1977 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 25 

BIOASSAY OF 

CHLORAMBEN 

FOR POSSIBLE CARCINOGEN ICITY 

CAS No. 133-90-4 

NCI-CG-TR-25 

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

Public Health Service 

National Institutes of Health 
BIOASSAY OF
 

CHLORAMBEN
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogen Bioassay and Program Resources Branch
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

DREW Publication No. (NIH) 77-825
 

BIOASSAY OF
 

CHLORAMBEN
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

CONTRIBUTORS; This report presents the results of the bioassay
 

of chloramben for possible carcinogenicity, conducted for the
 

Carcinogen Bioassay and Program Resources Branch, Carcinogenesis
 

Program, Division of Cancer Cause and Prevention, National Cancer
 

Institute (NCI), Bethesda, Maryland. The bioassay was conducted
 

by Gulf South Research Institute, New Iberia, Louisiana, initially under direct contract to NCI and currently under a
 

subcontract to Tracer Jitco, Inc., prime contractor for the NCI
 

Carcinogenesis bioassay program.
 

The experimental design was determined by Drs. J. H.
 Weisburger^'^ and R> R< Bates^'^; the doses were selected by
 

Drs. T. E. Shellenberger^'5, J. H. Weisburger, and R. R. Bates.
 

Animal treatment and observation were supervised by Drs. T. E.
 Shellenberger and H. P. Burchfield , with the technical
 

assistance of Ms. D. H. Monceaux^ and Mr. D. Broussard .
 

Histopathology was performed by Drs. E. Bernal^ and B. Buratto*
 

at Gulf South Research Institute, and the diagnoses included in
 

this report represent the interpretation of these pathologists.
 

Pathologists from NCI and Tracer Jitco have reviewed selected
 

slides and concur with the overall pathologic evaluation of the
 

study.
 

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

Mason Research Institute6. Statistical analyses were performed
 

by Dr. J. R. Joiner', using methods selected for the bioassay
 

program by Dr. J. J. Gart . Chemicals used in this bioassay were
 

analyzed under the direction of Dr. H. P. Burchfield, and the
 

analytical results were reviewed by Dr. S. S. Olin .
 

iii
 
This report was prepared at Tracer Jitco under the direction of
 

NCI. Those responsible for the report at Tracer Jitco were
 

Dr. Marshall Steinberg , Director of the Bioassay Program; Dr. J.
 

F. Robens', toxicologist; Ms. L. A. Waitz' and Ms. Y. E.
 

Presley , technical writers; and Dr. E. W. Gunberg', technical
 

editor.
 

The following scientists at the National Cancer Institute 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. Thomas W. Orme
 

Dr. Robert A. Squire9
 

Dr. Jerrold M. Ward
 

^•Carcinogenesis Program, Divison of Cancer Cause and Prevention,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

%ow with the Office of the Commissioner, Food and Drug
 

Administration, Rockville, Maryland.
 

^Gulf South Research Institute, Atchafalaya Basin Laboratories,
 

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

%ow with the National Center for Toxicological Research,
 

Jefferson, Arkansas.
 

6EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

^Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

iv
 
^Mathematical Statistics and Applied Mathematics Section, Field
 

Studies and Statistics Branch, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

^Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay of technical-grade chloramben for possible carcinogenicity was conducted by administering the test material in feed to
 

Osborne-Mendel rats and B6C3F1 mice. Groups of 50 rats and 50
 

mice of both sexes were administered chloramben at one of two
 

doses, either 10,000 or 20,000 ppm. The rats were treated for 80
 

weeks, then observed for 32 or 33 weeks; the mice were treated
 

for 80 weeks, then observed for 11 or 12 weeks. Matched controls
 

consisted of groups of 10 untreated rats and 10 untreated mice of
 

each sex; pooled controls, used for statistical evaluation,
 

consisted of these matched controls combined with 75 untreated
 

male and 75 untreated female rats or 70 untreated male and 70
 

untreated female mice from similarly performed bioassays of six
 

other test chemicals. Surviving rats were killed at 112 or 113
 

weeks; surviving mice were killed at 91 or 92 weeks.
 

Body weights and mortality of the treated animals were not
 

markedly affected by chloramben under the conditions of the 

bioassay. The various clinical signs observed were common to 

both treated and control groups. 

In male rats, hemangiomas occurred at a significantly higher
 

incidence in the low-dose animals than in the pooled controls
 

(controls 0/73, low-dose 5/48, P - 0.009). This lesion was not
 

considered to be related to the administration of chloramben,
 

since the tumor did not occur at a significantly higher incidence
 

in the high-dose group than in the pooled-control group, and the
 

incidences did not show a significant dose-related trend.
 

In both male and female mice, the incidences of hepatocellular
 

carcinoma showed significant dose-related trends using pooled
 

controls (for males: controls 9/69, low-dose 16/48, high-dose
 

14/48, P - 0.029; for females: controls 2/67, low-dose 7/48,
 

high-dose 10/50, P = 0.004). Direct comparisons showed
 

significantly higher incidences of the tumor in the low-dose
 

males (P - 0.008) and in the high-dose females (P - 0.003) than
 

in the pooled controls. Probability levels of P - 0.028 in high-


dose males and P = 0.027 in low-dose females were attained. In
 

male mice, however, the incidence of hepatocellular carcinoma was
 

vii
 
considered to be only marginally associated with the
 

administration of chloramben because of the variations in the
 

spontaneous incidence of this lesion in male mice encountered at
 

this laboratory.
 

In conclusion, under the conditions of this bioassay, there were
 

no tumors in Osborne-Mendel rats that were significantly related
 

to administration of the chemical. In B6C3F1 female mice,
 chloramben was carcinogenic, producing hepatocellular carcinomas
 

in treated animals.
 

viii
 
TABLE OF CONTENTS
 

Page
 

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 6
 

F. Designs of Chronic Studies 7
 

G. Clinical and Pathologic Examinations 10
 

H. Data Recording and Statistical Analyses 11
 

III. Results - Rats 17
 

A. Body Weights and Clinical Signs (Rats) 17
 

B. Survival (Rats) 17
 

C. Pathology (Rats) 20
 

D. Statistical Analyses of Results (Rats) 21
 

IV. Results - Mice 23
 

A. Body Weights and Clinical Signs (Mice) 23
 

B. Survival (Mice) 23
 

C. Pathology (Mice) 26
 

D. Statistical Analyses of Results (Mice) 26
 

V. Discussion.. 29
 

VI. Bibliography 31
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed Chloramben in the Diet 33
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed Chloramben in the Diet 35
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Fed Chloramben in the Diet 38
 

ix 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Fed Chloramben in the Diet 41 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Chloramben in the Diet 43 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Chloramben in the Diet 46 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Chloramben in the Diet 49 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Chloramben in the Diet.... 51 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Chloramben in the Diet.. 55 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Chloramben in the Diet 59 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Chloramben in the Diet.... 61 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Chloramben in the Diet.. 63 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Chloramben in the Diet 67 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Chloramben in the Diet 69 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Chloramben in the Diet 73 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Chloramben in the Diet 79 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Chloramben in the Diet 81 

X 
Page 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Chloramben in the Diet 83 

Appendix G Analysis of Formulated Diets for 

Concentrations of Chloramben 85 

TABLES 

Table 1 Design of Chloramben Chronic Feeding Studies 

in Rats 8 

Table 2 Design of Chloramben Chronic Feeding Studies 

in Mice 9 

FIGURES 

Figure 1 Growth Curves for Rats Fed Chloramben 

in the Diet 18 

Figure 2 Survival Curves for Rats Fed Chloramben 

in the Diet 19 

Figure 3 Growth Curves for Mice Fed Chloramben 

in the Diet 24 

Figure 4 Survival Curves for Mice Fed Chloramben 

in the Diet 25 

xi
 

I. INTRODUCTION
 

Chloramben (CAS 133-90-4; NCI C00055) has been used since 1958 as
 

a preemergent herbicide to control shallow, germinating,
 

broadleaf weeds and annual grasses. Applied as a spray at the
 

time of planting, chloramben remains effective in the soil for
 

several weeks until crops have become well established (Amchem
 

Products, 1976). It is currently registered for use in the
 

cultivation of several vegetable crops. The residue tolerance on
 

most of these vegetables is 0.1 ppm (EPA, 1974 and 1975).
 

Chloramben is not known to be persistent in the environment
 

(Edwards, 1976). Soils sampled 3 months after treatment showed
 

no detectable toxic residues (Burgis, 1972), although a
 

metabolite, N-(3-carboxy-2,5dichlorophenyl)glucosylamine, has
 

been found in treated plants (Swanson et al. , 1966). Chloramben
 

is of low mammalian toxicity; the oral LD5Q in rats is 5,260
 

mg/kg (Spencer, 1973). The chemical was selected for testing
 

because its extensive use as a herbicide results in human
 

exposure.
 

1 

II. MATERIALS AND METHODS
 

A. Chemical
 

Chloramben, which is the generic name for 3-amino-2,5-dichlorobenzoic acid, was obtained in several batches from Amchem
 

Products, Inc., Ambler, Pennsylvania, as technical-grade
 

AmibenT-M-. These batches were used sequentially, in the general
 

order in which they were obtained. The purity of these batches,
 

according to the manufacturer, ranged from 90-95%. Analyses at
 

Gulf South Research Institute (melting point; elemental analysis;
 

infrared, ultraviolet, nuclear magnetic resonance, and mass
 

spectrometry; thin-layer and gas-liquid chromatography) confirmed
 

the identity of these batches, and analyses were consistent with
 

the manufacturer's assay. No attempt was made to identify or
 

quantitate impurities. The chemical was stored at approximately
 

4°C in the original container.
 

B. Dietary Preparation
 

(R)
 All diets were formulated using finely ground Wayne Lab Blox
 

(Allied Mills, Inc., Chicago, 111.) to which was added the
 

required amount of chloramben for each dietary concentration. A
 

given amount of the test material was first hand-mixed with an
 

approximately equal amount of feed. This mixture was then added
 

slowly with mechanical mixing to a larger quantity of feed to
 

3 
give the desired concentration of the material. Acetone
 

(Mallinckrodt Inc., St. Louis, Mo.) and corn oil (Louana ,
 

Opelousas Refinery Co., Opelousas, La.) were then added to the
 

feed, each in an amount corresponding to 2% of the final weight
 

of feed. The diets were mixed mechanically for not less than 25
 

minutes to assure homogeneity of the mixture and evaporation of
 

the acetone. Formulated diets were stored at approximately 17°C
 

until used, but no longer than 1 week.
 

The stability of chloramben in feed was tested by determining the
 

concentration of the chemical in formulated diets at intervals
 

over a 7-day period. Diets containing 10,000 or 20,000 ppm
 

chloramben showed no change in concentration on standing at
 

ambient temperature for this period.
 

