National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 9 

1977 

BIOASSAY OF 

HEPTACHLOR 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 76-44-8 

NCI-CG-TR-9 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

HEPTACHLOR
 

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) 77-809
 

BIOASSAY OF
 

HEPTACHLOR
 

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 heptachlor 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.
 

Weisburger^' 2 and R. R. Bates^-. The doses were selected by Drs.
 

T. E. Shellenburger^'^, J. H. Weisburger and R. R. Bates. Animal
 

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

Shellenburger and H. P. Burchfield^, with the technical
 

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

Necropsies were performed under the supervision of Drs. E.
 

Bernal^ and B. Buratto-^. The histopathologic evaluation was
 

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

Renne->»9 and Dr. J. F. Ferrell-*, and the diagnoses included in
 

this report represent the interpretation of these pathologists.
 

Pathologists from Tracor Jitco and NCI reviewed selected slides
 

and concurred with the overall pathologic evaluation of the
 

study.
 

Compilation of individual animal survival and summary tables was
 

performed by EG&G Mason Research Institute?. Pathology tables
 

iii
 
were prepared oy Dr. R. A. Renne. Statistical analyses were
 

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

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

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

toxicologists, Drs. W. £. 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. D. Reichardt
 

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

of the participating organizations^»-^»^»^.
 

^Carcinogenesis Program, Division of Cancer Cause and Prevention,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

^Now with the Naylor Dana Institute 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.
 

^Now 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.
 

^Now with Battelle Pacific Northwest Laboratories,
 

Battelle Boulevard, Richland, Washington.
 

iv
 
SUMMARY
 

A bioassay of technical-grade heptachlor 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
 

heptachlor for 80 weeks, then observed for 30 weeks. Doses for
 

females were first increased, but because of toxic effects the
 

doses were then reduced twice for both male and female rats
 

during the remaining course of the tests. Time-weighted average
 

doses used for the male rats were 38.9 and 77.9 ppm; for the
 

females, 25.7 and 51.3 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 110-111 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 a concurrent control group was started with
 

each. Because of toxic effects, doses were reduced once for the
 

males at 17-18 weeks after the initiation of tests; twice for the
 

females, at 17 and 30 weeks, after the initiation of tests. The
 

time-weighted average doses used for the male mice were 6.1 and
 

13.8 ppm; for the females, 9 and 18 ppm. Matched controls
 

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

controls consisted of the matched—control groups combined with 90
 

untreated male and 70 untreated female mice from similar 

bioassays of five other compounds. All surviving mice were 

killed at 90-91 weeks. 

The effects of heptachlor 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 average body
 

weights of rats treated with high doses were consistently lower
 

than those of untreated controls, while body weights of low—dose
 

rats were unaffected. Body weights of mice given either high or
 

low doses showed little or no differences from those of control
 

mice; however, other adverse clinical signs were found in
 

high-dose mice, predominantly in the females.
 

V 
The effects of heptachlor on survival rates indicated that
 

mortality was dose-related for both female rats and female mice,
 

but not for males of either species. However, a substantial
 

proportion of all groups of animals survived to an age at which
 

tumors could be expected to appear.
 

In mice, hepatocellular carcinoma showed a highly significant
 

dose-related trend in both males (matched controls 5/19, low dose
 

11/46, high dose 34/47, P = 0.001) and females (control 2/10, low
 

dose 3/47, high dose 30/42, P < 0.0001). When pooled controls
 

were used for the comparison, the significance of the trend in
 

males increased to P < 0.0001. Comparably high levels of
 

significance were attained when the data were subjected to
 

life-table adjustment. No other tumors were found in mice in
 

sufficient numbers to justify analysis.
 

In marked contrast to the findings observed in mice, no hepatic
 

tumors were observed in rats administered heptachlor. There was
 

significant statistical evidence for the induction of proliferative lesions of follicular cells of the thyroid in treated
 

female rats, but this finding was discounted because the rates of
 

incidence were comparatively low and are known to be variable in
 

control rat populations.
 

