCERTAINPRIMARY OP METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: JLL TUMORS EXCEPT SECONEARY TUMORS
 

**
 

Animals are in fact early terminal sacrifices, but will
 

appear as scheduled sacrifices due to system interpretation.
 

# SECONDAR Y TUMORS : METASTATIC TUMOES OR TOHORS INVASIVE INTO AN ADJACENT ORGAN 

57 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED ANILAZINE IN THE DIET
 

59
 
TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED ANILAZINE IN THE DIET 

ANIMAL S INITIALL Y I N S m U D Y

ANIKAL S NECROPSIE D

ANIMAL S EXAMINE D HI STOPATHOLOGIC ALL Y

INTEGUMENTAR Y SYSTE M 

NON E 

PESPIRATOR Y SYSTE M 

#LUN G

HEMORRHAG E 

HEMA^OPOIETIC SYSTE M 

#BON E MAPBO W

ATROPHY , NOS 

HYPOPLASIA , HEMATOPOIETTC 

#SPLEE N

CONGESTION , NOS

 

INFLAMMATION , ACU^E

ATROPHY , NOS 

HEMATOPOIESI S 

tMANDIBHLA R L. NOD E

INFLAMMATION , ACUTE

HYPERPLASIA , NOS

 

#MESENTERIC L. NOD E

PERIARTERITI S 

*RENAL LYMP H NOD E

CYST, NOS 

CIPCULATORY SYSTEM
 

#HEART
 

THROMBOSIS. NOS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 2 5 5C 50 

 2 5 50 50 

 2 5 50 50 

 (25) (U9 ) (49) 

1 (2%) 

 (24 ) (50) (48) 

1 (2% ) 

1 (2%) 

 (25) (46) (50) 

2 (4% ) 

1 (4X ) 

1 (2*) 

1 (2») 3 (6X) 

 (23) (47) (46) 

1 (4%) 

2 (14%) 

 (23) (47) (46) 

1 (2%) 

 (23) (47) (46) 

1 (2%) 

(25) (50) (50) 

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

61
 
TABLE C1. MALE RATS: NONNEOPLASTIC

fMYOCARDIUM
 

INFLAMMATION, ACUTE
 

DIGESTIVE SYSTEM
 

#SALIVARY GLAND
 

INFLAMMATION, NOS
 

HYPERPLASIA, NOS
 

#LIVEE
 

INFLAMMATION, NOS
 

NECROSIS, CENTRAL
 

METAMORPHOSIS FATTY
 

FOCAL CELLULAR CHANGE
 

ANGIECTASIS
 

HEMATOPOIESIS
 

ILIV2R/CENTRILOBULAR
 

DEGENERATION, NOS
 

*BILE DUCT
 

CYST, NOS
 

HYPERPLASIA, NOS
 

#PANCREAS
 

PERIARTERITIS
 

ATROPHY, NOS
 

ATROPHY, FOCAL
 

•STOMACH
 

INFLAMMATION, NOS
 

INFLAMMATION, ACUTE
 

ULCER, ACUTE
 

INFLAMMATION, CHRONIC
 

ULCER, CHRONIC
 

URINARY SYSTEM
 

#KIDNEY
 

INFLAMMATION, CHRONIC
 

ENDOCRINE SYSTEM
 

*PITUITARY
 

CYST. NOS
 

 LESIONS (CONTINUED) 

MATCHED
 

CONTROL
 

(25)
 

(25)
 

11 (44%)
 

11 (44%)
 

(24)
 

1 (4%)
 

3 (13X)
 

1 (4%)
 

(24)
 

(25)
 

5 (20%)
 

(24)
 

2 (8X)
 

1 (4«)
 

(24)
 

1 (4%)
 

1 (4%)
 

1 (•»*)
 

1 (<»%)
 

(25)
 

14 (56%)
 

(25)
 

LOW DOSE
 

(50)
 

1 (2%)
 

•
 

(47)
 

21 (45%)
 

21 (45%)
 

(48)
 

1 (256)
 

1 (2%)
 

3 (6%)
 

3 (6%)
 

(48)
 

(50)
 

18 (36*)
 

(47)
 

1 (2%)
 

1 (2%)
 

(50)
 

2 (4%)
 

1 (2%)
 

(49)
 

36 (73X)
 

(44)
 

1 12%)
 

HIGH DOSE
 

(50)
 

(49)
 

12 (24%)
 

12 (24X)
 