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

diets, the concentration of chloramben was determined in
 

different batches of formulated diets during the chronic study.
 

The results are summarized in Appendix G. At each dietary
 

concentration, the mean of the analytical concentrations for the
 

samples tested was within 0.5% of the theoretical concentration,
 

and the coefficient of variation was 4.9%. Thus, the evidence
 

indicates that the formulated diets were prepared accurately.
 

4 
C. Animals
 

Rats and mice of both sexes, obtained through contracts of the
 

Division of Cancer Treatment, National Cancer Institute, were
 

used in these bioassays. The rats were of the Osborne-Mendel
 

strain obtained from Battelle Memorial Institute, Columbus, Ohio,
 

and the mice were B6C3F1 hybrids obtained from Charles River
 

Breeding Laboratories, Inc., Wilmington, Massachusetts. Upon
 

arrival at the laboratory, all animals were quarantined for an
 

acclimation period (rats for 10 days, mice for 12 days) and were
 

then assigned to control and test groups.
 

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
 

changed 10-12 times per hour. Fluorescent lighting provided
 

illumination 10 hours per day. Food and water were supplied ad
 

libitum.
 

The rats were housed individually in hanging galvanized steel
 

mesh cages, and the mice were housed in plastic cages with filter
 

bonnets, five per cage for females, and two or three per cage for
 

males. Initially, rats were transferred once per week to clean
 

cages; later in the study, cages were changed every 2 weeks.
 

5 
Mice were transferred once per week to clean cages with filter
 

bonnets; bedding used for the mice was Absorb-Dri® (Lab Products,
 

Inc., Garfield, N.J.). For rats, absorbent sheets under the
 

cages were changed three times per week. Feeder jars and water
 

bottles were changed and sterilized three times per week.
 

Cages for control and treated mice were placed on separate racks
 

in the same room. Animal racks for both species were rotated
 

laterally once per week; at the same time, each cage was changed
 

to a different position in the row within the same column. Rats
 

receiving chloramben, along with their respective matched
 

controls, were housed in a room by themselves. Mice receiving
 

chloramben were maintained in a room housing mice administered
 

chlorothalonil (CAS 1897-45-6), picloram (CAS 1918-02-1), or
 

endrin (CAS 72-20-8), together with their respective matched
 

controls.
 

E. Subchronic Studies
 

Feeding studies using rats and mice were conducted to estimate
 

the maximum tolerated doses of chloramben, on the basis of which
 

low and high concentrations (hereinafter referred to as "low
 

doses" and "high doses") were determined for administration in
 

the chronic studies. In the subchronic studies, chloramben was
 

added to the animal feed in twofold increasing concentrations,
 

6 
ranging from 62.5 to 2,000 ppm for rats and 1,250 to 30,000 ppm
 

for mice. Control and treated groups each consisted of five male
 

and five female animals. The chemical was provided in feed to
 

the treated groups for 6 weeks, followed by 2 weeks of
 

observation. Because there were no deaths and no effects on body
 

weights in the rats at 62.5 to 2,000 ppm, indicating that the
 

maximum tolerated dose had not been reached, a second study was
 

performed on the rats using doses ranging from 2,000 to 32,000
 

ppm.
 

At 16,000 ppm none of the rats died, and there was no effect on
 

body weights; at 32,000 ppm, the treated animals lost weight.
 

The low and high doses for the chronic studies using rats were
 

set at 10,000 and 20,000 ppm.
 

There were no marked adverse effects on mice receiving dietary
 

concentrations as high as 30,000 ppm. The low and high doses for
 

the chronic studies using mice were set at 10,000 and 20,000 ppm,
 

consistent with those set for rats.
 

F. Designs of Chronic Studies
 

The designs of the chronic studies are shown in tables 1 and 2.
 

Since the numbers of animals in the matched-control groups were
 

small, pooled-control groups also were used for statistical
 

7 
Table 1. Design of Chloramben Chronic Feeding Studies in Rats
 

Sex and 

Treatment 

Initial 

No. of 

Chloramben 

in Diet 

Time c 

Treated 

m Study 

Untreated11 

Group Animala§. (ppm) (weeks ) (weeks ) 

Male 

Matched-Control 10 0 113 

Low-Dose 50 10,000 80 

0 32 

High-Dose 50 20,000 80 

0 33 

Female 

Matched-Control 10 0 113 

Low-Dose 50 10,000 80 

0 33 

High-Dose 50 20,000 80 

0 33 

a

All animals were 35 days of age when placed on study.
 

^When diets containing chloramben were discontinued, treated rats and
 

their matched controls were fed diets without corn oil for 3 weeks,
 then control diets (2% corn oil added) for an additional 29 or 30
 

weeks.
 

8 
Table 2. Design of Chloramben Chronic Feeding Studies in Mice
 

Sex and 

Treatment 

Initial 

No. of 

Chloramben 

in Diet 

Time o 

Treated 

m Study 

Untreatedb 

Group Animals^ (ppm) (weeks ) (weeks ) 

Male 

Matched-Control 10 0 91 

Low-Dose 50 10,000 80 

0 11-12 

High-Dose 50 20,000 80 

0 12 

Female 

Matched-Control 10 0 91 

Low-Dose 50 10,000 80 

0 11 

High-Dose 50 20,000 80 

0 12 

a

All animals were 35 days of age when placed on study.
 

^When diets containing chloramben were discontinued, treated mice and
 

their matched controls received the control diets (2% corn oil added)
 

until termination of the study.
 

9 
comparisons. Matched controls from the current study of
 

chloramben were combined with matched controls from studies
 

performed on malathion (CAS 121-75-5), tetrachlorvinphos (CAS
 

961-11-5), toxaphene (CAS 8001-35-2), lindane (CAS 58-89-9),
 

endrin, and chlorothalonil. The pooled controls for statistical
 

tests using rats consisted of 75 males and 75 females; using
 

mice, 70 males and 70 females. The studies of chemicals other
 

than chloramben were also conducted at Gulf South Research
 

Institute and overlapped the chloramben study by at least 1 year.
 

The matched-control groups for the different test chemicals were
 

of the same strain and from the same supplier, and they were
 

examined by the same pathologists.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily 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 were killed 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,
 

10
 
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 were
 

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,
 

11
 
clinical observations, survival, animal 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.
 

The data of the experiments in this bioassay program are subjected to the statistical analyses described in the subsequent
 

paragraphs of this section. The analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival are estimated by the product-limit
 

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

report in the form of graphs. Animals are statistically censored
 

as of the time they are missing or are dead due to other than
 

natural causes; animals dying from natural causes are
 

statistically uncensored. Statistical analyses for a possible
 

dose-related effect on survival employ 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 are reported for all tests except the
 

departure from linearity test, which is noted when its two-tailed
 

P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions is given as
 

12
 
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 include only those animals for which such sites
 

are examined histologically. However, when macroscopic examination is required to detect lesions and when this examination is
 

followed by histologic sampling (e.g., skin or mammary tumors),
 

or when lesions could appear at multiple sites (e.g., lymphomas),
 

the denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of the incidences of
 

'.$
 

tumors is to determine whether animals receiving the test
 

chemical develop a significantly higher proportion of tumors than
 

do control animals. Statistical analyses of the incidences of
 

specific types of tumors are made using the one-tailed Fishar
 

exact test (Cox, 1970) to compare a control group with group;: of
 

treated animals at each dose. When results for a number of
 

treated groups (k) are compared simultaneously with those for a
 

control group, a correction which ensures 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. When appropriate the correction is discussed
 

in the narrative section, but it is not used in the tables, wnrire
 

the Fisher exact P values are shown.
 

13
 
The Cochran-Armitage test for linear trend in proportions, with
 

con inuity correction (Armitage, 1971), is also used. Under the
 

as; ^notion of a linear trend, this test determines if the slope
 

of r.he dose-response curve is different from zero at the oneta ed 0.05 level of significance. Unless otherwise noted, the
 

di ,:t.ion of the significant trend is a positive dose
 

; e,- itionship. This method also provides a two-tailed test of
 

de -cLure from linear trend.
 

An Luernative analysis is applied when early deaths result from
 

ca t s that are not associated with the formation of tumors. In
 

th, aaa.lv sis, deaths that occur before the first tumor is
 

observed are excluded by basing the statistical tests on animals
 

th-i. survive at least as long as 52 weeks, unless a tumor is
 

fo. n.d at the anatomic site of interest before week 52. When such
 

an ariy tumor is found, comparisons are based exclusively on
 

an •'••His that, survive at least as long as the animal in which the
 

i 3 •.c tumor is found. Once this reduced set of data is obtained,
 

i;h. standard procedures for analyses of the incidence of tumors
 

(Fisher exact test, Cochran-Armitage test, etc.) are followed.
 

V.hi ., appropriate, life-table methods are applied to the incidence
 

of iriers. Curves of the proportions surviving without a tumor
 

bei;:>: observed are computed according to Saffiotti et al. (1972),
 

jh times at which animals die naturally or are killed are
 

14
 
entered as the time point of tumor observation. Cox's methods of
 

comparing these curves are used for two groups, and Tarone's
 

extension to testing for linear trend is used for three groups.
 

The tests for the incidence of tumors using life-table methods
 

are one-tailed and, unless otherwise noted, in the direction of a
 

positive dose relationship. Significant departures from
 

linearity (< 0.05, two-tailed test) are also noted.
 

The approximate 95% confidence interval for the relative risk
 

between each of the treated groups and its control is calculated
 

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

relative risk is Pt/Pc where pt is the true binomial probability
 

of the incidence of a specific type of tumor in a treated 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 treated group and that in a control group is expressed
 

by a relative risk of unity. Values in excess of unity represent
 

the condition of a larger proportion in the treated group than in
 

the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk are included in the tables of statistical analyses.
 

The interpretation of the limits is that in approximately 95% of
 

a large number of similar experiments, the true ratio of the risk
 

15
 
in a treated group of animals to that in a control group would be
 

within the interval calculated from the experiment. When the
 

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

occurrence of 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) will also obtain. When the
 

lower limit is less than unity and the upper limit is greater
 

than unity, the former" indicates the absence of a significant
 

result while the latter 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.
 

16
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

The mean body weights of the high-dose male and female rats were
 

slightly lower than those of the corresponding matched controls
 

(figure 1). The weights of the low-dose males were, for unknown
 

reasons, slightly higher than those of the male controls; weights
 

of low-dose females were essentially unaffected by chloramben.
 