It is concluded that under the conditions of this bioassay,
 

heptachlor 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 the 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) 29
 

IV. Results - Mice 33
 

A. Body Weights and Clinical Signs (Mice) 33
 

B. Survival (Mice) 33
 

C. Pathology (Mice) 38
 

D. Statistical Analyses of Results (Mice) 40
 

V. Discussion 43
 

VI. Bibliography 49
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms and
 

Other Proliferative Lesions in Rats Fed
 

Heptachlor in the Diet 51
 

Table Al Summary of the Disposition of Tissues 53
 

Table A2 Proliferative Lesions of the Thyroid 54
 

Table A3 Other Neoplasms of the Endocrine System.... 55
 

vii
 
p

£S.e 

Table A4 Neoplasms of the Digestive System 56 

Table A5 Neoplasms of the Reproductive System 

and Mammary Gland 57 

Table A6 Neoplasms of the Cardiovascular System 59 

Table A7 Neoplasms of the Hematopoietic System 60 

Table A8 Miscellaneous Neoplasms 61 

Appendix B Summary of the Incidence of Neoplasms and 

Other Proliferative Lesions in Mice Fed 

Heptachlor in the Diet 63 

Table Bl Summary of the Disposition of Tissues 65 

Table B2 Proliferative Lesions of the Liver 66 

Table B3 Miscellaneous Neoplasms 67 

Appendix C Summary of the Incidence of Nonneoplastic Lesions in Rats Fed 

Heptachlor in the Diet 69 

Table Cl Summary of the Incidence of Nonneoplastic Lesions in Male Rats Fed 

Heptachlor in the Diet 71 

Table C2 Summary of the Incidence of Nonneoplastic Lesions in Female Rats Fed 

Heptachlor in the Diet 76 

Appendix D Summary of the Incidence of Nonneoplastic Lesions in Mice Fed 

Heptachlor in the Diet 81 

Table Dl Summary of the Incidence of Nonneoplastic Lesions in Male Mice Fed 

Heptachlor in the Diet 83 

Table D2 Summary of the Incidence of Nonneoplastic Lesions in Female Mice Fed 

Heptachlor in the Diet 86 

viii 
Page 

Appendix E Statistical Analyses of Neoplasms 

in Rats Fed Heptachlor in the Diet 89 

Table El Statistical Analysis of Neoplasms of 

the Liver in Rats Fed Heptachlor in 

the Diet (using matched control) 91 

Table E2 Statistical Analysis of Neoplasms of 

the Liver in Rats Fed Heptachlor in 

the Diet (using pooled control) 92 

Table E3 Statistical Analysis of Neoplasms of 

Thyroid Follicular Cells in Rats 

Fed Heptachlor in the Diet 

(using matched control) 93 

Table E4 Statistical Analysis of Neoplasms of 

Thyroid Follicular Cells in Rats 

Fed Heptachlor in the Diet 

(using pooled control) 94 

Table E5 Statistical Analysis of Neoplasms of 

Thyroid C-Cells in Rats Fed Heptachlor 

in the Diet (using matched control) 95 

Table E6 Statistical Analysis of Neoplasms of 

Thyroid C-Cells in Rats Fed Heptachlor 

in the Diet (using pooled control) 96 

Table E7 Statistical Analysis of Neoplasms of the 

Pituitary Gland in Rats Fed Heptachlor 

in the Diet (using matched control) 97 

Table E8 Statistical Analysis of Neoplasms of the 

Pituitary Gland in Rats Fed Heptachlor 

in the Diet (using pooled control) 98 

Table E9 Statistical Analysis of Neoplasms of 

the Mammary Gland in Female Rats Fed 

Heptachlor in the Diet (using matched 

control) 99 

Table ElO Statistical Analysis of Neoplasms of 

the Mammary Gland in Female Rats Fed 

Heptachlor in the Diet (using pooled 

control) 100 

ix
 
P

?JB£ 

Table Ell Statistical Analysis of Neoplasms of 

the Uterus in Female Rats Fed Heptachlor 

in the Diet (using matched control) 101 

Table El2 Statistical Analysis of Neoplasms of 

the Uterus in Female Rats Fed Heptachlor 

in the Diet (using pooled control) 102 

Appendix F Statistical Analyses of Neoplasms in 

Mice Fed Heptachlor in the Diet 103 

Table Fl Statistical Analysis of Neoplasms of 

