National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 8 

1977 

BIOASSAY OF 

CHLORDANE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 57-74-9 

NCI-CG-TR-8 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

CHLORDANE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogen Bioassay and Program Resources Branch
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

DHEW Publication No. (NIH) 77-808
 

BIOASSAY OF
 

CHLORDANE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Program, Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

CONTRIBUTORS; This report presents the results of the bioassay
 

of chlordane for possible carcinogenicity, conducted by the
 

Carcinogen Bioassay and Program Resources Branch, Carcinogenesis
 

Program, Division of Cancer Cause and Prevention, National Cancer
 

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

at Gulf South Research Institute, New Iberia, Louisiana,
 

initially under direct contract to the NCI and currently under a
 

subcontract to Tracer Jitco, Incorporated, prime contractor for
 

the NCI carcinogen bioassay program.
 

The experimental design was determined by Drs. J. H.
 

Weisburgerl>2 and R. R. Bates^. The doses were selected by Drs.
 

T. E. Shellenburger3'^, j. H. Weisburger and R. R. Bates. Animal
 

treatment and observations were supervised by Drs. T. E.
 

jhellenburger and H. P. Burchfield3, with the technical
 

•assistance of Ms. D. H. Monceaux-* and Mr. D. Broussard3.
 

Necropsies were performed under the supervision of Drs. E.
 

Vernal3 and B. Buratto3. The histopathologic evaluation was
 

conducted at Experimental Pathology Laboratories by Dr. R. A.
 

Renne^»9 and or. j. p. Ferrell^, and the diagnoses included in
 

:his report represent the interpretation of these pathologists.
 

?athologists from Tracer Jitco and NCI reviewed selected slides
 

and concurred with the overall pathologic evaluation of the
 

:5tudy.
 

Compilation of individual animal survival and summary tables was
 

performed by EG&G Mason Research Institute?; pathology tables
 

iii
 
were prepared by Dr. R. A. Renne; and statistical analyses were
 

performed by Mr. J. Nam^ and Dr. J. J. Gart^.
 

This report was prepared at Tracor Jitco under the direction of
 

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

toxicologists, Drs. W. E. MacDonald and J. F. Robens; the
 

chemist, Dr. S. S. Olin; the technical editor, Dr. E. W. Gunberg;
 

and the technical writers, Dr. G. L. Miller, Mr. W. E. Reichardt,
 

and Ms. Y. E. Presley. The final report was reviewed by members


 1 S A
 of the participating organizations11

'J> -'» °.
 

^Carcinogenesis Program, Division of Cancer Cause and Prevention,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

^Now with the Naylor Dana Institite for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla, New
 

York.
 

^Gulf South Research Institute, Atchafalaya Basin Laboratories,
 

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

with the National Center for Toxicological Research,
 

Jefferson, Arkansas.
 

^Experimental Pathology Laboratories (EPL), 17 Pine Street,
 

Herndon, Virginia.
 

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

Rockville, Maryland.
 

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

Rockville, Maryland.
 

^Mathematical Statistics and Applied Mathematics Section,
 

Field Studies and Statistics, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

%ow with Battelle Pacific Northwest Laboratories,
 

Battelle Boulevard, Richland, Washington.
 

iv
 
SUMMARY
 

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

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

sex were administered low or high concentrations of the chlordane
 

for 80 weeks, then observed for 29 weeks. Because of toxic
 

effects, doses were reduced for both male and female rats during
 

the course of the tests. Time-weighted average doses used for 

the male rats were 203.5 and 407.0 ppm; for the females, 120.8 

and 241.5 ppm. Matched controls consisted of groups of 10 

untreated rats of each sex; pooled controls consisted of the
 

matched-control groups combined with 50 untreated male and 50
 

untreated female rats from similar bioassays of five other
 

compounds. All surviving rats were killed at 109 weeks.
 

Groups of 50 mice of each sex were administered the test material
 

at low or high concentrations for 80 weeks, then observed for 10
 

weeks. The low- and high-dose groups were tested at different
 

calendar times, but each of the treated groups was tested along
 

with a concurrent control. Because of toxic effects, doses were
 

reduced for female mice during the course of the tests; however,
 

it was possible to increase the doses for the male mice. The
 

time-weighted average doses used for the male mice were 29.9 and
 

56.2 ppm; for the females, 30.1 and 63.8 ppm. Matched controls
 

consisted of groups of 10 untreated mice of each sex; pooled
 

controls consisted of the matched-control groups combined with 70
 

untreated male and 80 untreated female mice from similar
 

bioassays of five other compounds. All surviving mice were
 

killed at 90-91 weeks.
 

The effects of chlordane on body weights and other clinical signs
 

in rats and mice indicated that the dosages used were near the
 

maximum permissible. This was evident in that the average body
 

weights of the high-dose male and female rats were consistently
 

lower than those of the untreated controls, while differences
 

between the low-dose and control rats were negligible. Body
 

weights of mice given either low or high doses showed little or
 

no effect of the chlordane; however, other adverse clinical signs
 

were seen with greater frequency in treated than in control mice.
 
The effects of chlordane on survival rates indicated that
 

mortality was dose-related for female rats and for male mice.
 

However, a substantial proportion of most groups of animals
 

survived to an age at which tumors could be expected to appear;
 

male control rats, for unknown reasons, showed an abnormally low
 

survival rate.
 

Hepatocellular carcinoma showed a highly significant dose-related
 

trend for mice, using either matched controls (for males,
 

controls 2/18, low dose 16/48, high dose 43/49, P < 0.0001; for
 

females, controls 0/19, low dose 3/47, high dose 34/49, P <
 

0.0001) or pooled controls (for males, controls 17/92, P <
 

0.0001; for females, controls 3/78, P < 0.0001). These high
 

levels of significance were maintained when hepatocellular
 

carcinoma was combined with nodular hyperplasia or when the data
 

were subjected to life-table adjustment. No other tumors were
 

found in mice in sufficient numbers to justify analysis.
 

In contrast to findings with mice, hepatocellular carcinoma
 

failed to appear at a significant rate of incidence in rats
 

administered chlordane. Further, the number of lesions of the
 

liver in rats did not become significant with the addition of
 

nodular neoplasia or with the application of life-table
 

adjustment to the data.
 

There was significant statistical evidence for the induction in
 

treated male rats of proliferative lesions of follicular cells of
 

the thyroid and of malignant fibrous histiocytoma, but these
 

findings were discounted because the rates of incidence were
 

comparatively low and/or are known to be variable in control rat
 

populations.
 

It is concluded that under the conditions of this bioassay
 

chlordane is carcinogenic for the liver in mice.
 

vi
 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 5
 

A. Chemical 5
 

B. Dietary Preparation 6
 

C. Animals 7
 

D. Animal Maintenance 7
 

E. Subchronic Studies 8
 

F. Design of Chronic Studies 10
 

G. Clinical and Pathologic Examinations 14
 

H. Data Recording and Statistical Analyses .... 15
 

III. Results - Rats 21
 

A. Body Weights and Clinical Signs (Rats) .... 21
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 24
 

D. Statistical Analyses of Results (Rats) .... 32
 

IV. Results - Mice 37
 

A. Body Weights and Clinical Signs (Mice) .... 37
 

B. Survival (Mice) 37
 

C. Pathology (Mice) 42
 

D. Statistical Analyses of Results (Mice) .... 44
 

V. Discussion 47
 

VI. Bibliography 51
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms and
 

Other Proliferative Lesions in Rats Fed
 

Chlordane in the Diet 55
 

Table Al Summary of the Disposition of Tissues 57
 

Table A2 Proliferative Lesions of the Thyroid 58
 

Table A3 Other Endocrine Neoplasms 59
 

vii
 
Page 

Table AA Primary Hepatic Neoplasms 60 

Table A5 Primary Renal Neoplasms 61 

Table A6 Neoplasms of the Reproductive System 62 

Table A7 Neoplasms of the Mammary Gland 63 

Table A8 Miscellaneous Neoplasms 64 

Appendix B Summary of the Incidence of Neoplasms and 

Other Proliferative Lesions in Mice Fed 

Chlordane in the Diet 67 

Table Bl Summary of the Disposition of Tissues 69 

Table B2 Proliferative Hepatic Lesions 70 

Table B3 Miscellaneous Neoplasms 71 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Chlordane in the Diet. . . 73 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Chlordane 

in the Diet 75 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Chlordane 

in the Diet 80 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Chlordane 

in the Diet 85 

Table Dl Summmary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Chlordane 

in the Diet 87 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Chlordane 

in the Diet 91 

Appendix E Statistical Analyses of Neoplasms 

in Rats Fed Chlordane in the Diet 93 

viii 
Page 

Table El Statistical Analysis of Neoplasms 

of the Liver in Rats Fed Chlordane 

in the Diet (using matched control) 95 

Table E2 Statistical Analysis of Neoplasms 

of the Liver in Rats Fed Chlordane 

in the Diet (using pooled control) 96 

Table E3 Statistical Analysis of Neoplasms of Thyroid 

Follicular Cells in Rats Fed Chlordane 

in the Diet (using matched control) 97 

Table E4 Statistical Analysis of Neoplasms of Thyroid 

Follicular Cells in Rats Fed Chlordane 

in the Diet (using pooled control) 98 

Table E5 Statistical Analysis of Neoplasms of 

Thyroid C-cells in Rats Fed Chlordane 

in the Diet (using matched control). ..... 99 

Table E6 Statistical Analysis of Neoplasms of 

Thyroid C-cells in Rats Fed Chlordane 

in the Diet (using pooled control) 100 

Table E7 Statistical Analysis of Malignant 

Fibrous Histiocytoma in Rats Fed 

Chlordane in the Diet (using matched 

control) 101 

Table E8 Statistical Analysis of Malignant 

Fibrous Histiocytoma in Rats Fed 

Chlordane in the Diet (using pooled 

control) 102 

Table E9 Statistical Analysis of Neoplasms of the 

Pituitary Gland in Rats Fed Chlordane 

in the Diet (using matched control) 103 

Table E10 Statistical Analysis of Neoplasms of the 

Pituitary Gland in Rats Fed Chlordane 

in the Diet (using pooled control) 104 

Table Ell Statistical Analysis of Neoplasms of the 

Mammary Gland in Female Rats Fed 

Chlordane in the Diet (using matched 

control) 105 

ix
 
Page 

Table E12 Statistical Analysis of Neoplasms of the 

Mammary Gland in Female Rats Fed 

Chlordane in the Diet (using pooled 

control) 106 

Table E13 Statistical Analysis of Neoplasms of the 

Uterus in Female Rats Fed Chlordane 

in the Diet (using matched control) 107 

Table E14 Statistical Analysis of Neoplasms of the 

Uterus in Female Rats Fed Chlordane 

in the Diet (using pooled control) 108 

Appendix F Statistical Analyses of Neoplasms in Mice 

Fed Chlordane in the Diet 109 

Table Fl Statistical Analysis of Neoplasms of the 

Liver in Male Mice Fed Chlordane 

in the Diet (using matched control) Ill 

Table F2 Statistical Analysis of Neoplasms of the 

Liver in Male Mice Fed Chlordane 

in the Diet (using pooled control) 112 

Table F3 Statistical Analysis of Neoplasms of the 

Liver in Female Mice Fed Chlordane 

in the Diet (using matched control) 113 

Table F4 Statistical Analysis of Neoplasms of the 

Liver in Female Mice Fed Chlordane 

in the Diet (using pooled control) HA 

Appendix G Analysis of Formulated Diets For 

Concentration of Chlordane 115 

TABLES 

Table 1 Design of Chlordane Chronic Feeding Study 

in Rats 11 

Table 2 Design of Chlardane Chronic Feeding Study 

in Mice 12 

x
 
Page 

FIGURES 

Figure 1 Growth Curves for Rats Fed Chlordane 

in the Diet 22 

Figure 2 Survival Curves for Rats Fed Chlordane 

in the Diet 23 

Figure 3 Growth Curves for Mice Fed Chlordane 

in the Diet 38 

Figure 4 Growth Curves for Female Mice Fed Chlordane 

in the Diet 39 

Figure 5 Survival Curves for Male Mice Fed Chlordane 

in the Diet 40 

Figure 6 Survival Curves for Female Mice Fed Chlordane 

in the Diet 41 

xi
 

I. INTRODUCTION
 

Chlordane is a member of the cyclodiene group of chlorinated
 

insecticides, which includes aldrin, dieldrin, endrin,
 

heptachlor, and endosulfan. It was introduced in 1945 and was
 

the first chlorinated cyclodiene developed for insect control
 

(Chlordane Formulation Guide, 1962). It is effective on a wide
 

variety of insects of agricultural, industrial, and domestic
 

importance. The compound was registered for use on more than 40
 

vegetable and 27 fruit crops. About a third of the amount used
 

in the United States is applied to pests of the home, garden
 

lawn, and turf (EPA, 1976).
 

On November 18, 1974, the EPA issued an order of intent to cancel
 

the registration and use of pesticides containing chlordane,
 

except for subsurface ground insertion against termites and for
 

dipping of nonfood plants (EPA, 1974). On December 24, 1975, the
 

EPA issued an order that suspended the registered uses of
 

products containing chlordane against ticks and chiggers, against
 

pests of home, garden, lawn, and turf, as an ingredient in shelf
 

paper, and (effective August 1, 1976) as a control against
 

cutworms on corn (EPA, 1976). Exempted from this order were the
 

use of chlordane to control root weevils infecting Florida citrus
 

crops, and certain other uses.
 
Chlordane persists in the environment, lasting 10 years or more
 

in treated soils (EPA, 1974). Translocation to untreated areas
 

occurs through soil erosion and dusting. Plants grown in treated
 

soil, particularly root crops, also may absorb chlordane residues
 

and thus enter the food chain.
 

Estimates of human exposure through the diet, based on Food and
 

Drug Administration (FDA) market basket surveys conducted from
 

June 1968 through April 1970, showed only trace amounts of
 

chlordane in a few root crops, such as potatoes, which
 

contributed less than 0.001 mg chlordane per person per day
 

(Duggan and Corneliussen, 1972). The FAO/WHO (1968) tentative
 

acceptable daily intake of chlordane in man is 0.001 mg/kg/day, a
 

value considerably higher than the actual human exposure reported
 

by the FDA. Exposure may also occur with persons applying
 

chlordane and with workers producing the compound and its
 

formulations.
 