The treated animals were generally comparable to the controls in
 

appearance and behavior throughout the bioassay. During the
 

second 6 months, clinical signs including epistaxis, diarrhea,
 

and hematuria were noted at low incidences. During the second
 

year, clinical signs including rough and discolored hair coats,
 

dermatitis, pale mucous membranes, tachypnea, ataxia of hind
 

legs, hyperactivity, and vaginal bleeding were noted with
 

increasing frequency. Several animals appeared emaciated.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival of male and female rats receiving chloramben at the
 

doses used in this experiment, together with those of the
 

controls, are shown in figure 2. The Tarone test results for
 

positive dose-related trend in mortality over the period are not
 

17
 
Figure 1. Growth Curves for Rats Fed Chloramben in the Diet
 

18
 
Figure 2. Survival Curves for Rats Fed Chloramben in the Diet
 

19 
significant in either sex. In male rats, 61% of the high-dose
 

group, 74% of the low-dose group, and 40% of the controls lived
 

to the end of the study. In females, 68% of the high-dose group,
 

58% of the low-dose group, and 50% of the controls lived to the
 

end of the study. A pooled-control group was used, providing
 

adequate numbers of control animals for meaningful statistical
 

analyses oi: the incidences of late-developing 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 tumors occurred randomly in both the control and
 

treated rats. For the most part, these lesions are not uncommon
 

in this strain of rat independent of any treatment. In addition
 

to the neoplastic lesions, a number of nonneoplastic lesions also
 

were observed in both the treated and control groups. In
 

general, these nonneoplastic lesions are routinely encountered in
 

aged rats of this strain.
 

C-cell adenomas of the thyroid occurred among both treated male
 

and female rats, whereas adrenal cortical adenomas occurred only
 

among treated females. There was a higher incidence of hyper-


plastic changes of both follicular cells and C cells of the
 

20
 
thyroid in the treated rats than in the controls, particularly in
 

the treated males. The incidences of C-cell adenomas of the
 

thyroid and cortical adenomas of the adrenal in the treated
 

groups are not unusual in untreated rats of this strain.
 

Although follicular-cell hyperplasia of the thyroid occurred only
 

in the treated animals and not in the controls, the incidence of
 

the lesion was not dose related. These thyroid lesions suggest
 

that the test chemical may have a goitrogenic effect, but
 

insufficient numbers of controls were available to draw firm
 

conclusions.
 

In the judgment of the pathologists, chloramben did not induce
 

tumors in rats during this study.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those specific primary tumors that were
 

observed in at least 5% of one or more treated groups of either
 

sex.
 

In male rats, the Cochran-Armitage test for positive dose-related
 

linear trend in proportions for hemangioma, using pooled
 

controls, has a probability level of P = 0.074; but a significant
 

departure from linear trend (P = 0.042) is indicated, due to the
 

21
 
higher incidence in the low-dose group than in the high-dose
 

group. The Fisher exact test shows a significantly higher
 

incidence of this tumor in the low-dose group (P = 0.009), but
 

not in the high-dose group, than in the pooled controls. The 95%
 

confidence interval shows a lower limit greater than one for the
 

relative risk comparing the low-dose group with the control
 

group. This indicates that there is a theoretical possibility of
 

the induction of tumors by the test chemical. Because of the
 

lack of statistical significance of the incidence of hemangioma
 

in the high-dose group, the true significance of this tumor in
 

male rats is questionable.
 

In female rats, the statistical test results on hemangioma are
 

not significant at the 0.05 level. There are no other incidences
 

of tumors at any specific site in either sex which are
 

significant using either the Cochran-Armitage test or the Fisher
 

exact test.
 

22
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

The mean body weights of the treated male and female mice were
 

essentially unaffected by chloramben (figure 3).
 

The treated animals were generally comparable to the controls in
 

appearance and behavior throughout the bioassay. During the
 

second year, clinical signs including alopecia, rough hair coats,
 

hyperactivity, dyspnea, abdominal distention, and hunched
 

appearance were observed. Many males, treated and control, were
 

observed fighting. The equilibrium of one low-dose male appeared
 

to have been impaired.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival of male and female mice receiving chloramben at the
 

doses used in this experiment, together with those of the
 

controls, are shown in figure 4. The Tarone test results for
 

positive dose-related trend in mortality over the period are not
 

significant in either sex. In male mice, 90% of the high-dose
 

group, 84% of the low-dose group, and 80% of the controls lived
 

to the end of the study. Similarly, in female mice, 90% of the
 

high-dose group, 76% of the low-dose group, and 90% of the
 

23
 
Figure 3. Growth Curves for Mice Fed Chloramben in the Diet
 

24
 
Figure 4. Survival Curves for Mice Fed Chloramben in the Diet
 

25 
controls lived to the end of the study. Survival of both sexes
 

was adequate for meaningful statistical analyses of the incidence
 

of tumors in these mice.
 

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.
 

For the most part, the lesions were of the type commonly encountered in this strain of mice. The incidence of hepatocellular
 

carcinoma in both male and female treated mice was higher than
 

that in the controls (males: controls 2/10 [20%], low-dose 16/48
 

[33%], high-dose 14/48 [29%]; females: controls 0/9 [0%],
 

low-dose 7/48 [15%], high-dose 10/50 [20%]). Spontaneous
 

hepatocellular carcinoma is not uncommon in this strain of mouse,
 

particularly in the males. In the judgment of the pathologists,
 

the incidence in males was insufficient to indicate a clear
 

relationship to treatment; however, the relationship between
 

treatment with chloramben and the incidence of hepatocellular
 

carcinoma appears to be significant in female mice.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

26
 
of the incidences of those specific primary tumors that were
 

observed in at least 5% of one or more treated groups of either
 

sex.
 

In male mice, the Cochran-Armitage test for positive dose-related
 

trend in the proportions of hepatocellular carcinoma is significant (P = 0.029) using the pooled controls, and the Fisher exact
 

test shows higher incidences of this tumor in both the low-dose
 

group (P = 0.008) and the high-dose group (P = 0.028) than in the
 

pooled controls. The probability level of 0.028 is above the
 

level of 0.025 required for significance by the multiple
 

comparison procedure for the Fisher exact test. The 95%
 

confidence intervals using the pooled controls have lower limits
 

greater than one.
 

The significant results in the males are confirmed in the
 

females, since the Cochran-Armitage test result for positive
 

dose-related trend in the proportions of hepatocellular carcinoma
 

is significant (P = 0.004) using the pooled controls, and the
 

Fisher exact test shows significantly higher incidences of thit
 

tumor in both the low-dose group (P = 0.027) and the high-dose
 

group (P = 0.003) than in the pooled controls. The probability
 

level of 0.027 is above the level of 0.025 required for
 

significance by the multiple comparison procedure for the Fisher
 

exact test. The 95% confidence intervals for the relative risk
 

27
 
comparing the high-dose group with the pooled controls have lower
 

limits greater than one. These tests show that, statistically,
 

there is an association between chloramben treatment and the
 

occurrence of hepatocellular carcinoma in mice at the doses used
 

in this experiment.
 

28
 
V. DISCUSSION
 

Mean body weights and rates of mortality of the treated animals
 

were not markedly affected by chloramben under the conditions of
 

the bioassay. The various clinical signs observed were common to
 

both treated and control groups. Survival was adequate for
 

meaningful statistical analyses of the incidence of tumors.
 

Thus, the concentrations of chloramben used in both rats and
 

mice, i.e., 10,000 and 20,000 ppm, can be considered to be only
 

slightly toxic. However, these concentrations are high when
 

compared with the possible exposure of humans to residues of the
 

herbicide.
 

In rats, hemangiomas occurred at a significantly higher incidence
 

in low-dose males (5/48 [10%]) than in pooled controls (0/73);
 

however, the incidence of this tumor was not significant for
 

high-dose males compared with pooled controls, and the dose-


related trend was not statistically significant. In addition,
 

the pathologists did not consider the hemangioma to be related to
 

administration of the chemical. Thus, the occurrences of
 

hemangiomas are not considered to be related to the administration of chloramben.
 

In both male and female mice, the incidences of hepatocellular
 

carcinoma showed significant dose-related trends using pooled
 

29
 
controls (for males: controls 9/69, low-dose 16/48, high-dose
 

14/48, P = 0.029; for females: controls 2/67, low-dose 7/48,
 

high-dose 10/50, P = 0.004). Direct comparisons showed
 

significantly higher incidences of the tumor in the low-dose
 

males (P = 0.008) and in the high-dose females (P = 0.003) than
 

in the pooled controls. Probability levels of P = 0.028 in high-


dose males and P = 0.027 in low-dose females were attained. Very
 

few related lesions were observed. Two additional male animals,
 

but only one female, had hepatocellular adenoma or neoplastic
 

nodule. No hyperplastic lesions of the liver were observed in
 

either sex.
 

The variability in the incidence of hepatocellular carcinoma
 

among historical control mice at Gulf South Research Institute
 

was considered. In a few control groups in the bioassay program
 

at this testing laboratory, as many as 3/10 or 4/10 male mice had
 

hepatocellular carcinoma. The mean of the incidences for the
 

male controls at Gulf South Research Institute was 16.8%.
 

Because of the variation (0-40%) in the historical incidences of
 

spontaneous hepatocellular carcinomas in control male mice at
 

this laboratory, the incidences of these lesions in treated male
 

mice reported in this study are considered as marginal. This is
 

consistent with the pathologists' view that the incidence in
 

males was insufficient to indicate a clear relationship to
 

30
 
treatment. Control groups of female mice had no more than 2/9
 

animals with hepatocellular carcinoma, and the mean of the
 

incidences for all females in the historical group was 2.3%.
 

In conclusion, under the conditions of this bioassay, chloramben
 

was not carcinogenic in either sex of the Osborne-Mendel rats.
 

Hemangiomas were present at a slightly higher incidence in low-


dose male rats than in pooled controls. However, the bioassay
 

does not conclusively demonstrate the relationship of these
 

lesions to treatment. In B6C3F1 male mice, the incidence of
 

hepatocellular carcinoma was considered as only marginally
 

associated with the administration of chloramben. In B6C3F1
 

female mice, chloramben was carcinogenic, producing
 

hepatocellular carcinomas in treated animals.
 

31
 

VI. BIBLIOGRAPHY
 

Amchem Products, AmibenT'M; Preemergence Herbicide, Amchem
 

Products, Inc., Ambler, Pa., accepted 1976.
 

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

& Sons, Inc., New York, 1971, pp. 362-265.
 

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

Panel on Carcinogenicity of the Cancer Research Commission
 

of the UICC, Vol. JJ. International Union Against Cancer,
 

Geneva, 1969.
 

Burgis, D. S., Herbicide tests on pepper transplants and seeded
 

peppers. Proc. Fla. State Hort. Soc. 84:183-186, 1972.
 

Cox, D. R., Regression models and life tables. J. K. Statist.
 

Soc. B 34 (2):187-220, 1972.
 

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

1970, pp. 48-52.
 