the Liver in Male Mice Fed Heptachlor 

in the Diet (using matched control) 105 

Table F2 Statistical Analysis of Neoplasms of 

the Liver in Male Mice Fed Heptachlor 

in the Diet (using pooled control) 106 

Table F3 Statistical Analysis of Neoplasms of 

the Liver in Female Mice Fed Heptachlor 

in the Diet (using matched control) 107 

Table F4 Statistical Analysis of Neoplasms of 

the Liver in Female Mice Fed Heptachlor 

in the Diet (using pooled control)... 108 

Appendix G Analysis of Formulated Diets for 

Concentration of Heptachlor 109 

TABLES 

Table 1 Design of Heptachlor Chronic Feeding 

Studies in Rats 11 

Table 2 Design of Heptachlor Chronic Feeding 

Studies in Mice 12 

FIGURES 

Figure 1 Growth Curves for Rats Fed Heptachlor 

in the Diet 22 

Figure 2 Survival Curves for Rats Fed 

Heptachlor in the Diet 23 

X 
Pa8i
 

Figure 3 Growth Curves for Male Mice Fed 

Heptachlor in the Diet 34 

Figure 4 Growth Curves for Female Mice Fed 

Heptachlor in the Diet 35 

Figure 5 Survival Curves for Male Mice Fed 

Heptachlor in the Diet 36 

Figure 6 Survival Curves for Female Mice Fed 

Heptachlor in the Diet 37 

xi
 

I. INTRODUCTION
 

Heptachlor is a member of the cyclodiene group of chlorinated
 

insecticides (aldrin, dieldrin, endrin, chlordane, heptachlor,
 

and endosulfan) that were developed in the 15 years following
 

World War II. It was registered as a commercial pesticide in
 

1952 for foliar, soil, and structure applications and for
 

malarial control programs (EPA, 1972; Casarett and Doull, 1975);
 

after I960 it was used primarily in soil applications against
 

agricultural pests and to a lesser extent against termites.
 

On November 18, 1974, the EPA issued a notice of intent to cancel
 

the registration and use of pesticides containing heptachlor,
 

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 heptachlor 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
 

was the use of heptachlor for treatment of seeds, control of ants
 

on Hawaiian pineapples, and control of the narcissus bulb fly.
 

The persistence of heptachlor in the environment has been well
 

documented. Human exposure occurs as a result of mobilization of
 

1 
the compound in crops, water, and atmosphere. Zero tolerance was
 

established for residues on 16 vegetables, 4 fruits, and 12 field
 

crops used for animal consumption; also, for residues in meat and
 

milk (EPA, 1972). Tolerance of 0.1 ppra was set for four
 

additional vegetables. An acceptable daily human intake of 0.005
 

mg/kg was established for heptachlor and its epoxide (FAO/WHO,
 

1967). Tne average daily intake of both compounds from 1965 to
 

1970 was 0.00003 mg/kfi, well below the acceptable daily intake
 

(Duggan and Corneliussen, 1972). When assays for heptachlor were
 

performed on tissue samples in 1970, mean concentrations of 0.08
 

ppm were detected in 96% of 3,451 hospital patients tested.
 

Similar observations were made in 1971 and 1972, with some
 

individual concentrations ranging as high as 1.68 ppm.
 

Heptachlor has also been shown to be transported across the
 

placenta and to pass into breast milk (EPA, 1974).
 

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 tneir Relationship to
 

Environmental Health, a committee of the Department of Health,
 

Education and Welfare. This review pointed out the need for an
 

evaluation of the carcinogenicity of several pesticides, including heptachlor (Secretary's Commission on Pesticides, 1969).
 

Because of known biological effects of low doses of neptachlor
 

over extended periods of time (FAD/WHO, 1967), the persistence of
 

2 
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.
 