In 1969 the National Cancer Institute (NCI) was requested to
 

review the biological and environmental data on pesticides by the
 

Secretary's Commission on Pesticides and Their Relationship to
 

Environmental Health, a committee of the Department of Health,
 

Education and Welfare. This review (Secretary's Commission on
 

Pesticides, 1969) pointed out the need for an evaluation of the
 

carcinogenicity of several pesticides, including certain members
 
of the cyclodiene group. Because of known biological effects of
 

low doses of chlordane over extended periods of time (Ingle,
 

1965), the persistence of the compound in the environment, and
 

the probability of continued human exposure, this insecticide was
 

selected by the NCI for inclusion in the carcinogen bioassay
 

program.
 

II. MATERIALS AND METHODS
 

A. Chemical
 

The material tested was analytical-grade chlordane obtained from
 

Velsicol Chemical Corporation. The Velsicol preparation is a
 

developmental product and is considerably purer than the
 

commercial technical grade. According to the manufacturer's
 

analysis, the batch used for the bioassay contained 94.8%
 

chlordane (71.7% alpha- or cis-chlordane and 23.1% gamma- or
 

trans-chlordane), 0.3% heptachlor, 0.6% nonachlor, 1.1% hexachlorocyclopentadiene, 0.25% chlordene isomers, and other
 

chlorinated compounds. Chemically, chlordane is the product of
 

the addition of chlorine to chlordene; chlordene is the Diels-


Alder product of hexachlorocyclopentadienol and cyclopentadiene.
 

Under the conditions employed for gas chromatography at Gulf
 

South Research Institute, two major peaks were observed, probably
 

corresponding to alpha- and gamma-chlordane. The analyses did
 

not, however, detect the minor components, such as heptachlor and
 

nonachlor. From the mass spectrum, the group of parent ions
 

around m/e 410 was consistent with the molecular formula of
 

chlordane. The elemental analysis was correct for chlordane.
 

Other physical, spectral, and chromatographic data were
 

consistent with the manufacturer's analysis.
 
The chlordane used for the tests was stored in a refrigerator.
 

B. Dietary Preparation
 

All diets were formulated using Wayne^ Lab Blox Meal (Allied
 

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

amount of chlordane for each dietary concentration. The test
 

compound was first dissolved in a small amount of acetone
 

(Mallinckrodt Chemical Works, St. Louis, Mo.), which was added to
 

the feed. Corn oil equal to 2% of the final feed weight was then
 

added, primarily as a dust suppressant. Diets for control
 

animals were the same as those for treated animals except for the
 

absence of chlordane. The diets were mixed mechanically to
 

assure homogeneity and to allow the evaporation of the acetone.
 

The corn oil (Louana®) was produced by Opelousas Refinery Co.,
 

Opelousas, Louisiana. The prepared diets were analyzed regularly
 

throughout the test to assure uniformity. Water and the
 

formulated diets were made available ad libitum to the
 

experimental animals and were replaced three times per week.
 

The stability of chlordane in feed was checked by analyzing
 

formulated diets for the concentration of chlordane at intervals
 

over a 7-day period. Diets containing 30 and 100 ppm chlordane
 

showed no change on standing at ambient temperature for this
 

period.
 
Theoretical chlordane concentrations in formulated diets were
 

checked analytically at intervals during the chronic study to
 

assess the accuracy of the diet preparation and the homogeneity
 

of the mixtures. Results are summarized in Appendix G. At each
 

dietary level from 30 to 800 ppm, the mean of the analytical
 

concentrations for the samples checked was within 5% of the
 

theoretical concentration, and the coefficient of variation was
 

never more than 5.6%. Thus, the evidence indicates that the
 

formulated diets were accurately prepared and were homogeneous.
 

C. Animals
 

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

Division of Cancer Treatment, NCI, were used in these tests. The
 

rats were the Osborne-Mendel strain obtained from Battelle
 

Memorial Institute, Columbus, Ohio, and the mice were B6C3F1
 

hybrids obtained from Charles River Breeding Laboratories, Inc.,
 

Wilmington, Mass. Upon arrival at the laboratory, all animals
 

were quarantined for 14 days, as a laboratory acclimation period,
 

and then assigned to treatment and control groups.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. Incoming air was filtered through fiberglass air
 

conditioner filters that were changed monthly. The total air in
 
each room was changed 10-12 times per hour. Fluorescent lighting
 

provided illumination 10 hours per day. The rats were housed
 

individually in suspended wire cages; the mice, in plastic cages
 

equipped with filter caps. Female mice were housed five per cage
 

and males two or three per cage. Initially, rats were
 

transferred weekly to clean cages; later in the study, clean
 

cages were provided biweekly. Mice were transferred weekly to
 

clean cages with filter bonnets. Fresh bedding (Absorb-Dri®, Lab
 

Products) was provided two times a week for male mice and three
 

times a week for females. Food (Wayne&) Lab Blox Meal) and water
 

were consumed ad libitum. Feeder jars and water bottles were
 

changed and sterilized three times per week. Animal racks were
 

rotated laterally for both species at weekly intervals. Rats
 

receiving chlordane, along with their matched controls, were
 

housed in a room by themselves. Mice being treated with
 

chlordane were maintained in a room housing other mice being
 

treated with toxaphene, heptachlor, and chlordecone. Cages for
 

control and treated mice were placed on separate racks in the
 

same room.
 

E. Subchronic Studies
 

Feeding studies were conducted with rats and mice to estimate
 

maximum tolerated doses of chlordane, on the basis of which high
 

and low concentrations (hereinafter referred to as "high doses"
 
and "low doses") were determined for administration in the
 

chronic studies. The low doses given in the chronic studies were
 

1/2 the high doses. In the subchronic studies, chlordane was
 

provided in feed to groups of five male and five female rats and
 

mice for 6 weeks, followed by a 2-week period of observation.
 

Twofold increasing concentrations from 50 to 1,600 ppm were used
 

for male rats; 100 to 1,600 ppm, for females. Concentrations
 

from 20 to 640 ppra were used for both male and female mice.
 

Weights of animals and consumption of feed were measured weekly,
 

and deaths were noted.
 

All male rats fed 1,600 ppm died during the second week of the
 

study, while those fed 800 ppm showed no deaths and their gain in
 

weight and consumption of feed were low only for the first week
 

of treatment. Four of five female rats died, however, when given
 

800 ppm. When given 400 ppm or less, the gain in weight and
 

consumption of feed in the treated females were generally similar
 

to those of the controls. Based on these findings, low and high
 

doses of 400 and 800 ppm, respectively, were selected for the
 

chronic studies in the male rats; 200 and 400 ppm, for the
 

females.
 

With mice fed 320 ppm, two of five males and all females died; at
 

160 ppm again two of five males died, but no females; at 80 ppm,
 

no males or females died. Gain in weight and consumption of
 
feed, however, were unaffected in either male or female mice by
 

any of the doses tested. Based on the mortality data, concentrations of 40 and 80 ppm were selected as low and high doses for
 

the chronic studies in male mice; 80 and 160 ppm, for the
 

females.
 

F. Design^ of_ Chronic Studies
 

Designs of the chronic studies with rats and mice, including test
 

and matched control groups, are presented in tables 1 and 2.
 

When tests with the rats were initiated at doses indicated by the
 

subchronic studies, toxic effects were observed; doses were,
 

therefore, reduced during the course of the tests. When tests
 

with the mice were initiated at doses indicated by the subchronic
 

studies, high mortality resulted, and the original high-dose
 

groups were discarded at 14 weeks. The 40-ppm males and 80-ppm
 

females then became the high-dose groups, and the study was
 

continued using the original control mice (10 males, 10 females).
 

A new low—dose group was started at 20 ppm for males and 40 ppm
 

for females, along with additional control mice (10 males, 10
 

females). After 1 week, the doses for the males could be
 

increased, while those for the females had to be reduced, due to
 

toxic effects.
 

*
 

Since the matched-control groups for both rats (10 males, 10
 

females) and mice (20 males, 10 females) were small, pooled-


control groups were formed by combining matched controls from
 

10
 
Table 1. Design of Chlordane Chronic Feeding Studies in Rats
 

Sex and
 

Treatment
 

Group
 

MALE
 

Matched-Control
 

Low-Dose
 

High-Dose
 

FEMALE
 

Matched-Control


Low-Dose


High-Dose
 

Initial Chlordane 

No. of in Diet 

Animals3 (ppm) 

10 

50 400 

100 

50 

0 

50 800 

200 

100 

0 

 10 0 

 50 200 

100 

50 

0 

50 400 

200 

100 

0 

Time on Study Time-Weighted
 

Treated Untreated^1 Average Dosesc
 

(weeks) (weeks) (ppm)
 

109
 

33 203.5
 

14
 

33
 

0 29
 

33 407.0
 

14
 

33
 

0 29
 

0 109
 

33 120.8
 

14
 

33
 

0 29
 

33 241.5
 

14
 

33
 

0 29
 

a

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

^When diets containing chlordane were discontinued, treated rats and
 

their matched controls were fed plain feed diets (without corn oil) for
 

12 weeks, then control diets (2% corn oil added) for an additional 17
 

weeks.
 G

Time-weighted average dose = E(dose in ppm x no. of days at that dose)
 

I(no. of days receiving each dose)
 

11
 
Table 2. Design of Chlordane Chronic Feeding Studies in Mice
 

Sex and 

Treatment 

Group 

Initial Chlordane

No. of in Diet

Animals3 (ppm)

 Time on Study Time-Weighted 

 Treated Untreated*5

 Average Dosec 

 (weeks) (weeks) (ppm) 

MALE 

Matched-Control 20C 0 0 91 

Low-Dose 50 20 1 29.9 

30 79 

0 0 10 

High-Dose 50 40 15 56.2 

60 65 

0 0 10 

FEMALE 

Matched-Control 20d 0 0 91 

Low—Dose 50 40 1 30.1 

30 79 

0 0 10 

High-Dose 50 80 15 63.8 

60 65 

0 0 10 

a

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

bwhen diets containing chlordane were discontinued, mice received
 

the control diet (2% corn oil added) until termination.
 c

Time-weighted average dose = £(dose in ppm x no. of days at that dose)
 

Z(no. of days receiving each dose)
 

"Initially 10 animals of each sex were placed on test as matched
 

controls; however, when the study was restarted, 10 additional
 

animals of each sex were placed on test as matched controls.
 

12
 
similar bioassays of other compounds with the matched-controls
 

for chlordane. The periods during which the bioassays of the
 

different compounds were performed overlapped one another for at
 

least a year. For rats, the animals that comprised the
 

pooled-control groups consisted of groups of 10 male and 10
 

female controls taken from tests performed on dieldrin,
 

chlordane, heptachlor, dichlorvos, and dimethoate; this resulted
 

in pooled-control groups containing 60 males and 60 females. For
 

mice, the animals that comprised the pooled-control groups
 

consisted of groups of 20 male and 20 female controls taken from
 

tests performed on dieldrin and chlordane, 20 male and 10 female
 

controls from tests on aldrin and heptachlor, and 10 male and 10
 

female controls from tests on dichlorvos and dimethoate; this
 

resulted in pooled-control groups containing 100 males and 80
 

females. All treated and pooled-control animals were placed on
 

study as weanlings at 35 days of age except for the matched-


control rats for dichlorvos. Because dichlorvos was the last
 

compound of this series to be bioassayed, there were the following slight differences in the dichlorvos matched-control rats
 

that were pooled for use as controls in the chlordane study: (1)
 

half of the animals of each sex were started on test at 43 days
 

of age and half at 36 days of age; (2) they were obtained from
 

the Charles River Breeding Laboratories, Inc. and were the
 

progeny (third generation) of a group of Osborne-Mendel rats
 

13
 
which were purchased from the Battelle Memorial Insitute,
 

Columbus, Ohio. Thus there was probably no significant genetic
 

drift that might influence the incidence of tumors.
 

G- Clinical^and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity,
 

weighed at regular intervals, and palpated for masses at each
 

weighing. Those animals appearing moribund at the time of
 

clinical examination were killed and necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of all major tissues, organs, or gross lesions. The
 

following tissues and organs were taken from killed animals and,
 

where feasible, from animals found dead: skin, mammary gland,
 

brain, pituitary, mandibular nodes, salivary glands, thyroid,
 

parathyroid, trachea, lung, heart, diaphragm, stomach, small
 

intestine, large intestine, pancreas, adrenal, kidney, liver,
 

spleen, urinary bladder, prostate or uterus, testis or ovary, and
 

bone. Tissues were preserved in 10% buffered formalin, embedded
 

in paraffin, sectioned, routinely stained with hematoxylin and
 

eosin, and examined histopathologically. An occasional section
 

was subjected to special staining techniques for more definitive
 

diagnosis.
 

A few tissues were not examined for some animals, particularly
 

14
 
for those that showed early deaths. Also, some animals were
 

missing, cannibalized, 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 lesions 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 for 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, animal weight, and
 

individual pathologic results, as recommended by the
 

International Union Against Cancer (Berenblum, 1969). Data
 

tables were generated for verification of data transcription and
 

for statistical review.
 

Survival curves were computed using standard life-table methods,
 

e.g., Kaplan and Meier (1958) or Armitage (1971). Deaths which
 

were labeled accidental or scheduled sacrifice were excluded from
 

the numerator but not the denominator, i.e., they were treated as
 

censored observations. All other deaths were counted as
 

uncensored observations in the numerator. Statistical tests of
 

15
 
differences between groups were computed using the methods of Cox
 

(1972). When two groups were to be compared, the method
 

explicitly given by Cox was employed. When three groups were
 

compared, an extension by Tarone (1975) of Cox's method was used;
 

this was a test for linear trend in survival rate among the
 

control, low-dose, and high-dose groups. In all instances the P
 

value was given for a one-tailed test. Unless otherwise noted,
 

the P value was given in terms of a positive relation to dose.
 

If there was a significant departure from the linear relation,
 

this was so noted. Combined tests on groups treated at different
 

times were carried out by methods formally equivalent to those of
 

Mantel (1963).
 

The incidence of neoplastic or nonneoplastic lesions is given as
 

the proportion of the number of animals bearing such lesions at a
 

specific anatomic site (numerator) to the number of animals
 

examined pathologically at the site (denominator). For the
 

organs and tissues in which most of the lesions appeared, the
 

denominators included only those animals for which such sites
 

were examined histologically. For tissues that required gross
 

observation for detection of lesions (e.g., skin or mammary
 

tumors), for lesions that appeared at several sites (e.g.,
 

lymphomas), or for tissues that were examined histologically only
 

when lesions were detected grossly, the denominators consisted of
 

the numbers of animals necropsied.
 

16
 
The analysis of tumor incidence took two forms: (a) comparisons
 

of the number of animals with a given tumor as a proportion of
 

those examined for that type; (b) comparisons of the groups with
 

regard to both the number of animals with tumors and the times
 

(in weeks) at which all of the examined animals died.
 