Edwards, C. A., Persistent Pesticides in the Environment. CRC
 

Press, Cleveland, Ohio, 1976, pp. 107-108.
 

Environmental Protection Agency, Chloramben. EPA Compendium of
 

Registered Pesticides, U. S. Government Printing Office,
 

Washington, D.C., 1975, I-C-13.1.
 

Environmental Protection Agency, Chloramben. EPA Compendium of
 

Registered Pesticides, U.S. Government Printing Office,
 

Washington, D. C., 1974, I-C-13.2.
 

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.
 

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

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

1958.
 

Leigh, E. S. J., Jr. and Lisk, D. L., Excretory pathway of Amiben
 

in a lactating cow. J. Agr. Food Chem. 18:482-484, 1970.
 

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

J., Carcinogenesis bioassay data system. Comp. and Biomed.
 Res. 7:230-248, 1974.
 

333 
Miller, R. G., Jr. , Simultaneous Statistical Inference,
 

McGraw-Hill, 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 administrations of benzo (a)
 

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

Spencer, E. Y. , Guide to Chemicals Used in Crop Protection,
 

Research Institute, University of Western Ontario, London,
 

Ontario, 1973, p. 90.
 

Squire, R. A. and Levit<_, M., Report of a workshop on
 

classification of specific hepatocellular lesions in rats.
 

Cancer Res. 35;3214-3223. 1975.
 

Swanson, C. R., Kadunce, R. E., Hodgson, R. H., and Frear, D. S. ,
 

Amiben metabolism in plants, I. Isolation and identification
 

of an N-glucosyl complex. Weeds 14;319-323, 1966.
 

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

Biometrika 62(3);679-682. 1975.
 

34
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED CHLORAMBEN IN THE DIET
 

35
 

TABLE A1. 

SUMMARY OF THE INCiDENCE OF NEOPLASMS IN MALE RATS 

FED CHLGRAMBEW fN THE DIET 

CONTRO L LOW DOSE 

ANIMAL S INITIALL Y T N STUD Y 10 50 

ANIMAL S NECROPSIE D 9 U8 

ANIMAL S EXAMINE D HIST O PA THOLOGIC A LL Y 9 141 

INTEGUMENTAR Y SYSTE M 

*SKI N (9! («8) 

FIBROU S HISTIOCYnW A 

FIBROU S HTSTIOCYrnHA , MALIGNAN T 2 ('4S)

 

*SURCU T TISSU E (9) (H8 )

 

L^IOMYOM A 1 (2% ) 

RESPIRATOR Y SYSTE M 

NON E 

[l^KATOPOIETI TM SYSTFt'. 

*MULTIPL P ORGAN S (9 ) (48 ) 

'IPANUT.OCYTI C LEUKE1I A 1 (2% ) 

tSPLE^ N (~!) (47 )

 

HFHANGION A 3 (6S) 

CTPC'JI.ATOR Y SYSTE M 

*HFAR T m (!*7) 

ADENOCAPC I NO«iA . NO S 

JIGBSTTV F SYSTE M 

tLIVR (9) (46 ) 

NFOPLASTI C NODUL 1 7 l* (9% ) 

H ^ M ANGTOM A 1 (2% )

 

tPAMCRFATI C DUCT (8) (U6 )

 

ADENOCAR:; I NOPIA ^ NO S . 1 J13SL .

 

# NUMBER OF ANIMAL S WITH TISSUE ^XAMINED MICROSCOPI- ALLY
 

* NU-IB^R OF ANIMAL S NFCPOPSIED
 

HI3H DOSE 

50 

49 

HI 

It9) 

1 (2*) 

(»9) 

(«9 ) 

(47 ) 

2 ('*«) 

|U8) 

1 (2$ ) 

(«6) 

1 (2« ) 

1 (2% ) 

(48) 

37
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOB DOSE 

_..i 

URINAR Y SYSTEM 

•KIDNEY (9) (47)

 

t HAMARTOM A 

ENDOCRINE SYSTFM 

• PITUITAR Y (9) (UU ) 

CARCINOMA , NOS 

CHROMOPHOBE ADENOBA 1 (11%) 5 (1U) 

•ADS^NAL (8) (U6) 

CORTICAL ADENOH A 1 (2*) 

PHEOCHROMOCYTOM A 1 (13%) 

•THYROID (8) (47) 

C-CELL ADENOM A 4 (9«) 

• PANCREATIC ISLETS (8) (46) 

ISLFT-CELL ADFNOMA 2 (4X) 

REPRODUCTIV E SYSTEM 

*«AMMAR Y GLAN D (9) (48) 

INFILTRATIN G DUCT CARCINOM A 

"IBROMA 

LIPOH A 1 (2%) 

NERVOU S SYSTE M 

• BRAI N (9) (47) 

MFNINGIOM A 1 (2X) 

SPECIAL SENSE ORGANS 

NONF 

HUSCTJLOSKELFTAL SYSTEM 

*SKULL (9) (48)

 

QSTEOMA 

# NUMBER 0° ANIMALS WITH TISSOF EXAMINED HICROSCOPICALLY 

* NUMBE R OF ANIMAL S NFCPOPSIED 

t NONNEOPLASTIC PROLIFERATIVE LKSION 

COMPOSED OF LIPOCYTES, TDBOLAIt STRUCTURES, 

AND FIBROB1ASTS IN VARYING PMOfOKTIONS. 

HIGH DOSE 

(«8) 

2 (4X» 

(40) 

1 (3*) 8 (20X) 

(48) 

(48) 

3 (6X) 

(48) 

2 (4X) 

(49) 

1 (2X| 

1 <2X) 

1 (2X) 

(48) 

(49) 

i_12SL38

 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

1CONTROL LOW DOSE HIGH DOSE
 

BODY CAVITIES
 

*MESFNT?RY (9) (l»8) («9)
 

HAMARTOMA 1 (2X)
 

ALL OTHF.F SYSTEMS
 

*MULTIPLF ORGANS (9) (48) (U9>
 

OSTFOSARCOMA 1 (2%)
 

DIAPHRAGM
 

HEMANGIONA 1
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 10 50 50
 

NATURAL DEATHS 1 5 5
 

MORIBUND SACRIFICE 5 9 15
 

SCHEDULESCHEDULED SACRIFIC SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICSACRIFICE H4 36 330
 

A N I M AA N I M AL MISSING
 

3 INCLUDE INCLUDESS AUTOLYZE AUTOLYZED ANIMALS
 

TUMOR SUMMARY 

TOTAL ANIMALS HITH PRIMARY TUM3RSB*

TOTAL PRIMARY TUM3RS

2

3-

22

27

221

28 

N

TOTAL ANIMALS WITH BENIGN TUMOR TUMORSS

TOTAL BFNIGM TCMORS

2 .

2

16

20

16 

23 

TOTAL ANIMALS WITH MALIGNAN MALIGNANT TUMORS

TOTAL MALIGNAN MALIGNANT TUMORS

1

1

3

3

4 

4 

H 

H 

TOTAL ANIMALS HITH SECONDAR SECONDARY TUMORS! 

TOTAL SECONDAR SECONDARY TUMORS 

TOTAL ANIMALS WITH TUMORS ONCF.RTAIH 0NCF.RTAIN-­

BENIGN OR MALIGNAN MALIGNANTT

TOTAL UNCERTAIUNCERTAIN TUMORS

itH

4

1 1

1 1

TOTAL ANIMALS WITH TUMORS UNCERTAIN UNCERTAIH-­

PRIMARY OR HETASTATI HETASTATIC 

TOTAL UNCERTAI UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

t SECONDARY TUMORS: HETASTATIC TUMORS OR TUMORS INVASIVE INTO A H ADJACENT ORGAN
 

39
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

FED CHLORAMBEN IN THE DIET 

ANIMALS INITIALLY IN STUPY
 

ANIHALS N'CROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTFGUMENTAPY SYSTEM
 

*SKIN
 

SQOAMOOS CELL CARCINOMA
 

FIBROMA
 

LEIOMYOMA
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETTC SYSTEM
 

tSPLEEN
 

HEMANGIOMA
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER
 

HEOPLASTIC NODDLE
 

URINARY SYSTEM
 

tKIDNEY
 

TUBULAR-CELL ADENOMA
 

_ tI£2S4g£QHi
 

CONTROL LOW DOSE HIGH DOSE 

10 50 50 

10 50 50 

10 49 50 

(10) (50) (50) 

1 (10%) 

1 (2%) 

1 (2X) 

(10) (U9) (50) 

1 (2«) 

(10) (<»8) (50) 

2 (4X) i (2X) 

(10) («9) (50) 

1 (2%}

i-J2iL__^ — 

* NOMBER OF ANIMALS WITH TISSOE EXAMINED HICROSCOPICALLY
 

* BOMBER OF ANIMALS NECROPSIED
 

40
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOB DOSE 

ENDOCRINE SYSTEH 

tPITUITARY (9) (45) 

CARCINOMA, NOS 3 (7*) 

ADENOMA, NOS 1 (11*) 

CHROMOPHOBE ADENOM A 1 (11X) 11 (2UX ) 

tADRENAL (10) (49) 

CORTICAL ADENOM A 3 (6*) 

fTHYROI D 

C-CF.LL ADENOMA 

(8) (48) 

4 (8*) 

tPANCREATIC ISLETS 

ISLET-CELL ADENOM A 

(9) (US) 

REPRODUCTIVE SYSTEM 

*MAMMAR Y GLAND (10) (50) 

ADENOCARCINOBA , ND S 1 (2*) 

FIBROM A 

LIPOM A 

FIBROADENOM A 2 (20*) 7 (14X) 

*UTERDS (10) («•"») 

ADENOCARCINOMA , NO S 1 (2*) 

F.NDOMETRIA L STROHA L POLYP 3 (7X) 

ENDOMETRIA L STROMA L SARCOM A 

tOTERUS/ENDOMETRIOM (10) (IH») 

PAPILLAR Y ADENOCARCINOM A 

NFRVOUS SYSTEM 

#BPAI N (10) (48) 

CARCINOMA , NOS, INVASIV E 

GLIOMA , NO S 1 (2*) 

SPECIAL SENSE ORGAN S 

*FAR CANA L (10) (50) 

LETOMYOM A 1 (2*) 

MUSCULOSKELFTA L SYSTEM 

__NON£ 

# NUMBER OF ANIMALS HIPH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBFR OF ANIMALS NECPOPSIED
 

HIGH

(H6) 

1

6

(50) 

a

(50) 

U

(50) 

1

(50) 

 DOSE 

 (2*) 

 (13*) 

 (8*) 

 (8*) 

 (2*) 

1 (2X) 

1

7

(47) 

2

2

(47) 

1

(50) 

1

(50) 

 (2*) (1**) 

 (4X) 

 (4X) 

 (2X) 

 (2X) 