3 

II. MATERIALS AND METHODS
 

A. Chemical
 

The material tested was technical-grade heptachlor obtained from
 

Velsicol Chemical Corporation (Chicago, 111.) in one batch for
 

use in the chronic study. Heptachlor is made by allylic
 

chlorination of chlordene, the Diels-Alder adduct of hexachlorocyclopentadiene and cyclopentadiene. According to the manufacturer, a typical analysis of the technical-grade product is
 

73% heptachlor, 22% trans-chlordane, and 5% nonachlor. Analysis
 

of the bioassay batch at Gulf South Research Institute by melting
 

point, elemental analysis, and spectroscopic and chromatographic
 

techniques confirmed the identity °f the material.
 

After completion of the bioassay, this batch was reanalyzed at
 

Midwest Research Institute. Gas-liquid chromatographic (glc)
 

comparison with the sample that was used for the bioassay showed
 

the two samples to be essentially the same. All major components
 

were present in similar proportions, and all minor components at
 

a level > 0.1% appeared in both samples. Using authentic
 

proportions of each component for standardization, it was
 

determined by glc that the technical-grade heptachlor contained
 

approximately 72 + 3% heptachlor, 18% trans-chlordane, 2% cis5

 
chlordane, 2% nonachlor, 1% chlordene 0.2% hexachlorobutadiene,
 

and smaller amounts of 10-15 other compounds.
 

B. Dietary Preparation
 

All diets were formulated weekly using Wayne&/ Lab Blox Meal
 

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

required amount of heptachlor for each dietary concentration.
 

Small amounts of acetone (Mallinckrodt Chemical Works, St. Louis,
 

Mo.) were used as an aid to uniform dispersion of the test
 

compound in the feed. The diets were mixed mechanically to
 

assure homogeneity and to allow for evaporation of the acetone.
 

Corn oil equal to 2% of the final weight of feed 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
 

heptachlor. The corn oil (LouanaS)) was produced by Opelousas
 

Refinery Co., Opelousas, La. Formulated diets were stored at
 

approximately 17°C until used, but no longer than 1 week. Water
 

and the formulated diets were made available ad libitum to the
 

experimental animals and were replaced three times per week.
 

The stability of heptachlor in feed was checked by analyzing
 

formulated diets for the concentration of heptachlor at intervals
 

over a 7-day period. Diets containing 5 and 20 ppm heptachlor
 

showed no change on standing at ambient temperature for this
 

period.
 

6 
Theoretical heptachlor 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 concentration, ranging from 5 to 160 ppm, the mean of the
 

analytical concentrations for the samples checked was within 10%
 

of the theoretical concentration, and the coefficient of
 

variation was not more than 6.9% at any level. 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 procured 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 randomly selected and assigned to cages and treatment groups.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. Incoming air was filtered through fiberglass air condi7

 
tioner 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 hanging galvanized steel-mesh cages; the mice, in
 

plastic cages equipped with filter caps, five mice per cage for
 

females and two or three per cage for males. 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^H 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 heptachlor, along with their controls, were housed
 

in a room by themselves. Mice receiving heptachlor were
 

maintained in a room housing mice administered chlordane,
 

toxaphene, 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 the
 

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

and low concentrations (hereinafter referred to as "high doses"
 

8 
and "low doses") were determined for administration in the
 

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

1/2 of the high doses. In the subchronic studies, heptachlor 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 20 ppm through 320 ppm,
 

were used. Weights of animals and consumption of feed were
 

measured weekly, and deaths were noted.
 

With rats given 320 ppm, two out of five males and all of five
 

females died. At 80 or 160 ppm, no males died, and their average
 

weekly gain in weight and consumption of feed were similar to
 

those of untreated controls. Four of five females died, however,
 

when given 160 ppm. At 80 ppm, the treated females showed less
 

gain in weight than the controls for the first week of treatment,
 

with comparable gains thereafter. Based on these findings, low
 

and high doses of 80 and 160 ppm, respectively, were selected for
 

the chronic studies in the male rats; 40 and 80 ppm, for the
 

females.
 

With mice fed 80 ppm, all males died during the second and the
 

third weeks, and two of the females died during the sixth week,
 

while those mice fed concentrations of only 20 or 40 ppm showed
 

no deaths and no adverse effects with respect to either body
 

weight or feed consumption. The concentrations of 20 and 40 ppm
 

9 
were selected, therefore, as low and high doses for chronic
 

studies in both male and female mice.
 

