National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 62 

1978 

BIOASSAY OF 

ENDOSULFAN 

FOR POSSIBLE CARCINOGENICITY
 

CAS No. 115-29-7 

NCI-CG-TR-62 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

ENDOSULFAN
 

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) 78-1312
 

REPORT ON THE BIOASSAY OF ENDOSULFAN
 

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 endosulfan 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
 

the test chemical is not a carcinogen because the experiments are
 

conducted under a limited set of circumstances· Positive results
 

demonstrate that the test chemical is carcinogenic for animals under
 

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

The actual determination of the risk to man from animal carcinogens
 

requires a wider analysis.
 

CONTRIBUTORS: This bioassay of endosulfan was conducted by Hazleton
 

Laboratories America, Inc., Vienna, Virginia, initially under direct
 

contract to the NCI and currently under a subcontract to Tracor Jitco,
 

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

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

principal investigators for the contract were Dr. Μ. Β. Powers (3),
 

Dr. R. W. Voelker (3), Dr. W. A. Olson (3,4) and Dr. W. M. Weatherholtz (3). Chemical analysis was performed by Dr. C. L. Guyton (3,
 

5) and the analytical results were reviewed by Dr. N. Zimmerman (6);
 

the technical supervisor of animal treatment and observation was Ms.
 

K. J. Petrovics (3).
 

Histopathologic examinations were performed by Dr. R. H. Habermann (3) and reviewed by Dr. R. W. Voelker (3) at the Hazleton Laboratories America, Inc., and the diagnoses included in this report
 

represent the interpretation of these pathologists. Histopathology
 

findings and reports were reviewed by Dr. R. L. Schueler (7).
 

Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (8)'; the
 

statistical analysis was performed by Mr. W. W. Belew (6) and Dr. J.
 

R. Joiner (7), using methods selected for the Bioassay Program by Dr.
 

J. J. Gart (9).
 

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

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

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

Ms. P. Walker (6), and the technical editor, Ms. P. A. Miller (6).
 

The final report was reviewed by members of the participating organizations.
 

The statistical analysis was reviewed by members of the Mathematical Statistics and Applied Mathematics Section of the NCI: Dr.
 

J. J. Gart (9), Mr. J. Nam (9), Dr. H. M. Pettigrew (9), and Dr. R.
 

E. Tarone (9).
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

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

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

Squire (1,10), and Dr. J. Μ. Ward (1).
 

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 Now with the Naylor Dana Institute for Disease Prevention, American Health Foundation, Hammon House Road, Valhalla, New York.
 

3.	 Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

4.	 Now with the Center for Regulatory Services, 2347 Paddock Lane,
 

Reston, Virginia.
 

5.	 Now with Rhodia, Inc., 23 Belmont Drive, Somerset, New Jersey.
 

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

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

9. Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

10· Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland·
 

SUMMARY
 

A bioassay of technical-grade endosulfan for possible carcinogenicity was conducted using Osborne-Mendel rats and B6C3F1 mice.
 

Endosulfan was administered in the feed, at either of two concentrations, to groups of 50 male and 50 female animals of each species.
 

The time-weighted average high and low dietary concentrations of
 

endosulfan were, respectively, 952 and 408 ppm for the male rats, and
 

445 and 223 ppm for the female rats. In mice the high and low time-


weighted average concentrations were, respectively, 6.9 and 3.5 ppm
 

for the males and 3.9 and 2.0 ppm for the females. Twenty animals of
 

each sex and species were placed on test as controls. The bioassay
 

of high dose male rats was terminated during week 82, and the bioassay
 

of low dose male rats was terminated during week 74. After a 78-week
 

period of chemical administration, observation of female rats continued for 33 additional weeks and observation of mice continued for 14
 

additional weeks.
 

At the doses administered to rats in this study endosulfan was
 

toxic, inducing a high incidence of toxic nephropathy in both sexes
 

and testicular atrophy in males.
 

In both species high early mortality was observed in the male
 

groups and no conclusions concerning the carcinogenicity of endosulfan can be drawn from this part of the bioassay· However, survival
 

among females of both species was sufficient for meaningful statistical evaluation of the incidence of late-developing tumors. It is
 

concluded that under the conditions of this bioassay, technical-grade
 

endosulfan was not carcinogenic in female Osborne-Mendel rats or in
 

female B6C3F1 mice.
 

v i i 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 3
 

A. Chemicals 3
 

B. Dietary Preparation 3
 

C· Animals 4
 

D· Animal Maintenance 4
 

E. Selection of Initial Concentrations 5
 

F. Experimental Design 7
 

G· Clinical and Histopathologic Examinations 11
 

H· Data Recording and Statistical Analyses 13
 

III. CHRONIC TESTING RESULTS: RATS 18
 

A· Body Weights and Clinical Observations 18
 

B. Survival 20
 

C. Pathology 22
 

D. Statistical Analyses of Results 23
 

IV. CHRONIC TESTING RESULTS: MICE 38
 

A. Body Weights and Clinical Observations 38
 

B. Survival 38
 

C. Pathology 41
 

D. Statistical Analyses of Results 42
 

V. DISCUSSION 48
 

VI. BIBLIOGRAPHY 50
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH ENDOSULFAN A-l
 

APPENDIX Β SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH ENDOSULFAN B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH ENDOSULFAN C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH ENDOSULFAN D-l
 

ix
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 GROWTH CURVES FOR ENDOSULFAN CHRONIC STUDY 

RATS 19 

2 SURVIVAL· COMPARISONS OF ENDOSULFAN CHRONIC 

STUDY RATS 21 

3 GROWTH CURVES FOR ENDOSULFAN CHRONIC STUDY 

MICE 39 

4 SURVIVAL COMPARISONS OF ENDOSULFAN CHRONIC 

STUDY MICE 40 

LIST OF TABLES 

Table Number , Page 

1 DESIGN SUMMARY FOR OSBORNE-MENDEL RATS— 

ENDOSULFAN FEEDING EXPERIMENT 8 

2 DESIGN SUMMARY FOR B6C3F1 ΜICE—ENDOSULFAN 

FEEDING EXPERIMENT 9 

3 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

ENDOSULFAN 24 

4 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH ENDOSULFAN 27 

5 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

ENDOSULFAN WHICH SURVIVED AT LEAST 52 WEEKS 32 

6 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH ENDOSULFAN WHICH SURVIVED AT LEAST 52 

WEEKS 34 

7 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

ENDOSULFAN 43 

χ 
LIST OF TABLES (Concluded) 

Table Number Page 

8 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH ENDOSULFAN ^ 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH ENDOSULFAN A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH ENDOSULFAN A-6 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH ENDOSULFAN B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH ENDOSULFAN B-6 

CI SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH ENDOSULFAN C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH ENDOSULFAN C-7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH ENDOSULFAN D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH ENDO­ SULFAN D-6 

X I 

I. INTRODUCTION
 

In the late 1960s scientists at the National Cancer Institute
 

noted that a group of pesticides used extensively in agriculture had
 

not been adequately tested for carcinogenicity. In 1969 the Report
 

of the Secretary's Commission on Pesticides and their Relationship
 

to Environmental Health further emphasized the need for chronic
 

toxicity studies of certain specific pesticides (U.S· Department of
 

Health, Education and Welfare, 1969). As a result, a chronic biossay
 

of endosulfan (NCI No. CO0566), one of the chemicals included in
 

this group, was conducted at Hazleton Laboratories America, Inc.
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this pesticide is 6,7,8,9,10,10-hexachloro-l,5,5a,6,
 

9,9a-hexahydro~6,9-methano-2,4,3-benzodioxathiepin-3-oxide. Endosulfan, a mixture of two geometric isomers, is a synthetic chlorinated
 

cyclodiene and was introduced in 1956 as an experimental broad
 

spectrum insecticide (Spencer, 1968). In 1971 farmers in the United
 

States used roughly one million pounds of endosulfan (Andrelinas,
 

1974). About 20 percent of this quantity was used on Irish potatoes,
 

with substantial application on other vegetables, tobacco, apples and
 

other fruits, and nuts. By 1973, when endosulfan was registered for
 

use on 59 agricultural crops, California alone used over 0.8 million
 

pounds (Stanford Research Institute, 1974). Endosulfan residues in
 

*The CAS registry number is 115-29-7,
 

1 
1973 averaged 0.001 ppm in potatoes, 0.019 ppm in leafy vegetables,
 

and traces were found in a variety of fruits. Overall, endosulfan
 

residues ranged from trace amounts to 0.439 ppm in over 8 percent of
 

all food composites sampled during that year (Johnson and Manske,
 

1976).
 

2 
II. MATERIALS AND METHODS
 

A· Chemicals
 

One batch of technical-grade endosulfan (Thiodan ) was purchased
 

by Hazleton Laboratories America, Inc., Vienna, Virginia, from Niagara
 

Chemical Division (now Agricultural Chemical Division of FMC Corporation)· The manufacturer's analysis indicated a purity of 98·80
 

percent.
 

It should be noted that the presence of two geometric isomers
 

(Thiodan I and Thiodan II) of the compound would tend to make it difficult to interpret results obtained by all but the most sophisticated
 

analytical procedures. Gas-liquid chromatography (GLC) indicated two
 

major peaks (presumed to represent the two isomers) comprising over 95
 

percent of the total area. Five minor peaks detected indicated the
 

presence of at least five impurities. Similar results were obtained
 

upon GLC analysis after one year·
 

Throughout this report the term endosulfan is used to represent
 

this technical-grade material·
 

B· Dietary Preparation
 

The basal laboratory diet for both control and dosed animals
 

consisted of 2 percent Duke's corn oil (S. F. Sauer Company) by
 

weight added to Wayne Lab-Blox meal (Allied Mills, Inc.). Fresh
 

mixtures of endosulfan in corn oil were prepared each week and stored
 

in the dark. The mixtures of endosulfan in corn oil were incorporated
 

into the appropriate amount of the basal laboratory diet in a twin-


shell blender fitted with an accelerator bar.
 

3
 
C. Animals
 

Two animal species, rats and mice, were used in the carcinogenicity bioassay. The Osborne-Mendel rat was selected on the basis of a
 

comparative study of the tumorigenic responsiveness to carbon tetrachloride of five different strains of rats (Reuber and Glover, 1970).
 

The B6C3F1 mouse was selected because it has been used by the NCI for
 

carcinogenesis bioassays and has proved satisfactory in this capacity.
 

Rats and mice of both sexes were obtained through contracts with
 

the Division of Cancer Treatment, National Cancer Institute. The
 

Osborne-Mendel rats were procured from the Battelle Memorial Institute,
 

Columbus, Ohio, and the B6C3F1 mice were obtained from the Charles
 

River Breeding Laboratories, Inc., Wilmington, Massachusetts. Upon
 

receipt, animals were quarantined for at least 10 days, observed for
 

visible signs of disease or parasites, and assigned to the various
 

dosed and control groups.
 

D. Animal Maintenance
 

All animals were housed by species in temperature- and humidity-


controlled rooms. The temperature range was 20° to 24°C and the
 

relative humidity was maintained between 45 and 55 percent. The air
 

conditioning system in the laboratory provided filtered air at a rate
 

of 12 complete changes of room air per hour. Fluorescent lighting
 

was provided on a 12-hour-daily cycle. The rats were individually
 

housed in suspended galvanized-steel wire-mesh cages with perforated
 

floors. Mice were housed by sex in groups of ten in solid-bottom
 

4
 
polypropylene cages equipped with filter tops. Sanitized cages with
 

fresh bedding (Sanichips®, Shurfire) were provided once each week for
 

mice. Rats received sanitized cages with no bedding with the same
 

frequency· Food hoppers were changed and heat-sterilized once a week
 

for the first 10 weeks and once a month thereafter. Fresh heat-


sterilized glass water bottles were provided three times a week.
 