41
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTfNUED) 

CONTROL LOW DOSE HIGH DOSE
 

BODY CAVITIES
 

NONF
 

ALL OTHER SYSTEMS
 

*MULTIPL* ORGANS
 

FIBROUS HISTIOCYTDMA, MALIGNANT
 

HESOTHTXIOMA, NOS
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULFD SACRIFICE
 

ACCIDENTALLY KILLED
 

TFRMINAL SACRIFICE
 

ANIMAL MISSING
 

9 INCLUDES AUTOLY2ED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUM3HS*
 

TOTAL PRIMARY TUM3RS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SFCONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN UNCERTAIN-­

PRIMARY OR METASTATI METASTATIC
 

TOTAL UNCERTAI UNCERTAIN TUMORS
 

(10)
 

1 (10%)
 

10
 

5
 

5
 

6
 

6
 

4
 

4
 

2
 

2
 

(50)	 (50)
 

1 (2X) 1 (2*)
 

1 (2X)
 

50 50
 

3 4
 

18 12
 

29	 34
 

34 30
 

41 35
 

27 26
 

30 29
 

8 5
 

8 5
 

1
 

1
 

3 1
 

3 1
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TUMORS: METASTATIC TUMORS OB TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

42
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED CHLORAMBEN IN THE DIET
 

43
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED CHLORAMBEN IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISrOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEK
 

#LUNG
 

HEPATOCELLULAR CARCINOMA, HETAST
 

ALVEOLAR/BRONCHIOLAR ADENOMA
 

ALVEOLAB/BRONCHIOLAR CARCINOHA
 

HEMATOPOIETIC SYSTEM
 

*MOLTIPLE ORGANS
 

PLASMA-CELL MYELOMA
 

•KIDNEY
 

MALIGNANT LYMPHOMA, SOS
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

fLIVER
 

HEPATOCELLULAR ADENOMA
 

HEOPLASTIC NODDLE
 

HEPATOCELLULAR CARCINOMA
 

OBINARY SYSTEM
 

__NONE
 

CONTROL
 

10
 

10
 

10
 

(10)
 

(10)
 

(9}
 

(10)
 

2 (20%)
 

LOW DOSE	 HIGH DOSE
 

50	 50
 

«9	 '49
 

l»9	 48
 

(49)	 (48)
 

5	 (10X)
 

3 J6X)
 

1 l2X)
 

(U9) («9)
 

1 (2X>
 

(i>8) (18i
 

1 {2I|
 

1 (2%)
 

(US) (*fi%
 

1 (2X)
 

1 (2X)
 

16 (331)	 u (29*y
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOHBER OF ANIMALS HECROPSIED
 

45
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ENDOCRI?- - SYSTE H 

*PITUII> ay (10) (U1 ) (39) 

ADENOMA , NO S 1 (3%) 

#THYPC ' • > (9) («2 ) (1*7) 

FOLI.TCOLAB-CELL ADENOM A 1 (2*) 

BEPFODUCTTV S SYSTEM 

tTSSTIfi (10) (<»3) (<*7) 

SEM" 'W/DYSGERHINOM* 1 (2%) 

NERVOU S iSTSH 

NON r 

SPECIAL '.SSSF ORGANS 

NON E 

MnsetiLosr:Fi,ETAL SYSTEM 

NONE 

BOD T CAV rTIES 

NON E 

A L L OTH V • SYSTEMS 

*HnLTIP'i-E ORGANS (10) (U9) (49) 

„ £H3i;iJ.«S_KISTIOCITOSAJL_a4LlS»a&I J-i2iL__. 

S NUSBEB OF ANIHALS WITH TISSOE EXAMINED MICROSCOPICALLY
 

* SOHBKt OF AHIMALS NECROPSIED
 

46
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)

 

CONTROL
 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

KORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING 

10 

2 

8 

9 INCLUDES AUTOLYZED ANIMALS 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 2
 

TOTAL PRIMARY TUMORS 2
 

TOTAL ANIMALS WITH BENIGN TDHORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIHALS WITH MALIGNANT TUHORS 2
 

TOTAL MALIGNANT TUMORS 2
 

TOTAL ANIHALS WITH SECONDARY TDHORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS ONCERTAIHBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUHORBS UNCERTAIN
 UNCEHTAIN­-

PRIMAR
 PBIMARY OR BETASTATi BETASTATi;
 

TOTAL UNCERTAI UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

LOB DOSB HIGH DOSB 

50 

2 

5 

50 

2 

3 

l»3 45 

20 

21 

1 

1 

18 

19 

5 

5 

20 

22 

5 

5 

16 

17 

1 

1 

* SECONDARY TUMORS: METASTATIC TUMORS OH TUMORS INVASIVE IHTO AH ADJACENT ORGAN
 

47
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

FED CHLORAMBEN IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTDP ATHdLOGICALLY
 

INTEGUMENTARY SYSTEH
 

S
 

NONE
 

y
 

RESPIRATORY SYSTEM
 

*LtJNG
 

ALVEOLAR/BRONCHIOLAR ADEMOHA
 

HEMATOPOIETIC SYSTEH
 

*MULTIPLE ORGANS
 

MALIGNANT LYHPHOHA, NOS
 

GRANOLOCYTTC LEUKEMIA
 

•SPLEEN
 

HEHANGIOSAHCONA
 

ILIVER
 

GP.ANDLOCYTIC LEUKEMIA
 

CIRCULATORY SYSTEM
 

NONF
 

DIGESTIVE SYSTEM
 

tLIVER
 

HEPATOCELLULAR ADENOHA
 

HEPATOCELLDLAR CARCINOMA
 

f STOMACH
 

ES.EILL.OMA^.NQS
 

CONTROL
 

10
 

10
 

10
 

(10)
 

(10)
 

(10)
 

(9)
 

(9)
 

(10)
 

LOB DOSE HIGH DOSE
 

50	 50
 

50	 50
 «9	 50
 

(48)	 (50)
 

1 (2X) 1 (2X»
 

(50)	 (50)
 

1 (2*)
 

1 (2X)
 

(i»8) (»9)
 

1 (2X)
 

(48)	 (50)
 

1 (2X)
 

(«8) (50)
 

1 (2X)
 

7	 (15X) 10 (20X)
 

(48)	 (49)
 

1-I2SL—.
 

t	 NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

48
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE
 

URINARY SYSTEM
 

HONE
 

ENDOCRINE SYSTEM
 

NONE
 

SEPRODOCTIVF SYSTEM
 

IOVARY (7) (H6) <»8)
 

CYSTADENOHA, NOS 1 (2X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

HUSCOLOSKELETAL SYSTEM
 

HONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

SITE UNKNOWN
 1_
 . HJflAJGIOSJmeoqA
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

49 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE
 

ANIMAL DISPOSITIOH SUHSARY
 

ANIHALS INITIALLY IN STODY 10 50 50
 

NATURAL DEATH8 1 1
 

HORIBOND SACRIFICE 1 11 4
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 9 38 45
 

ANIHAL HISSING
 

9 INCLUDES AUTOLYZED ANIHALS
 

TOHOR SUHHARY
 

TOTAL AHIHALS WITH PRIMARY TUHORS* 12 13
 

TOTAL PRIHARY TOHOHS 13 14
 

TOTAL ANIHALS WITH BENIGN TUMORS H 1
 

TOTAL BEHIGN TUMORS H 1
 

TOTAL ANIHALS HITH MALIGNANT TUMORS 9 12
 

TOTAL MALIGNANT TUMORS 9 13
 

TOTAL ANIMALS WITH SECONDARSECONDARY TUHORSO
t


TOTAL SECONDARSECONDARY TUMOROH
S
 

TOTAL ANIHALS WITH TUH08S UNCERTAIN UNCERTAIN-­

BENIGN OR MALIGNAN MALIGNANT
 

TOTAL UNCERTAI UNCERTAINN TUMOROH
S
 

TOTAL ANIHALS WITH TUHORM
S UNCEHTAIN UNCEHTAIN-­

PBIHARY OR HBTASTATI HBTASTATIC
 

TOTAL UNCERTAIUNCERTAIN TOHORUM
S


* FBIHART TUH08S: ALL TOHORS EXCEPT SKCOBDART TUHORS
 

* SECONDARY TUHORS: HETASTATIC TUHORS OR TUMORS IITASITB INTO AN ADJACENT ORGAN
 

50 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED CHLORAMBEN IN THE DIET
 

51
 

TABLE C1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED CHLORAMBEN IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

HHIHM.S INITIALLY IN STUDY 10 50 50 

AHIHALS NECROPSIED 9 48 49 

AHIHALS EXAMINED HISTDPATHOLOGICALLY 9 47 47 

INTEGUMENTAR Y SYSTEM 

*SKIN (9) (48) (49) 

INFLAMMATION , NOS 1 (2X) 

GRANULOMA , NOS 1 (11X) 

RESPIRATORY SYSTEM 

tLUNS (9) (47) (48) 

ATELECTASIS 1 (11X) 1 (2%) 

fLUNG/ALVEOLI (9) (47) (48)

 

EMPHYSEMA, NOS 1 (2*)

 

CALCIFICATION, FOCAL 1 (2*)

 

CALCIFICATION, HETASTATIC 2 (4*)

 

HEMATOPOIETIC SYSTEH 

tSPLEEN (7) (47) (47) 

FIBPOSIS, FOCAL 1 (2X) 

HFHATOPOIESIS 1 (2») 

fLYHP H NODE (9) (40) (43) 

DILATATION , NOS 1 (2*) 

CIRCULATORY SYSTEM 

#HEAR T (9) (47) (48)

 

THROMBOSIS, NOS 1 (11%)

 

MEDIAL CALCIFICAPIDN 1 (2X)

 

tMYOCARDIU M (9) (47) (48) 

IfiFLAJSSmQN^CHRONIC. _I.i2XL_ 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED SICROSCOPICALLY
 

* NUHBFR OF ANIHALS NECROPSIED
 

53 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

HIGH DOSE
 

1 (2X)
 

1 (2X)
 

<t (8X)v
 

(U9)
 

1 (2X)
 

1 (2%)
 

(1*9)
 

(U9)
 

(U8)
 

(«6)
 

2 (U%)
 

1 (2%)


U (9%)
 

(»6)
 

{«»)
 

(»<»)


1 (2%)
 

(44)
 

(45)
 

INFLAMMATION, CHRONIC FOCAL
 

FIBROSIS
 

FIBROSIS, FOCAL
 

*AORTA
 

ANEURYSB
 

ARTERIOSCLEROSIS, NOS
 

MEDIAL CALCIFICATION
 

*CORONARY ARTERY
 

MEDIAL CALCIFICATION
 

*SPLENIC ARTERY
 

MEDIAL CALCIFICATION
 

DIGESTIVE SYSTEM
 

tSUBMAXILLARY GLAND
 

FIBROSIS, FOCAL
 

tLIVER
 

FIBPOSIS
 

DEGENERATION, BALLOONING
 

DEGENERATION PARENCHYMATOUS
 

NECROSIS, FOCAL
 

METAMORPHOSIS FATTY
 

ANGIECTASIS
 

fLIVER/CENTRILOBULAR
 

NECROSIS, NOS
 

fSTOMACH
 

CALCIFICATION, NOS
 

CALCIFICATION, RETASTATIC
 

HYPERKFRATOSIS
 

*GASTRIC MUCOSA
 

CALCIFICATION, NOS
 

CALCIFICATION, HETASTATIC
 

IGASTRIC SUBMQCOSA
 

CALCIFICATION, NOS
 

#SMALL INTESTINE
 

NECROSIS...^!