(49)
 

1 (2%)
 

4 (8%)
 

2 (4%)
 

1 (2%)
 

4 (8X)
 

(49)
 

1 (2%)
 

(50)
 

1 (2%)
 

15 (30%)
 

(50)
 

1 (2%)
 

(48)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

(49)
 

36 (73*)
 

(46)
 

-*» J9*l
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

62
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 LOW DOSE HIGH DOSE
 CONTROL
 

HYPEBPLASIA, FOCAi	 2 (8X)
 

#ADRENAL (23) (49) (49)
 

HEMORRHAGE 1 (2%)
 

HEBORRHAGIC CYST 1 (2X)
 

#ADRENAL CORTEX (23) (49) (49)
 

ANGIECTASIS 2 (4X)
 

#ADRENAL MEDULLA (23) (49) (49)
 

HYPERPLASIA, NOS 1 (2*)
 

#THYROID (23) (38) (47)
 

HYPERPLASIA, C-CELL 3 (13S) 1 (3%) 3 (6X)
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND (25) (50) (50)
 

INFLAMMATION, CHRONIC 1 (2»)
 

*PREPUTIAL GLAND (25) (50) (50)
 

HYPEEPLASIA, NOS 1 (2*)
 

#PROSTATE (18) (45) (45)
 

INFLAMMATION, ACUTE 1 (2X)
 

INFLAMMATION, ACUTE SDPPURATIVE 1 (2%)
 

ABSCESS, NOS 1 (6%)
 

*TE?TIS (24) (49) (49)
 

ATROPHY, NOS 2 (4»)
 

NERVOUS SYSTEM
 

tBRAIN (25) (47) (49)
 

HYDROCEPHALUS, NOS 2 (4%)
 

INFLAMMATION, NOS 1 (28)
 

•CEREBELLUM	 (25) (47) (49)
 

GLIOSIS 1 (2%)
 

SPECIAL SENSE ORGANS
 

NONE
 

# NUKBE P OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

63 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

.LOW DOSE HIGH DOSE 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

MESENTERY (25) (50) (50) 

ABSCESS, NOS 1 (US) 

All OTHER SYSTEMS 

ADIPOSE TISSUE 

INFLAMMATION , CHRONIC 1 3 1 

SPECIAL MORPHOLOG Y SUMMAR Y 

NONE 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECEOPSIED 

64 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED ANILAZINE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALL Y IN STUDY 25 50 50 

ANIMAL S NECBOPSIED 25 50 50 

ANIMAL S EXAMINE D HIETOPATHOLOGICALLY 25 50 50 

INTEGUMENTAR Y SYSTEM 

NONE 

RESPIRATORY SYSTEM 

#LUNG (24) (49) (50)
 

HYPEEPLASIA, NOS 1 (2*)
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN (25) (47) (50)
 

THROMBOSIS, NOS 1 (2X)
 

HYPERPLASIA, NOS 1 (2*)
 

HEMATOPOIESIS 1 (2X)
 

tMANDIBULAR L. NODE (23) (43) (44)
 

INFLAMMATION, ACUTE 4 (9X)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#SALIVARY GLAND (22) C*8)

INFLAMMATION, NOS 7 (32X) 23 (48*) 20 (41X) 

DEGENERATION, NOS 1 (5%) 

HYPERPLASIA, NOS 8 (36*) 23 (48X) 20 (41X) 

ULIVER (25) (U9) 

INFLAMMATION. ACOTB 

f NUMBER OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBED OF ANIMALS NECROPSIED 

65 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 LOW DOSE HIGH DOSE
 CONTROL
 

METAMORPHOSIS FATTY 5 (10X) 1 (2X)
 

BASOPHILIC CYTO CHANGE 2 (8X) 3 (6%) 6 (12X)
 

FOCAL CEILOLAR CHANGE 1 (4X) 2 (4X) 3 (6X)
 

HEM ATOPO TESTS 2 (4X) 2 (4X)
 

REGENERATION, NOS 1 (2%)
 

*BILE DUCT (25) (50) (50)
 

HYPERPLASIA, NOS 1 (IX) 4 (8X> 6 (12S)
 

#PANCREAS (24) (49) (50)
 

ATROPHY, NOS 1 (2X) 2 (4X)
 

#STOMACH (24) (48) (48)
 

ULCER, CHRONIC 1 (4X)
 

URINARY SYSTEM
 

#KIDNEY (24) (48) (49)
 