Food and water were available ad libitum.
 

The endosulfan-treated and control rats were housed in the same
 

room with other rats receiving diets treated with trifluralin (1582­

09-8); dioxathion (78-34-2); dicofol (115-32-2); nitrofen (1836-75-5);
 

and mexacarbate (315-18-4). All mice used in the endosulfan study
 

were housed in the same room with other mice receiving diets treated
 

with trifluralin (1582-09-8); dioxathion (78-34-2); sulfallate (95­

06-7); p,pf-DDT (50-29-3); methoxychlor (72-43-5); ρ,ρ'-DDE (72-55-9);
 

ρ,ρ'-TDE (72-54-8); dicofol (115-32-2); pentachloronitrobenzene (82­

68-8); clonitralid (1420-04-8); nitrofen (1836-75-5); chlorobenzilate
 

(510-15-6); mexacarbate (315-18-4); amitrole (61-82-5); acetylaminofluorene (53-96-3); and safrole (94-59-7).
 

E.	 Selection of Initial Concentrations
 

In order to establish the maximum tolerated doses of endosulfan
 

for administration to treated animals in the chronic studies, sub-


chronic toxicity tests were conducted with both rats and mice.
 

Animals of each species were distributed among six groups, each
 

*
 

CAS registry numbers are given in parentheses·
 
consisting of five males and five females· Endosulfan was premixed
 

with a small amount of corn oil. The mixture was then incorporated
 

into the basal laboratory diet and fed ad libitum to five of the six
 

rat groups at concentrations of 178, 316, 562, 1000, and 1780 ppm and
 

five of the six mouse groups at concentrations of 3.2, 5.6, 10, 18,
 

and 32 ppm. The sixth group of each species served as a control
 

group, receiving only the basal laboratory diet. The dosed dietary
 

preparations were administered for a period of 6 weeks, followed by a
 

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

diet of corn oil and laboratory chow.
 

A dosage inducing no mortality and resulting in a depression in
 

mean group body weight of approximately 20 percent relative to controls was selected as the initial high dose. When weight gain criteria were not applicable, mortality data alone were utilized.
 

In the male rats a dose-related depression in mean group body
 

weight was observed at concentrations of 562 ppm and above. At 562
 

and 1000 ppm, the mean group body weight depression was 9 and 20
 

percent, respectively. Three male rats died at 1780 ppm. In the
 

female rats no dose-related depression in mean group body weight was
 

observed, but one animal died at 316 ppm and four animals died at 562
 

ppm. The initial high concentrations selected for the chronic study
 

were 562 ppm for the male rats and 178 ppm for the female rats.
 

In mice, depression in mean group body weight, although not
 

clearly dose-related, was observed at concentrations of 5.6 ppm and
 

6
 
above. One male died at 10 ppm and one female died at 5.6 ppm· Mortality increased with concentration in both sexes. The initial high
 

concentrations selected for the chronic study were 6 ppm for the male
 

and 3 ppm for the female mice.
 

F.	 Experimental Design
 

The experimental design parameters for the chronic study (species, sex, group size, concentrations administered, duration of
 

treated and untreated observation periods, and the time-weighted
 

average concentrations) are summarized in Tables 1 and 2.
 

At initiation of the study the treated and control rats were all
 

approximately 7 weeks old. The concentrations of endosulfan initially
 

utilized for males were 562 and 281 ppm. Throughout this report the
 

male rats initially receiving the former concentration are referred to
 

as the high dose group while the males initially receiving the latter
 

concentration are referred to as the low dose group. In week 4, high
 

and low concentrations were increased to 700 and 350 ppm, respectively;
 

in week 11 the concentrations were increased to 900 and 450 ppm, and
 

in week 21 the concentrations were raised to 1200 and 600 ppm. These
 

increases in concentrations were made because of the animals1

 apparent
 

tolerance of the chemical, but at the concentration of 1200 ppm, L3/50
 

male rats died. In week 44, administration of the compound was discontinued for 1 week, followed by 4 weeks of dosing. Cyclic administration at concentrations of 1200 and 600 ppm continued for a second
 

5-week cycle. In week 54 the high and low concentrations were reduced
 

7
 
TABLE 1
 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS
 

ENDOSULFAN FEEDING EXPERIMENT
 

MALE 

CONTROL 

LOW DOSE 

INITIAL 

GROUP 

SIZE 

io 

50 

HIGH DOSE6 50 

FEMALE 

CONTROL 

LOW DOSE 

ϊό 

50 

HIGH DOSE 50 

ENDOSULFAN
 

CONCENTRATION3
 

0
 

281
 

350
 

450
 

600
 

600°
 

450°
 

0
 

450°
 

0
 

562
 

700
 

900
 

1200
 

1200C
 

900°
 

0
 

0
 

89
 

150
 

225
 

300
 

300C
 

225c
 

0
 

178
 

300
 

450
 

600
 

600c
 

450C
 

0
 

Concentrations in party pet million·
 

Time-weighted average concentration =


TIME-WEIGHTED 

AVERAGE 

OBSERVATION PERIOD CONCENTRATION 

TREATED UNTREATED OVER A 78-WEEK 

(WEEKS) (WEEKS) PERIODb 

110 0
 

3 408
 

7
 

10
 

23
 

8 2
 

8 2
 

10
 

4 1
 

3
 

3 952
 

7
 

10
 

23
 

8 2
 

8 2
 

10
 

110 0
 

3 223
 

7
 

10
 

23
 

8 2
 

20 5
 

33
 

3 445
 

7
 

10
 

23
 

8 2
 

20 5
 

33
 

Σ(concentrationn X weeks received received)


— — —78 weeks ­

78 weeks
 

 (concentratio
 

These concentrations were cyclically administered with a pattern of 1
 

dosage-free week followed by 4 weeks of dosing at the level indicated.
 

These animals were sacrificed after 81 weeks.
 

These animals were sacrificed after 73 weeks·
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

ENDOSULFAN FEEDING EXPERIMENT
 

INITIAL OBSERVATION PERIOD TIME-WEIGHTED 

GROUP ENDOSULFAN TREATED UNTREATED AVERAGE 

SIZE CONCENTRATION8 (WEEKS) (WEEKS) CONCENTRATION 

MALE
 

CONTROL 20 0 91 0
 

LOW DOSE 50 3.0 5 3.5
 

3.5 73
 

0 14
 

HIGH DOSE 50 6.0 5 6.9
 

7.0 73
 

0 14
 

FEMALE
 

CONTROL 20 0 91 0
 

LOW DOSE 50 1.5 5 2
 

2.0 73
 

0 14
 

HIGH DOSE 50 3.0 5 3.9
 

4.0 73
 

0 14
 

aConcentrations in parts per million·
 

b Σ(concentration X weeks received)
 Time-weighted average concentration =
 (weeks receiving chemical)
 
to 900 and 450 ppm, respectively, and the same method of cyclic
 

administration was continued· Ten weeks later, dosing of both high
 

and low dose male rats was discontinued and they received the basal
 

diet only. During week 74 of the experiment the surviving high dose
 

male rats were sacrificed. At this time the low dose males were
 

again placed on the cyclic feeding schedule at a concentration of 450
 

ppm. In week 82 the surviving males in this group were sacrificed.
 

For female rats, the concentrations of endosulfan initially administered were 178 and 89 ppm. Throughout this report those female
 

rats initially receiving the former concentration are referred to as
 

the high dose group and those initially receiving the latter concentration are referred to as the low dose group. In week 4 the high
 

and low concentrations were increased to 300 and 150 ppm, respectively. In week 11 the concentrations were again increased, to 450
 

and 225 ppm, and in week 21 the concentrations were raised to 600
 

and 300 ppm. In week 44, 23 weeks after the last increase, administration of the compound was discontinued for 1 week, followed by 4
 

weeks of dosing. This cyclic administration, designed to decrease
 

total compound intake due to apparent toxic effects of the chemical,
 

continued for the remainder of the dosing period, but in week 54, the
 

high and low concentrations were reduced to 450 and 225 ppm, respectively. Chemical administration to the female rats continued at
 

these concentrations, on a cyclic basis, until week 78. They were
 

10
 
then observed for an additional 32 weeks before sacrifice at termination of the bioassay.
 

At initiation of the study the mice were all approximately 6
 

weeks old· The concentrations initially administered to the male
 

mice were 6 and 3 ppm. Throughout this report those male mice initially receiving the former concentration are referred to as the high
 

dose group and those initially receiving the latter concentration are
 

referred to as the low dose group. In week 6 of the experiment, the
 

high and low concentrations were increased to 7 and 3.5 ppm, respectively. These concentrations were maintained for the remainder of
 

the dosing period. Female mice received initial concentrations of 3
 

and 1.5 ppm. Throughout this report those female mice initially receiving the former concentration are referred to as the high dose
 

group and those initially receiving the latter concentration are referred to as the low dose group. High and low concentrations were
 

increased during week 6 to 4 and 2 ppm, respectively, and these
 

levels were maintained until the end of the dosing period. After the
 

78-week dosing period, all groups were observed for 12 additional
 

weeks.
 

G» Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. From the first day, all animals were inspected daily
 

for mortality. Body weights, food consumption, and data concerning
 

appearance, behavior, signs of toxic effects, and incidence, size,
 

11
 
and location of tissue masses were recorded at weekly intervals for
 

the first 10 weeks and at monthly intervals thereafter· The presence
 

of tissue masses was determined by observation and palpation of each
 

animal.
 

A necropsy was performed on each animal regardless of whether it
 

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

bioassay. The animals were euthanized by exsanguination under sodium
 

pentobarbital anesthesia, and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examination
 

of major tissues, organs, or gross lesions taken from sacrificed animals and, whenever possible, from animals found dead.
 

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

lymph nodes, thymus, heart, salivary gland, liver, gallbladder and
 

bile duct (mice), pancreas, esophagus, stomach, small intestine, large
 

intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, brain, uterus, mammary gland, and ovary.
 

Tissues for which slides were prepared were preserved in 10 percent buffered formalin, embedded in paraffin, sectioned, and stained
 

with hematoxylin and eosin prior to microscopic examination. An
 

occasional section was subjected to special staining techniques for
 

more definitive diagnosis.
 

A few tissues were not examined for some animals, particularly for
 

those that died early. Also, some animals were missing, cannibalized,
 

12
 
or judged to be in such an advanced state of autolysis as to preclude
 

histopathologic interpretation. Thus, the number of animals for
 

which particular organs, tissues, or lessions were examined microscopically varies and does not necessarily represent the number of
 

animals that were placed on experiment 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 statistical techniques described in this section. Those analyses of the experimental results
 

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

13
 
on survival used the method of Cox (1972) for testing two groups for
 

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

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0·05 may be made· The Bonferroni inequality
 

14
 
(Miller, 1966, pp. 6-10) requires that the P-value for any comparison
 

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

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

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

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

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

Under the assumption of a linear trend, this test determined if the
 

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


tailed 0.05 level of significance. Unless otherwise noted, the direction of the significant trend was a positive dose relationship.
 

This method also provides a two-tailed test of departure from linear
 

trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

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

Cochran-Armitage tests, etc») were followed.
 

15
 
When appropriate, life-table methods were used to analyze the
 

incidence of tumors. Curves of the proportions surviving without an
 

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

during which animals died naturally or were sacrificed was entered as
 

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

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

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

is defined as ρ / where ρ is the true binomial probability of the
 

t c t
 

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

16
 
analyses. The interpretation of the limits is that in approximately
 

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

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

would be within the interval calculated trap* the experiment. When
 

the lower limit of the confidence interval 13 greater than one, it
 

can be inferred that a statistically significant result; (fl Ρ < 0.025
 

one-tailed test when the control incidence i^ tip£ zero, Ρ < 0.050
 

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

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

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

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

detected under the conditions of this tesf.
 