CONTROL
 

(9)
 

1 (11%)
 

(9)
 

(9)
 

(9)
 

(9)
 

1 (11%)
 

1 (11%)
 

2 (22%)
 

1 (11%)
 

(9)
 

1 (11%)
 

(9)
 

(9)
 

1 (11%)
 

(9)
 

(9)
 

LOU DOSE
 

1 (2%) ,
 6 (13%)^
 

(«8)
 

2 (<**)
 

(48)
 

1 (2%)
 

(«8)
 

1 (2%)
 

(47)
 

1 (2%)
 

(46)
 

2 (»X)
 

2 (H%)
 

6 (13X)
 

1 (2%)
 

(U6)
 

(«6)
 

1 (2»)
 

1 (2%)
 

1 (2%)
 

(U6)
 

1 (2%)
 

(46)
 

1 (2%)
 

(45)
 

1.J2SL JLJ2IL
.
 

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

54
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CUNTINUED)

 

CONTROL LOW DOSE 

fCECOH (7) (4U) 

INFLAMMATION , ACUTE 1 (2%) 

URINAR Y SYSTEM 

^KIDNEY 

GLOMERULONEPHRITIS, NOS 

INFLAMMATION , CHRONIC 

FIBROSIS, DIFFDSE 

NEPHROSIS, NOS 

CALCIFICATION, HETASTATIC 

(9) 

2

3

 (22%) 

 (33*) 

(l»7) 

1

6

1

15

1

 (2%) 

 (13J) 

(2%) 

 {32*)^ 

(2%) 

ENDOCRINE SYSTEM 

tPITUITARY (9) (<»»)

CYST, NOS 11 (25»Jl/ 

HYPERPLASIA, NOS 2 (5X)^ 

HYPERPLASIA, FOCAL H (9%)^/ 

ANGIECTASIS 1 (11X) 

tADRENA L (8) (46) 

HEHOPRHASB 1 (2*) 

IADRENAL CORTEX (8) («6) 

DEGENERATION, CYSTIC 1 (2%) 

NECROSIS, FOCAL 

METAMORPHOSIS FATTY 1 (2*) 

HYPERPLASIA, FOCAL 1 (2*) 

ANGIECTASIS 

#THYROID (8) (HI) 

EPIDERMAL INCLUSION CYST 1 (2%) 

CYSTIC FOLLICLES 3 <6«r 

ATROPHY, NOS 5 (11X)V 

HYPERPLASIA, C-CELL 8 (17XH 

HYPERPLASIA, FOLLICtJLAR-CELL 9 (19X)V' 

tPARATHYROID («) (28) 

HYPEHPLASIA, NOS 1 («> 

HYPERPLASIA, SECONDARY 5 (18X)vx 

HYPERPLASIA, DIFFUSE 1 (<•«) 

REPRODUCTIVE SYSTEM 

*HAHHARY GLAND (9) (48) 

ai2IS£liSIAi_HQ5 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* HUHBER OF ANIMALS HECROPSIED
 

HIGH

(43) 

(«8) 

22

3

(ttO) 

5

1

(»8) 

(«8) 

1

1

(i»8) 

1

6

»

(33) 

3

(49) 

 DOSE 

(H6%) 

 (6X) 

(13X) V 

 (3X) 

 (2X) 

 (2X) 

 (2X) 

 (13X) 

 (8X) 

 (9X»­

2_19SL_ 

55
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOB DOSE
 

DYSPLASIA, DOS
 

#PROSTATF (9) (t5)
 

EDEMA, NOS
 

INFLAMMATION, SUPPURATIVE 1 (2X)
 

INFLAMMATION ACUTE AND CHRONIC 1 (2«)
 

#TESTIS (9(9)) C»6 (46)
)


EDEMA, NOS
S

PERIARTERITIS 1 (2* (2*)
)

NECROSISNECROSIS, FIBRINOI FIBRINOIDD 1 (11X (11X)
 )

ATROPHY, NOSS 1 (11X (11X)) 14 (30J
*)


'""
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCOLOSKELETA
 HUSCOLOSKELETAL SYSTE SYSTM
 

*FEHDR (9) (48)
)

OSTEOPOROSIS 3 (6<)(6*)
vv/
 /

BODY CAVITIES
 

*HESENTERY (9(9)) (48))
 

PERIARTERITIS 11 (2X (2X)
)

ALL OTHER SYSTEM
 SYSTEHS


ADIPOSE TISSUE
 

FIBROSIS 1
 1

SPECIAL MORPHOLOG MORPHOLOGY SUMMARY
 

NO LESIOH REPORTE REPORTE DD 3 5

AUTO/NECROPSY/NO HISTO 1

MJTQ1ISISZN APTQLISISZMOQ HECB3PS HECH3PSY !„J __ . 22

t NUHBBR OF ANIMAL S BITH TISSUE EXAMINED HICROSCOPICALLT
 

* BOMBER OF ANIMALS NECBOPSIED
 

HIGH DOSE
 

1 (2*)
 

(46)
 

1 (2X)
 

1 (2*)


(48 (48))
 

1 (2* (2«
)
 )

11 (2X(2X))
 

7(15X (15X)


(49 ()
 

(«9)
 

3 (6X6X)) v
 

«4
 

2


-11
 ­

56
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS FED CHLORAMBEN IN THE DIET 

CONTROL LOB DOSE 

ANIHALS INITIALLY IN STUDY 10 50 

ANIBALS NECROPSIED 10 50 

ANIBALS EXAMINE D HISTOPATHOLOGICALLT 10 49 

INTE60BENTARY SISTEH 

*SDBCnT TISSUE (10) (50) 

GRANULOBA , NOS 1 (2X) 

RESPIRATORY SYSTEM 

NONE 

HEMATOPOIETTC SYSTEB 

#SPLEFN (10) (U9)

 

HEBATOPOIESIS 

tLYMPH NODE (9) (<n>

INFLABBATION, NOS 1 (2X) 

CIRCULATORY SYSTEH 

tHEART (10) (49)

 

HEHORRHAGE 

#BYOCARDIOB (10) (H9)

 

FIBROSIS, ?OCAL 

DIGESTIVE SYSTEB 

tSALIVAR Y GLAN D (10) (U8) 

INFLABBATION , ACOTE NECROTIZING 1 (2*) 

tLIVER (10) (i»8)

 

LHFI,AlIflA.lI2Hi-i£aiI_SUEEaSillVE . 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUHBFR OF ANIMALS NECROPSIED
 

HIGH DOSE 

50 

50 

50 

(50) 

(50) 

2 (UX) 

(»D 

(50) 

1 (2X) 

(50) 

1 (2X) 

(i>9) 

(50) 

__l_i2SL_ 

57
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE 

DEGENERATION PARENCHYHATOUS 3 (30X)

 

HFTAHORPHOSIS FATTY 1 (2X)'

 

ATYPIA, NOS 1 (10X)

 

HYPERPLASIA. DIFFUSE 1 (10X) 

ANGIFCTASIS 2 (20X)

 

*BILE DUCT (10) (50) 

IHFLAHMATION , ACOTE SUPPORATIVF 

INFLAHHATION, CHROHIC FOCAL H (8%r^ 

HYPERPLASIA, FOCAL 

tPANCREAS (9) (US)

 

CALCDLUS, NOS 

DILATATION/DOCTS 

INFLAHHATION, CHRONIC 

#STOHACH (10) (US) 

OLCER, NOS 1 (2X) 

BROSION 1 (2X) 

ORINAR Y SYSTEM 

tKIDNEY (10) (U9) 

HYDRONEPHROSIS 1 (2X) 

GLOMFRULONEPHRITIS, NOS 1 (10X) 

INFLAHHATION , CHRONIC 1 (10X) 5 (10X) 

ENDOCRINF SYSTEM 

tPITOITARY (9) (U5) 

CYST, NOS 2 («) 

CONGESTION, NOS 1 (2X) 

HFHORRHAG E 

HYPERPLASIA, NOS 2 (<*«) 

HYPERPLASIA, FOCAL 1 (2%) 

ANGIECTASIS 1 (11X) 

tADRENA L (10) (49) 

CYST, NOS 2 C»X) 

HEHORRHAGE 2 (t«) 

HEMORRHAGIC CYST 

DEGENERATION , CYSTIC 

ANGIFCTASIS i» (8X) 

#ADRFNAL CORTEX (10) (U9) Dttmnai«jus 

i NDMBER OF ANIMALS BITH TISSUE EXAHIHED MICROSCOPICALLY
 * NDMBFR OF ANIMALS NECROPSIED
 

HIGH DOSB 

(50) 

1 (2X) 

1 (2X) 

(50) 

1 (2X) 

1 (2X) 

1 (2X) 

(U 9) 

(50) 

2 (4X) 

(«6) 

2 (4X) 

1 (2X) 

2 <<»X) / 

i* (9X) ^ 

1 (2X) 

(50) 

3 (6X) V 

3 (6X)" 

5 (lOX) 1' * (8X) ­

(50) 

1-lllL58
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTBOL LOH DOSE 

HEMORRHAG E 1 <2X) 

DEGENERATION, NOS 

DEGENERATION , CYSTIC 

HYPERPLASIA, NOS 1 (2») 

HYPERPLASIA , FOCAL 

ANGIECTASIS U (8*)^ 

#THYROID 

HYPERPLASIA,

HYPERPLASIA ,

 C-CELL 

 FOLLICULAR-CELL 

(8)

2 (25X) 

(«8) 

3

6

 (6X) 

 (13X)­

REPRODUCTIVE SYSTEH 

*HABHAR Y GLAN D (10) (50) 

HYPERPLASIA, NOS a (8X) 

DYSPLASIA, NOS 

FIBROCYSTIC DISEASE 1 (10X) 

f UTERUS (10) (44) 

HEMORRHAGE 

*OVARY (10) («7) 

FOLLICOLAR CYST, NOS 1 (10X) 

NERVOUS SYSTEH 

tBRAIN <10) (48) 