INFLAMMATION, CHRONIC 8 (33X) 12 (25X) 6 (12%)
 

DEGENERATION, NOS 1 (2X)
 

DYSPLASIA, NOS 1 (2X)
 

#URINARY BLADDER (22) (46) (44)
 

HYPERPLASIA, EPITHELIAL 1 (2%)
 

ENDOCRINE SYSTEM
 

*PITUITARY (22) (43) (46)
 

CYST, NOS 2 (9X) 1 (2X) 4 (9X)
 

HEMORRHAGIC CYST 1 (2%) 1 (2X)
 

HYPERPLASIA, FOCAL 2 (9X) 1 (2%)
 

tADRENAL (24) (47) (48)
 

CYST, NOS 1 (2X)
 

DEGENERATION, NOS 1 (2X)
 

ANGIECTASIS 1 (2X)
 

tADRENAL CORTEX (24) (47) (48)
 

LIPOIBOSIS 3 (13X) 1 (2%)
 

ANGIECTASIS 1 (2X)
 

#THYROID (24) (35) (46)
 

HYPERPLASIAt C-CELL 2 (6X)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

66
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

REPRODUCTIVE SYSTEM 

*MAMMARY DOCT

HYPERPLASIA, NOS

ICTERUS

HENATOMA, NOS

METAPLASIA, SQOAMODS

 (25)

 (22)

 1 (5%) 

 (50)

 (46)

 1 (2X) 

 (50) 

1 (2%) 

 (43) 

NERVOUS SYSTEM 

#BRAIN

HYDROCEPHALUS, NOS

HEMORRHAGE

 (24)

 1 (4X)

 (49)

 2

 1

 (4X) 

 (2%) 

 (50) 

SPECIAL SENSE ORGANS 

NONE 

MUSCIILOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

ADIPOSE TISSUE 

STEATITIS

INFLAMMATION, ACUTE

INFLAMMATION, CHRONIC

 1 

1 

2 1 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 2 2 

* NUMBER OF ANIMALS WITH TISSUE EXAHINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

67
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE ADMINISTERED ANILAZINE IN THE DIET
 

69
 
TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED ANILAZINE IN THE DIET 

MATCHED 

CONTROL LOW DOSE 

ANIHALS INITIALLY IN STUDY 25 5fl 

ANIHALS NECROPSIED 23 49 

ANIMALS EXAMINED HISTOPATHOLOGIC ALLY 23 49 

INTEGUMENTARY SYSTEM
 

*SKIN (23) (49)
 

ABSCESS, NOS
 

BESPIRATORY SYSTEM
 

tlUNG (23) (48)
 

HYPERPLASIA. ALVEOLAR EPITHELIUM 3 (6X)
 

HEHATOPOIETIC SYSTEM
 

#SPLEEN (23) (49)
 

ANGIECTASIS
 

HYPERPLASIA, LYMPHOID 1 (2%)
 

HEMATOPOIESIS 1 (2%)
 

tLYMPH NOEE (18) (45)
 

CONGESTION, NOS
 

INFLAMMATION, NOS
 

HYPERPLASIA, LYMPHOID 1 (2%)
 

#MESENTERIC L. NODE (18) (45)
 

CONGESTION, NOS
 

#THYMUS (D (2)
 

HYPERPLASIA, LYMPHOID
 

CIRCULATORY SYSTEM
 

tHEART (23) (48)
 

__ PERIARTERITIS
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

3 (6%)
 

(50)
 

(50)
 

1 (2X)
 

(40)
 

1 OX)
 

1 (3%)
 

1 (3X)
 

(40)
 

1 (3X)
 

(3)
 

1 (33%)
 

(50)
 

71
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL 

LOW DOSE HIGH DOSE 

DIGESTIVE SYSTEM 

ILIVER 

NECROSIS, NOS 

NECROSIS, COAGULATIVE 

NECROSIS, HEflORRHAGIC 

HYPERPLASIA , NODULAR 

(23) 

2 (9X) 

1 (4%) 

(49) 

1

1

 (2%) 

 (2%) 

(50) 

2 (4X) 

*BILE DUCT 

HYPERPLASIA, CYSTIC 

(23) (49) (50) 

1 (2X) 

•PANCREAS 

INFLAMMATION, NOS 

(23) (49) 

1 (2X) 

tSMALL INTESTINE

ULCER, NOS

HYPERPLASTA, LYMPHOID

 (20)