17
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

No appreciable differences in mean body weight were observed
 

among female rats (Figure 1). From week 35 to week 100, the high
 

dose females did have a mean group body weight lower than the low
 

dose and control groups, but at the end of the study all three female
 

groups were similar in weight. A distinct dose-related depression in
 

mean body weight was evident in males as early as week 22. The disparity between the treated and control males increased until weeks 74
 

and 82 when the high and low dose groups, respectively, were sacrificed· Fluctuations in the growth curve may be due to mortality; as
 

the size of the group diminishes, the mean body weight may be subject
 

to wide variations·
 

No other characteristic clinical signs were observed during the
 

first 6 months of the study with the exception of occasional hunched
 

appearance and reddened or squinted eyes in a few treated rats· As
 

the study progressed, a hunched appearance was noted in increasing
 

numbers of treated rats.
 

Other clinical signs included abdominal urine stains and rough
 

fur. These signs were observed with a slightly greater frequency in
 

the treated groups than in the control groups over the duration of
 

the period of chemical administration but were noted at comparable
 

rates in treated and control female rats during the observation
 

period. Respiratory signs were observed at a low incidence in all
 

18
 
TIME ON TEST (WEEKS) 

FIGURE 1 

GROWTH CURVES FOR ENDOSULFAN CHRONIC STUDY RATS 

19 
groups. Signs often associated with aging in group-housed laboratory
 

rats were observed at a comparable rate in treated and control animals
 

during the last 6 months of the study· These signs included alopecia,
 

sores on the body and/or extremities, eye or nasal discharge, palpable
 

nodules, tissue masses, and swollen areas. Isolated observations in
 

one to three animals included head tilt, ataxia and circling.
 

B.	 Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and endosulfan-treated groups are shown in Figure 2.
 

For male rats the Tarone test for a positive dose-related trend
 

in mortality was highly significant (P < 0.001). The usefulness of
 

any analysis of late-developing tumors based upon the male rat data
 

was restricted due to the early ages at which the animals died. By
 

week 54, 52 percent of the high dose rats had died. The six high dose
 

males surviving past week 71 were sacrificed in week 74. The 10 low
 

dose males surviving past week 80 were sacrificed in week 82. Seven
 

of the 17 surviving males in the control group were sacrificed in week
 

74; of the 10 male controls remaining 5 survived until termination of
 

the study.
 

For female rats the Tarone test for a positive dose-related trend
 

in mortality was not significant. The low dose group had a high early
 

mortality, primarily due to the death of seven female rats in week 21.
 

Six of these animals were reported to have cerebral hemorrhage and the
 

seventh to have cerebral angiectasis; no other rats or mice included
 

20
 
TIME ON TEST (WEEKS)

 

FIGURE 2 

SURVIVAL COMPARISONS OF ENDOSULFAIM CHRONIC STUDY RATS 

21 
in this bioassay in any control or endosulfan-treated groups were
 

found to have cerebral lesions· These deaths do not appear, therefore, to be compound-related. Since 50 percent of the high dose
 

animals, 62 percent of the low dose animals, and 70 percent of the
 

controls survived to the termination of the study, adequate numbers
 

of female rats survived sufficiently long to be at risk from late-


developing tumors· Early deaths in the treated groups were not
 

tumor-related.
 

C.	 Pathology
 

Histopathologic findings on neoplasms in rats are tabulated in
 

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

tabulated in Appendix C (Tables C1 and C2).
 

Toxic nephropathy occurred in 47/50 low dose males, 43/47 high
 

dose males, 27/50 low dose females, and 29/50 high dose females.
 

Microscopically, toxic nephropathy was characterized by degenerative
 

changes in the proximal convoluted tubules at the junction of the
 

cortex and medulla, with cloudy swelling, fatty degeneration, and
 

necrosis of the tubular epithelium. Some affected tubules were
 

filled with hyalin casts. In occasional tubules, there were enlarged
 

dark-staining regenerative tubular epithelial cells. At this stage
 

the kidney often had infiltration of inflammatory cells, fibrosis,
 

and focal mineralization. Parathyroid hyperplasia associated with
 

renal lesions occurred in 21/48 low dose males, 18/47 high dose males,
 

and 1/49 low dose females. Medial calcification of the blood vessels,
 

22
 
perhaps related to the hyperplasia of the parathyroid, occurred frequently in the treated male rats.
 

Testicular atrophy occurred in 3/19 control, 18/47 low dose, and
 

24/47 high dose male rats, and was characterized by degeneration and
 

necrosis of the germinal cells lining the seminiferous tubules, multi-


nucleated cells (fusion bodies), and calcium deposition resulting in
 

aspermatogenesis·
 

Other inflammatory, degenerative, proliferative and neoplastic
 

lesions in the control and treated animals were similar in number and
 

kind to lesions that occur naturally in aged Osborne-Mendel rats·
 

In this study, endosulfan induced testicular atrophy in male rats
 

and toxic nephropathy with secondary parathyroid hyperplasia in male
 

and female rats. However, there was no histopathologic evidence of
 

carcinogenicity.
 

D.	 Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or endosulfandosed groups and where such tumors were observed in at least 5 percent
 

of the group. Due to high early mortality, additional time-adjusted
 

analyses were conducted for selected tumors in both males and females
 

(Tables 5 and 6) .
 

23
 
TABLE 3 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN MALE RATS TREATED WITH ENDOSULFANa 

TOPOGRAPHY: MORPHOLOGY 

POOLED 

CONTROL 

MATCHED 

CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Hematopoietic

Lymphoma 

 Values 0 

 System: Malignant 

2/40(0.05) 

N.S. 

2/20(0.10) 

N.S. 

2/50(0.04) 

N.S. 

1/49(0.02) 

N.S. 

Relativ e Risk (Pooled Control) d 

Lower Limit 

Upper Limit 

—.— ___ 

— 

— 

0.800 

0.060 

10.643 

0.408 

0.007 

7.568 

to Relativ e Risk (Matched Control) 

Lower Limit 

Upper Limit 

— 

— 

— 

0.400 

0.032 

5.277 

0.204 

0.004 

3.754 

Weeks to Firs t Observed Tumor 49 49 28 40 

Kidney: Mixed Tumor Malignant 

 Values 0 

Relativ e Risk (Pooled Control) 

Lower Limit 

Upper Limit 

Relativ e Risk (Matched Control) d 

Lower Limit 

Upper Limit 

2/40(0.05) 

N.S. 

2/20(0.10) 

 = 0.044(N) 

2/50(0.04) 

N.S. 

0.800 

0.060 

10.643 

0.400 

0.032 

5.282 

0/47(0.00) 

N.S. 

0.000 

0.000 

2.870 

0.000 

0.000 

1.428 

Weeks to Firs t Observed Tumor 110 110 82 
TOPOGRAPHY: MORPHOLOGY
 

Pituitary: Chromophobe Adenoma
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (Continued)
 

POOLED MATCHED
 

CONTROL CONTROL
 

6/39(0.15) 3/19(0.16)
 

 = 0.003(N) = 0.010(N)
 

96 98
 

2/40(0.05) 1/20(0.05)
 

N.S. N.S.
 

110 110
 

LOW
 

DOSE
 

1/49(0.02)
 

 - 0.028*(Ν)
 

0.133
 

0.003
 

1.032
 

0.129
 

0.003
 

1.517
 

63
 

0/48(0.00)
 

N.S.
 

0.000
 

0.000
 

2.812
 

0.000
 

0.000
 

7.780
 

_.__
 

HIGH
 

DOSE
 

0/45(0.00)
 

 = 0.008*(Ν)
 

 = 0.023**(Ν)
 

0.000
 

0.000
 

0.539
 

G.000
 

0.000
 

0.694
 

0/47(0.00)
 

N.S.
 

0.000
 

0.000
 

2.871
 

0.000
 

0.000
 

7.942
 
TABLE 3 (Concluded)
 

Treated groups received time-weighted average doses of 408 or 952 ppm in feed.
 

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

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

the corresponding control group when Ρ < 0.05; otherwise, not significant (N.SO is indicated.
 

The probability level for the Fisher exact test for the comparison of a treated group with the
 

pooled control group (*) or the matched control group (**) is given beneath the incidence of tumors in that treated group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated. For
 

both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 
TABLE 4 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN FEMALE RATS TREATED WITH ENDOSULFAN* 

POOLED MATCHED LOW HIGH 

TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL DOSE DOSE 

Subcutaneous Tissue: Fibroma 2/40(0.05) 1/20(0.05) 1/50(0.02) 0/50(0.00) 

 Values N.S. N.S. N.S . N.S. 

Relative Risk (Pooled Control) 0.400 0.000 

Lower Limit 0.007 0.000 

Upper Limit 7.421 2.702 

Relative Risk (Matched Control) 0.400 0.000 

Lower Limit - — 0.005 0.000 

Upper Limit 30.802 7.475 

Weeks to First Observed Tumor 55 110 111 

Subcutaneous Tissue; Fibrosarcoma 2/40(0.05) 2/20(0,10) 1/50(0.02) 0/50(0.00) 

 Values0 N.S. = 0.035(N) N.S. N.S. 

Relative Risk (Pooled Control) — 0.400 0.000 

Lower Limit — 0.007 0.000 

Upper Limit — 7.421 2.702 

Relative Risk (Matched Control) — 0.200 0.000 

Lower Limit — 0.004 0.000 

Upper Limit — 3.682 1.345 

Weeks to First Observed Tumor 110 110 1 11 
___ 

00 

TOPOGRAPHY: MORPHOLOGY 

Pituitary : Chromophobe Adenoma 

 Values0 

Relative Risk (Pooled Control) 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Thyroid: Follicular-Cell Adenoma or 

Follicular-Cell Carcinoma*5 

 Values0 

Relative Risk (Pooled Control) 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

TABLE 4 (Continued ) 

POOLED 

CONTROL 

20/39(0.51) 

 = 0.001(N) 

79 

2/39(0.05) 

N.S. 

110 

MATCHED 

CONTROL 

11/19(0.58) 

 = 0.002(N) 

—
 

—
 

—
 

—
 

—
 

— 

102 

2/19(0.11) 

N.S. 

110 

tow 

DOSE 

16/48(0.33) 

N.S. 

0.650 

0.376 

1.132 

0.576 

0.337 

1.138 

84 

1/48(0.02) 

N.S.1 

0.406 

0.007 

7.526 

0.198 

0.004 

3.635 

98 

HIGH
 

DOSE
 

9/48(0.19) 

 = 0.001*(N) 

 = 0.003** (Ν) 

0.366 

0.172 

0.735 

0.324 

0.159 

0.725 

1 0 0 

1/48(0.02) 

N.S. 

0.406 

0.007 

7.526 

0.198 

0.004 

3.635 

1 11 
____ 

___ 

TOPOGRAPHY: MORPHOLOGY 

Mammary Gland: Adenocarcinoma NOS 

 Values 0 

Relativ e Risk (Pooled Control) 

Lower Limit 

Upper Limit 

Relativ e Risk (Matched Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Mammary Gland: Fibroadenoma 

 Values 0 

Relativ e Risk (Pooled Control) d 

Lower Limit 

Upper Limit 

Relativ e Risk (Matched Control) d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

TABLE 4 (Continued ) 

POOLED 

CONTROL 

2/40(0.05) 

N.S. 

110 

10/40(0.25) 

N.S. 

— 

— 

_ — 

— 

— 

109 

MATCHED 

CONTROL 

2/20(0.10) 

N.S. 