In the first analyses, exact (or conditional) tests for
 

proportions were used as given by Cox (1970). For the comparison
 

of two groups, this is simply the Fisher exact test. When three
 

groups are compared, this is the exact test for a linear trend in
 

4
 

the logistic scale. All tests were one-tailed and, unless
 

otherwise noted, in the direction of a positive relationship to
 

dose. If there was a significant departure from linearity, this
 

was so noted. Combined tests over groups run at different times
 

were also performed using exact methods.
 

For some of the important tumor sites, the exact analysis was
 

applied in an additional way. This analysis eliminated from the
 

denominators for any of the groups being compared all animals
 

which died or were killed at a time before the first animal was
 

found to have a tumor at that site.
 

The second analysis of tumor incidence used life-table methods.
 

Curves of the proportion surviving without tumor being observed
 

were computed using life-table methods (e.g., Saffiotti et al.,
 

1972). The times at which animals were killed were entered as
 

17
 
the last time point of tumor observation. Cox's methods of
 

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

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

All tests of tumor incidence using life-table methods were
 

one-tailed and in the direction of a positive dose relationship
 

unless otherwise noted. If there was a significant departure
 

from linearity this is so noted. Combined tests on groups
 

treated at different times were carried out using methods
 

formally equivalent to Mantel (1963).
 

All P values are given on a per comparison basis rather than on
 

an experiment-wise basis. If the latter is desired, one may
 

utilize the Bonferroni inequality and multiply any given P value
 

by the total number of comparisons of interest to arrive at an
 

experiment-wise P value (Wilks, 1962).
 

Analyses that were applied to the comparisons including pooled
 

controls were similar to those used in the comparisons involving
 

matched controls.
 

The original data were converted by computer program from days on
 

study to weeks on study for the individual animal data. Since
 

the weeks on study were given in whole integers only, it was
 

possible for animals dying or being killed 1 to 4 days apart to
 

be reported as a week apart. These small possible discrepancies
 

were not of major importance in the statistical analyses except
 

18
 
when deaths of a large number of animals were involved in a given
 

week. This could happen at the termination of the test. The
 

animals in one dose group could be sacrificed 4 days later than
 

the others. Thus their time on study differs by 1 week. These
 

were all grouped to one time interval in the statistical tests.
 

19
 

III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

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

consistently lower than those of the untreated controls, while
 

differences between low-dose rats and the controls were slight or
 

absent (figure 1).
 

During the first year of study, the appearance and behavior of
 

the treated rats were generally comparable to those of the
 

control rats. At week 44 a majority of the high-dose females had
 

generalized tremors along with loss of weight, but the tremors
 

were not noted again. Adverse clinical signs in all treated
 

groups occurred frequently during the first year of study and
 

gradually increased in frequency during the second year. These
 

signs included loss of weight, rough and discolored hair coats,
 

palpable masses, and in some cases multiple tumors. Surviving
 

rats at the termination of the study (109 weeks) were generally
 

in a poor physical condition.
 

B. Survival - (Rats)
 

Curves showing the probability of survival of treated and control
 

rats are shown in figure 2. For male rats there was no
 

indication of a significant difference in survivorship among the
 

21
 
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Figure 1. Growth Curves for Rats Fed
 

Chlordane in the Diet
 

22
 
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three groups. For the female rats there was a highly significant
 

dose-related trend (P - 0.003) in mortality from other than
 

accidental kills or scheduled sacrifice.
 

C. Pathology (Rats)
 

Uistopathologic findings are summarized in Appendix A, tables
 

A1-A8, covering neoplasms and other proliferative lesions, and in
 

Appendix C, tables Cl and C2, covering nonneoplastic lesions.
 

Numerous inflammatory, degenerative, and proliferative lesions
 

commonly seen in aged rats occurred with approximately equal
 

frequency in drug-treated and control animals. These included
 

aggregates of alveolar macrophages in the lungs, pericholangitis
 

and biliary hyperplasia, chronic nephritis with tubular
 

dilatation and epithelial hyperplasia of the renal pelvis,
 

chronic cystitis with varying degrees of hyperplasia of urinary
 

vesical epithelium, subacute to chronic prostatitis, and atrophy
 

of seminiferous epithelium of the testes.
 

Table A2 summarizes the incidence of proliferative lesions of the
 

thyroid gland. Both follicular-cell and C-cell lesions were
 

observed. Areas of follicular-cell hyperplasia were characterized by follicles with larger lumens than normal, lined by follicular epithelial cells which were either stratified to form an
 

irregular epithelial border several cells thick or projecting
 
into the lumen by papillary infolding of simple cuboidal or
 

columnar epithelial cells which were larger and had more
 

basophilic cytoplasm than the surrounding normal thyroid
 

follicular cells. Compression of adjacent tissue was minimal
 

other than that caused by distention of the follicular lumen.
 

Follicular architecture (size and shape of lumen, thickness of
 

the layer of follicular cells, and amount of stroma) was
 

heterogenous and resembled somewhat the adjacent normal thyroid.
 

As a result, the lesion blended in gradually with the adjacent
 

normal tissue, especially when compression was minimal. There
 

was no evidence of encapsulation by connective tissue. In some
 

cases, multiple foci of hyperplasia were present.
 

In comparison with those lesions classified as hyperplastic, the
 

follicular-cell adenomas of the thyroid (Bloodworth, 1968;
 

Meissner and Warren, 1968) characteristically were more
 

distinctly circumscribed, had some evidence of encapsulation by
 

connective tissue, and had a follicular architecture more
 

distinct from adjacent normal thyroid parenchyma. Follicularcell adenomas were usually single.
 

Follicular-cell carcinomas were characterized by a variable
 

mixture of solid masses of follicular cells and numerous closely
 

packed small follicles. In both patterns the follicular cells
 

were basophilic, and hyperchromatic nuclei and mitotic figures
 

25
 
were numerous. Evidence of connective tissue and/or inflammatory
 

response and necrosis was often present. Although some
 

carcinomas were large enough to be noted at necropsy, others were
 

noted only on microscopic examination. Metastasis was not
 

observed.
 

Proliferative C-cell lesions presented a spectrum of histologic
 

characteristics ranging from very minimal increases in the number
 

of C-cells interspersed among normal thyroid follicles, with no
 

compression or distortion of follicular architecture, to grossly
 

visible tumors that invaded adjacent tissue. This spectrum of
 

lesions was easy to classify at each end (minimal hyperplasia and
 

overt carcinoma), but differentiation between severe hyperplasia
 

and adenoma and between adenoma and carcinoma was difficult. The
 

following criteria were used in classifying proliferative lesions
 

of C-cells in these rats:
 

Hyperplasia
 

(1)	 Increased numbers of C-cells are interspersed among
 

follicles but are not compressing follicles or
 

distorting follicular architecture.
 

(2)	 Proliferating C-cells are polyhedral to spherical, with
 

pale eosinophilic cytoplasm and spherical nucleus,
 

usually centrally located.
 

Adenoma
 

(1)	 A discrete mass of C-cells widely separates follicles,
 

although some isolated single follicles may be present
 

within the mass.
 

26
 
(2)	 Spindling or lengthening of C-cells to form interlacing
 

bundles may occur.
 

(3)	 There may be an increase in basophilia of C-cells.
 

(4)	 Invasion of thyroid capsule, adjacent tissue, or
 

lymphatics is lacking.
 

(5) Encapsulation is rare.
 

Carcinoma
 

(1)	 There is invasion of thyroid capsule, adjacent tissue,
 

or vessels.
 

(2)	 There may be metastasis.
 

(3)	 Spindling and increased basophilia of neoplastic C-cells
 

are usually prominent features in carcinoma, but are not
 

necessary for diagnosis of malignancy.
 

(4)	 Increased mitotic activity is rare.
 

Although no metastasis of C-cell carcinoma was observed in rats
 

from this study, metastatic C-cell carcinomas have been found in
 

rats of the same strain (Osborne-Mendel) from other similar
 

studies.
 

The incidence of other endocrine neoplasms is summarized in Table
 

A3. Pituitary adenomas occurred frequently in all groups,
 

whereas neoplasms of the adrenal gland and pancreatic islets
 

occurred infrequently.
 

Table A4 summarizes the incidence of primary hepatic neoplasms.
 

These included a number of lesions, classified as "neoplastic
 

nodules," which consisted of nodules of enlarged hepatocytes
 

27
 
compressing adjacent tissue, and which were similar in
 

microscopic appearance to nodules produced experimentally in rat
 

livers by known carcinogens. Such nodules have recently been
 

defined morphologically and given the designation of neoplastic
 

nodules (Squire and Levitt, 1975); as such, they have been
 

categorized and coded as neoplasms when observed in this study.
 

The incidence of primary renal tumors is summarized in table A5.
 

Those renal neoplasms classified as "malignant mixed tumors"
 

contained neoplastic tissue components having the microscopic
 

appearance of renal mesenchymal stroma, adipose tissue, and
 

primitive renal epithelium, in various proportions.
 

Table A6 summarizes the incidence of neoplasms of the reproductive system. Endometrial stromal polyps represented the most
 

frequently occurring neoplasm of the reproductive tract.
 

Numerous mammary fibroadenomas, some of which were multiple, were
 

observed in both test and control females (table A7).
 

Various other types of neoplasms occurred infrequently in both
 

test and control rats. The incidence of these neoplasms is
 

summarized in table A8. Several tumors occurred in the skin,
 

subcutis, and/or skeletal muscles. With the exception of two
 

obvious carcinomas, one osteosarcoma, and two lipomas, all of
 

these tumors had a somewhat similar microscopic appearance. The
 

proliferating cells in these tumors were apparently histiocytes,
 

28
 
in some cases appearing as a pleomorphic population of round or
 

polyhedral cells having large vesicular nuclei with prominent
 

nucleoli, forming numerous multinucleated giant cells surrounded
 

by varying amounts of stroma. Other tumors in the spectrum
 

appeared as interlacing bundles of oval cells having the
 

appearance of fibroblasts. In some areas, the histologic
 

appearance was similar to a granulomatous inflammatory response
 

within fascia and subcutis. Similar lesions occurred in and
 

around the mesentery, pancreas, spleen, and, less frequently, the
 

pleura. Staining representative sections of all morphologic
 

variants of these lesions with Gomori's method for reticulum
 

demonstrated the presence of numerous reticulin fibrils intertwined among the proliferating cells in all lesions except those
 

resembling fibrosarcomas, in which there was little intercellular
 

reticulin. There appeared to be a histologic spectrum of lesions
 

ranging from a proliferation of plump histiocytes, some forming
 

multinucleated giant cells, with a mixture of granulocytic and
 

mononuclear inflammatory cells and foci of acute necrosis, to an
 

obviously malignant neoplastic process with pleomorphic cells,
 

numerous mitoses, and extensive invasion of adjacent normal
 

muscle and parenchymatous organs. The relatively high incidence
 

of involvement of tissues around the pancreas and mesenteric
 

vessels and the presence of degenerating arteries in some nodules
 

of proliferating tissue suggest the possible relationship of
 

29
 
these lesions to periarteritis nodosa, a common lesion in aging
 

rats often involving pancreatic and mesenteric vessels. However,
 

these proliferative lesions occurred in many cas«s in this study
 

in the absence of histologically evident arteritis. Another
 

possibility is the presence of some infectious agent or foreign
 

body in tissues of these rats, which elicited a granulomatous
 

response leading to a neoplastic proliferation of histiocytic
 

cells. However, no organism or foreign body was observed
 

microscopically in acid-fast, PAS-, GMS-, or Gram-stained tissue
 

sections from representative lesions. Examinations of representative tissues by electron microscopy supported the conclusion that
 

the basic proliferating cell in all these lesions was the
 

histiocyte, regardless of the variability in cellular architecture, the degree of inflammatory reactions, or the tissues
 

involved. Recent reports in the literature have described
 

neoplasms of histiocytes in humans, (Fu et al., 1975; Stout and
 

Lattes, 1967), including the extreme variability in the histologic picture they may present with light microscopy, (Vilanova and
 

Flint, 1974), and involvement of visceral organs (Chawla et al.,
 

1975; Okayasu et al., 1975). The proliferative lesions described
 

above in this study fit the light microscopic and ultrastructural
 

criteria for "fibrous histiocytomas" as given in the cited literature and are classified as such in this report.
 

Tables A1-A8 summarize the incidence of all neoplasms observed in
 

30
 
this study. There was an increased incidence of total follicularcell neoplasms (adenomas and carcinomas) in high-dose rats of
 

both sexes when compared with the incidence in controls of the
 

same sex (6/31 [19%] in high-dose males versus 4/51 [8%] in
 

pooled controls and 0/6 in matched controls; 6/32 [19%] in
 

high-dose females versus 3/59 [5%] in pooled controls and 0/10 in
 

matched controls). The biologic significance of these increases
 

is difficult to assess. Experimental induction of thyroid
 

hyperplasia in rats and birds by chronic exposure to chlorinated
 

hydrocarbons has been described in the literature (c-loriarty,
 

1975); however, no thyroid neoplasms induced by chlordane were
 

mentioned in the studies cited. A factor which complicates
 

interpretation is the large number of test rats from which no
 

section of thyroid was available for microscopic examination.
 

The data in the present study, although somewhat suggestive, do
 

not, in the judgment of the pathologist, appear to be sufficient
 

to indicate clearly a carcinogenic effect of chlordane on the
 

thyroid follicular cells of rats.
 

There were other instances where neoplasms occurred only in test
 

animals or with increased frequency when compared to control
 

groups; however, the nature, incidence, and severity of the
 

lesions observed provide no clear evidence of carcinogenic effect
 

of chlordane in rats.
 

31
 
D. Statistical Analyses of Results (Rats)
 

Statistical analyses of neoplasms in rats fed chlordane are given
 

in Appendix E, tables El - E14.
 

Only one hepatocellular carcinoma was found among the 170
 

chlordane-treated animals of both sexes whose livers were
 

examined (table El). When hepatocellular carcinoma was combined
 

with neoplastic nodules, a significant linear trend still was not
 

found in either sex by either statistical test. Large numbers of
 

female rats were found with neoplastic nodules in the low-dose
 

group (23%) compared with the controls (10%). This difference,
 

however, was not significant by either statistical test. Comparisons using the pooled controls reinforced most of these conclusions (table E2). However, the incidence of neoplastic nodules
 

in low-dose females (23%) was significantly higher than in the
 

pooled controls (8%) by either test, with P < 0.05.
 

There were several follicular-cell carcinomas of the thyroid
 

found among males and females of the treated groups, but there
 

were not enough to reach statistical significance (table E3).
 

When carcinomas were combined with adenomas and comparisons were
 

made with matched controls, significant linear trend was found in
 

the males but not in the females. When comparisons were made
 

with pooled controls, however, the combination of carcinomas and
 

32
 
adenomas yielded a significant linear trend in the females but
 

not in the males (figure E4).
 