 1 (5X) 

 (44) (1*3) 

1 (2X) 

URINARY SYSTEM 

tKIDNEY

INFLAMMATION, CHRONIC

 (23) (49)

 1 (2%) 

 (50) 

IUBINARY BLADDER

INFLAMMATION, CHRONIC

 (18) (47)

 1 (2%) 

 (49) 

ENDOCRINE SYSTEM 

NONE 

REPBODUCTIVE SYSTEM 

• PROSTATE

HEMORRHAGE

 (18) (44)

 1 (2%) 

 (48) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*HARDERIAN GLAND 

INFLABHATIOH, NOS 

(23) (49) (50) 

f NUMBER OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY 

* NDMBER OF ANIHALS UECROPSIED 

72
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

JoNTROL LOW DOSE HIGH DOSE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*PEPITONEOK (23) (49) (50)
 

INFLAMMATION, GRANULOMATOUS 2 (4X)
 

AIL OTHER SYSTEMS
 

*«ULTIPLE ORGANS (23) (49) (50)
 

INFLAMMATION, GRANULOHATOUS 1 (2%)
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 6 19 19
 

ACCIDENTAL DEATH 1
 

AOTOLYSIS/NO NECROPSY 1 1
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECPOPSIED
 

NOTE: ANIMAL t049 IS IN FACT ANV EARLY TSAC BUT HILL APPEAR AS SSAC DUE TO SYSTEM
 

INTERPRETATION.
 

73
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED ANILAZINE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIHALS INITIALLY IN STUDY 25 50 50 

ANIMAL S NECROPSIED 25 HI 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 25 47 50 

INTEGUMENTARY SYSTEM 

*SKIN (25) (<47) (50)
 

INFLAMMATION, FOCAL 1 (2X)
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN (25) (47) (49)
 

HYPERPLASIA, LYMPHOID . 1 (2*)
 

CIRCULATORY SYSTEM
 

•MYOCARDIUM	 (25) (47) (50)
 

INFLAMMATION, FOCAL 1 (4%)
 

*PULMONARY ARTERY (25) (47) (50)
 

SCLEROSIS 4 (16X) 3 (6X)
 

DIGESTIVE SYSTEM
 

•LIVER	 (24) (45) (48) 

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

INFLAMMATION. ACUTE 1 (2X) 

INFLAMMATION, GRANULOMATOUS 2 (8X) 

NECROSIS, NOS 1 (2X) 

FOCAL CELLULAR CHANGE 1 (2*) 

fPEYERS PATCH (24) (41) (44)

 

HYPERPLASI1U LTHgHOIp
 

* NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

74 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

 LOW DOSE HIGH DOSE CONTROL

IJEJUNUH (24) (41) (44) 

HYPEHPLASIA, 1YHPHOID 1 (2X) 

tILEOH (24) (41) (44) 

HYPERPLASIA, LYHPHOID 2 (5X) 

ORINAR Y SYSTEH 

IKIDNEY (25) (47) (50)
 

NECROSIS, DIFFUSE 1 (2%)
 

ENDOCRINE SYSTEH
 

#PITUITARY (23) (43) (44)
 

HEMORRHAGIC CYST 1 (2%)
 

HYPERPLASIA, BOS 1 (2%)
 

•THYROID (25) (44) (48)
 

CYSTIC FOLLICLES 1 (2%)
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND (25) (47) (50)
 

GALACTOCELE 1 (2%)
 

tUTERUS/ENDOHITRIUM (25) (43) (U8)
 

HYPESPLASIA, NOS 1 (2X) 1 (2X)
 

HYPERPLASIA, CYSTIC 1 (4%) 2 (««)
 

*OVA5Y (25) (46) (49)
 

CYST, NOS 1 (4%) 2 (4X) 1 (2X)
 

FOLLICULAR CYST, NOS 1 (2%) 2 (4X)
 

HEMORPHAGIC CYST 1 (2X)
 

INFLAMMATION, NOS 1 (2X)
 

EHDOMETRIOSIS 1 (2%)
 

NERVOOS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

HOVE
 

# NUMBE R OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

75 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

*PERITONEUM

INFLAMMATION, ACUTE

INFLAMMATION, GRANULOMiTOUS

 (25) p»7)

 1 <2X) 

 (50) 

1 (2*) 

ALt OTHER SYSTEMS 

NONE 

SPECIAL HORPHOLOGY SOBHARY 

NO LESION REPORTED 1H 15

AUTOLYSIS/NO NECROPSY 3 

* NDMBER OF ANIMALS HITH TISSUE IXAHINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

 22 

76
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

RATS ADMINISTERED ANILAZINE IN THE DIET
 

77
 
Table El. Analyses of the Incidence of Primary Tumors in Kale Rats
 

Administered Anilazine in the Diet3
 

Topography: Morphology

Matched 

 Control 

Lung: Alveolar/Bronchiolar 

Adenoma or Carcinoma13 2/25 (8) 

P Valuesc « d N.S. 