110 

5/20(0.25) 

N.S. 

110 

LOW 

DOSE 

3/50(0.06) 

N.S. 

1.200 

0.145 

13.831 

0.600 

0.076 

6.860 

111 

13/50(0,26) 

N.S. 

1.040 

0.475 

2.376 

1.040 

0.416 

3.342 

98 

HIGH 

DOSE 

1/50(0.02) 

N.S. 

0.400 

0.007 

7.419 

0.200 

0.004 

3.682 

111 

11/50(0.22) 

N.S. 

0.880 

0.381 

2.082 

0.880 

0.336 

2.905 

97 
TOPOGRAPHY:MORPHOLOGY
 

Uterus: Endometrial Stromal Polyp
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Malignant
 

Lymphoma
 

 Values0
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (Continued)
 

POOLED MATCHED 

CONTROL CONTROL 

2/40(0.05) 1/20(0.05) 

N.S. N.S. 

___ 

110 110
 

0/40(0.00) 0/20(0.00)
 

N.S. N.S.
 

 - 0.019 = 0.047
 

LOW
 

DOSE
 

3/49(0.06)
 

N.S.
 

1.224
 

0.148
 

14.113
 

1.224
 

0.108
 

62.958
 

110
 

3/50(0.06)
 

N.S.
 

Infinite
 

0.485
 

Infinite
 

Infinite
 

0.250
 

Infinite
 

71
 

HIGH
 

DOSE
 

1/49(0.02)
 

N.S.
 

0.408
 

0.007
 

7.568
 

0.408
 

0.005
 

31.414
 

111
 

0/50(0.00)
 

N.S.
 
31 

TABLE 4 (Concluded)
 

Treated groups received time-weighted average doses of 223 or 446 ppm in feed.
 

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

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

the corresponding control group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated·
 

The probability level for the Fisher exact test for the comparison of a treated group with the
 

pooled control group (*) or the matched control group (**) is given beneath the incidence of tumors in that treated group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated. For
 

both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

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

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

group when Ρ < 0.05.
 
___ 

___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH ENDOSULFAN
 

WHICH SURVIVED AT LEAST 52 WEEKSa
 

TOPOGRAPHY :MORPHOLOGY
 

Pituitary: Chromophobe Adenoma
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 hO
 

Kidney: Mixed Tumor, Malignant
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

POOLED
 

CONTROL
 

6/36(0.17)
 

 = 0.010(N)
 

— —
 

-_—
 

2/37(0.05)
 

N.S.
 

110
 

MATCHED
 

CONTROL
 

3/16(0.19)
 

 = 0.020(N)
 

—
 

—
 

—
 

—
 

98
 

2/17(0.12)
 

N.S.
 

110
 

LOW
 

DOSE
 

1/44(0.02)
 

 = 0.030(N)
 

0.136
 

0.003
 

1.053
 

0.121
 

0.002
 

1.409
 

63
 

2/45(0.04)
 

N.S.
 

0.822
 

0.062
 

10.895
 

0.378
 

0.030
 

4.953
 

82
 

HIGH
 

DOSE
 

0/24(0.00)
 

 = 0.039(Ν)
 

0.000
 

0.000
 

0.908
 

0.000
 

0.000
 

1.066
 

0/24(0.00)
 

N.S.
 

0.000
 

0.000
 

5.073
 

0.000
 

0.000
 

2.319
 
TABLE 5 (Concluded)
 

Treated groups received time-weighted average doses of 408 or 952 ppm in feed.
 

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

c

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

the corresponding control group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated.
 

The probability level for the Fisher exact test for the comparison of a treated group with the
 

pooled control group (*) or the matched control group (**) is given beneath the incidence of tumors in that treated group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated. For
 

both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

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

CO
 
___ 

TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH ENDOSULFAN
 

WHICH SURVIVED AT LEAST 52 WEEKSa
 

TOPOGRAPHY: MORPHOLOGY
 

Subcutaneous Tissue: Fibrosarcoma
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

POOLED
 

CONTROL
 

2/39(0.05)
 

N.S.
 

.
 

110
 

20/38(0.53)
 

 = 0.018(N)
 

—
 

79
 

MATCHED
 

CONTROL
 

2/20(0.10)
 

N.S.
 

110
 

11/19(0.58)
 

 - 0.015(N)
 

— —
 

—
 

—
 

—
 

—
 

—
 

102
 

LOW
 

DOSE
 

1/38(0.03)
 

N.S.
 

0.513
 

0.009
 

9.434
 

0.263
 

0.005
 

4.801
 

111
 

16/36(0.44)
 

N.S.
 

0.844
 

0.498
 

1.418
 

0.768
 

0.452
 

1.472
 

84
 

HIGH
 

DOSE
 

0/36(0.00)
 

N.S.
 

0.000
 

0.000
 

3.625
 

0.000
 

0.000
 

1.852
 

9/34(0.26)
 

 - 0.025(N)
 

 = 0.021(N)
 

0.503
 

0.240
 

0.980
 

0.457
 

0.225
 

0.999
 

100
 
TABLE 6 (Concluded)
 

Treated groups received time-weighted average doses of 223 or 445 ppm in feed.
 

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

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

the corresponding control group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated.
 

The probability level for the Fisher exact test for the comparison of a treated group with the
 

pooled control group (*) or the matched control group (**) is given beneath the incidence of tumors in that treated group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated. For
 

both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group. .
 
In addition to the control group specifically assigned in the
 

experimental design to the endosulfan bioassay (referred to in this
 

section as the "matched" control), a pooled control group was used
 

for statistical purposes· The pooled group combined the controls
 

from the studies of endosulfan and mexacarbate. All of these control
 

rats were of the same strain, were housed in the same room, were
 

tested at approximately the same time, and were examined by the same
 

pathologists.
 

For both male and female rats, neither the Cochran-Armitage
 

tests nor the Fisher exact tests indicated a significant positive
 

association between dosage and tumor incidence at the 0.05 level for
 

any type of tumor. For male rats the incidence of neoplasms was
 

almost uniformly higher in the matched control group than in the high
 

and low dose groups. This was probably due to the absence of dosed
 

animals surviving long enough for late-developing tumors to occur.
 

For both male and female rats all Cochran-Armitage tests indicated a significant negative association between dosage and the incidence of pituitary chromophobe adenomas. For males, however, the
 

time-adjusted Fisher exact tests did not support this finding since
 

the marginal results were not significant under the Bonferroni criterion. For females the time-adjusted Fisher exact tests indicated
 

that the high dose incidence was significantly lower than either the
 

pooled (P = 0.025) or the matched (P = 0.021) control. It must be
 

noted, however, that the incidence in the controls was somewhat higher
 

36
 
than the 40/160 (25 percent) observed in the historical Osborne-Mendel
 

untreated control females maintained at Hazleton Laboratories for the
 

NCI Bioassay Program.
 

For malignant mixed tumors of the kidney in males and for fibrosarcomas of the subcutaneous tissue in females, the Cochran-Armitage
 

test indicated a significant negative association between dosage and
 

incidence. In both cases, however, no Fisher exact tests were significant.
 

To provide additional insight into the statistical implications
 

of the biological findings, 95 percent confidence intervals on the relative risk have been estimated and entered in the tables based upon
 

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

in Tables 3, 4, 5, and 6, the value one is included; this indicates
 

the absence of statistically significant results. It should also be
 

noted that many of the confidence intervals have an upper limit
 

greater than one, indicating the theoretical possibility of tumor
 

induction in rats by endosulfan that could not be established under
 

the conditions of this test.
 

37
 
IV· CHRONIC TESTING RESULTS: MICE
 

A· Body Weights and Clinical Observations
 

No appreciable differences in mean body weight were observed
 

among the control and endosulfan-treated female mice (Figure 3)· The
 

same held true for the male mice until after week 73, where the curve
 

indicates a gain in body weight for control males, while weights for
 

both of the treated groups remained approximately the same· Since
 

mice do not normally gain body weight at that age, the increase in
 

weight of male controls is apparently an artifact, indicating that
 

the 5 male controls surviving beyond week 73 were heavier than the
 

11 male controls that died in that week· Fluctuations in the growth
 

curve may be due to mortality; as the size of the group diminishes,
 

the mean body weight may be subject to wide variations.
 

There were no definite compound-related effects on appearance and
 

behavior in any of the treated groups during the study· Signs often
 

observed in group-housed laboratory mice of this hybrid strain were
 

observed at a similar frequency in treated and control groups throughout the study. These signs included sores on the body (particularly
 

in males), alopecia, hunched appearance, penile, anal, or vulvar
 

irritation, rough fur, bloated appearance, and palpable nodules or
 

tissue masses.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and endosulfan-treated groups are shown in Figure 4.
 

38
 
TIME ON TEST (WEEKS)

 

FIGURE 3 

GROWTH CURVES FOR ENDOSULFAN CHRONIC STUDY MICE 

39 
TIME ON TEST (WEEKS)

 

FIGURE 4 

SURVIVAL COMPARISONS OF ENDOSULFAN CHRONIC STUDY MICE 

40 
Although high mortality was noted in male mice, the Tarone test
 

for association between increased dosage and elevated mortality was
 

not significant. The meaning of these results is difficult to interpret because of the deaths of 11 high dose males in week 19 and of 11
 

control males in week 73. There is an indication that the deaths in
 

the control group may have been due to fighting among the animals.
 

No common cause of death was found for the high dose males. Ten percent of the high dose, 38 percent of the low dose, and 15 percent of
 

the control group lived to the end of the test. Early deaths in the
 

males were not tumor-related.
 

In female mice the Tarone test did not indicate a significant
 

association between increased dosage and elevated mortality. Survival of the female mice was extremely good with 96 percent of the
 

high dose, 94 percent of the low dose, and 85 percent of the controls
 

living to termination of the bioassay. Thus, adequate numbers of
 

females survived sufficiently long to be at risk from late-developing
 

tumors·
 

C. Pathology
 

Histopathologic findings on neoplasms in mice are tabulated in
 

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

tabulated in Appendix D (Tables Dl and D2).
 

Hepatocellular carcinomas occurred in 1/20 (5 percent) control
 

males, 6/49 (12 percent) low dose males, 2/50 (4 percent) high dose
 

males, and 1/50 (2 percent) high dose females. Osteosarcomas occurred
 

in one high dose male and one low dose female.
 

41
 
Inflammatory, degenerative, proliferative, and other neoplastic
 

lesions as seen in the control and treated animals were similar in
 

number and kind to lesions occurring naturally in aged B6C3F1 mice·
 

This histopathologic examination provided no evidence for the
 

carcinogenicity of endosulfan in B6C3F1 mice.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

mice are summarized in Tables 7 and 8· The analysis is included for
 

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

tumors were observed in at least one of the control or endosulfandosed groups and where such tumors were observed in at least 5 percent of the group.
 

In addition to the control group specifically assigned in the
 

experimental design to the endosulfan bioassay (referred to in this
 

section as the "matched11 control), a pooled control group was used
 

for statistical purposes. This pooled group combined the controls
 

from the studies of endosulfan and mexacarbate. All of these control
 

mice were of the same strain, were housed in the same room, were
 

tested at approximately the same time, and were examined by the same
 

pathologists.
 

For both male and female mice, neither the Cochran-Armitage
 

tests nor the Fisher exact tests indicated a significant positive
 

association between dosage and tumor incidence at the 0.05 level for
 

any type of tumor. Because of the extreme early mortality in male
 

42
 
TABLE 7 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN MALE MICE TREATED WITH ENDOSULFAN' 

POOLED MATCHED LOW HIGH 

TOPO GRAPHY: MORPHOLOGY CONTROL CONTROL DOSE DOSE 

Subcutaneous Tissue: Fibrosarcoma 3/35(0.09) 3/20(0.15) 1/49(0.02) 2/50(0.04) 

 Values0 N.S. N.S. N.S. N.S. 