Several C-cell carcinomas of the thyroid were found, but three
 

were in the female control group (table E5). No statistically
 

significant results were found here. When C-cell adenomas were
 

added to the carcinomas, a significant result was found in the
 

life-table-adjusted test for the female rats. This finding was
 

vitiated by the fact that there was a significant departure from
 

the linear trend. The control group and the high-dose group had
 

similar results, and the "significant" linear trend was mainly a
 

comparison of the low dose with the high dose. In the males a
 

significantly negative trend was found for the life-tableadjusted test (P = 0.02). Analyses using the pooled controls
 

yielded similar results (table E6), since the category of
 

carcinoma yielded no significant differences of interest. Among
 

females the combination of carcinoma and adenoma yielded results
 

of an erratic character: the response to the high dose was
 

significantly higher than that to the pooled controls, while the
 

response to the low dose was significantly lower.
 

Thus, the thyroid results were ambiguous for the follicular-cell
 

and C-cell tumors of the thyroid. Although dose-response
 

relationships were found to be consistently "positive for
 

follicular-cell tumors of both male and female rats, the negative
 

33
 
relationship among males for C-cell tumors was somewhat offset by
 

an indication of a positive relationship among females.
 

There was a highly significant finding of dose-related trend of
 

incidence of malignant fibrous histiocytoraa in male rats (P =•
 

0.0007 by the life-table-adjusted test), but there was no
 

indication of such tumor induction in females (table E7).
 

Comparisons using pooled controls reinforced these findings
 

(table E8).
 

An appreciable number of animals in both the matched control and
 

treated groups had pituitary tumors, but with no significant
 

dose-related trends in either sex by either statistical test
 

(table E9). The pooled controls yielded only one adenocarcinoma
 

and one carcinoma among the male rats and none among the female
 

rats; therefore, no analyses were carried out for this category
 

of tumors.
 

Comparisons using pooled controls (table E10) generally agreed
 

with those using matched controls. The only exception was in the
 

category in which adenoma was added, where the female low-dose
 

group showed a significantly lower incidence than the pooled
 

controls by both tests (P < 0.01).
 

When different categories of mammary tumors were considered
 

together, a large number of such tumors were found in female
 

34
 
rats. No significant differences were found, however, between
 

treated animals and matched controls (table Ell). The incidence
 

in the low-dose group significantly exceeded that in the pooled
 

controls (P < 0.05) in the category which included adenomas,
 

fibroadenomas, and fibromas (table £12).
 

The high-dose female group had two animals with sarcomas of the
 

uterus, but this was not a large enough response to achieve
 

significance (tables E13 and E14).
 

35
 

IV. RESULTS - MICE
 

A* Body Weights and Clinical Signs (Mice)
 

The average body weights of mouse groups receiving low and high
 

doses did not differ appreciably from those of the untreated
 

groups during the course of the tests (figures 3 and 4).
 

During the first year of the study the appearances and behaviors
 

of the treated and control mice were generally comparable. At
 

week 20, generalized tremors were observed in the high-dose male
 

and female mice even though, as noted above, their weights were
 

hardly affected. After 50 weeks of treatment, alopecia (general
 

and/or local) and a hunched appearance were noted in a few
 

animals. Abdominal distention was observed in all groups but was
 

predominant in the females.
 

B. Survival (Mice)
 

Curves for the probability of survival of treated and control
 

mice are shown in figures 5 and 6. For male mice there were
 

significant differences between the low-dose and control groups
 

(P = 0.02) and high-dose and control groups (P = 0.01+) in
 

mortality from other than accidental death or scheduled
 

sacrifice. This may reflect toxicity of the treatment in the
 

male mice. On the other hand, there was no such indication at
 

37
 
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39
 
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all in female mice, where the survivals of the treated groups and
 

their respective control groups were virtually identical.
 

C. Pathology (Mice)
 

Histopathologic findings are summarized in Appendix B, tables
 

B1-B3, covering neoplasms and other proliferative lesions, and in
 

Appendix D, tables Dl and D2, covering nonneoplastic lesions.
 

A variety of inflammatory, degenerative, and proliferative
 

lesions occurred with approximately equal frequency in
 

chlordane-treated and control mice. These included purulent
 

oophoritis, cystic ovaries, cystic endometrial hyperplasia and/or
 

suppurative endometritis, lymphoid hyperplasia of the spleen,
 

chronic nephritis, and amyloidosis of the kidney.
 

With the exception of primary hepatic neoplasms (table B2), a low
 

overall incidence of neoplasms occurred in both control and test
 

groups of mice in this study. Hepatocellular carcinomas occurred
 

in 17/92(18%) of pooled-control males, 2/18(11%) of matched-


control males, 16/48(33.3%) of low-dose males, 43/49(88%) of
 

high-dose males; 3/78(4%) of pooled-control females, 0/19 of
 

matched-control females, 3/47(6%) of low-dose females, and
 

34/49(69%) of high-dose females. The incidence of hepatocellular
 

carcinomas, therefore, was clearly increased in both males and
 

females at the higher dosage level; a less distinct increase in
 
incidence was present in the low-dose male group. The incidence
 

in low-dose females (6%) was slightly higher than in the control
 

females, but lower than in the control males.
 

There was a wide range of histologic variation from normal liver
 

among the hepatocellular carcinomas (Lemon, 1967). Some of these
 

lesions consisted of nodules of hepatocytes with only moderate
 

variation from adjacent normal hepatic parenchyma in staining
 

characteristics, size and shape of cells, and lobular architecture. Although these tumors clearly compressed adjacent normal
 

parenchyma, they usually did not appear to be growing by
 

invasion. At the other end of the spectrum were tumors with
 

clearly anaplastic cytologic characteristics, hepatocytes in
 

solid sheets or haphazardly arranged in papillary or pseudoacinar
 

patterns, with no resemblance to normal lobular architecture
 

within the tumor. Metastasis of hepatocellular carcinoma to the
 

lung occurred in two high—dose males and three high-dose females.
 

Hepatic lesions that were classified as nodular hyperplasia
 

(table B2) consisted of small nodules of proliferating
 

hepatocytes, which compressed adjacent hepatic parenchyma but
 

which did not have sufficient abnormality of cellular morphology
 

or lobular architecture to warrant diagnosis of neoplasia.
 

The incidence of primary pulmonary neoplasms was slightly
 

increased in male mice (table B3), but the amount of increase was
 

43
 
insufficient to indicate clearly a carcinogenic effect on the
 

lung.
 

Alveolar-cell adenomas (Baillif and Jones, 1973) consisted of
 

discrete, densely cellular nodules of small, round epithelial
 

cells arranged in rows or nests, compressing adjacent pulmonary
 

parenchyma. The neoplastic cells had spherical, central nuclei
 

and eosinophilic cytoplasm. Carcinomas of alveolar cells were
 

larger and less discrete; nuclei of tumor cells were much more
 

pleomorphic and hyperchromatic, and the nuclearrcytoplasmic ratio
 

was much greater in the carcinomas. The neoplastic cells in the
 

carcinomas were larger than in the adenomas: their cytoplasm was
 

indistinct and sometimes contained clear vacuoles.
 

Several other types of neoplasms involving various tissues
 

occurred infrequently (table B3), with no distinct differences in
 

incidence between test and control groups.
 

D. Statistical Analyses of Results (Mice)
 

Statistical analyses of neoplasms in mice fed chlordane are given
 

in Appendix F, tables Fl - F4.
 

The small size of the control group notwithstanding, a highly
 

significant difference in hepatocellular carcinoma was found
 

between the high-dose male group and its control (table Fl) by
 

the simple proportion analysis (P < 0.0001) and by the life-table44
 
adjusted analysis (P < 0.0001). The low-dose group was marginally significantly different (P - 0.05+) from its control with the
 

life-table-adjusted test. Comparison with the pooled controls
 

(table F2) reinforced all of these results. In particular, the
 

low-dose group had significantly more hepatocellular carcinoma
 

than the pooled controls (P =• 0.04 and 0.001) by each of the two
 

tests.
 

In female mice, the difference between the high-dose and control
 

groups with respect to the incidence of hepatocellular carcinoma
 

(table F3) was highly significant (P - 0.0001 and 0.0002). The
 

difference between the low-dose and control groups was not
 

significant, although it was always in the direction of a
 

positive response. The results were similar for comparisons
 

using pooled controls (table F4).
 

At no other site was a sufficient number of animals with tumors
 

found to warrant statistical analysis.
 

45
 

V. DISCUSSION
 

Chlordane is a member of the organochlorine group of pesticides
 

that can be classed as neurotoxins (Brooks, 1975). Acute
 

exposure to chlordane stimulates the control nervous system, but
 

the compound is less toxic than other cyclodiene pesticides.
 

Tremors, a manifestation of the neurotoxicity that is
 

characteristic of such compounds, developed in female high-dose
 

rats at week 44 of treatment with chlordane and in both male and
 

female high-dose mice at week 20.
 

The effects of chlordane on body weights and other clinical signs
 

in rats and mice indicated that the dosages used were near the
 

maximum permissible. This was evident in that the average body
 

weights of the high-dose male and female rats were consistently
 

lower than those of the untreated controls, while differences
 

between the low-dose and control rats were negligible. Adverse
 

clinical signs in treated rats included rough and discolored hair
 

coats and the occurrence of palpable masses. For mice given
 

either low or high doses, body weights showed little or no effect
 

of the chlordane; however, adverse clinical signs of alopecia,
 

hunched appearance, and, predominantly in females, bloating or
 

abdominal distention were observed with greater frequency in
 

treated than in untreated mice.
 

47
 
The effects of chlordane on survival rates showed a dose-related
 

decrease only in the females; in the males, the survival of the
 

controls, for unknown reasons, was abnormally low. For mice,
 

mortality rates were increased in low- and high-dose males but
 

not in females. Except for the male control rats, a substantial
 

proportion of all groups survived to an age at which tumors could
 

be expected to appear.
 

Hepatocellular carcinoma was induced at a highly significant rate
 

of incidence in mice given chlordane (Appendix F, tables F1-F4),
 

particularly at the high dose, using either matched controls or
 

pooled controls. At the low dose, the incidence of the tumor
 

was significant only for male mice, using pooled controls. The
 

significance became greater when animals dying prior to
 

appearance of the first tumor in any of the groups were
 

eliminated from the denominators in the calculations. Finally,
 

dose-related trends for hepatocellular carcinoma were
 

consistently significant, using either matched or pooled controls
 

for both males and females.
 

In contrast to findings with mice, the incidence of hepatocellular carcinoma in rats (Appendix E, tables El and E2) was
 

extremely low in both treated and control animals. Lesions of
 

the liver in rats did not become statistically significant with
 

48
 
the addition of nodular neoplasia to carcinoma or with the
 

application of life-table adjustment to the data.
 

A significant dose-related trend in follicular-cell lesions of
 

the thyroid was found, however, in the rats (tables E3 and E4).
 

Although the trend for follicular-cell carcinoma alone was not
 

significant, that for follicular-cell carcinoma combined with
 

adenoma was significant or nearly significant. The trend for
 

thyroid follicular-cell lesions was significant for females, but
 

not for males, when pooled controls were used for comparison
 

instead of the matched controls. A significant dose-related
 

trend in malignant fibrous histiocytoma in male rats also was
 

found, with the use of either matched or pooled controls (tables
 

E7 and E8). There was, however, a large number of test rats from
 

which no sections of thyroid were available for microscopic
 

examination, and, in the judgment of the pathologist, the nature,
 

incidence, and severity of neither the thyroid lesions nor the
 

histiocytotnas were sufficient to indicate clearly a carcinogenic
 

effect of chlordane in rats.
 

Chlordane is metabolized primarily to the epoxide, oxychlordane,
 

which is the principal form in which the insecticide is stored in
 

body fat (Barnett and Dorough, 1974). Minor quanitites of other
 

metabolites are excreted in the urine and feces.
 

49
 
Livers of rats fed diets containing 10 to 320 ppm (Ambrose et
 

al., 1953), 2.5 to 25 ppm (Ortega et al., 1957), or 5 to 300 ppm
 

(Ingle, 1965) chlordane for periods up to 2 years were reported
 

to show abnormalities of hepatocytes, such as the appearance of
 

intracytoplasmic bodies, enlargement of nuclei and nucleoli, and
 

hypertrophy; however, no hepatocytic neoplasms were described.
 

Brooks (1975) has questioned whether such lesions represent
 

damage to the liver or simply adaptational response to challenge
 

by foreign compounds.
 

In the present bioassay, chlordane was hepatocarcinogenic in mice
 

but not in rats. In rats, chlordane induced statistically
 

significant increases in proliferative follicular-cell lesions of
 

the thyroid in both males and females and in malignant fibrous
 

histiocytomas in the males. The relationship of these lesions to
 

administration of the compound is not clear, however, since the
 

spontaneous incidence of the lesions has varied throughout the
 

bioassay program.
 

50
 
VI. BIBLIOGRAPHY
 

Ambrose, A. M., Christensen, H. E., Robbins, D. J., and Rather,
 

L. J. Toxicological and pharmacological studies on
 

chlordane. Arch. Indust. Hyg. Occupational Med. ^:197-210,
 

1953.
 

Armitage, P. Statistical Methods in Medical Research, J. Wiley &
 

Sons, New York, 1971, ch. 14.
 

Baillif, R. N., and Jones, E. L. Pulmonary adenomatosis in aging
 

mice. J. Comp. P_ath_._ JJ3:597-603, 1973.
 

Barnett, J. R. and Dorough, H. W. Metabolism of chlordane in
 

rats. J. Agr. Food Chem. ^2:612-619, 1974.
 

Berenblum, I., ed. Carcinogenicity Testing, U.I.C.C. Technical
 

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

Geneva, 1969.
 

Bloodworth, J. B. M., Jr. Endocrine Pathology, Williams and
 

Wilkins, Baltimore, 1969.
 

Brooks, G. T. Chlorinated Insecticides, Vol. 2. CRC Press,
 

Inc., Cleveland, Ohio, 1975.
 

Chawla, S. K., Lopresti, P. A., Burdman, D., Sileo, A., Govoni,
 

A. F., and Smulewicz, J. J. Diffuse small bowel involvement
 

in malignant histiocytosis. American Journal of
 

Gastoenterology. 6_3:129, 1975.
 

Chlordane Formulation Guide, Velsicol Chemical Corp., Chicago,
 

1962, pp. 502-535.
 

Cox, D. R. Analysis of Binary Data, Methuen, London, 1970, chs.
 

4 and 5.
 

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

Soc. B. _34:187-220, 1972.
 