Relative Riskf 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 95 

VO llematopoietic System: 

Undifferentiated Leukemiab 2/25 (8) 

P Valuesc » d N.S. 

Relative Risk^ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 95 

Low
 

Dose
 

0/49 (0)
 

N.S.
 

0.000
 

0.000
 

1.718
 

——
 

1/50 (2)
 

N.S.
 

0.250
 

0.004
 

4.616
 

92
 

High
 

Dose
 

3/49 (6)
 

N.S.
 

0.765
 

0.095
 

8.775
 

104
 

1/50 (2)
 

N.S.
 

0.250
 

0.004
 

4.616
 

94
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Anilazine in the Dieta
 

(continued)
 

Matched Low High
 

Topography; Morphology Control Dose Dose
 

Pituitary: Adenoma or
 

Carcinoma, NOSb 6/25 (24) 16/44 (36) 17/46 (37)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf 1.515 1.540
 

Lower Limit 0.663 0.683
 

Upper Limit 4.167 4.213
 

Weeks to First Observed Tumor 95 75 61
 

00
 

o
 Thyroid: C-cell Adenoma^ 1/23 (4) 4/38 (11) 1/47 (2)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf 2.421 0.489
 

Lower Limit 0.263 0.007
 

Upper Limit 115.840 37.631
 

Weeks to First Observed Tumor 104 103 104
 
00 

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

Administered Anilazine in the Diet3
 

(continued)
 

Topography: Morphology


Thyroid: Papillary Adenoma


P Valuesc>d


Relative Risk^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Pancreatic Islets: Islet-cell
 

Adenomab


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 0/23 (0)
 

 N.S.
 

—
 

 1/24 (4)
 

 N.S.
 

 104
 

Low
 

Dose
 

2/38 (5)
 

N.S.
 

Infinite
 

0.184
 

Infinite
 

104
 

4/47 (9)
 

N.S.
 

2.043
 

0.220
 

98.366
 

94
 

High
 

Dose
 

0/47 (0)
 

N.S.
 

;-;
 

—
 

5/50 (10)
 

N.S.
 

2.400
 

0.294
 

111.118
 

92 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Anilazine in the Dieta
 

(continued)
 

Matched Low High
 

Topography; Morphology Control Dose Dose
 

Testis: Interstitial-cell Tumorb 20/24 (83) 43/49 (88) 44/49 (90)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf 1.053 1.078
 

Lower Limit 0.870 0.892
 

Upper Limit 1.340 1.345
 

Weeks to First Observed Tumor 95 77 92
 

oo aDosed groups received 500 or 1,000 ppm in feed.
 

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

c

Beneath the incidence of tumors in the matched-control group is the probability level for the Cochran-


Armitage test when P < 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 < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

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

Topography ; Morphology
 

Hematopoietic System:
 

Undif ferentiated Leukemia^5
 

P Values0 »d
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

GO
 

OJ Hematopoietic System:
 

Lyraphoma or Leukemia"
 

P Values0 >d
 

Relative Riakf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Administered Anilazine in the Diet3
 

Matched Low 

Control Dose 

3/25 (12) 3/50 (6) 

N.S. N.S. 

0.500 

0.073 

3.524 

77 69 

3/25 (12) 3/50 (6) 

N.S. N.S. 

0.500 

0.073 

3.524 

77 69 

High
 

Dose
 

1/50 (2)
 

N.S.
 

0.167
 

0.003
 

1.971
 

102
 

2/50 (4)
 

N.S.
 

0.333
 

0.030
 

2.753
 

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

Administered Anilazine in the Diet3
 

(continued)
 

Matched Low High
 

Topography; Morphology Control Dose Dose
 

Pituitary: Carcinoma, NOSb 1/22 (5) 3/43 (7) 1/46 (2)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk^ 1.535 0.478
 

Lower Limit 0.134 0.006
 

Upper Limit 78.651 36.761
 

Weeks to First Observed Tumor 83 83 104
 

oo Pituitary: Adenoma
 

or Carcinoma, NOS*> 12/22 (55) 25/43 (58) 33/46 (72)
 

P Values0 »d M.S. N.S. N.S.
 