Relative Risk (Pooled Control) 0.238 0.467 

Lower Limit 0.005 0.041 

Upper Limit 2.832 3.879 

Relative Risk (Matched Control) d 0.136 0.267 

Lower Limit 0.003 0.024 

u> Upper Limit 1.599 2.191 

Weeks to First Observed Tumor 73 73 92 86 

Liver: Hepatocellular Carcinoma 1/35(0.03) 1/20(0.05) 6/49(0.12) 2/50(0.04) 

 Values0 N.S. N.S. N.S. N.S. 

Relative Risk (Pooled Control) 4.286 1.400 

Lower Limit 0.557 0.076 

Upper Limit 192.714 80.948 

Relative Risk (Matched Control) 2.440 0.800 

Lower Limit 0.332 0.045 

Upper Limit 110.166 46.273 

Weeks to First Observed Tumor 73 73 92 75 
TABLE 7 (Concluded)
 

Treated groups received time-weighted average doses of 3.5 or 6.9 ppm in feed.
 

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

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

the corresponding control group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated.
 

The probability level for the Fisher exact test for the comparison of a treated group with the
 

pooled control group (*) or the matched control group (**) is given beneath the incidence of tumors in that treated group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated. For
 

both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

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

TABLE 8
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH ENDOSULFAN*
 

TOPOGRAPHY: MORPHOLOGY 

Lung: Alveolar/Bronchiolar Carcinoma 

 Values0 

Relative Risk (Pooled Control) 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Ln
 

Lung: Alveolar/Bronchiolar Adenoma or 

Alveolar/Bronchiolar Carcinoma*5 

 Values0 

Relative Risk (Pooled Control) d 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

POOLED
 

CONTROL
 

1/40(0.03) 

N.S. 

91 

3/40(0.08) 

N.S. 

87 

MATCHED
 

CONTROL
 

1/20(0.05) 

N.S. 

91 

2/20(0.10) 

 = 0.040(Ν) 

—
 

—
 

— 

—
 

—
 

91 

LOW
 

DOSE
 

1/50(0.02) 

N.S. 

0.800 

0.011 

61.572 

0.400 

0.005 

30.802
 

92
 

5/50(0.10) 

N.S . 

1.333 

0.278 

8.155 

1.000 

0.184 

10.007
 

92
 

HIGH
 

DOSE
 

0/50(0.00) 

N.S. 

0.000 

0.000 

14.930 

0.000 

0.000 

7.475 

0/50(0.00) 

N.S. 

0.000 

0.000 

1.328 

0.000 

0.000 

1.345 

— — — 
TABLE 8 (Concluded)
 

Treated groups received time-weighted average doses of 2.0 or 3.9 ppm in feed.
 

POOLED MATCHED LOW HIGH 

TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL DOSE DOSE 

Hematopoietic System: Malignant 

Lymphoma^ 9/40(0.22) 5/20(0.25) 10/50(0.20) 5/50(0.10) 

 Values0 N.S. N.S. N.S. N.S. 

Relative Risk (Pooled Control) 0.889 0.444 

Lower Limit 0.362 0.127 

Upper Limit 2.241 1.356 

Relative Risk (Matched Control) 0.800 0.400 

Lower Limit 0.296 0.107 

Upper Limit 2.690 1.584 

Weeks to First Observed Tumor 39 39 83 40 

a

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

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

the corresponding control group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated.
 The probability level for the Fisher exact test for the comparison of a treated group with the
 

pooled control group (*) or the matched control group (**) is given beneath the incidence of tu­ mors in that treated group when Ρ < 0.05; otherwise, not significant (N.S.) is indicated. For
 

both Cochran-Armitage and Fisher exact test a negative designation (N) indicates a lower inci­ dence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 
mice, a time-adjusted analysis was performed in which animals that
 

died before 52 weeks of study were eliminated; again no statistically
 

significant positive results were observed· This bioassay, therefore,
 

provided no statistical evidence for the carcinogenicity of endosulfan
 

in B6C3F1 mice.
 

For female mice the Cochran-Armitage test indicated a significant
 

(P = 0.040) negative association between administration and the incidence of alveolar/bronchiolar neoplasms· The Fisher exact tests, however, were not significant·
 

To provide additional insight into the statistical implications
 

of the biological findings, 95 percent confidence intervals on the
 

relative risk have been estimated and entered in the tables based
 

upon the observed tumor incidence rates· In many of the intervals
 

shown in Tables 7 and 8, the value one is included; this indicates
 

the absence of statistically significant results. It should also
 

be noted that many of the confidence intervals have an upper limit
 

greater than one, indicating the theoretical possibility of tumor
 

induction in mice by endosulfan that could not be established under
 

the conditions of this test·
 

47
 
V. DISCUSSION
 

In rats high early mortality, associated with toxic nephropathy,
 

occurred in both treated male groups. The six high dose males surviving past week 71 were sacrificed in week 74. The 10 low dose males
 

surviving past week 80 were sacrificed in week 82· Probably as a result of the low survival rate, the incidence of neoplasms was almost
 

uniformly higher in the control group than in the high and low dose
 

groups. Survival of female rats was considered sufficient for statistical analysis of late-appearing tumors.
 

At the doses fed to rats in this study, endosulfan was toxic,
 

inducing a high incidence of toxic nephropathy in both sexes and testicular atrophy in males. The poor survival in male rats was associated with secondary parathyroid hyperplasia and mineralization
 

(calcium deposits) in several tissues, both lesions resulting from
 

the effects of chronic renal failure.
 

High early mortality was observed among male mice. Eleven high
 

dose males and two low dose males died in week 19. Median survival
 

on test was 69 weeks for high dose males, 75 weeks for low dose males,
 

and 73 weeks for control males. Survival at termination of the bioassay was 10 percent (5/50) in the high dose males, 39 percent (19/50)
 

in the low dose males, and 15 percent (3/20) in the control group
 

males. Among the female mice, survival in all groups was high, providing sufficient animals for statistical analysis of late-appearing
 

tumors. No significant increases in tumor incidence were observed
 

48
 
among dosed mice of either sex· The toxic nephropathy observed in
 

both sexes of rats and the testicular atrophy observed in male rats
 

were not seen in mice.
 

No conclusions concerning the carcinogenicity of endosulfan can
 

be drawn from the bioassay of male rats or male mice because of the
 

abbreviated life spans of these animals· However, survival among females of both species was sufficient for meaningful statistical evaluation of the incidence of late-developing tumors· It is concluded
 

that under the conditions of this bioassay, technical-grade endosulfan
 

was not carcinogenic in female Osborne-Mendel rats or in female B6C3F1
 

mice.
 

49
 
VI. BIBLIOGRAPHY
 

Andrelinas , P.Α., Farmers' Use of Pesticides in 1971. Economic Research Service, U.S. Department of Agriculture. Economic Report
 

No. 252, Washington, D.C., 1974.
 

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS)
 

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

Chemical Society, Washington, D.C., 1977.
 

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

Co., Ltd., London, 1970.
 

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

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

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

Tests, Confidence Limits, and Adjustments for Stratification."
 

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

Johnson, R.D. and D.D. Manske, "Residues in Foods and Feeds." Pesticide Monitoring Journal 9(4):157, 1976.
 

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

Observations." Journal of the American Statistical Association
 

^:457-481, 1958.
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

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

Co., New York, 1966.
 

Reuber, M.D., and E.L. Glover, "Cirrhosis and Carcinoma of the Liver
 

in Male Rats Given Subcutaneous Carbon Tetrachloride." Journal
 

of the National Cancer Institute 44:419-423, 1970.
 

50
 
Saffiotti, U., R. Montesano, A.R. Sellakumar, F. Cefis, and D.G.
 

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

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

Ferric Oxide.11 Cancer Research 32:1073-1079, 1972.
 

Spencer, E.Y., Guide to the Chemicals Used in Crop Production, Fifth
 

edition. Research Branch, Canadian Department of Agriculture,
 

Publication 1093, 1968.
 

Stanford Research Institute, "Stage I Chemical Dossier 6,7,8,9,10,10­

Hexachloro-l,5,5A,6,9,9A-Hexahydro-6,9~Methano-2,4,3-Benzodioxathipin-3-0xide." Menlo Park, California, 1974.
 

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

62_:679-682, 1975.
 

U.S. Department of Health, Education, and Welfare, Report of the
 

Secretary's Commission on Pesticides and their Relationship
 

to Environmental Health. Parts I and II. TJS No. PB-251-630,
 

Washington, D.C., 1969.
 

51
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH ENDOSULFAN
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH ENDOSULFAN 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 

INTEGUMENTARY SYSTEM 

•SKIN 

SEBACEOUS ADENOMA 

RESPIRATORY SYSTEM 

#LUNG 

FIBROSARCOMA, METASTATIC 

HEMATOPOIETIC SYSTEM 

•MULTIPLE ORGANS 

MALIG.LYMPHOMA, HISTIOCYTIC

LYMPHOCYTIC LEUKEMIA 

•SUBCUT TISSUE/AXILLA 

MALIG.LYMPHOMA, HISTIOCYTIC

#SPLEEN 

HEMANGIOSARCOMA 

#CERVICAL LYMPH NODE 

MALIG.LYMPHOMA, LYMPHOCYTIC

CIRCULATORY SYSTEM 

«ENDOCARDIUM 

SARCOMA, NOS

 

DIGESTIVE SYSTEM 

NONE 

# NUMBER OF ANIMALS WITH TISSUE

* NUMBER OF ANIMALS NECROPSIED 

 TYPE 

 TYPE 

 TYPE 

CONTROL (VEH) 

01-M065 

20 

20 

20 

(20) 

(20) 

1 (5%) 

(20) 

2 (10%) 

1 (5%) 

(20) 

(20) 

1 (5%) 

(20) 

(20) 

LOU DOSE HIGH DOSE 

01-M066 01-M067 

50 50 

50 4 9 

50 4 7 

(50) (49) 

1 (2%) 

(50) (47) 

(50) (49) 

1 (2*) 

(50) 

1 (2%) 

(49) (46) 

(41) (40) 

1 (2*) 

(50) (47) 

1 <2X) 

 EXAMINED MICROSCOPICALLY 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
URINARY SYSTEM 

#KIDNEY 

TUBULAR-CELL ADENOCARCINOMA 

MIXED TUMOR, MALIGNANT 

HEMANGIOSARCOMA 

ENDOCRINE SYSTEM 

#PITUITARY 

CHROMOPHOBE ADENOMA 

tTHYROID 

FOLLICULAR-CELL ADENOMA 

#PANCREATIC ISLETS 

ISLET-CELL ADENOMA 

REPRODUCTIVE SYSTEM 

•MAMMARY GLAND 

FIBROADENOMA 

•PROSTATE 

LIPOMA 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

*MUSCLE OF BACK

 

FIBROSARCOMA 

BODY CAVITIES 

NONE 

TABLE Al (CONTINUED) 

CONTROL (VEH) 

Q1-M06 5 

(20) 

2 (10%) 

(19) 

3 (16%) 

(20) 

1 (5%) 

(20) 

3 (15%) 

(20) 

(13) 

(20) 

1 (5%) 

LOW DOSE HIGH DOSE 

01-M066 01-M067 

(50) (^7) 

1 (2%) 1 (2%) 

2 (4%) 

1 (2%) 

0*9) (45)

 

1 (2%) 

(48) (47) 

(50) (47) 

(5 0) (49) 

1 (2%) 

(29) (19)

 

1 (3%) 