F. Design of Chronic Studies
 

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

and matched-control groups, are shown in tables 1 and 2. When
 

tests with the rats were initiated at doses indicated by the
 

subchronic studies, toxic effects were observed with the males,
 

requiring reduction of the dose after 31 weeks and again after 45
 

weeks; with the female rats, the dose was increased at 22 weeks,
 

but development of toxicity required subsequent reductions in
 

dose at 31 and 45 weeks. When tests with mice were initiated at
 

doses indicated by the subchronic studies, high mortality
 

resulted in the males, and the original high-dose group of males
 

was discarded. The males receiving 20 ppm then became the
 

high-dose group, and the study was continued, keeping the
 

original 10 control males. A new low-dose group of male mice was
 

started at 10 ppm, along with an additional group of 10 male
 

controls. The total number of male controls thus became 20. Due
 

to further appearance of toxic effects, doses for males were
 

reduced once, at 17-18 weeks, and doses for females were reduced
 

twice, at 17 weeks and again at 30 weeks.
 

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

females) and mice (20 males, 20 females) were small, pooled10
 
Table 1. Design of Heptachlor Chronic Feeding Studies in Rats
 

Sex and
 

Treatment
 

Group
 

MALE
 

Matched-Control
 

Low— Dose
 

High-Dose
 

FEMALE
 

Matched-Control
 

Low— Dose
 

High-Dose
 

Initial
 

No. of
 

Animals3
 

10
 

50
 

50
 

10
 

50
 

Heptachlor
 

in Diet
 

(pom)
 

0
 

80
 

20
 

10
 

0
 

160
 

40
 

20
 

0
 

0
 

40
 

60
 

20
 

10
 

0
 

80
 

120
 

40
 

20
 

0
 

Time o 

Treated 

n Study 

Untreated15 

Time-Weighted 

Average Dosec 

(weeks ) (weeks) (ppm) 

0 111 

31 38.9 

14 

35 

0 30 

31 77.9 

14 

35 

0 30 

0 111 

22 25.7 

9 

14 

35 

0 30 

22 

9 51.3 

14 

35 

0 30 

a

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

H/hen diets containing heptachlor were discontinued, treated male rats and
 

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

11 weeks, then control diets (2% corn oil added) for an additional 18
 

weeks; treated female rats and their matched controls were fed plain feed
 

diets for 9.5 weeks, then control diets for an additional 20 weeks.
 c

Time-weighted average dose = Z(dose in ppm^x no._ j?f days at that dose)^
 

Z(no. of days receiving each dose)
 

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

Sex and
 

Treatment
 

Group
 

MALE
 

Matched-Control
 

Low-Dose
 

High-Dose
 

FEMALE
 

Matched-Control
 

Low-Dose
 

High-Dose
 

Initial
 

No. of
 

Animalsa
 

20d
 

50
 

50
 

10
 

50
 

50
 

Heptachlor
 

in Diet
 

(ppm)
 

0
 

10
 

5
 

0
 

20
 

10
 

0
 

0
 

20
 

10
 

5
 

0
 

40
 

20
 

10
 

0
 

Time on
 

Treated
 

(weeks)
 

0
 

18
 

62
 

0
 

17
 

50
 

0
 

0
 

17
 

13
 

50
 

0
 

17
 

13
 

50
 

0
 

Study Time-Weighted
 

Untreatedb Average Dosec
 

(weeks) (ppm)
 

90-91
 

6.1
 

10
 

13.8
 

10
 

90-91
 

9.0
 

10
 

18.0
 

10
 

a

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

"When diets containing heptachlor were discontinued, mice received control
 

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

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

£(no. of days receiving each dose)
 

"Initially 10 males were placed on test as matched controls; however, when
 

the study was restarted, 10 additional male animals were placed on test
 

as matched controls.
 