Relative Riskf 1.066 1.315
 

Lower Limit 0.673 0.868
 

Upper Limit 1.867 2.168
 

Weeks to First Observed Tumor 83 50 89
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

(continued) 

Topography; Morphology 

Thyroid: C-cell Adenoma" 

F Valuesc » d 

Relative Risk^ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

00 

Ul 

Mammary Gland:

P Values0'd 

 Fibroadenoma" 

Relative Riskf 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Administered Anilazine in the Dieta
 

Matched

Control

 Low 

 Dose 

0/24 (0) 1/35 (3) 

N.S. N.S. 

Infinite 

0.038 

Infinite 

82 

1/25 (4) 3/50 (6) 

N.S. N.S. 

1.500 

0.130 

77.150 

104 76 

High
 

Dose
 

3/46 (7)
 

N.S.
 

Infinite
 

0.323
 

Infinite
 

96
 

7/50 (14)
 

N.S.
 

3.500
 

0.494
 

154.214
 

98
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Anilazine in the Diet3
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Uose
 

Uterus: Endometrial Stromal Polypb 6/22 (27) 13/46 (28) 11/43 (26)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Riskf 1.036 0.930
 

Lower Limit 0.438 0.378
 

Upper Limit 2.945 2.734
 

Weeks to First Observed Tumor 95 76 89
 

oo
 Uterus: Endometrial Stromal
 

Polyp or Sarcomab

 6/22 (27) 14/46 (30) 11/43 (26)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Riskf 1.116 0.938
 

Lower Limit 0.481 0.378
 

Upper Limit 3.131 2.734
 

Weeks to First Observed Tumor 95 76 89
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Anilazine in the Diet3
 

(continued)
 

a

Uosed groups received 500 or 1,000 ppm in feed.
 

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

c

Beneath the incidence of tumors in the matched-control group is the probability level for the Cochran-


Armitage test when P < 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 < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

"A negative trend (K) indicates a lower incidence in a dosed group than in a control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

'•The 95/' 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 ANILAZINE IN THE DIET
 

89
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Anilazine in the Diet3
 

Topography: Morphology


Integumentary System:
 

Sarcoma, NUS, of the
 

Subcutaneous Tissue13


P Values0 »d


Departure from Linear Trend6


Relative Risk/
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Lung: Alveolar/Bronchiolar
 

Carcinoma13


P Valuesc>d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 0/23 (0)
 

 N.S.
 

P = 0.038
 

—
 

 2/23 (9)
 

 N.S.
 

 107
 

Low
 

Dose
 

3/49 (6)
 

N.S.
 

Infinite
 

0.291
 

Infinite
 

78
 

2/48 (4)
 

N.S.
 

0.479
 

0.037
 

6.328
 

83
 

High
 

Dose
 

0/50 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

2/50 (4)
 

N.S.
 

0.460
 

0.036
 

6.082
 

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

Administered Anilazine in the Dieta
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma*3 4/23 (17) 7/48 (15) 6/50 (12)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk/ 0.839 0.690
 

Lower Limit 0.243 0.186
 

Upper Limit 3.600 3.075
 

Weeks to First Observed Tumor 83 83 102
 

Hematopoietic System: Lymphoma 2/23 (9) 1/49 (2) 4/50 (8)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Riskf 0.235 0.920
 

Lower Limit 0.004 0.145
 

Upper Limit 4.326 9.724
 

Weeks to First Observed Tumor 78 103 95
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Anilazine in the Dieta
 

(continued) 

Topography: Morphology

Matched 

 Control 

Liver: liepatocellular Carcinomab 9/23 (39) 

P Valuesc » d N.S. 

Departure from Linear Trende P = 0.007 

Relative Riskf 

Lower Limit 

Upper Limit 

VO 

u> 

Weeks to First Observed Tumor

Liver: Hepatocellular 

Adenoma or Carcinona^

 68 

 9/23 (39) 

P Valuesc » d N.S. 