(50) (49) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

A-4
 
TABLE Al (CONCLUDED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-M06 5 01-M06 6 01-M06 7 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 20 50 50 

NATURAL DEATH@ 7 40 44 

MORIBUND SACRIFICE 1 

SCHEDULED SACRIFICE 7 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 5 10 6 

ANIMAL MISSING 

@ INCLUDES AUTOLYZLD ANIMALS 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS* 12 8 3 

TOTAL PRIMARY TUMORS 14 10 3 

TJTAL ANIMALS WITH BENIGN TUMORS 6 4 

TOTAL BEtfIGN TUMORS 7 4 

TOTAL ANIMALS WITH MALIGNANT TUMORS 7 6 3 

TOTAL MALIGNANT TUMORS 7 6 3 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 

TOTAL SECONDARY TUMORS 1 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT

 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC

 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

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

A-5 
TABLE A2 

SUMMARY OF THF INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED WITH ENDOSULFAN 

CONTROL(VEH) LOW DOSE HIGH DOSE 

01-F065 01-F068 01-F069 

ANIMALS INITIALLY IN STUDY 20 5 0 50 

ANIMALS NECROPSIED 20 50 5 0 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 20 5 0 5 0 

INTEGUMENTARY SYSTEM 

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

FIBROMA 1 (5%) 1 (2%) 

FIBROSARCOMA 2 (10%) 1 (2*) 

RESPIRATORY SYSTEM 

#LUNG (20) (50) (50) 

MIXED TUMOR, METASTATIC 1 (2%) HEMANGIOSARCOMA 1 (2%) 

HEMATOPOIETIC SYSTEM 

•MULTIPLE ORGANS (20) (50) (50) 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 2 (4%) 

#SPLEEN (20) (50) (50) 

HEMANGIOSARCOMA 1 (5%) 1 (2%) 

#CERVICAL LYMPH NODE (20) (48) (40) MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

#LIVER 

HEPATOCELLULAR CARCINOMA 

(20) (5 0) 

1 (2%) 

(49) 

1 (2%) 

# PANCREAS 

FIBROSARCOMA, METASTATIC 

HEMANGIOSARCOMA, METASTATIC .

(20) 

1 15XL _. 

(50) 

1 (2%) 

(48) 

# NUMBER

* NUMBER

 OF ANIMALS

 OF ANIMALS

 WITH TISSU E

 NECROPSIED 

EXAMINED MICROSCOPICALLY 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-6 
#LARGE INTESTINE

 

FIBROSARCOMA 

URINARY SYSTEM 

#KIDNEY 

TUBULAR-CELL ADENOMA 

LIPOSARCOMA 

MIXED TUMOR, MALIGNANT 

ENDOCRINE SYSTEM 

#PITUITARY 

CHROMOPHOBE ADENOMA 

#ADRENAL 

CORTICAL ADENOMA 

#THYROID 

FOLLICULAR-CELL ADENOMA 

FOLLICULAR-CELL CARCINOMA 

C-CELL ADENOMA 

REPRODUCTIVE SYSTEM 

•MAMMARY GLAND 

ADENOCARCINOMA, NOS 

FIBROADENOMA 

•VAGINA 

LEIOMYOMA 

#UTERUS 

LIPOMA 

LEIOMYOSARCOMA 

ENDOMETRIAL STROMAL POLYP 

#OVARY 

GRANULOSA-CELL TUMOR 

LIPOMA 

LEIOMYOSARCOMA^. METASTATIC

# NUMBER OF ANIMALS WITH TISSUE

* NUMBER OF ANIMALS NECROPSIED 

TABLE A2 (CONTINUED) 

CONTROL (VEH) LOW DOSE 

01-F065 01-F068 

(20) (50) 

1 (2%) 

(20) (5 0) 

1 (5%) 2 (4%) 

(19) (48) 

11 (58%) 1b (J3%) 

(20) (50) 

2 (4%) 

(19) (48) 

2 (11%) 

1 (2*,) 

(20) (5 0) 

2 (10%) 3 (6%) 

5 (25%) 13 (26%) 

(20) (50) 

(20) (49) 

1 (5%) 

1 (2%) 

1 (5%) 3 (6%) 

(20) (50) 

1 (2%) 

1 (5%) .

 _ . 1 12%L . 

 EXAMINED MICROSCOPICALLY 

HIGH DOSE 

01-F069 

(47) 

(50) 

1

1

1

(48) 

9

(50) 

(48) 

1

1

(50) 

1

11

(50) 

1

(49) 

1

(50) 

 (2%) 

 (2%) 

 (2%) 

 (19%) 

 (2%) 

 (2%) 

 (2%) 

 (22%) 

 (2%) 

 (2%) 

A-7 
TABLE A2 (CONTINUED) 

CONTROL (VEH) LO» DOSE 

01-F065 01-F068 

NERVOUS SYSTEM 

#BRAIN (20) (50) 

ASTROCYTOMA 1 (2*) 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

NGNE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 20 50

 

NATURAL DEATHd) 6 19

 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 14 31

 ANIMAL MISSING

 

3 INCLUDES AUTOLYZED ANIMALS 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

HIGH DOSE 

01-F069 

(49) 

1 (2%) 

50 

25 

25 

A-8 
TABLE A2 (CONCLUDED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-F065 01-F068 01-F069 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS* 15 30 24

 

TOTAL PRIMARY TUMORS 28 50 32

 

TOTAL ANIMALS WITH BENIGN TUMORS 14 23 19

 

TOTAL BENIGN TUMORS 22 35 2 4 

TOTAL ANIMALS WITH MALIGNANT TUMORS 6 1 2 7

 

TOTAL MALIGNANT TUMORS 6 14 8

 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 3 

TOTAL SECONDARY TUMORS 1 3 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 1

 

TOTAL UNCERTAIN TUMORS 1

 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC

 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

A-9 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH ENDOSULFAN
 

TABLE B1

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH ENDOSULFAN 

CONTROL (VEH) LOW DOSE 

02-M072 02-M073 

ANIMALS INITIALLY IN STUDY 20 5 0 

ANIMALS MISSING 1 

ANIMALS NECROPSIED 20 4 9 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 20 49 

INTEGUMENTARY SYSTEM 

• SKIN (20) (49) 

FIBROMA 

OSTEOSARCOMA, METASTATIC 

•SUBCUT TISSUE (20) (49) 

FIBROSARCOMA 3 (15%) 1 (2%) 

RESPIRATORY SYSTEM 

#LUNG (20) (49) 

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

HEMATOPOIETIC SYSTEM 

NONE 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

#LIVER (20) (49) 

HEPATOCELLULAR CARCINOMA 1 (5%) 6 (12%) 

URINARY SYSTEM 

NONE 

ENDOCRINE SYSTEM 

NONE 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE 

02-M074 

50 

50 

50 

(50) 

1 {2%) 

1 (2%) 

(50) 

2 (4%) 

(50) 

2 (4%) 

(50) 

2 (4%) 

B-3 
TABLE Bl (CONTINUED) 

HIGH DOSE 02-M074 

(50) 

1 (2%) 

(50) 

1 (2%) 

50 

4 5 

5 

LOW DOSE 

02-M073 

(U9) 

(4 9) 

5 0 

30 

19 

1 

REPRODUCTIVE SYSTEM 

•MAMMARY GLAND 

ADENOCARCINOMA, NOS

 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

*BONE/LOWER EXTREMITY 

OSTEOSARCOMA 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING 

3 INCLUDES AUTOLYZED ANIMALS 

CONTROL (VEH) 

02-M072 

(20) 

(20) 

2 0 

17 

3 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

B-4 
TABLE B1 (CONCLUDED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

02-M07 2 02-M07 3 02-M07U 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS* 4 8 7

 

TOTAL PRIMARY TUMORS 4 9

 

TOTAL ANIMALS WITH BENIGN TUMORS 1 3

 

TOTAL BENIGN TUMORS 1 3 

4 TOTAL ANIMALS WITH MALIGNANT TUMORS 7 5

 

TOTAL MALIGNANT TUMORS 4 8 6 

TOTAL ANIMALS WITH SECONDARY TUMORS# 1 

TOTAL SECONDARY TUMORS 1 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT

 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC

 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

B-5 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH ENDOSULFAN 

CONTROL(VEH) LOW DOSE HIGH DOSE 

02-F072 02-F075 02-F076 

ANIMALS INITIALLY IN STUDY 20 50 50 

ANIMALS NECROPSIED ** 20 50 50 ANIMALS EXAMINED HIST0PATH0L03ICALLY ** 20 50 50 

INTEGUMENTARY SYSTEM 

•SUBCUT TISSUE (20) (50) (50)

 

FIBROSARCOMA 1 (2%)

 HEMANGIOSARCOMA 1 (2%)

 

RESPIRATORY SYSTEM 

#LUNG (20) (5 0) (50)

 

ALVEOLAfi/BRONCHIOLAR ADENOMA 1 (5%) 4 (8%) 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (5%) 1 12%)

 

OSTEOSARCOMA, METASTATIC 1 (2%)

 

HEMATOPOIETIC SYSTEM 

•MULTIPLE ORGANS (2 0) (5 0) (50) 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 2 (10%) 6 (12%) 4 (8%) MALIG.LYMPHOMA, HISTIOCYTIC TYPE 3 (15%) 

#SPLEEN (20) (50) (50) 

HEMANGIOSARCOMA 1 (5%) 1 (2%) 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%) 1 (2%) 

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

 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%)

 

#LIVER (20) (50) (50)

 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%)

 

#SMALL INTESTINE (20) (50) (50) 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%)

 

CIRCULATORY SYSTEM 

NONE 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-6 
TABLE B2 (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

02-F072 02-F075 02-F076 

DIGESTIVE SYSTEM 

#LIVER (20) (50) (50) HEPATOCELLULAR CARCINOMA 1 (2%) 

URINARY SYSTEM 

NONE 

ENDOCRINE SYSTEM 

#PITUITARY (20) (<•<• ) (50) 

CHROMOPHOBE ADENOMA 1 (2%) 

REPRODUCTIVE SYSTEM 

•MAMMARY GLAND (20) (50) (50) 

ADENOCABCINOMA, NOS 1 (2%) 1 (2%) 

#UTERUS (20) (50) (50) 

HEMANGIOSARCOMA 1 (2%) 

#UTERUS/ENDOMETRIUM (20) (50) (50) ADENOCARCINOMA, NOS 1 (5%) 

#OVARY (20) (49) (50) 

GRANULOSA-CELL TUMOR 1 (2%) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

•FEMUR (20) (50) (50) 

OSTEOSARCOMA 1_12|L_ . 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

B-7 
TABLE B2 (CONCLUDED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

02-F072 02-F075 02-F076 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUdMARY 

ANIMALS INITIALLY IN STUDY 20 50 5 0

 

NATURAL DEATHS 3 3 2 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 17 4 7 4 8 

ANIMAL MISSING 

a> INCLUDES AUTOLYZED ANIMALS 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS* 7 18 8 

TOTAL PRIMARY TUMORS 9 19 11

 

TOTAL ANIMALS WITH BENIGN TUMORS 1 4 1 

TOTAL BENIGN TUMuRS 1 4 1

 

TOTAL ANIMALS WITH MALIGNANT TUMORS 6 14 6

 

TOTAL MALIGNANT TUMORS 8 15 9

 

TOTAL ANIMALS WITH SECONDARY TUMORS» 1

 

TOTAL SECONDARY TUMORS 1

 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 1

 

TOTAL UNCERTAIN TUMORS 1 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC

 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INT3 AN ADJACENT ORGAN
 

B-8
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH ENDOSULFAN
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS TREATED WITH ENDOSULFAN 

CONTROL(VEH) 