Duggan, R. E. and Corneliussen, P. E. Dietary intake of
 

pesticide chemicals in the United States (III) June
 

1968-April 1970. Pest Monit. J. j>:331-341, 1972.
 

51
 
Environmental Protection Agency. Pesticide products containing
 

heptachlor or chlordane. Intent to cancel registration,
 

li^erii 5£&iSter 39:41298-41300, 1974.
 

Environmental Protection Agency. Velsicol Chemical Co. et al.
 

Consolidated Heptachlor/Chlordane Hearing. Federal Re glister
 

41:7552-7585, 1976.
 

FAO/WHO. _19.67 Evaluations of Some Pesticide Residues in Food.
 

Rome, 1968.
 

Fu, Y. S., Babbiani, G., Kaye, G. I. and Lattes, R. Malignant
 

soft tissue tumors of probable histiocytic origin (Malignant
 

Fibrous Histiocytomas): General considerations and electron
 

microscopic and culture studies. Cancer 3^5:176, 1975.
 

Ingles, L. A Monograph on Chlordane Toxicolqgical and
 

Pharmacological Properties. University of Illinois, Urbana,
 

T965.
 

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

incomplete observations. J^ Am^ Statis^t^ Assoc. 53:457-481,
 

1958.
 

Lemon, P. G. Hepatic neoplasms of rats and mice. Pathology o>f_
 

Laboratory Rats and Mice, edited by Cotchin, E. and Roe, J.
 

F. C., Blackwell Scientific Publications, Oxford, England,
 

1967, pp. 25-26.
 

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

Peters, J. A. Carcinogenesis bioassay data system. J^
 

7:230 248 1974
 Cpm£.. Biomet^ Res^ ~ > ­

Mantel, N. Chi-square tests with one degree of freedom:
 

extensions of the Mantel-Haenszel procedure. J. Am^
 

Statist^ Assoc^ 58:690-700, 1963.
 

Meissner, W. A., and Warren, S. Tumors of the thyroid gland.
 

Atlas o£ TunKH: Pathology, Fascicle 4, A. F. I. P.,
 

Washington, D~.C., 1968.
 

Moriarty, F., ed. OESJiBJ^hJ^ojrine Insect^ic^ide£; Persistent
 

Organic Pollutants, Academic Press, London, 1975, p. 143.
 

Okayasu, I., Okayasu, N., Mori, W,, Miyazki, K., and Matsubara,
 

S. Reticulosarcomatosis originating from skin- A clinicopathologic study. Acta Pa^thplogica Jap^onica 25:201, 1975.
 

52
 
Ortega P., Hayes, W. J., Jr., and Durham, W. F. Pathologic
 

changes in the liver of rats after feeding low levels of
 

various insecticides. Arch. Path. 64; 6 14-622, 1957.
 

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

Kaufman, D. G. Respiratory tract carcinogenesis in hamsters
 

induced by different numbers of administration of benzo (a)
 

pyrene and ferric oxide. Cancer Res. 12: 1073-1079, 1972.
 

Secretary's Commission on Pesticides, U.S. Dept. of Health,
 

Education and Welfare. Report of the Secretary's Commission
 

on Pesticides and Their Relationship to Environmental
 

Health, U.S. Government Printing Office, Washington, D.C.,
 

1969.
 

Squire, R. A., and Levitt, M. Report of a workshop on
 

classification of specific heptocellular lesions in rats.
 

Cancer Res. _35:3214, 1975.
 

Stout, A. P., and Lattes, R. Tumors of the soft tissues. Atlas
 

of Tumor Pathology, Second Series, Fascicle 1, A. F.I. P.,
 

Washington, D.C., 1967, p. 107.
 

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

j>2:679-682, 1975.
 

Vilanova, J. R., and Flint, A. The morphological variation of
 

fibrous histiocytomas. Journal of Cutaneous Pathology
 

, 1974.
 

Wilks, S. S. i-lathematical Statistics, John Wiley and Sons,
 

Inc., rtew York, 1962, pp. 290-291.
 

53
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND OTHER
 

PROLIFERATIVE LESIONS IN RATS
 

FED CHLORDANE IN THE DIET
 

55
 

TABLE Al 

SUMMARY OF THE DISPOSITION OF TISSUES 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

Animals Initially in Study 60 10 50 50 60 10 50 50 

Animals Necropsied 58 8 44 43 60 10 49 44 

Animals Examined 

Histopathologically 58 44 43 60 10 49 42 
TABLE A2 

PROLIFERATIVE LESIONS OF THE THYROID 

in 

OO 

Follicular-cell Carcinoma 

Follicular-cell Adenoma 

C-cell Carcinoma 

C-cell Adenoma 

Pooled 

Control 

(51) 

1 

3 

1 

3 

MALE RATS 

Matched

Control 

(6) 

0 

0 

0 

0 

 Low 

Dose 

(34) 

1 

0 

1 

4 

High 

Dose 

(31) 

2 

4 

2 

3 

Pooled 

Control 

(58) 

1 

2 

5 

7 

FEMALE RATS 

Matched Low 

Control 

(10) 

0 

0 

3 

0 

Dose 

(43) 

2 

2 

0 

3 

High 

Dose 

(32) 

2 

4 

3 

7 

Follicular-cell Hyperplasia 

C-cell Hyperplasia 

0 

29 

0 

4 

1 

15 

4 

6 

4 

30 

1 

4 

4 

22 

5 

10 

Numbers in parentheses represent the numbers of tissues examined microscopically. 
TABLE A3
 

OTHER ENDOCRINE NEOPLASMS
 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

ADRENAL (55) (7) (39) (36) (56) (9) (48) (36) 

Cortical Carcinoma 1 0 0 0 0 0 1 1 

Cortical Adenoma 2 0 1 0 0 0 0 0 

vo PANCREATIC ISLETS (52) (7) (42) (36) (60) (10) (45) (40) 

Adenoma 1 0 0 0 1 0 0 1 

PITUITARY (48) (6) (34) (34) (52) (10) (46) (37) 

Carcinoma 2 0 1 1 0 0 1 0 

Adenoma 14 0 23 11 

Numbers in parentheses represent the numbers of tissues examined microscopically. 
TABLE A4
 

PRIMARY HEPATIC NEOPLASMS'
 

MALE RATS FEMALE RATS 

Pooled 

Control 

(58) 

Matched 

Control 

(8) 

Low 

Dose 

(44) 

High 

Dose 

(40) 

Pooled 

Control 

(59) 

Matched 

Control 

(10) 

Low 

Dose 

(47) 

High 

Dose 

(39) 

Hepatocellular Carcinoma 1 0 1 0 0 0 0 0 

Neoplastic Nodule 2 0 1 0 5 1 11 6 

Hemangiosarcoma 3 0 0 0 0 0 0 1 

Numbers in parentheses represent the numbers of tissues examined microscopically.
 
TABLE A5 

PRIMARY RENAL NEOPLASMS' 

Malignant Mixed Tumor 

Tubular Adenoma 

Pooled 

Control 

(57) 

0 

3 

MALE RATS 

Matched 

Control 

(8) 

0 

1 

Low 

Dose 

(43) 

4 

0 

High 

Dose 

(42) 

0 

0 

Pooled 

Control 

(58) 

1 

0 

FEMALE RATS 

Matched 

Control 

(10) 

0 

0 

Low 

Dose 

(49) 

0 

0 

High 

Dose 

(42) 

0 

0 

Numbers in parentheses represent the numbers of tissues examined microscopically. 
TESTIS


Interstitial-cell Tumor


OVARY
 

Granulosa-cell Tumor
 

UTERUS
 

Sarcoma, N.O.S.
 

Hemangio sarcoma
 

Adenocarcinoma
 

Endometrial Stromal Polyp
 

TABLE A6 

NEOPLASMS OF THE REPRODUCTIVE SYSTEM3 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

___ ___ ___ (58) (8) (43) (42) —_ 

0 O i l 

(58) (9) (48) (36) 

1 0 0 1 

(56) (9) (47) (33) 

0 0 0 2 

0 0 1 0 

0 0 1 0 

6 0 3 1 

Numbers in parentheses represent the numbers of tissues examined microscopically.
 
TABLE A7
 

NEOPLASMS OF THE MAMMARY GLAND
 

MALE RATS FEMALE RATS 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

MAMMARY GLAND 

Carcinoma 

(58) 

0 

(8) 

0 

(44) 

0 

(43) 

0 

(60) 

2 

(10) 

1 

(49) 

2 

(44) 

3 

Adenoma 0 0 0 0 1 0 10 4 

Fibroadenoma 0 0 0 0 8 1 6 3 

Fibroma 1 0 0 0 1 0 1 0 

Lipoma 0 0 0 1 0 0 0 0 

The adjacent number in parentheses represents the number of animals necropsied in that group,
 

rather than the number of tissues examined microscopically.
 
TABLE A8 

MISCELLANEOUS NEOPLASMS0 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

Malignant Lymphoma (58)b 

1 

(8)b 

1 

(44)b 

0 

(44)b 

1 

(60)b 

2 

(io)b 

0 

(49)b 

0 

(44)b 

0 

Malignant Fibrous 

Histiocytoma (any site) 2 0 1 7 1 1 1 1 

SKIN/SUBCUTIS 

Squamous-cell Carcinoma 

(58)b 

0 

<8)b 

0 

(44)b 

0 

(43)b 

1 

(60)b 

0 

(io)b 

0 

(49)b 

0 

(44)b 

0 

Carcinoma, N.O.S. 0 0 1 0 0 0 0 0 

Lipoma 0 0 1 0 1 0 0 0 

LUNG (58) (8) (43) (39) (58) (10) (48) (48) 

Alveolar-cell Adenoma 0 0 1 0 0 0 0 0 

BRAIN (57) (8) (44) (42) (59) (10) (49) (41) 

Glioma 1 1 0 0 0 0 1 0 
TABLE A8
 

MISCELLANEOUS NEOPLASMS'
 

(continued)
 

MALE RATS FEMALE RATS
 

Pooled Matched Low High Pooled Matched Low High
 

Control Control Dose Dose Control Control Dose Dose
 

b b b b b b
 DIAPHRAGM (58) (8) (44) (43)b (60) (io)b 

(49) (44)


Osteosarcoma 0 0 0 1 0 0 0 0
 

Ui SPLEEN (56) (8) (44) (38) (55) (10) (49) (38)
 

Hemangioma 0 0 0 0 0 0 1 0
 

Numbers in parentheses represent the numbers of tissues examined microscopically.
 

The adjacent number in parentheses represents the number of animals necropsied in that group,
 

rather than the number of tissues examined microscopically.
 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND OTHER
 

PROLIFERATIVE LESIONS IN MICE
 

FED CHLORDANE IN THE DIET
 

67
 

TABLE Bl 

SUMMARY OF THE DISPOSITION OF TISSUES 

MALE MICE FEMALE MICE 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

ON 

VO 

Animals Initially in Study 

Animals Necropsied 

Animals Examined 

Histopathologically 

100 

92 

92 

18 

18 

18 

50 

49 

48 

50 

49 

49 

80 

79 

79 

20 

20 

20 

50 

50 

50 

50 

50 

50 
TABLE B2
 

PROLIFERATIVE HEPATIC LESIONS'
 

MALE MICE FEMALE MICE 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

LIVER 

Hepatocellular Carcinoma 

(92) 

17 

(18) 

2 

(48) 

16 

(49) 

43 

(78) 

3 

(19) 

0 

(47) 

3 

(49) 

34 

Nodular Hyperplasia 3 2 7 0 2 1 3 3 

Diffuse Hyperplasia 3 3 3 0 1 0 3 3 

Hepatocytomegaly 0 0 1 1 1 0 0 0 

Numbers in parentheses represent the numbers of tissues examined microscopically.
 
TABLE B3 

MISCELLANEOUS NEOPLASMS 

MALE MICE FEMALE MICE 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

LUNG (91) (17) (46) (46) (79) (20) (47) (47) 

Alveolar-cell Carcinoma 0 0 1 1 0 0 0 0 

Alveolar-cell Adenoma 6 1 5 2 0 0 0 1 

HEMATOPOIETIC SYSTEM (92)b (18)b (49)b (49)b (79)b (20)b (50)b (50)b 

Malignant Lymphoma 5 1 1 0 8 1 1 0 

THYROID (79) (17) (36) (35) (72) (16) (43) (35) 

Follicular-cell Adenoma 1 0 0 1 1 0 0 0 

KIDNEY (92) (18) (48) (47) (78) (20) (46) (48) 

Tubular Adenoma 0 0 0 1 0 0 0 0 

MAMMARY GLAND (92)b (18)b (49)b (49)b (79)b (20)b (50)b (50)b 

Fibroadenoma 0 0 0 0 0 0 1 0 

Neurof ibroma 0 0 0 0 0 0 1 0 
TABLE B3
 

MISCELLANEOUS NEOPLASMS
 

(continued)
 

MALE MICE FEMALE MICE
 

Pooled Matched Low High Pooled Matched Low High
 

Control Control Dose Dose Control Control Dose Dose
 

b b b b
 LACRIMAL GLAND (92)b (18) (49) (49)b (79)b (20)b (50) (50)


Adenoma 1 0 0 0 1 1 0 0
 

NJ
 

Numbers in parentheses represent the numbers of tissues examined microscopically.
 