12
 
control groups were formed oy combining matched controls from
 

similar bioassays of other compounds with the matched controls
 

for heptachlor. 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 aldrin, dieldrin,
 

chlordane, heptachlor, dichlorvos, and dimethoate; this gave
 

pooled-control groups containing 60 males and 60 females,
 

respectively. 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 gave pooled-control groups
 

containing 100 males and 80 females, respectively. 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 slight differences in the
 

dichlorvos matched-control rats that were pooled for use as
 

controls in the heptachlor 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
 

13
 
Breeding Laboratories, Inc. and were the progeny (third
 

generation) of a group of Osborne-Mendel rats which were
 

purchased from the Battelle Memorial Institute, 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, 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.
 

14
 
A few tissues were not examined for some animals, particularly
 

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 Statigtical 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 uncen15
 
sored observations in the numerator. Statistical tests of
 

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 that 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
 

16
 
when lesions were detected grossly, the denominators consisted of
 

the numbers of animals necropsied.
 

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, the exact (or conditional) test for
 

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

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

groups were compared, this was the exact test for a linear trend
 

in 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.
 

17
 
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
 

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
 

18
 
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
 

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 (Ratsl
 

Average'""body weights of high-dose male rats were consistently
 

lower than those of untreated controls; there was less effect in
 

high-dose females. Low doses had, however, essentially no effect
 

on body weights (figure 1).
 

Adverse clinical signs in the treated and control groups were
 

noted at a low or moderate incidence during the first year and
 

gradually increased in frequency during the second year of study.
 

These signs in individual animals included loss of weight, rough
 

and discolored hair coats, and palpable masses. After 80 weeks
 

vaginal bleeding was observed in some females (both high— and
 

low-dose). Several palpable subcutaneous masses in the abdominal
 

region, probably mammary tumors, were noted in the low-dose
 

females. Surviving animals at termination of the study (111
 

weeks) were generally in a poor physical condition.
 

B>

 Survival (Rats)
 

Curves showing the probability of survival of treated and control
 

rats are presented in figure 2. Although a positive dose-


relation was observed for mortality in male rats, the result was
 

not statistically significant (P = 0.17). For female rats,
 

21
 
Figure 1. Growth Curves for Rats Fed Heptachlor in the Diet
 

22
 
Figure 2. Survival Curves for Rats Fed Heptachlor in the Diet
 

23
 
however, the linear trend test for mortality was significantly
 

positive (P = 0.04). This was due mainly to the difference in
 

mortality between the low- and high-dose animals.
 

C. Pathology (Rats)
 

Histopathologic findings are tabulated 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 the
 

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. 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
 

24
 
into the lumen by papillary infolding of simple cuboidal or
 

columnar epithelial cells which were usually larger and had more
 

basophilic cytoplasm than the surrounding normal 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 heterogeneous 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,
 

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

Meissner and Warren, 1968) characteristically were more
 

distinctly circumscribed, had some evidence of connective-tissue
 

encapsulation, and had a follicular architecture more distinct
 

from adjacent normal thyroid parenchyma. Follicular-cell
 

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 with hyperchromatic nuclei, and numerous mitotic
 

25
 
figures were present. Evidence of connective tissue and/or
 

inflammatory response and necrosis was often present. No
 

metastasis of a follicular-cell carcinoma occurred.
 

Proliferative C-cell lesions presented a spectrum of histologic
 

characteristics ranging from very minimal increases in numbers of
 

C-cells interspersed among normal thyroid follicles, with no
 

compression or distortion of follicular architecture, to grossly
 

visible tumors that invaded adjacent tissue and in one case
 

metastasized to the lung. 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
 

A.	 Hyperplasia
 

1.	 Increased numbers of C-cells are interspersed
 

among follicles but are not compressing follicles
 

or distorting follicle architecture.
 

2.	 Proliferating C-cells are polyhedral to spherical
 

with pale eosinophilic cytoplasm and spherical
 

nucleus usually centrally located.
 

B.	 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 be present,
 

3.	 A increas n e in basophilia of C-cells may be
 

present.
 

4.	 No invasion of thyroid capsule, adjacent tissue,
 

or lymphatics is present.
 

5.	 Encapsulation is rare.
 

C.	 Carcinoma
 

1.	 Invasion of thyroid capsule, adjacent tissue, or
 

vessels is present.
 