01-M065 

LOW DOSE 

01-M066 

HIGH DOSE 

01-M067 

ANIMALS

ANIMALS

ANIMALS

 INITIALLY IN STUDY 

 NECROPSIED 

 EXAMINED HISTOPAIHOLOGICALLY **

20 

20 

 20 

5 0 

50 

5 0 

50 

4 9 

4 7 

INTEGUMENTARY SYSTEM 

*SKIN 

EPIDERMAL INCLUSION CYST 

(20) (5 0) 

1 (2%) 

(49) 

•SUBCUT TISSUE 

ABSCESS, NOS 

(20) 

1 (5%) 

(50) (49) 

RESPIRATORY SYSTEM 

#TRACriEA 

INFLAMMATION, NOS 

(20) (48) (47) 

1 (2%) 

tLUNG 

PNEUMONIA, CHRONIC

CALCIUM DEPOSIT 

 MURINE 

(20) 

11 (55%) 

(50) 

7

11

 (14%) 

 (22%) 

(47) 

17

5

 (36%) 

 (11%) 

HEMATOPOIETIC SYSTEM 

#BONE MARROW 

METAMORPHOSIS FATTY 

(20) 

1 (5%) 

(48) (47) 

#SPLEEN 

THROMBUS, ORGANIZED 

ABSCESS, NOS 

HEMATOPOIESIS 

(20) 

3 (15%) 

(49) 

2 (4%) 

(46) 

1

1

 (2%) 

 (2%) 

#CERVICAL LYMPH NODE 

INFLAMMATION, NOS 

(20) 

2 (10%) 

(4 1) (40) 

«THYMUS 

INFLAMMATION, NOS 

(13) 

1 (8%) 

(31) (20) 

CIRCULATORY SYSTEM 

#HEART 

CALCIUM DEPOSIT 

CALCIFICATION^. NOS 

(20) 

1 (5%) 

(50) 

9 (18%) 

.

(47) 

10

 __ 1

 (21%) 

(2%) _ ­ . 

# NUMBER OF ANIMALS WITH TISSU E EXAMINED

* NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

C-3 
TABLE CI (CONTINUED) 

HIGH DOSE 

01-M067 

(47) 

2 (4%) 

2 (4%) 

(49) 

22 (45%) 

(49) 

(49) 

1 (2%) 

23 (47%) 

(49) 

4 (8%) 

(49) 

2 (4%) 

(47) 

1 (2%) 

3 (6%) 

1 (2%) 

7 (15%) 

1 (2%) 

2 (4%) 

(49) 

2 (4%) 

(47) 

1 (2%) 

(47) 

21 (45%) 

1 (2%) 

(43) 

#MYOCARDIUM 

INFLAMMATION, NOS

 

FIBROSIS 

DEGENERATION, NOS

 

•AORTA 

MEDIAL CALCIFICATION 

•CORONARY ARTERY 

MEDIAL CALCIFICATION 

•MESENTERIC ARTERY 

THROMBUS, ORGANIZED 

MEDIAL CALCIFICATION 

•SPERMATIC ARTERY 

MEDIAL CALCIFICATION 

•PROSTATIC ARTERY 

MEDIAL CALCIFICATION 

DIGESTIVE SYSTEM 

#LIVER 

INFLAMMATION, NOS

 

ABSCESS, NOS 

PELIOSIS HEPATIS 

METAMORPHOSIS FATTY 

CALCIUM DEPOSIT 

ANGIECTASIS 

*BILE DUCT 

HYPERPLASIA, NOS

 

#PANCREAS 

PERIARTERITIS 

#STOMACH 

INFLAMMATION, NOS

 

CALCIUM DEPOSIT 

CALCIFICATION, NOS

 

#COLON 

NEMATODIASIS 

# NUMBER OF ANIMALS WITH TISSUE

* NUMBER OF ANIMALS NECfiOPSIED 

CONTROL (VEH) 

01-M065 

(20) 

1 (5%) 

(20) 

1 (5%) 

(20) 

(20) 

1 (5%) 

(20) 

(20) 

(19) 

1 (5%) 

3 (16%) 

1 (5%) 

(20) 

2 (10%) 

(20) 

(20) 

1 (5%) 

(20) 

LOW DOSE 

01-M066 

 EXAMINED MICROSCOPICALLY 

(50) 

3

2

(50) 

29

(50) 

1

(50) 

28

(5 0) 

6

(50) 

2

(49) 

1

15

2

(50) 

4

(50) 

2

(50) 

1

31

(50) 

1

 (6%) 

 (4%) 

 (5836) 

 (2%) 

 (56%) 

 (12%) 

 (4%) 

 (2%) 

 (31%) 

 (4%) 

 (8%) 

 (4%) 

 (2%) 

 (62%) 

 (2%) 

C-4 
TABLE CI (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-M065 01-M06 6 01-M067 

URINARY SYSTEM 

#KIDNEY (20) (50) (47) 

HYDRONEPHROSIS 1 (2%) 1 (2%) 

PYELONEPHRITIS, NOS 1 (5%) 

INFLAMMATION, CHRONIC 8 (40%) 42 (8436) 34 (72%) 

NEPHROPATHY, TOXIC 47 (94%) 43 (91%) 

CALCIUM DEPOSIT 1 (5%) 29 {58%) 22 (47%) 

CALCIFICATION, NOS 1 (2%) 

PIGMENTATION, NOS 2 (4%) 

#URINARY BLADDER (20) (49) (47)

 

CALCIUM DEPOSIT 1 (2%)

 

ENDOCRINE SYSTEM 

#PITUITARY (19) (49) (45)

 

CYST, NOS 1 <2X)

 

#ADRENAL (20) (50) (47)

 

ANGIECTASJS 1 (5%) 

•THYROID (20) (48) (47) 

FOLLICULAR CYST, NOS 6 (30%) 2 (4%) 

HYPERPLASIA, FOLLICULAR-CELL 1 (2%) 

•PARATHYROID (19) (48) (47) 

HYPERPLASIA, NOS 21 (44%) 18 (38%) 

REPRODUCTIVE SYSTEM 

*PENIS (20) (50) (49)

 

EPIDERMAL INCLUSION CYST 1 (2%)

 

•PROSTATE (13) (29) (19) 

INFLAMMATION, NOS 1 (5%) 

CALCIUM DEPOSIT 1 (3%) 

•TESTIS (19) (47) (47) 

CALCIUM DEPOSIT 5 (11%) 1 (2%) 

ATROPHY, NOS 3 (16%) 18 (38%) 24 (51%) 

•EPIDIDYMIS (20) (50) (49)

 

GRANULOMA, SPERMATIC 1 (2%)

 

NECROSIS, FAT 1 15%) 1 (2%)

 

• NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-5
 
TABLE CI (CONCLUDED) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE OfiGANS 

•EYE/LACRIMAL GLAND 

INFLAMMATION, NOS 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

•PERITONEUM 

INFLAMMATION, NOS 

•PERICARDIUM 

INFLAMMATION, NOS

 

•MESENTERY 

PERIARTERITIS 

ALL OTHER SYSTEMS 

DIAPHRAGM 

INFLAMMATION, NOS

 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 

AUTOLYSIS/NECROPSY PERF/NO HISTO 

AUTOLYSIS/NO NECROPSY PERFORMED 

CONTROL (VEH) 

01-M065 

(20) 

(20) 

(2 0) 

1 (5%) 

(20) 

1 

LOW DOSE 

01-M066 

(50) 

1 (2%) 

(50) 

(50) 

2 (4%) 

(50) 

2 (4X) 

1 

HIGH DOSE 

01-M067 

(49) 

(49) 

1 {2%) 

(49) 

(49) 

1 (2%) 

2 

1 

C-6
 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS TREATED WITH ENDOSULFAN 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-F065 01-F068 01-F069 

ANIMALS INITIALLY IN STUDY 20 50 50 

ANIMALS NECROPSIED 20 50 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 20 50 50 

INTEGUMENTARY SYSTEM 

•SKIN (20) (50) (50) 

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

ABSCESS, NOS 1 (2%) 1 (2%) 

•SU3CUT TISSUE (2 0) (50) (50) 

ABSCESS, NOS 1 (2%) 

RESPIRATORY SYSTEM 

#TRACHEA (19) (48) (50) 

INFLAMMATION, NOS 1 (2%) 

#LUNG (20) (50) (50) 

THROMBUS, ORGANIZED 1 (5%) 

PNEUMONIA, CHRONIC MURINE 14 (70%) 33 (66%) 37 (7 4%) 

CALCIUM DEPOSIT 1 (5%) 

HEMATOPOIETIC SYSTEM 

#SPLEEN (20) (5 0) (50) 

HEMATOPOIESIS 1 (5%) 2 (4%) 5 (10%) 

tCERVICAL LYMPH NODE (20) (48) (40) 

INFLAMMATION, NOS 2 (5%) 

ANGIECTASIS 2 (4%) 2 (5%) 

#THYMUS (18) (4 3) (36) 

CYST, NOS 1 (3%) 

CIRCULATORY SYSTEM 

# MYOCARDIUM (20) (5 0) (50) 

INFLAMMATION,. NOS _ 2 14£L . 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 
TABLE C2 (CONTINUED) 

CONTROL (VEH) 

01-F065 LOW DOSE 01-F068 HIGH DOSE 01-F069 

•ENDOCARDIUM 

INFLAMMATION, NOS 

(20) (50) (50) 

1 (2%) 

•AORTA 

PERIARTERITIS 

MEDIAL CALCIFICATION 

(20) 

1 (5%) 

(50) 

1

1

 (2%) 

(2%) 

(50) 

DIGESTIVE SYSTEM 

#LIVER 

INFLAMMATION, NOS 

PELIOSIS HEPATIS 

METAMORPHOSIS FATTY 

FOCAL CELLULAR CHANGE 

ANGIECTASIS 

(2 0) 

1 (5%) 

2 (10%) 

(5 0) 

1 (2%) 

3 (6%) 

4 (8%) 

1 (2%) 

5 (10%) 

(49) 

2

2

1

 (4%) 

 (4%) 

 (2%) 

*BILE DUCT 

DILATATION, NOS 

HYPERPLASIA, NOS 

(20) 

4 (20%) 

(50) 

1

7

 (2%) 

 (14%) 

(50) 

2

5

 (4%) 

 (10%) 

# PANCREAS 

PERIARTERITIS 

(20) 

1 (5%) 

(50) 

1 (2%) 

(48) 

2 (4%) 

#STOMACH 

ULCER, FOCAL 

CALCIUM DEPOSIT 

(20) (50) 

2 (4%) 

(49) 

3

1

 (6%) 

 (2%) 

URINARY SYSTEM 

#KIDNEY 

HYDRONEPHROSIS 

PYELONEPHRITIS, NOS 

INFLAMMATION, CHRONIC 

NEPHROPATHY, TOXIC 

CALCIUM DEPOSIT 

(2 0) 

1 (5%) 

13 (65%) 

1 (5%) 

(5 0) 

1

7

*27

3

 (2%) 

 (14%) 

 (54%) 

 (6%) 

(50) 

1

5

29

1

 (2%) 

 (10%) 

 (58%) 

 (2%) 

ENDOCRINE SYSTEM 

#ADRENAL 

ANGIECTASIS 

(20) 

3 (15%) 

(50) 

9 (18%) 

(50) 

3 (b%) 

•ADRENAL CORTEX 

DEGENERATION, NOS _ _

(20) 

_„ ! J2O%1 _ 

(50) 

4 J8%]L . _

{r>0) 

_ 2_14%L „ . 