The adjacent number in parentheses represents the number of animals necropsied in that group,
 

rather than the number of tissues examined microscopically.
 

i
 

"For purposes of this summary, the following lesions are grouped under malignant lymphoma:
 

lymphocytic lymphosarcoma, lymphosarcoma, and reticulum-cell sarcoma (any site).
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED CHLORDANE IN THE DIET
 

73
 

TABLE C1 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED CHLORDANE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIHAL S INITIALLY I H STUDY 10 50 50 ANIMAL S NECROPSIED 8(100*) 144(10011) 47(100X) 

ANIMALS EXAMINE!) HISTOPATHOLOGICALLY & 44 47 ANIHAL S JIIH NON-TUMO R PATHOLOGY 8 (100*) 43 (98*) 38 (SIX) 

INTEGUMENTAR Y SYSTEM * 1 (13X) 3 (7X) 

SOBCUT TISSUE 1 3

 

GBANULOMA 1

 

GRANULATIO N TISSUE 1 2 

RESPIRATOR Y SYSTEM 3 (38*) 25 (57») 15 (32X) 

TBACHEA 1 3 INFLAMMATIO N CHBONIC 1 3

 

LUN G 3 23 15 

EfiPHYSEBA 2 

»T£LECTASIS 2 

INFLAHHAIION INItHSTIlIAL 1 

PNEUMONI A ASPIRATIO N 1 

CHOLiSTEBOL DEPOSITION 3 1 

ALVEOLAR HACBOPHAGES 2 21 15 LYHPHOI D HYPEBPLASIt 1 1 

CIRCULATOR Y SYSTEM 21 (43t) 15 (32X) 

J1YOCA8DIUH 21 14 INFLAMMATION 3 

FlbSOSIS 21 14 

ENDOCARDIU M 1 

FIBBOSIS 1 

AOBTA 

ARTERIOSCLEROSIS 

CORONAR Y ARTER Y 

• SYSTE M PERCKNTA3ES ARE BASED ON THE NUMBE R DP 4NI.B A.L.S._8SC.B.2ES.iE.2 

75 
TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONT.) 

PULMONARY ABTERY
 

INFLAMMATION
 

INFLAMMATION ACUTE
 

MESENTEBIC ARTEBY
 

PEBIABTEEITIS
 

DIGESTIVE SYSTEM »


SALIVARY GLAND
 

FIBBOSIS
 

LIVER


CONGESTION
 

METAMORPHOSIS FATTY
 

CYTOPLASHIC VACUOLIZATION


HEPATOCYTOHEGALY


HYPBBPLASIA DIFFUSE
 

ANGIECTASIS


BILE DUCT
 DILATATION
 

INFLAMMATION


FIBBOSIS
 

HYPEBPLASIA


PANCREAS
 

ECTOPIA
 

INFLAMMATION ACUTE
 

FIBHOSIS
 

PEBIASTBHITIS
 

PANCREATIC ACINUS


ATROPHY


STOMACH
 

EROSIVE INFLAMMATION
 

CALCIFICATION DYSTBOPHIC
 

GASTRIC MUCOSA
 

£At£J£iC4lIS!i
 

*SYSPE M PERCENTA3ES ABE BASED

CONTROL
 

6 (75X)
 

5
 

3
 

4
 

1
 

4
 

2
 

4
 

1
 

1
 

 ON THE NUMBE R 3F

LOU DOSE HIGH DOSE
 

1 1
 

1
 

1
 

1
 

1
 

40 (91X) 31 (66X)
 

1
 

1
 

28 16
 

1
 

1
 

19 3
 

25 13
 

2
 

4 5
 

35 25
 

1
 

18 20
 

7 3
 

32 19
 

8 7
 

1
 

1
 

5 6
 

2 3
 

4 4
 

4 4
 

2 2
 

1 2
 

1
 

1 3
 

1 1
 

 SS£S4Ii£-!!S£S2SSIED 

76 
TABLE G1 MALE RATS: tyOMUEOPLASTtC LESIONS (CUNT.) 

CONTROL LOB DOSE 

URINAR Y SYSTEH * 3 (38X) 38 (86X) 

KIDNEY 3 38

 

INFLAHHATION INTERSTITIAL 1

 

INFLAMHATION CHROMIC 3 35

 

PYELONEPHRITIS CHHOMIC 2 

HEHATOPOIESIS 1

 

KIDNfiY/PELVIS 2 

INFLAMMATION CHRONIC 

HIPERPLASIA EPITHELIAL 2 

URINAR Y BLADDER 5

 

INFLAMMATIO N CHRONIC 2 

HYPERPLASIA EPITHELIAL 5

 

EHDOCRIME SYSTEM 5 (63X) 31 (70J) 

PITOITARY 1 6

 

CYST 2

 

HEMORRHAGE 1

 

ATROPHY 

HYPEEPLASIA CHROHOPHOBE-CELL 1 3 

ANGIBCTASIS 1 

ADRENAL CORTEX 1 17

 

CYTOHEGALY 1 16 

PHA60CYTIC CELL 

HYPERPLASIA FOCAL 1

 

THYROID U 21

 

ULTIMOBRANCHIAL CYST 2

 

CYSTIC FOLLICLES 5 

ATROPHY 

HYPERPLASIA C-CELL t 15

 

HYPERPLASIA FOLLICULAE-CELL 1

 

PARATHYROID 1 u

 HYP£BPLASIA 1 3

 

HYPERPLASIA DIFFUSE 1

 

PANCREATIC ISLETS 2 

____ JUUIEUStt______________________________............ .2 

*SYSTEH PERCENTAGES ARE BASED ON THE NDHBER OF 

HIGH DOSS 

32 (68*) 

32 

31 

1 

5 

1 

4 

5 

5 

19 (40X) 

3 

1 

1 

1 

8 

6 

1 

1 

13 

3 

2 

1 

6 

4 

3 

3 

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

CONTRO L L01 DOSE HIG H DOSE 

HEMATOFOIETIC SYSTEM * 8 {18X) 9 (19«) 

SPLEEN 7 9 

HEHOSIDEROSIS 3 2 

HYPEBPLASIA EETICULUB-CELL 2 

H3HATOPOIESIS a 7 

LYMPH NODE 1 1 

GBAHULOM A 1 

LYHPHOID HYPERPLASIA 1 

CERVICAL LYMP H NODL 1 

INFLAMMATIO N 1

 

REPRODUCTIVE SYSTEM J (38)1) 33 (75X) 26 (55X) 

PROSTATE 1 3 5 

INFLAMMATION SUPPUBATIVE 1 3 5 

INFLAHflATIO N ACTIVE CHRONIC 1 

METAPLASIA SQUAMOUS 1 3 

TESTIS 3 32 2K EDtM A 1 1 

PE5IARTERI1IS 1 6 

ATROPH Y 3 32 2" 

NERVOU S SYSTEM 

NON E 

MUSCULOSKELETAL SYSTEM 1 (2*) 

FASCIA 1 

INFLAM3ATIO N 1 

SPECIAL ShNSt OBGAN S 1 (2«) 

E YE 1 

_ CAI^SA.CT 1 

*SYSrE.1 PERCENTAGES ABE BASED ON THE NUMBER OF A.NigA_LS_.N.E.C.ROPSiED
 

78
 
TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS(CONT-) 

CONTROL LOI DOSE HIGH DOSE 

ALL OTHEB SYSTEMS * 2 (25*) 10 (23*) 15 132%) 

FOOT 1 

GRANULATIO N TISSUE 1 

NO ASSOCIATED OBGA N 2 7 11 

NO LESION BEPOBTED 1 4 

ACCIDENTA L DEATH 4 

AUTOLYSIS/NO NECBOPSY PEBFOBUED 2 6 3 

ADIPOSE TISSUE 1 

INFLAMMATIO N 1 

HESENTEEY 2 3 

HEMORRHAG E 1 

PEBIABTEBITIS 1 1 

ABTEBIOSCLEBOSIS 1 

fifiC.6S5I3-F.il 1 

*SYSTEM PERCENTAGE S ABE BASED Oil THE NUMBER 3F 4SIB4i§. 

79 
TABLE C2 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS FED CHLORDANE IN THE DIET 

CONTROL LOi DOSE HI3H DOSE 

ANIMALS INITIALLY IN STUDY 10 50 50 

ANIMAL S NECROPSIED 10(100*) 49 (100X) 4t| (100%) 

ANIHALS EXAMINED HISTOPATHOLOGICALLY 10 49 42 

ANIMAL S HUH NON-TUHOB PATHOLOGY 10 (100X) 48 (98*) 141 (93X) 

INTEGOaENTARY SYSTEM 

NONE 

BESPIBATOBY SYSTEM 2 (20%) 18 (37X) 20 (45X) 

TRACHEA 1
 

INFLAMMATION CHBONIC 

LUNG 2 16 20 

EMPHYSEM A 4 

ATELECTASIS it 

CONGESTION 1 

INFLAMMATION INTERSTITIAL 1 

CHOLESTEROL DEPOSITION 2 2 

ALVEOLAR MACBOPHAGES 2 14 20 

HYPEBPLASIA ALVEOLAB-CELL 1 

LYMPHOID HYPEfiPLASIA 1 

LUNG/ALVEOLI 5 2

 

INFLAMMATIO N SUPPUBATIV E 5 2

 

CIBCOLATOBY SYSTEM 3 (30X) 9 (18*) 5 (11X) 

HEART 3 

FIBBOSIS 3

 

BYOCABOIUM 3 6 5 

FIBBOSIS 3 5 5 

DEGENEBATION 1 

CALCIFICATION 1 

ENCOCABDIDH 1

 

_ FJL£ROSIS__ _ _ __ 1

 

•SYSTEM PERCENTAGES ABE BAS£D ON THE NUMBER 3P A£iMA.L.S_NSC80j;S.IE.D
 

80
 
TABLE C2 FEMALE RATS: NONBIEOPLASTIC LESIONS (CONT4 

CONTR3L LOK DOSE HIGH DOSE
 

DIGESTIVE SYSTEM * 9 (90%) 42 (86*) 38 (86%)
 

LIVES 6 28 28
 

CONGESTION 1
 

FIBBOSIS FOCAL 1
 

DEGENERATION 1
 

NECROSIS 1 1
 

NECROSIS FOCAL 6 U
 

METAMORPHOSIS FATTY 1
 

CYTOPLASMIC VACUOLIZATION 6 8 17
 

HEPATOCYTOIiEGALY 6 20 22
 

HYPEEPLASIA DIFFUSE 1
 

ANGIECTASIS 2 16
 

LIVEB/HEPATOCYTES 1
 

CYTOPLASMIC VACUOLIZATION 1
 

BILE DUCT 5 38 30
 

DILATATION 3 1
 

INFLAdMATION 3 2b 19
 

FIBHOSIS 1 13 9
 

HYPEEPLASIA U 31 24
 

PANCREAS 1 6 1
 

INFLAMMATION CHRONIC 1
 

GRANOLOMA 1
 

FIBR03I5 1 5
 

PANCREATIC ACINUS 3
 

ATROPHY 3
 

UBINARY SYSIEK 9 (90S) 27 (55%) 30 (68%)
 

KIDNEY 6 21 28
 CYST 1
 

INFLAMMATION INTERSTITIAL 1 1
 INFLAHHATION ChHONIC 5 20 26
 FIBBOSIS FOCAL 1
 

KIDNEY/PELVIS 3 7 3
 HYPERPLASIA EPITHELIAL 3 7 3
 

URINARY BLADDER 3 3 1
 

- _HY££..BPLA,SIA_E.EJJ.HhI.IA.L_ _ !_ 1 1
 

*SYSTEM PERCENTAGE S ARE BASi.0 ON THE NUHBER 3F ig!3&L.S._NEQEO£§iED 

81 
TABLE C2 FEMALE RATS; NONNEOPLASTIC LESIONS (CONT.) 

CONTROL LOU DOSS HIGH DOSE 

EHDOCBINE SYSTEM * 8 (BOH) 33 (67X| 25 (57X) 

PITUITARY 3 7 6 

CONGESTION 1 

HEHOERHAGE 1 

HBHOSIDEBOSIS 1 

HYPEBPLASIA FOCAL 1 

HYPERPLASIA CHROHOPHOBE-CELL 2 5 5 

ADRENAL 2 5 

DEGENERATION 1 

ANGIECTASIS 1 5

 

ADRENA L CORTEX 3 12 12 

METAHOBPHOSIS FATTY 1 

CYTOHEGALY 2 11 8 

HYPERPLASIA 1 1 

HYPERPLASI A FOCAL 2 

ANGIECTASIS 1 

THYROI D 5 25 15 

ULTIMOBRANCHIA L CYST 1 1 

CYSTIC FOLLICLES 4 

SCAR 1 

ATROPH Y FOCAL 1 

HYPERPLASI A C-CELL 14 22 11 

HYPEBPLASIA FOLLICULAR-CELL 1 4 5 

PARATHYROI D 1

 

HYPERPLASI A 1 

PANCREATIC ISLETS 1

 

HYPERPLASI A 1 

HEMATOPOIETIC SYSTEM 4 (40X) 13 (27%) 12 (27X) 

i

INFLAMMATIO N CYSTIC 1 

HEMOSIDiiROSIS 3 5 6 

HYPERPLASI A BETICULUM-CELL 1 

HEMATOPOIESIS 2 2 5 

SPLEEN « 8 11 

CjJRVICA L LYMP H NOD E 1 

*SYSTEM PERCENTAGi S ARE BASED ON THE NUMBE R 3F iSiSl.L.S_J!£J.H2PS.ISe 

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

CONTBOL LOU DOSE HISH DOSE 

THYHUS

CYST

ATBOPHY

 1

 4 

•

 1 

1 

BEPBODUCIIVE SYSTEM

MAMMAR Y GLAN D

HYPEBPLASIA

 * 1 (10X)

 1 

1 

3 <6S) 6 (11*) 

UIEBas/eNDOHGTBIUfl

INFLAMMATION SUPPUBATI¥B

HYPEBPLASIA

HYPEBPLASIA CYSTIC

 3

 1

 1

 2

 5 

1 

1 

<i 

OVABY

GBANULATIOH TISSUE

 1 

1 

HBBVOUS SYSTEM 

NON£ 

HUSCDLOSKELETAL SYSTEM 

NON E 

SPECIAL SENSE OBGANS 

NONE 

ALL OTHh H SYSTEMS

ABDOMINAL CAVITY

GBANOLOM A

 1 (2%) 10 (23*) 

1 

1 

NO ASSOCIATED QBGAN

NO LfiSIOti EEPOBTED

AUIOLYSIS/NSCBOPSY PEBF/NO HISIO

i210iIs.iS^B2_fifiCBQ£SY._E££E2RttfiC

 1

 1

 9 

1 

2 

S 

*SYSTBH PEBC£NTAGES ABE BASED ON THE NUMBER Of A.N1JA.L.S._JIEC.B.QP.S.££P. 