2.	 Metastasis may be present.
 

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.
 

The incidence of other endocrine neoplasms is summarized in table
 

A3. Pituitary adenomas occurred frequently in all groups;
 

neoplasms of adrenal, parathyroid, and pancreatic islets occurred
 

infrequently.
 

Table A4 summarizes the incidence of primary neoplasms of the
 

digestive system. These included a number of hepatic lesions
 

classified as neoplastic nodules, which consisted of nodules of
 

enlarged hepatocytes compressing adjacent tissue, and 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
 

27
 
nodules (Squire and Levitt, 1975); as such, they have been
 

categorized and coded as neoplasms when observed in this study.
 

Table A5 summarizes the incidence of neoplasms of the
 

reproductive system and mammary gland. Endometrial stromal
 

polyps represented the most frequently occurring neoplasms of the
 

reproductive tract. Numerous mammary fibroadenomas, some of
 

which were multiple, were observed in test and control females.
 

Several other neoplasms occurred infrequently in various tissues
 

from test and control rats; the incidence of these neoplasms is
 

summarized in tables A6, A7, and A8. Neoplasms similar to the
 

malignant fibrous histiocytomas observed in this study (table A8)
 

have been described previously (NCI, Bioassay of Chlordane for
 

Possible Carcinogenicity).
 

There was a distinct increase in the incidence of follicular-cell
 

neoplasms, including both adenomas and carcinomas in high-dose
 

female rats (15/38 [39%] versus pooled controls (3/58 [5%]) and
 

matched controls(l/9 [11%]); a smaller increase in the total
 

incidence of follicular-cell neoplasms was present in low-dose
 

males (11/38 [29%]) versus pooled control 4/51 [8%] and matched
 

controls (1/8 [13%]). 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
 

28
 
(Moriarty, 1975); however, no thyroid neoplasms induced by
 

heptachlor were mentioned in the studies cited. 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 heptachlor 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 heptachlor in rats.
 

D

• Statistical Analyses of Resu 11aJRat.s)
 

Statistical analyses of neoplasms in rats fed heptachlor are
 

given in Appendix E, tables E1-E12. No hepatocellular carcinomas
 

were found among the 187 heptachlor-treated animals 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. 

These findings were confirmed by comparisons using the pooled 

controls (table E2). 

The data for proliferative thyroid lesions were divided into
 

follicular-cell and C-cell types. Several follicular-cell
 

29
 
carcinomas were found among the treated male and female rats, but
 

none of the findings were statistically significant (table E3).
 

When carcinoma was combined with adenoma, a significantly
 

positive linear trend was found among the female rats by each
 

test (P = 0.002 and P = 0.001). This result was not confirmed in
 

the male animals. Comparisons with pooled controls for the
 

females yielded significant results for carcinomas and confirmed
 

the results when adenomas were added (table E4). In male rats
 

the low-dose group had significantly more carcinomas and/or
 

adenomas than the pooled controls; however, here the high dose
 

and pooled controls had the same percentages.
 

A few C-cell thyroid carcinomas were found among the treated male
 

rats, but the results were not significant (table E5). Only one
 

C-cell thyroid carcinoma was found among the female rats; this
 

was in the control group. The life-table adjusted analyses could
 

not be validly applied here because of the small number of
 

tumors. The exact test was more appropriate, and it yielded a
 

nonsignificant result (P = 0.10). When the carcinoma was
 

combined with adenoma, the results for the male rats remained
 

nonsignificant, but the results for the females were significant
 

in the negative direction by both tests. The pooled comparisons
 

for male rats, in general, paralleled the results with matched
 

controls. Significantly negative trends were found for the com30
 
parison involving pooled female controls for the carcinoma-only
 

criterion (table E6).
 

The results of analyses of thyroid lesions were ambiguous in that
 

the various criteria for female rats yielded (a) a significantly
 

positive linear dose effect for follicular-cell lesions, and (b)
 

a significantly negative linear dose effect for C-cell lesions.
 