# NUMBER OF ANIMALS

* NUMBER OF ANIMALS

 WITH TISSUE

 NECBOPSIED 

 EXAMINED MICROSCOPICALLY 

C-8 
#THYROID

 

FOLLICULAR CYST, NOS

 

HYPERPLASIA, C-CELL 

«PARATHYROID 

HYPERPLASIA, NOS

 

REPRODUCTIVE SYSTEM 

•VAGINA 

INFLAMMATION, NOS 

#UTERUS 

HYDROMETRA 

INFLAMMATION, NOS

 

#UTERUS/ENDOMETRIUM 

HYPERPLASIA, CYSTIC 

#OVARY 

CYST, NOS

 

HEMORRHAGIC CYST 

NERVOUS SYSTEM 

#BRAIN 

HEMORRHAGE 

ANGIECTASIS 

SPECIAL SENSE ORGANS 

*EYE 

INFLAMMATION, NOS

 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

•PERICARDIUM 

INFLAMMATION- NOS

 

# NUMBER OF ANIMALS WITH TISSUE

* NUMBER OF ANIMALS NECROPSIED 

TABLE C2 (CONTINUED) 

CONTROL (V$H) LOW DOSE 

01-F065 01-F068 

(19) (48) 

2 (11%) 2 (4%) 

1 (2%) 

(18) (49) 

1 (2%) 

(20) (50) 

1 (5%) 2 (4%) 

(20) (49) 

4 (20%) 5 (10%) 

1 (5%) 1 (2%) 

(20) (49) 

1 (5%) 1 (2%) 

(20) (50) 

1

 (5%) 1 (2%) 

1 (2%) 

(2 0) (5 0) 

6 (12%) 

1 (2%) 

(2 0) (50) 

1 (5%) 

(20) (50) 

 EXAMINED MICROSCOPICALLY 

HIGH DOSE 

01-F069 

(48) 

(48) 

(50) 

(49) 

3 (6%) 

(49) 

(50) 

1 (2%) 

(49) 

(50) 

(50) 

1 (2%) 

C-9 
TABLE C2 (CONCLUDED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-F065 01-F068 01-F069 

•MESENTERY (20) (50) (50) 

PERIARTERITIS 1 <5X) 1 (2%) 

ALL OTHIR SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 3 5 

C-IO 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH ENDOSULFAN
 

TABLE Dl 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE TREATED WITH FNDOSULFAN 

CONTROL (VEH) LOW DOSE 

02-M072 02-M073 

ANIMALS INITIALLY IN STUDY 20 5 0 

ANIMALS MISSING 1 

ANIMALS NECROPSIED 20 49 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 20 49 

INTEGUMENTARY SYSTEM 

*SKIN (2 0) (49) 

EPIDERMAL INCLUSION CYST 1 (5%) 1 (2%) INFLAMMATION, NOS 1 (2%) 

*SUBCUT TISSUE (20) (49) ABSCESS, NOS 2 (4%) 

RESPIRATORY SYSTEM 

#LUNG (20) (49) 

PNEUMONIA, CHRONIC MURINE 2 (10») 3 (bX) 

HEMATOPOIETIC SYSTEM 

#SPLEEN (20) (49) 

AMYLOIDOSIS 4 (20%) 2 (4%) HEMATOPOIESIS 1 (5%) 2 (4%) 

#MESLNTERIC L. NODE (20) (47) 

INFLAMMATION, NOS 1 (2%) 

CIRCULATORY SYSTEM 

#HEART (20) (49)

 

THROMBUS, ORGANIZED 

PERIARTERITIS 

#MYOCARDIUM (20) (49) 

. INFLAMMATION^. NOS. _ _ _ 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE 

02-M074 

50 

50 

50 

(50) 

2 (4%) 

(50) 

(50) 

2 (4%)

 

(50) 

3 (6%)

 

(49) 

1 (2%)

 

(50) 

2 (4%)

 

1 (2%) 

(50) 

1 L2%L _ 

D-3 
TABLE Dl (CONTINUED)
 

CONTROL (VEH) LOW DOSE 

02-H072 02-M073 

DIGESTIVE SYSTEM 

#LIVEB (20) (49) 

AMYLOIDOSIS 1 (5%) HYPERPLASIA, NODULAR 1 (5%) ANGIECTASIS 1 (2%) 

# PANCREAS (20) (49)

 

CYST, NOS 1 (5X)

 

ATROPHY, NOS 1 (5%)

 

«COLON (20) (48)

 

NEMATODIASIS 1 (5%)

 

URINARY SYSTEM 

#KIDNEY (20) (4 9) 

HYDRONEPHROSIS 12 (24%) 

CYST, NOS 3 (6%) 

PYELONEPHRITIS, NOS 2 (4%) 

INFLAMMATION, NOS 

INFLAMMATION, CHRONIC 12 (60%) 30 (61%) 

AMYLOIDOSIS 3 (15%) 3 (6%) 

CALCIUM DEPOSIT 

#URINARY BLADDER (20) (48)

 

CALCULUS, NOS 

INFLAMMATION, NOS 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

*PENIS (20) (49) 

PHIMOSIS 1 (2%) 

•PREPUTIAL GLAND (2 0) (49) 

INFLAMMATION, NOS 1 (2%) 

#PROSTATE (20) (45) 

INFLAMMATION- NOS 1 (2%) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

02-M074 

(50) 

1

(*9) 

(48) 

1

(50) 

1

2

1

27

4

1

(49) 

1

1

(50) 

(50) 

(49) 

 (2%) 

 (2%) 

 (2%) 

 (4%) 

 (2%) 

 (54%) 

 (8%) 

 (2%) 

 (2%) 

{2%) 

D-4
 
#TESTIS

 

INFLAMMATION, CHRONIC 

ATROPHY, NOS 

•EPIDIDYMIS 

GRANULOMA, SPERMATIC 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE OBGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

•MULTIPLE ORGANS 

AMYLOIDOSIS 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 

ANIMAL MISSING/NO NECROPSY PERF 

AUTO/NECROPSY PERF/HISTO PERF 

TABLE Dl (CONCLUDED) 

CONTROL (VEH) LOU DOSE HIGH DOSE 

02-M072 02-M073 02-M074 

(20) (49) (50) 

1 (2X) 

2 (4%) 3 (6%) 

(20) (49) (50) 

1 (2%) 

(20) (49) (50) 

6 (30%) 24 (49%) 24 (48%) 

1 8 11

 

1 

2 3 5

 

D-5 
TABLE D2
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE TREATED WITH ENDOSULFAN
 

CONTROL(VEH) 

02-F072 

LOW DOSE 

02-F075 

HIGH DOSE 

02-F076 

ANIMALS

ANIMALS

ANIMALS

 INITIALLY IN STUDY 

 NECROPSIED 

 EXAMINED HISTOPATHOIOG ICALLY**

20 

20 

 20 

5 0 

50 

5 0 

50 

5 0 

50 

INTEGUMENT Αί Υ SYSTEM 

NONE 

RESPIRATORY SYSTEM 

#LUNG 

PNEUMONIA, CHRONIC MURINE 

(20) (50) 

1 (2X) 

(50) 

1 (2%) 

HEMATOPOIETIC SYSTEM 

#CERVICAL LYMPH NODE 

HYPERPLASIA, NOS 

(20) (50) (50) 

1 (2%) 

#M£S£NTERIC L. NODE 

ANGIECTASIS 

(20) (50) 

2 ((IX) 

(50) 

1 (2%) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

#LIVER 

THROMBUS, ORGANIZED 

INFLAMMATION, NOS 

CALCIUM DEPOSIT 

HYPERPLASIA, NODULAR 

ANGIECTASIS 

HYPERPLASIA, LYMPHOID 

(20) 

2 (10%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

2 (4%) 

# PANCREAS 

INFLAMMATION, NOS 

DEGENERATION, NOS 

ATROPHY. NOS 

(19) 

1 (5%) 

(50) 

1 (2*) 

1 12%) 

(50) 

# NUMBER OF ANIMALS WITH TISSU E EXAMINED

* NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

D-6
 
TABLE D2 (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 02-F072 02-F075 02-F076 

#PANCREATIC DUCT (19) (50) (50) DILATATION, NOS 1 (2%) 

#STOMACH (20) (50) (50) 

HYPERKERATOSIS 1 (5%) 1 (2%) 

ACANTHOSIS 1 (5%) 1 (2%) 

#ILEUM (20) (50) (50) 

HYPERPLASIA, NOS 2 (4%) 

URINARY SYSTEM 

#KIDNEY (20) (5 0) (50) 

HYDRONEPHROSIS 2 (4*) 4 (8%) 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

#UTERUS (20) (50) (50) 

HYDROMETfiA 9 (18%) 7 (14% 

INFLAMMATION, NOS 3 (15%) 13 (26%) 13 (26% 

tUTERUS/ENDOMETRIUM (20) (50) (50) 

HYPERPLASIA, CYSTIC 8 (40%) 15 (30%) 20 (40% 

#OVARY (20) (49) (50) 

CYST, NOS 3 (15%) 11 (22%) 17 (34% 

INFLAMMATION, NOS 3 (15%) 12 (24%) 12 (24% 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

«HARDERIAN GLAND (2 0) (50) (50) 

HYPERPLASIA,, NOS 1 (2%) 1 12%) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-7 
TABLE D2 (CONCLUDED) 

CONTROL (VEH) 

02-F072 

LOH DOSE 

02-F075 

HIGH DOSE 

O2-F076 

MUSCULOSKELETAL

NONE 

 SYSTEM 

BODY CAVITIES 

•PERITONEUM 

INFLAMMATION, NOS 

(20) 

2 (10%) 

(50) (50) 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MOBPHOLOGY SUMMARY 

NO LESION REPORTED 

AUTO/NECROPSY PERF/HISTO PERF 

3 5 3 

1 

D-8 
Review of the Bioassay of Endosulfan* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

January 18, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976 under the authority of the National
 

Cancer Act of 1971 (P.L. 92-218). The purpose of the Clear­ inghouse is to advise on the National Cancer Institute's
 

bioassay program to identify and evaluate chemical carcino­ gens in the environment to which humans may be exposed. The
 

members of the Clearinghouse have been drawn from acadernia5
 

industry3 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 organic chemistry, bio­ statistics, biochemistry, 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 NCI bioasL
 

reports on chemicals studied for carcinogenicity. In this
 

context, below is the edited excerpt from the minutes of
 

the SubgroupTs meeting at which Endosulfan was reviewed.
 

After briefly describing the conditions of test, the
 

primary reviewer noted the high mortality in both the treats
 

male rats and mice, as well as among the control male mice.
 

However, the female rats and mice survived sufficiently
 

long to conclude that no carcinogenic effect was produced
 

by Endosulfan. The secondary reviewer concurred with this
 

critique.
 

A question was raised as to whether Endosulfan should
 

be considered for retest because of the high mortality
 

among the male animals. There was some uncertainty as to
 

the present extent of use of Endosulfan. After additional
 

discussion, the Subgroup recommended that Endosulfan be
 

considered by the Chemical Selection Working Group to
 

determine whether, under present selection criteria, it
 

warrants further study.
 

It was moved that the report be accepted as written.
 The motion was seconded and approved unanimously.
 

53
 
Members Present Were:
 

Arnold Brown (Acting Chairman), Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Charles Kensler, Arthur D. Little Company
 

Verald K. Rowe, Dow Chemical, U.S.A.
 

Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Louise Strong, University of Texas Health Sciences Center
 

Sidney Wolfe, Health Research Group
 

*	 Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the
 

review or other reasons. Thus, certain comments and
 

criticisms reflected in the review may no longer be
 

appropriate.
 

*U.S. GOVERNMENT PRINTING OFFICE: 1978 260-899/3058 1-3 54 


DHEW Publication (NIH) No. 78-1312