83
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED CHLORDANE IN THE DIET
 

85
 

TABLE 01

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE MICE FED CHLORDANE IN THE DIET 

HIGH DOSE LOW DOSE LOU DOSE HIGH D3SE 

CONTROL CONTROL 

ANIilALS INITIALLY IN STUD Y 20 10 50 50 

ANIHALS NECHOPSIED 10(100X ) 8(100%) t9(100X) 49(100X) 

ANIMAL S EXAMINE D HI STOP ATHOLOGICALL If 10 8 48 49 

ANIMAL S tllTH NON-TUMO B PATHOLOGY 2 (20X) 3 (38%) 14 (29X) 6 (12X ) 

INTEGUMEUIAH i SYSTEM 

NON E 

BESPIBAIC8Y SYSTEM * 1 (2*) 

LUN G 1 

CONGESTION 1 

CIRCULATOR Y SYSTEM 2 (UX ) 

HEAR T 1 

DEGENERATIO N PABENCHYMATOU S 1 

8YOCABEIU M 1 

INFLAHMATIO N CHRONIC 1 

DIGESTIVE SYSTEM 2 (20») 2 (25%) 10 (20*) 2 (IX) 

LIVER 2 1 10 1 

HEPATOCYTOMKGALY 

HYPBBPLASI A NODULA R 1 1 

1 1

7

HYPtBPLASiA DIFFUSE 3

STOMACH 

ULCtfl CHRONIC 

SMALL INTESTINE 

____ £2kX£ 

2 1 

*SY5XEtl PERCENTAGE S ABE BASED ON THE NUMBliB OF 

87 
TABLE D1 MALE MICE: NONNEOPLASTIC LESIONS (CONT.) 

HIGH DOSE

CONTROL

 LOW DOSE 

 CONTROL 

L O U DOSE HIGH D3SB 

UBINAB Y SYSTEM * 

KIDNE Y 

INFLAMMATION INTERSTITIAL 

2 (41)

2

2

 1 (2X) 

1 

1 

iNDOCBINi. SYSTEM 

THYROID 

ULTIdOBRANCHIAL CYST 

HYPEfiPLASIA FOU.ICULAB-CELL 

1 (2X)

1

1 

1 (2X) 

1 

1 

HEMATOPOJETIC SYSTEM 

SPLEEN 

LYMPHOI D HYPEHPLASIA 

LYMP H NODE 

INFLAMMATIO N 

HESENTEHIC LYJ1PHNODE 

EBECIA 

1 (10X) 

1 

1 

1 (13X) 

1 

1 

EEPBOEUCTIVE SYSTEM 

TESTIS 

ATROPH Y 

1 (2X)

1

I

 1 (2%) 

1 

1 

K&BVOUS SYSTEM 

NON E 

HUSCULOSKJiLETAL SYSTEM 

HONE 

SPECIAL SENSE ORGANS 

•SYSTEM PERCENTAGES ABE BASED OH THE NUMBER OF 

«8
 
TABLE D1 MALE MICE: NONNEOPLASTIC LESIONS (CONT.) 

HIGH DOSE LOW DOSE LOU DOSE HIGH D3SB 

CONTROL CONTROL 

ALL OTHiH SYSTEMS * 7 (70*) 5 (637.) 20 (U1X) 5 (10*) 

PEBIIONEUH 1 

INFLAMMATION 1 

NO ASSOCIATED OBGA N 7 l» 20 5 

NO LESION BEPOBffiD 7 <t 18 4 

AUTOLYSIS/NECBOPSY PEBF/NO HISTO 1 

AUTOLYSIS/NO NECROPSY PEBFOBMED 1 1 

*SYSTEM PERCENTAGES SHE BASED ON THE NUMBER OF ANIflA.iS._«.E.£fiOPS.J.£D 

89
 
TABLE D2 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE MICE FED CHLORDANE IN THE DIET 

LOW DOSE HIGH DOSE LOB DOSE HIGH DOSE 

CONTROL CONTROL 

ANIMALS INITIALLY III STUDY 10 10 50 50 

ANIMALS NECEOPSISD 10 (100X) 10(100%) 50(100X) 50(100X) 

ANIMALS EXAMINED HI STOPATHOLOGICALL Y 10 10 50 53 

ANIMALS KITH NON-TUMOR PATHOLOGY 7 (70S) 6 (60%) 28 (56X) 16 (32X) 

INTEGUMENTAR Y SYSTEM 

NONE 

RESPIRATOR Y SYSTEM * 2 (20X) 1 (2X) 1 (2X) 

LUN G 2 1 1 

CONGESTION PASSIVE 1 

EDEMA 1 

INFLAMMATION FOCAL 1 

HYPEBPLASIA ALVEOLAR-CELL 2 

CIRCULATORY SYSTEM 1 (2X) 

RENAL ARTERY 1

 

INFLAMMATION 1

 

DIGESTIVE SYSTEM 1 (10X) 1 (10%) 6 <12X) 9 (18X) 

LIVER 1 1 5 6 

INFLAMMATION 1 1 

INFLAMMATION FOCAL GRANULOMATOU S 1 

NECROSIS FOCAL 1 

HYPERPLASIA NODULA R 1 3 3 

HYPEBPLASIA DIFFUSE 3 3 

HEMATOPOIESIS 1 

PANCREAS 1 2 ' 

ATROPH Y 1 2 

STOMACH 1 

INFLAMMATION ACUT E _ . 1 

• SYSTEM PERCENTAGES ASE BASED ON THE NUMBER OF 41!Ifllii2_Jl££fi2E5LEC 

90 
TABLE D2 FEMALE MICE: NONNEOPLASTIC LESIONS (CONT.) 

LOW DOSE HIGH DOSE LOM DOSE HIGH DOSE 

CONTROL CONTROL 

UHINAH Y SYSTEM * 1 (10X) 1 (10%) 3 (6X) 2 '(.a*) 

KIDNE Y 1 1 3 2 

LYMPHOCYTIC INFLA M INFILTBATE 1 

INFLAMMATIO N INTERSTI1IAL 2 1 

INFLAMMATIO N GflANULOMATOU S 1 

AMYLOIDOSIS 1 1 

4ND3CBINE SYSTEM 1 (2*) 1 (2X) 

THYROI D 1 1 

HYPtBPLASIA FOLUCULAB-CELL 1 1 

HSSATOPOIETIC SYSTE M 2 (20*) 1 (10*) 3 (6*) 1 (2*) 

SPLEEN 2 3 1 

INFLAMMATIO N GRANULOMA1OU S 1 

LYMPHOI D HYPEBPLASI A 2 1 

HEMATOPOIESIS 2 

LYMP H NODJi 1 1 1 

CONGESTION CHHONIC PASSIVE 1 

INFLAMMATIO N 1 

INFLAMMATIO N GBAN ULOMATOUS 1 

RENA L LYMP H NODfi 1 

INFLAMMATIO N 1 

SEPBODUCTIVE SYSTEM 3 (30%) 5 (50*) 21 (<»2X) 3 (6X) 

UTEBU S 2 1 

INFLAMMATIO N 1 

INFLAMMATIO N SUPPUHA1IVE 2 

UTEBUS/ENDOMETBIUM 1 1 5 1 

INFLAMMATIO N SUPPUBATIV E 1 

HYPBBPLASI A CYSTIC 1 5 1 

OVABY/OVIOUCT 1 1 

INFLAMMATIO N 1 

— IflmflH.AT.ICfi ACUiE. SUPPUBiliVE J. 

*SYSTi,M PERCENTAGES ARE BASED ON THE NUMBE B OF AN154L.£_JJECBOPS.I.ED| 

91 
TABLE 02 FEMALE MICE: NONNEOPLASTIC LESIONS (CONT.) 

LOW DOSE 

CONTROL 

HIGH DOSE

CONTROL 

 LOB DOSE HI3H DOSE 

OVASY

CYST 

FOLLICULAB CYSI 

CORPUS LUTEUli CYST 

INFLAHHATION SUPPURATIVE 

INFLAMMATION ACUTE SUPPURATIVE

INFLAHHATION SOBACUTE

INFLAMMATION CHEONIC 

3 

2 

1 

4

1 

1

1 

1

 16

 3

4 

6 

1 

2 

1 

1 

NERVOUS SYSTEM 

NONE * 

HUSCULOSK£LETAL SYSTEH

SKELETAL HUSCLE 

DEGENERATION 

* 1 (2*) 

1 

1 

SPECIAL SENSE OSGAN5 

NON E 

ALL OTHER SYSTEMS 3 <30») 4 (40%) 17 (3«*) 9 (18») 

PERITONBUH 

INFLAHHATION ACUTE 

1 

NO ASSOCIATED ORGAN 

NO LESION REPORTED 

2 

2 

U 

4 

17 

17 

9 

9 

*SYSI£.B PERCENTAGES &RE BASED ON THE HUM BEE OF 

92
 
APPENDIX E
 

STATISTICAL ANALYSES OF NEOPLASMS
 

IN RATS FED CHLORDANE IN THE DIET
 

93
 

Table El. Statistical Analysis of Neoplasms of the Liver in Rats Fed
 

Chlordane in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Test for
 

No. Rats with Liver Tissue Examined Dose-related
 

Trend
 

Sex Control Low Dose High Dose (P)
 

Hepatocellular Carcinoma
 

Male 0/8 1/44 0/40
 

0.571
 

Female 0/10 0/47 0/39
 

0% 0% 0%
 —
 

Hepatocellular Carcinoma and/or Neoplastic Nodule
 

Male 0/8 2/44 0/40 _.


0% 5% 0% 0.39C


Female 1/10 11/47 6/39
 

10% 23% 15% 0.51C


fMales: 203.5 ppm; females: 120.8 ppm.
 

Males: 407.0 ppm; females: 241.5 ppm.
 

?P value given in the direction of a negative linear trend.
 

Tests After
 

Life-table
 

Adjustment
 

(P)
 

j
 

-d
 

 0.43
 

Validity of test is dubious because of the small number of animals with tumors.
 
Table E2. Statistical Analysis of Neoplasms of the Liver in Rats Fed
 

Chlordane in the Diet (using pooled control)
 

No. Rats with Lesion/ Exact Test for Tests After 

_ No. Rats with Liver Tissue Examined _ Dose-related Life-table 

, Trend Adjustment 

Sex Control Low Dose High Dose _ (P)_ (P) _ 

Hepatoaellular Carcinoma
 

Male 1/58 1/44 0/40
 

2% 2% 0% 0.42C
 

Female 0/59 0/47 0/39
 

0% 0% 0%
 ^^ -«i
 

Hepatooellular Carcinoma and/or Neoplastie Nodule
 

Male 3/58 2/44 0/40
 

5% 5% 0% 0.15 0.08
 

Female 5/59 ll/47d 6/39
 

8% 23% 15% 0.10 0.08
 

fMales: 203.5 ppm; females 120.8 ppm.
 

/•» Males: 407.0 ppm; females: 241.5 ppm.
 

7 value given in the direction of a negative linear trend.
 

Significantly higher than its pooled control in both test (P < 0.05).
 

validity of test is dubious because of the small number of animals with tumors.
 
Table E3. Statistical Analysis of Neoplasms of Thyroid Follicular Cells
 

in Rats Fed Chlordane in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Teat for Tests After
 

No. Rats with Thyroid Examined3 Dose-related Life-table
 

Trend Adjustment
 

Sex Control Low Dose High Dosec

 (P) (P)
 

Carcinoma
 

Male 0/6 1/34 2/31 A
 

0% 3% 6% 0.36
 

Female	 0/10(0/9) 2/43(2/38) 2/32(2/27) A
 

0% (0%) 5% (5%) 6% (7%) 0.37(0.35)
 

VO
 

—4
 

Carcinoma and/or Adenoma
 

Male 0/6 1/34 6/31
 

0% 3% 19% 0.02 0.01+
 

Female 0/10 4/43 6/32
 

0% 9% 19% 0.06 0.02
 

The early deaths are eliminated in those figures in parentheses, i.e., deaths before
 

the first tumor was found in any of the groups being compared to one another.
 

Males; 203.5 ppm; females: 120.8 ppm.
 

,Males: 407.0 ppm; females: 241.5 ppm.
 

Validity of test is dubious because of the small number of animals with tumors.
 
Table E4. Statistical Analysis of Neoplasms of Thyroid Follicular Cells
 

in Rats Fed Chlordane in the Diet (using pooled control)
 

No. Rats with Lesion/ Exact Test for Tests After
 

No. Rats with Thyroid Examined Dose-related Life-table
 

Trend Adjustment
 

Sex Control Low Dose High Dose (P) (P)
 

Carcinoma
 

Male 1/51 1/34 2/31 e
 

2% 3% 6% 0.23
 —
 

Female 1/58 2/43 2/32
 

2% 5% 6% 0.18 0.11
 

VO
 

oo
 Carcinoma and/of Adenoma
 

Male 4/51 1/34 6/31
 

8% 3% 19% 0.10 0.09
 

Female 3/58 4/43 6/32d
 

5% 9% 19% 0.03 0.01
 

fMales: 203.5 ppm; females: 120.8 ppm.
 

Males: 407.0ppm; females: 241.5 ppm.
 

"TData depart significantly from a linear trend (P < 0.05).
 

Significantly higher than its pooled control in both tests (P < 0.05).
 

Validity of test is dubious because of the small number of animals with tumors.
 
Sex
 

Carcinoma
 

Male
 

Female
 

vO
 

Carcinoma
 

Male
 

Female
 

Table E5. Statistical Analysis of Neoplasms of Thyroid C-cells in Rats
 

Fed Chlordane in the Diet (using matched control)
 

No. Rats with Lesion/
 

No. Rats with Thyroid Examined
 

Control
 

0/6
 

0%
 

3/10
 

30%
 

and/or Adenoma
 

0/6
 

0%
 

3/10
 

30%
 

Low Dose
 

1/34
 

3%
 

0/43
 

0%
 

5/34
 

15%
 

3/43
 

7%
 

Exact Test for


Dose-related


. Trend


High Dose (P)


2/31
 

6% 0.36


3/32 .
a
 9% 0.25C

'


5/31
 

16% 0.30


10/32
 d
 31% 0.16


 Tests After
 

 Life-table
 

 Adjustment
 

 (P)
 

—e
 

 .
 

0.23C'Q
 

 0.21
 

d
 0.046a
 

Males : 203.5 ppm; females: 120.8 ppm.
 

Males : 407.0 ppm; females: 241.5 ppm.
 

*TP value given in the direction of a negative linear trend.
 

Data depart significantly from a linear trend (P< 0.01).
 

Validity of test is dubious because of the small number of animals with tumors.
 
Table E6. Statistical Analysis of Neoplasms of Thyroid C-cells in Rats
 

Fed Chlordane in the Diet (using pooled control)
 

No. Rats with Lesion/ Exact Test for Tests After
 

No. Rats with Thyroid Examined Dose-related Life-table
 

Trend Adjustment
 

Sex Control Low Dose High Dose (P) (P)
 

Carcinoma
 

Male	 1/51 1/34 2/31
 

2% 3% 0.23
 

Female	 5/58 0/43J 3/32 

c.d
 c,d
 0%	 9% 0.36 0.48
 

o
 

o
 Careinoma and/or Adenoma
 

Male	 4/51 5/34 5/31
 

8% 15% 16% 0.16 0.16
 

Female	 11/58 3/43f 10/328
 

d	 p
 

19% 7%	 31% 0.18a 0.08
 

j^Males: 203.5 ppm; females: 120.8 ppm.
 

Males: 407.0 ppm; females: 241.5 ppm.
 

,P value given in the direction of a negative linear trend.
 

Data depart significantly from a linear trend (P < 0.05).
 

-Data depart significantly from a linear trend (P < 0.01).
 

Significantly lower than its pooled control in life-table adjusted test (P < 0.05).
 