HYDROCEPHALUS. NOS 1 (2X) 

SPECIAL SENSE ORGANS 

NONE 

HOSC0LOSKELETAL SYSTEH 

NONE 

BODY CAVITIES 

*HESENTERY (10) (50) 

PERIARTERITIS 2 («) 

ALL OTHER SYSTEHS 

IfiHE 

* NUMBER OF AHIHALS WITH TISSUE EXAHIHED HICBOSCOPICAILT
 

* BOBBER OF AHIHALS HECROPSIED
 

HIGH DOSE 

1 (2X) 

1 (2X) 

1 (2X) 9 (18X) V 

(50) 

6 (12X) 

3 (6X)i, 

(50) 

5 (10X)X 

3 (6X)v 

(47) 

1 (2X) 

(50) 

(50) 

(50) 

59 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

COHTBOL LOW DOSE HIGH DOSE 

SPECIAL HORPHOIOGY SUMMARY 

1 

NO LESIONN REPORTE REPORTED 5

AUTO/NECROPSY/NO HISTO 1 

* NUMBER OF ANIMALS WITH TISSUE EXAHIHED HICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

5 

60
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED CHLORAMBEN IN THE DIET
 

61
 

TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE MICE FED CHLORAMBEN IN THE DIET 

HIGH DOSE
 

50
 

49
 

48
 

(48)
 

(48)
 

1 (2X)
 

(44)
 

(46)
 

(48)
 

(48)
 

—	 ^
 

ANIMALS INITIALLY IN STUDY
 

ANIBALS NECROPSIED
 

AHIHALS EXAHINED HISTDPATHOLOGICALLY
 

INTEGOBENTARY SYSTEH
 

NONE
 

RESPIRATORY SYSTEH
 

tLUNG
 

CONGESTION, NOS
 

EDEBA, NOS
 

INFLABBATION, CHRONIC
 

HEBATOPOIETIC SYSTEH
 

*SPLEEN
 

ANGIECTASIS
 

#HESENTERIC L. NODE
 

INFLABBATION, GRAN3LOBATODS
 

CIPCULATORY SYSTEH
 

NONE
 

DIGESTIVE SYSTEB
 

tSALIVARY GLAND
 

INFLAHHATION, CHRONIC FOCAL
 

tLIVER
 

TNFARCT, NOS
 

URINARY SYSTEH
 

#KIDNEY
 

__ aiDRQUEPH.ROSI5 .
 

CONTROL LOB DOSE
 

10 50
 

10 49
 

10 49
 

(10)	 (49)
 

1 (10*)
 

1 (10X)
 

1 (2X)
 

(9) (48)
 

(8)	 (46)
 

1 (2X)
 

(10)	 (41)
 

1 (2X)
 

(10)	 (48)
 

1 <2X)
 

(9)	 (48)
 

J_(2Si
 

#	 NUMBER OF ANIMALS WITH TISSUE EXAHIHED MICROSCOPICALLY
 

* NDHBEE OF AHIHALS NECROPSIED
 

63
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOH DOSE HIGH DOSE
 

INFLAMMATION, CHRONIC 1 (2*)
 

INFLAMMATION, CHRONIC FOCAL 1 (2%)
 

ENDOCRINE SYSTEM
 

tADBENAL (10) (48) (08)
 

CTST, NOS 1 (2*)
 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

SOSCOLOSKELETAL SYSTEH
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEHS
 

NONE
 

SPECIAL BORPHOLOGY SOHHARY
 

NO LESION REPORTED 7 26 28
 

AUTO/NECROPSI/NO HISTO 1
 

AOTOLYSIS/NO NECROPSY 1 1
 

* NUMBER OF AHIHALS HITH TISSUE EXAHIHBD HICROSCOPICALLT
 

* NUHBER OF AHIHALS NECROPSIED
 

64
 
TABLE D2. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE MICE FED CHLORAMBEN IN THE DIET 

HIGH DOSE
 

50
 

50
 

50
 

(49)
 

1 (2»)
 

(49)
 

(50)
 

(50)
 

1.I2IL.
 

ANIHALS INITIALLY IN STUDY
 

ANIHALS NECROPSIED
 

ANIMALS EJAHINED HIST3P ATHOLOGICALLY
 

INTEGOBENTARY SYSTEB
 

NONE
 

RESPIRATORY SYSTEM
 

NOME
 

HBHATOPOIETIC SYSTEH
 

#SPLEEN
 

CONGESTION, NOS
 

INFLAHHATIOH, UOS
 

HYPERPLASIA, LYHPHOID
 

tLYHPH NODE
 

INFLABHATION, NOS
 

CIRCULATORY SYSTEH
 

NONE
 

DIGESTIVE SYSTEH
 

•LITER
 

INFLAHHATIOH, NOS
 

INFLAHHATIOH, ACUTE
 

GRANULOBA, 80S
 

HETAHORPHOSIS FATTY
 

HEHATOPOIESIS
 

*BILE DUCT
 

INFLAHJA^JOp. gOS
 

CONTROL LOB DOSE
 

10 50
 

10 50
 

10 49
 

(10)	 (<*8)
 

1 (2%)


2 (4%)
 

1 (2X)
 

(10)	 (U6)
 

1 (2%)
 

(9) (»8)


3 (6X)'
 

2 (IX)
 

1 (HX)
 

1 (2X)
 

1 (2X)
 

(10) (50)
 

f NDHBER OF ANIHALS 8ITH TISSUE EIAHIHED HICROSCOPI^ALLY 

* RUBBER OF ANIHALS BECROPSIED 

65 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTRO L LOW DOSE HIGH DOSE 

*PANCREATIC ACINU S (9) (48 ) (49)

 

ATROPHY, MOS 1 (2%)

 

URINAR Y SYSTEM 

IKIDNEY (10) (48) (50) 

HYDFONEPHROSI S 1 (2X) 

GLONERULONEPHRITIS, NOS 1 (10%) 

INFLAHMATION, FOCAL 1 (2%) 

INFLAMMATION , INTERSTITIA L 2 (4%) 

INFLAMMATION , CHRONIC FOCAL 1 (2X) 

ENDOCRINE SYSTEM 

fADR^NA L CORTEX (10) (47 ) (50) 

HYPERPLASIA , NOS 1 (2%) 

REPRODUCTIVE SYSTEM 

•UTERUS (10) (48) (48)

 

INFLAMMATION , NOS 1 (2*) 

#UTBRUS/ENDOHETRIUN (10) (48 ) (48) 

HYPEPPLASIA, CYSTIC 2 (20%) 1 (2X ) 1 (2X ) 

*OVARY (7) (<»6) (48) 

FOLLICULAR CYST, NOS 1 (14% ) 1 (2%) 2 (4«) 

INFLAMMATION , NOS 13 (28J) 3 (6X) 

INFLAMMATION , SUPPURATIV E 1 (14%) 1 (2%) 

NERVOU S SYSTEM 

*BRAIN (10) (47) (48) 

HYDROCFPHALUS, NOS 1 (2«) 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

—fiSHE 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

66
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOB DOSE HIGH DOSE 

BODY CAVITIES 

*PERITOHEOH 

IHFLAHHATIOH, GFAHOLOBATODS 

(10) (50) 

1 (2X) 

(50) 

ALL OTHER SYSTEHS 

HONE 

SPECIAL MORPHOLOGY SOHHABY 

NO LESION REPORTED 

AOTO/NECROPSY/NO HISTO 

6 18 

1 

# NDHBER 07 ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

30 

67
 
,.. *•"• 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED CHLORAMBEN IN THE DIET
 

69
 

__ __ 

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

Fed Chloramben in the Dieta
 

Topography: Morphology
 

All Sites: Hemangioma
 

P Values0 >d
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Neoplastic Nodule
 

P Values0>d
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

Matched
 

Control
 

0/9 (0.00)
 

N.S.
 

0/9 (0.00)
 

N.S.
 

Pooled
 

Control
 

0/73 (0.00)
 

N.S.
 

P = 0.042
 

1/72 (0.01)
 

N.S.
 

P = 0.035
 

Low
 

Dose
 

5/48 (0.10)
 

P = 0.009**
 

Infinite
 

0.269
 

Infinite
 

Infinite
 

1.903
 

Infinite
 

103
 

4/46 (0.09)
 

N.S.
 

Infinite
 

0.205
 

Infinite
 

6.261
 

0.641
 

301.199
 

112
 —
 

High
 

Dose
 

3/49 (0.06)
 

N.S.
 

Infinite
 

0.125
 

Infinite
 

Infinite
 

0.889
 

Infinite
 

113
 

1/46 (0.02)
 

N.S.
 

Infinite
 

0.011
 

Infinite
 

1.565
 

0.020
 

120.232
 

113
 
__ 

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

Fed Chloramben in the Diet3
 

(continued)
 

Topography: Morphology
 

Pituitary: Chromophobe Adenoma"
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) ^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Carcinoma, NOSb
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/9 (0.11)
 

N.S.
 

110
 

0/9 (0.00)
 

N.S.
 

—
 

Pooled
 

Control
 

7/63 (0.11)
 

N.S.
 

—
 

1/63 (0.02)
 

N.S.
 

—
 

Low
 

Dose
 

5/44 (0.12)
 

N.S.
 

1.023
 

0.145
 

46.226
 

1.023
 

0.271
 

3.472
 

96
 

0/44 (0.00)
 

N.S.
 

—
 

—
 

0.000
 

0.000
 

26.739
 

—
 

High
 

Dose
 

8/40 (0.20)
 

N.S.
 

1.800
 

0.310
 

77.552
 

1.800
 

0.616
 

5.325
 

101
 

1/40 (0.03)
 

N.S.
 

Infinite
 

0.013
 

Infinite
 

1.575
 

0.020
 

120.584
 

113
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Chloramben in the Diet3
 

(continued) 

Matched Pooled Low 

Topography: Morphology Control Control Dose 

Pituitary: Chromophobe Adenoma
 

or Carcinoma, NOS^ 1/9 (0.11) 8/63 (0.13) 5/44 (0.12)
 

P Values0>d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 1.023
 

Lower Limit 0.145
 

Upper Limit 47.226
 

Relative Risk (Pooled Control)f 0.895
 

Lower Limit 0.244
 

Upper Limit 2.867
 

Weeks to First Observed Tumor 110 96
 —
 

Thyroid: C-cell Adenoma" 0/8 (0.00) 2/63 (0.03) 4/47 (0.09)
 

P Values0>d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f Infinite
 

Lower Limit 0.182
 

Upper Limit Infinite
 

Relative Risk (Pooled Control)f 2.681
 

Lower Limit 0.401
 

Upper Limit 28.550
 

Weeks to First Observed Tumor 112
 —
 —
 

High
 

Dose
 

9/40 (0.23)
 

N.S.
 