Departure from Linear Trende P = 0.015 

Relative Riskf 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 68 

Low
 

Dose
 

5/49 (10)
 

P = 0.006(N)
 

0.261
 

0.081
 

0.771
 

95
 

6/49 (12)
 

P = 0.012(N)
 

0.313
 

0.109
 

0.872
 

95
 

High
 

Dose
 

12/50 (24)
 

N.S.
 

0.613
 

0.289
 

1.439
 

93
 

12/50 (24)
 

N.S.
 

0.613
 

0.289
 

1.439
 

93
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Anilazine in the Diet3
 

(continued)
 

a

Dosed groups received 500 or 1,000 ppm in feed.
 

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

c

Beneath the incidence of tumors in the matched-control group is the probability level for the Cochran-


Armitage test when P < 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 < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

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

Administered Anilazine in the Diet3
 

Topography: Morphology


Hematopoietic System:
 

Lymphoma or Leukemia*3


P Values0 >d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


>J3 ,
 01

 Pituitary: Chromophobe Adenoma0


P Valuesc>d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 3/25 (12)
 

 N.S.
 

 72
 

 1/23 (4)
 

 N.S.
 

 109
 

Low
 

Dose
 

9/47 (19)
 

N.S.
 

1.596
 

0.449
 

8.552
 

85
 

3/43 (7)
 

N.S.
 

1.605
 

0.140
 

82.217
 

107
 

High
 

Dose
 

10/50 (20)
 

N.S.
 

1.667
 

0.485
 

8.811
 

1/44 (2)
 

N.S.
 

0.523
 

0.007
 

40.132
 

109
 

86 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Anilazine in the l)ieta
 

(continued)
 

a

Dosed groups received 50U or 1,000 ppm in feed.
 

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

c

Beneath the incidence of tumors in the matched-control group is the probability level for the Cochran-


Armitage test when P < 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 < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

95% confidence interval of the relative risk between each dosed group and the control group.
 
APPENDIX C
 

ANALYSIS OF FORMULATED DIETS FOR
 

CONCENTRATIONS OF ANILAZINK
 

97
 
APPENDIX C
 

Analysis of Formulated Diets for
 

Concentrations of Anilazine
 

Duplicate LO->; samples of fornulated diets were extracted in 250
 

r.il of a 50:50 acetone:benzene solution and agitated mechanically
 

for 3-4 hours. Suitable diluted aliquots of the extract were
 

analyzed by "as-liquid chromatography usin;> an electron capture
 

detector. Spiked samples were worked up simultaneously with the
 

dosed feed samples and data from these analyses were used to
 

correct the recoveries from the dosed feed samples.
 

Theoretical No. of Sample Coefficient of Ranj;e (ppin) 

Concentration Samples Analytical Variation (7C) 

(ppm) riean (ppin) 

500 12 492 4.05 464-530
 

1,000 14 9H8 4.46 894-1056
 

98
 
Review of the Bioassay of Anilazine* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

June 29, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DREW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed.
 

The members of the Clearinghouse have been drawn from
 

academia, industry, organized labor, public interest groups,
 State health officials, and quasi-public health and research
 

organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and,
 collectively, provide expertise in chemistry, biochemistry,
 biostatistics, toxicology, pathology, and epidemiology.
 Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment
 

Subgroup of the Clearinghouse is charged with the responsibility of providing a peer review of reports prepared on
 

NCI-sponsored bioassays of chemicals studied for carcinogenic­ ity. It is in this context that the below critique is given
 

on the bioassay of Anilazine for carcinogenicity.
 

The reviewer agreed with the conclusion in the report
 

that Anilazine was not carcinogenic in treated rats or mice,
 under the conditions of test. After a brief description of
 

the experimental design, the reviewer commented on the small
 

number of control animals and low dose levels administered.
 

He said that the results from the subchronic study indicated
 

that higher dosages should have been used in the chronic
 

phase. The experimental flaw detracted from the value of
 

the bioassay. The reviewer moved that the report on the
 

bioassay of Anilazine be accepted as written. The motion
 

was approved without objection.
 

Clearinghouse Members present:
 

Arnold L. Brown (Chairman), Mayo Clinic
 

Paul Nettesheim, National Institute of Environmental
 

Health Sciences
 

Verne Ray, Pfizer Medical Research Laboratory
 

Verald K. Rowe, Dow Chemical U.S.A.
 Michael B. Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

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.
 

99
 

•V.U.5. GOVERNMEN T PRINTIN G OFFICE : 1978-260-899/3184
 
DHEW Publication No. (NIH) 78-1354