.Significantly higher than its pooled control in life-table adjusted test (P < 0.05),
 

Validity of test is dubious because of the small number of animals with tumors.
 
Table E7. Statistical Analysis of Malignant Fibrous Histiocytoma in
 

Rats Fed Chlordane in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Test for Tests After
 

No. Rats Necropsieda Dose-related Life-table
 

Trend Adjustment
 

Sex Control Low Dose High Dose0 (P) (P)
 

Male	 0/8(0/5) 1/44(1/37) 7/44(7/37)
 

0% (0%) 2% (3%) 16% (19%) 0.014(0.016) 0.007
 

Female	 1/10(1/10) 1/49(1/46) 1/44(1/36)
 A A
 

10% (10%) 2% (2%) 2% (3%) 0.32a

(0.37)a
 

The early deaths are eliminated in those figures in parentheses, i.e., deaths before
 

, the first tumor was found in any of the groups being compared to one another.
 

Males: 203.5 ppm; females: 120.8 ppm.
 

^Males: 407.0 ppm; females: 241.5 ppm.
 

P value given in the direction of a negative linear trend.
 

Validity of test is dubious because of the small number of animals with tumors.
 
Table E8. Statistical Analysis of Malignant Fibrous Histiocytoma in
 

Rats Fed Chlordane in the Diet (using pooled control)
 

No. Rats with Lesion/ Exact Test for Tests After
 

No. Rats Necropsied Dose-related Life-table
 

V Trend Adjustment
 

Sex Control Low Dose High Dose (P) (P)
 

Male 2/58 1/44 7/44c
 

3% 2% 16% 0.02 0.01
 

Female 1/60 1/49 1/44 A
 

2% 2% 2% 0.54 -­

^Males: 203.5 ppm; females : 120.8 ppm.
 

o
 Males: 407.0 ppm; females : 241.5 ppm.
 K>
 ^Significantly higher than its pooled control in both tests (P < 0.05).
 Validity of test is dubious because of the small number of animals with tumors.
 
Table E9. Statistical Analysis of Neoplasms of the Pituitary Gland in Rats
 

Fed Chlordane in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Test for Tests After 

No. Rats with Pituitary Examined' Dose-related Life-table 

Trend Adjustment 

Sex Control Low Dose High Dose (P) (P) 

Chromophobe Adenocareinoma ov Carcinoma
 

Male	 0/6(0/5) 1/34(1/32) 1/34(1/31)
 

g
 0% (0%) 3% (3%) 3% (3%) 0.64(0.64)
 

Female	 0/10(0/9) 1/46 (1/41) 0/37(0/32) A A
 

0% (0%) 2% (2%) 0% (0%) 0.60 (0.61)
 

o
 

OJ Chromophobe Adenooaroinoma ov Carcinoma and/or Chromophobe Adenoma
 

Male	 0/6 10/34 8/34
 

0% 29% 24% 0.39 0.23
 

Female	 6/10 8/46 11/37
 d p
 'e
 60%	 17% 30% 0.26d 0.38d)f
 

The early deaths are eliminated in those figures in parentheses, i.e., deaths before
 

the first tumor was found in any of the groups being compared to one another.
 

Males: 203.5 ppm; females: 120.8 ppm.
 

TMales: 407.0 ppm; females: 241.5 ppm.
 

P value given in the direction of a negative linear trend.
 

,-Data depart significantly from a linear trend (P < 0.01).
 

Data depart significantly from a. linear trend (P < 0.05).
 

Validity of test is dubious because of the small number of animals with tumors.
 
Table E10. Statistical Analysis of Neoplasms of the Pituitary Gland
 

in Rats Fed Chlordane in the Diet (using pooled control)
 

No. Rats
 

No . Rats with
 

Sex
 

Chpomophobe
 

Male
 

Female
 

o	 fMales:


Males:


Control
 

with Lesion/ Exact Test for 

Pituitary Examined Dose-related 

V Trend 

Low Dose High Dose (P) 

Adenoaaroinoma OP Carcinoma and/or Adenoma
 

16/48
 

33%
 

23/52
 

44%
 

 203.5 ppm; females:


 407.0 ppm; females:


10/34 

29% 

8/34 

24% 0.20 

8/46e 

17% 

11/37 

30% 0.04C'd 

 120.8 ppm. 

 241.5 ppm. 

_.P value given in the direction of a negative linear trend.
 

Data depart significantly from a linear trend (P < 0.01).
 6

Significantly lower than its pooled control in both tests (P < 0.01).
 

Tests After
 

Life-table
 

Adjustment
 

(P)
 

0.18C
 

'd
 0.13C

Table Ell. Statistical Analysis of Neoplasms of the Mammary Gland in
 

Female Rats Fed Chlordane in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Test for Tests After 

No. Rats Necropsieda Dose-related Life-table 

Control Low Dose High Dose Trend Adjustment 

(0 ppm) (120.8 ppm) (241.5 ppm) (P) (P) 

Carcinoma or Adenocarcinoma
 

1/10(1/10) 2/49(2/49) 3/44(3/40)
 

10% (10%) 4% (4%) 7% (8%) 0.63(0.59) 0.41
 

Carcinoma or Adenocarcinoma and/or Adenoma, Fibroadenoma or Fibroma
 

2/10 18/49 8/44
 V)


20% 37% 18% 0.17 0.34°
 

The early deaths are eliminated in those figures in parentheses, i.e., deaths before
 

the first tumor was found in any of those groups being compared to one another.
 

P value is given in the direction of a negative linear trend.
 
Table E12. Statistical Analysis of Neoplasms of the Mammary Gland in
 

Female Rats Fed Chlordane in the Diet (using pooled control)
 

- » j._ ­- ­

Control

(0 ppm)

No. Rats with Lesion/

No. Rats Necropsied

 Low Dose High Dose

 (120.8 ppm) (241.5 ppm)

 Exact Test for

 Dose-related

 Trend

 (P)

 Tests After 

 Life-table 

 Adjustment 

 (P) 

Carcinoma or Adenocarainoma 

2/60 2/49 3/44
 

3% 4% 7% 0.27 0.17
 

Carainoma or Adenooaroinoma and/or Adenoma, F-ibroadenoma or Fibroma
 

10/60 18/49 8/44
 

17% 37% 18% 0.30a 0.16b
 

,Data depart significantly from a linear trend (P < 0.01).
 

Data depart significantly from a linear trend (P < 0.05).
 

Significantly higher than its pooled control in both tests (P < 0.05).
 
Table E13. Statistical Analysis of Neoplasms of the Uterus in Female
 

Rats Fed Chlordane in the Diet (using matched control)
 

No. Rats with Lesion/
 

No. Rats with Uterus Examined3
 

Control
 

(0 ppm)
 

Sarcoma
 

0/9(0/8)
 

0% (0%)
 

Sarcoma and/or
 

0/9
 

0%
 

Adenocarcinoma
 

0/9(0/8)
 

0% (0%)
 

Sarcoma and/or
 

0/9
 

0%
 

Sarcoma and/or
 

0/9
 

0%
 

Low Dose High Dose
 

(120.8 ppm) (241.5 ppm)
 

0/47(0/35) 2/33(2/22)
 

0% (0%) 6% (9%)
 

Endometrial Stromal Polyp
 

3/47 3/33
 

6% 9%
 

1/47(1/41) 0/33(0/23)
 

2% (2%) 0% (0%)
 

Adenocarcinoma
 

1/47 2/33
 

2% 6%
 

Adenocarcinoma and/or Endometrial
 

4/47 3/33
 

9% 9%
 

Exact Test for
 

Dose-related
 

Trend
 

(P)
 

0.13(0.11)
 

0.28
 

•u v
 

0.63D(0.68)D
 

0.27
 

Stromal Polyp
 

0.36
 

Tests After
 

Life-table
 

Adjustment
 

(P)
 

c
 

0.12
 

0.17
 

The early deaths are eliminated in those figures in parentheses, i.e., deaths before
 

the first tumor was found in any of the groups being compared to one another.
 

? value is given in the direction of a negative linear trend.
 

Validity of test is dubious because of the small number of animals with tumors.
 
Table E14. Statistical Analysis of Neoplasms of the Uterus in Female
 

Rats Fed Chlordane in the Diet (using pooled control)
 

No. Rats with Lesion/ Exact Test for Tests After
 

No. Rats with Uterus Examined Dose-related Life-table
 

Control Low Dose High Dose Trend Adjustment
 

(0 ppm) (120.8 ppm) (241.5 ppm) (P) (P)
 

Sarcoma
 

0/56 0/47 2/33b
 

0% 0% 6% 0.06 — c
 

Sarcoma and/or Endometrial Stromal Polyp
 

6/56 3/47 3/33
 

o 11% 6% 9% 0.36a

 0.44
 

00
 

Sarcoma and/or Adenocarainoma
 

0/56 1/47 2/33b

 c
 

0% 2% 6% 0.07
 

Sarcoma and/or Adenocarcinoma and/or Endometrial Stromal Polyp
 

6/56 4/47 3/33
 

11% 9% 9% 0.41 0.47
 

,P value is given in the direction of a negative trend.
 

Significantly higher than its pooled control in life-table adjusted test (P < 0.05)
 

Validity of test is dubious because of the small number of animals with tumors.
 
APPENDIX F
 

STATISTICAL ANALYSES OF NEOPLASMS
 

IN MICE FED CHLORDANE IN THE DIET
 

109
 

__ 

Table Fl. Statistical Analysis of Neoplasms of the Liver in Male Mice
 

Fed Chlordane in the Diet (using matched control)
 

No. Mice with Lesion/ Fisher Exact Test for Tests After
 

No . Mice with Liver Tissue Examined Exact Dose-related Life-table
 

Control Low Dose High Dose Test Trend Adjustment
 

(0 ppm) (29.9 ppm) (56.2 ppm) (P) (?) (P)
 

Hepatoaellu'lar Carcinoma
 

1/10(1/10) 43/49(43/47)
 

10% (10%) 88% (91%) < 0.0001 (=0.0001) < 0.0001
 

1/8(1/8) 16/48(16/36)
 

12% (12%) 33% (44%) 0.23(0.10) 0.05+
 —
 

Combined <0.0001fc 0.0001) < 0.0001
 

The early deaths are eliminated in those figures in parentheses, i.e., deaths before
 

the first tumor was found in any of the groups being compared to one another,
 
Table F2. Statistical Analysis of Neoplasms of the Liver in Male Mice
 

Fed Chlordane in the Diet (using pooled control)
 

No. Mice with Lesion/ Fisher Exact Test for Tests After
 

No. Mice with Liver Tissue Examined Exact Dose-related Life-table
 

Control
 

(0 ppm)
 

Hepatoae1lulor
 

17/92
 

18%
 

17/92
 

18%
 

17/92
 

18%
 

Low Dose High Dose Test Trend Adjustment 

(29.9 ppm) (56.2 ppm) (P) (P) (P) 

Carcinoma 

43/49 

88% < 0.0001 < 0.0001 — 

16/48 — 33% 0.04 0.001 

16/48 43/49 

33% 88% < 0.0001a < 0.0001 

Data depart significantly from a linear trend (P < 0.01).
 
Table F3. Statistical Analysis of Neoplasms of the Liver in Female Mice
 

Fed Chlordane in the Diet (using matched control)
 

No. Mice with Lesion/

No. Mice with Liver Tissue Examined

Control 

(0 ppm) 

Low Dose 

(30.1 ppm) 

High Dose

(63.8 ppm)

 Fisher

 Exact

 Test

 (P)

 Exact Test for 

 Dose-related 

 Trend 

 (P) 

Tests After 

Life-table 

Adjustment 

(P) 

Hepatoaellular Caroinoma 

0/9(0/9) 

0% (0%) — 

— 

34/49(34/47) 

69% (72%) 0.0001(0.0001) 0.0002 

0/10(0/9)

0% (0%)

 3/47(3/46)

 6% (7%) 

—

0.58(0.55) v 

Combined 0.0001(0.0001) 0.0002 

The early deaths are eliminated in those figures in parentheses, i.e., deaths before
 

the first tumor was found in any of the groups being compared to one another.
 

Validity of test is dubious because of the small number of animals with tumors.
 
__ 

__ 

Table F4. Statistical Analysis of Neoplasms of the Liver in Female Mice
 

Fed Chlordane in the Diet (using pooled control)
 

No. Mice with Lesion/ Fisher Exact Test for Tests after
 

No. Mice with Liver Tissue Examined Exact Dose-related Life-table
 

Control Low Dose High Dose Test Trend Adjustment
 

(0 ppm) (30.1 ppm) (63.8 ppm) (P) (P) (P)
 

Hepatocellular Cccccinoma
 

3/78 34/49
 

4% 69% < 0.0001 < 0.0001
 —
 

3/78 3/47
 

4% 6% 0.41 0.26
 —
 

3/78 3/47 34/49
 

4% 6% 69% <o.oooia

 <o.oooia
 

T)ata depart significantly from a linear trend (P < 0.01).
 
APPENDIX G
 

ANALYSIS OF FORMULATED DIETS
 

FOR CONCENTRATION OF CHLORDANE
 

115
 

APPENDIX G
 

Analysis of Formulated Diets for
 

Concentration of Chlordane
 

A 100 g sample of the diet mixture was shaken with 12 ml acetone
 

at room temperature for 16 hrs., then filtered through Celite
 

with acetone washes, and reduced in volume to 10 ml. After
 

appropriate dilutions, the solution was quantitatively analyzed
 

for chlordane by gas-liquid chromatography (electron-capture
 

detector, 10% DC-200 on Gas-Chrom Q column). Recoveries were
 

checked with spiked samples, and external standards' were used for
 

calibration.
 

Theoretical
 

Dietary
 

Level (ppm)
 

30
 

40
 

50
 

60
 

80
 

100
 

160
 

200
 

400
 

800
 

No. of
 

Samples
 

21
 

4
 

10
 

12
 

4
 

12
 

3
 

8
 

9
 

5
 

Sample
 

Analytical
 

Mean (ppm)
 

30.1
 

39.4
 

49.2
 

59.8
 

78.7
 

100.0
 

154.0
 

198.0
 

391.0
 

802.0
 

Coefficient of
 

Variation (%)
 

5.6
 

3.5
 

3.1
 

3.2
 

3.9
 

4.4
 

3.8
 

1.8
 

3.6
 

2.9
 

Range (ppm)
 

27.6-34.6
 

38.1-40.6
 

46.0-51.8
 

55.0-62.5
 

74.2-80.6
 

93.5-111.5
 

150-161
 

195-204
 

374-412
 

770-833
 

117
 HJ.S. GOVERNMENT PRINTING OFFICE: 1977 241-161/3056 1-3 

DHEW Publication No. (NIH) 77-808