2.025
 

0.362
 

86.067
 

1.772
 

0.658
 

4.785
 

113
 

3/48 (0.06)
 

N.S.
 

Infinite
 

0.115
 

Infinite
 

1.969
 

0.235
 

22.746
 

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

Fed Chloramben in the Diet3
 

(continued)
 

Matched Pooled Low High 

Topography: Morphology Control Control Dose Dose 

Adrenal: Cortical Adenoma 0/8 (0.00) 2/70 (0.03) 1/46 (0.02) 0/48 (0.00) 

P Values0 » d N..S. N.S. N.S. N.S. 

__ 

Relative Risk (Matched Control )f Infinite 

Lower Limit 0.010 — Upper Limit Infinite — 

Relative Risk (Pooled Control)f 0.761 0.000 

Lower Limit 0.013 0.000 

Upper Limit 14.128 4.926 

Weeks to First Observed Tumor 112 — — — 

a

Treated groups received doses of 10,000 or 20,000 ppm.
 

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

c

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

the comparison of that treated group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated 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 treated group and the specified
 

control group.
 
__ __ 

__ 

__ 

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

Fed Chloramben in the Diet3
 

Topography: Morphology
 

All Sites: Hemangiomab
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Neoplastic Nodule"
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

0/10 (0.00)
 

N.S.
 

0/10 (0.00)
 

N.S.
 

—
 

Pooled
 

Control
 

0/74 (0.00)
 

N.S.
 

1/73 (0.01)
 

N.S.
 

—
 

Low
 

Dose
 

0/50 (0.00)
 

N.S.
 

—
 

—
 

—
 

—
 

—
 

2/48 (0.04)
 

N.S.
 

Infinite
 

0.068
 

Infinite
 

3.042
 

0.163
 

175.641
 

113
 

High
 

Dose
 

1/50 (0.02)
 

N.S.
 

Infinite
 

0.012
 

Infinite
 

Infinite
 

0.078
 

Infinite
 

113
 

1/50 (0.02)
 

N.S.
 

Infinite
 

0.012
 

Infinite
 

1.460
 

0.019
 

112.322
 

113
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Chloramben in the Diet3
 

(continued)
 

Topography: Morphology
 

Pituitary: Chromophobe Adenoma"
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Carcinoma, NOSb
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/9 (0.11)
 

N.S.
 

91
 

0/9 (0.00)
 

N.S.
 

—
 

Pooled
 

Control
 

12/65 (0.18)
 

N.S.
 

—
 

1/65 (0.02)
 

N.S.
 

—
 

Low
 

Dose
 

11/45 (0.24)
 

N.S.
 

2.200
 

0.414
 

92.100
 

1.324
 

0.578
 

2.950
 

75
 

3/45 (0.07)
 

N.S.
 

Infinite
 

0.135
 

Infinite
 

4.333
 

0.359
 

222.252
 

106
 

High
 

Dose
 

6/46 (0.14)
 

N.S.
 

1.174
 

0.181
 

52.803
 

0.707
 

0.234
 

1.867
 

94
 

1/46 (0.02)
 

N.S.
 

Infinite
 

0.011
 

Infinite
 

1.413
 

0.018
 

108.514
 

113
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Chloramben in the Diet3
 

(continued)
 

Topography: Morphology
 

Pituitary: Chromophobe Adenoma
 

or Carcinoma, NOSb
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-cell Adenoma'5
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/9 (0.11)
 

N.S.
 

94
 

0/8 (0.00)
 

N.S.
 

——
 

Pooled
 

Control
 

13/65 (0.20)
 

N.S. (N)
 

—
 

1/61 (0.02)
 

N.S.
 

—
 

Low
 

Dose
 

14/45 (0.31)
 

N.S.
 

2.800
 

0.550
 

114.824
 

1.556
 

0.748
 

3.206
 

75
 

4/48 (0.09)
 

N.S.
 

Infinite
 

0.179
 

Infinite
 

5.083
 

0.527
 

244.874
 

91
 

High
 

Dose
 

7/46 (0.16)
 

N.S.
 

1.370
 

0.224
 

60.268
 

0.761
 

0.276
 

1.873
 

94
 

4/50 (0.08)
 

N.S.
 

Infinite
 

0.170
 

Infinite
 

4.880
 

0.503
 

235.283
 

111
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Chloramben in the Diet3
 

(continued)
 

Topography: Morphology
 

Adrenal: Cortical Adenoma"
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control )f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Pooled Low High 

Control Control Dose Dose 

0/10 (0.00) 1/70 (0.01) 3/49 (0.06) 4/50 (0.08) 

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

Infinite Infinite 

0.137 0.206 

Infinite Infinite 

4.286 5.600 

0.358 0.575 

220.214 269.964 __ __ 

70 70 

2/10 (0.20) 11/74 (0.15) 7/50 (0.14) 7/50 (0.14) 

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

0.700 0.700 

0.173 0.173 

6.482 6.482 

0.942 0.942 

0.330 0.330 

2.455 2.455 

45 49 87 — 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Chloramben in the Dieta
 

(continued)
 

Matched Pooled Low High
 

Topography: Morphology Control Control Dose Dose
 

Uterus: Endometrial Stromal
 

Polyp6 0/10 (0.00) 6/69 (0.09) 3/44 (0.07) 2/47 (0.04)
 

P Values0 »d N.S. N.S. N.S. N.S.
 

Relative Risk (Matched Control)f Infinite Infinite
 

Lower Limit 0.152 0.069
 

Upper Limit Infinite Infinite
 

Relative Risk (Pooled Control)f 0.784 0.489
 

Lower Limit 0.132 0.049
 

Upper Limit 3.449 2.589
 

Weeks to First Observed Tumor 108 77
 —
 —


a

Treated groups received doses of 10,000 or 20,000 ppm.
 

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

c

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

the comparison of that treated group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated 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 treated group and the specified
 

control group.
 

APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE FED CHLORAMBEN IN THE DIET
 

81
 

__ __ 

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

Fed Chloramben in the Diet3
 

Matched Pooled Low
 

Topography: Morphology Control Control Dose
 

Lung: Alveolar /Bronchiolar
 

Adenoma or Carcinoma 0/10 (0.00) 4/66 (0.06) 0/49 (0.00)
 

P Valuesc»d	 P = 0.043 N.S. N.S.
 

Relative Risk (Matched Control)f
 

Lower Limit
 —
 

Upper Limit
 —
 

—
 

Relative Risk (Pooled Control)f 0.000
 

Lower Limit 0.000
 

Upper Limit 1.455
 

Weeks to First Observed Tumor	 87
 

Liver: Hepatocellular Carcinoma"5 2/10 (0.20) 9/69 (0.13) 16/48 (0.33)
 

P Values0 »d	 N.S. P = 0.029 P = 0.008**
 

Relative Risk (Matched Control)f 1.667
 

Lower Limit 0.515
 

Upper Limit 13.777
 

Relative Risk (Pooled Control)f 2.556
 

Lower Limit 1.164
 

Upper Limit 5.926
 

Weeks to First Observed Tumor 91	 87
 —
 

High
 

Dose
 

4/48 (0.08)
 

N.S.
 

Infinite
 

0.215
 

Infinite
 

1.375
 

0.266
 

7.004
 

92
 

14/48	 (0.29)
 

P = 0.028**
 

1.458
 

0.439
 

12.223
 

2.236
 

0.984
 

5.321
 

64
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed Chloramben in the Dieta
 

(continued)
 

a

Treated groups received doses of 10,000 or 20,000 ppm.
 

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

c

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

the comparison of that treated group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated 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 treated group and the specified
 

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

Fed Chloramben in the Diet3
 

Topography: Morphology 

Matched 

Control 

Pooled 

Control 

Low 

Dose 

High 

Dose 

Lung: Alveolar/Bronchiolar 

Adenoma or Carcinoma 0/10 (0.00) 3/69 (0.04) 1/48 (0.02) 1/50 (0.02) 

P Values0>d N.S. N.S. N.S. N.S. 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Infinite 

0.012 

Infinite 

Infinite 

0.012 

Infinite 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

0.479 

0.009 

5.734 

0.460 

0.009 

5.510 

Weeks to First Observed Tumor 

__ __ 

91 92 

Liver: Hepatocellular Carcinoma 0/9 (0.00) 2/67 (0.03) 7/48 (0.15) 10/50 (0.20) 

P Values0 »d N.S. P = 0.004 P = 0.027** P = 0.003** 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Infinite 

0.413 

Infinite 

Infinite 

0.607 

Infinite 

Relative Risk Pooled Control)f 

Lower Limit 

Upper Limit 

4.885 

0.982 

46.360 

6.700 

1.515 

60.419 

Weeks to First Observed Tumor 

__ _.,. 91 78 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Chloramben in the Diet3
 

(continued)
 

a

Treated groups received doses of 10,000 or 20,000 ppra,
 

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

G

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

the comparison of that treated group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated 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 treated group and the specified
 

control group.
 
APPENDIX G
 

ANALYSIS OF FORMULATED DIETS FOR CONCENTRATIONS
 

OF CHLORAMBEN
 

87
 

APPENDIX G
 

Analysis of Formulated Diets for
 

Concentrations of Chloramben
 

A 10-g sample of the formulated diet was shaken with 125 ml of
 

methanol at room temperature for 16 hours, then filtered through
 

Celite with methanol washes, and reduced in volume to a
 

theoretical chloramben concentration of about 400 ng/ml.
 

The chloramben then was converted to its methyl ester for gas-


liquid chromatographic (glc) analysis by a modification of the
 

procedure of Leigh and Lisk (1970). To a 1-ml aliquot of the
 

above extract in a 10-ml volumetric flask was added 3 ml of 14%
 

BF3 : CH3OH. After 2 hours at 75°C, the flask was cooled and
 

2 ml of hexane was added. An aqueous solution of Na2SO^ (2%, w/v)
 

was added to bring the total volume to 10 ml, and the sample was
 

shaken vigorously for 1 minute and then allowed to separate. The
 

(upper) hexane layer was quantitatively analyzed for chloramben
 

by glc (electron capture detector, 10% DC-200 on Gas Chrom Q
 

column), Recoveries were checked with chloramben-spiked samples
 

carried through the workup and analysis, and external standards
 

were used for calibration.
 

89
 
Theoretical 

Concentration 

in Diet (ppm) 

No. of 

Samples 

Sample 

Analytical 

Mean (ppm) 

Coefficient of 

Variation (%) 

Range (ppm) 

10,000 41 10,000 4.9% 9,100-11,330 

20,000 38 20,105 4.9% 18,200-22,000 

<rU.S.GOVERNMENTPRINTINGOFFICE: 1977- 241-161:3125 

90 
DHEW Publication No. (NIH) 77-825