The significantly positive relations for the follicular-cell
 

tumors reached higher degrees of significance for females (P =
 

0.002 and 0.001 for the two matched-control tests), while the
 

significantly negative relationships (P = 0.046 and 0.03) for the
 

C-cell tumors would be discounted on the basis of experiment-wise
 

error rates.
 

No carcinomas of the pituitary were found in any of the 149
 

treated animals or the 13 matched-control animals whose pituitary
 

glands were examined microscopically (table E7). When the
 

carcinoma was combined with adenoma, still no significant effect
 

was found. 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. A significantly negative result was found for female
 

rats for the combination of adenocarcinoma, carcinoma, and
 

adenoma (table E8), but this did not reach high levels of signi31
 
ficance except for one comparison involving trend among male rats
 

(P = 0.001).
 

When different categories of mammary tumors were considered
 

together, a large number of mammary tumors were found in the
 

female rats. No significant differences were found, however,
 

between treated animals and matched controls (table E9) In the
 

pooled-control analysis (table E10), only the low-dose group
 

yielded a positive significant result by one criterion.
 

With respect to tumors of the uterus, two endometrial stromal
 

sarcomas were found in the high—dose female group (table Ell).
 

This result was not significant. When polyps were added to the
 

criteria, no significance was found. Tests using pooled controls
 

yielded similar results (table E12).
 

32
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

There were no perceptible differences between the average body
 

weights of the treated mice and their corresponding controls
 

(figures 3 and 4).
 

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

the treated and control mice were generally comparable. Alopecia
 

(generalized and/or localized) and sores on the body were noted
 

in a number of mice in the treated and control groups. After 52
 

weeks of treatment, bloating or abdominal distention and alopecia
 

were the predominant observations in the high-dose females.
 

Rough coats, alopecia, sores, and palpable masses were noted in
 

an increasing number of mice in the treated and control groups
 

during the remainder of the study.
 

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 no
 

significant differences in survival between the high-dose group
 

and its control or the low-dose group and its control. In fact,
 

in each case the control groups had survival rates slightly lower
 

than those of the treated groups. In the female mice there is a
 

33
 
Figure 3. Growth Curves for Male Mice Fed Heptachlor in the Diet
 

34 
Figure 4. Growth Curves for Female Mice Fed Heptachlor in the Diet
 

35
 
Figure 5. Survival Curves for Male Mice Fed Heptachlor in the Diet
 

36 
Figure 6. Survival Curves for Female Mice Fed Heptachlor in the Diet
 

37
 
c

significant positive linear trend (P = 0.02), which is mainly a
 

reflection of the difference between the high- and low—dose
 

survivorship. This may indicate that heptachlor was toxic at the
 

high dose in female mice.
 

- Z2ill°.19£X..~lMi££l
 

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.
 

The most frequently occurring neoplasm was hepatocellular
 

carcinoma; the incidence of this and other proliferative
 

hepatocytic lesions is summarized in table B2. The morphology of
 

those lesions classified as hepatocellular carcinoma varied
 

widely. Some were present as one or more discrete nodules
 

containing solid cords and nests of well-differentiated but
 

hyperbasophilic hepatocytes with an increased nuclearrcytoplasmic
 

ratio. These lesions appeared to have enlarged by expansion,
 

with distinct compression but no obvious invasion of adjacent
 

normal hepatic parenchyma. Other hepatic neoplasms appeared as
 

very large masses which had completely replaced one or more
 

hepatic lobes and were composed of large, anaplastic hepatocytes,
 

forming confluent sheets, papillae, and pseudoacini, with large
 

foci of necropsis and complete loss of normal lobular
 

architecture. The morphologic appearance of the majority of
 

38
 
hepatocellular carcinomas fell somewhere between these two 

extremes. Pulmonary metastasis of hepatocellular carcinoma was 

observed in one low-dose male mouse. 

Hepatic lesions that were classified as nodular hyperplasia
 

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.
 

There was a striking increase in the incidence of hepatocellular
 

carcinoma in mice fed the high-dose concentration of hepatachlor
 

when compared with control groups: (34/47 [72%] in high-dose
 

males, 17/92 [18%] in pooled-control males, and 5/19 [26%] in
 

matched-control males; 30/42 [71%] in high-dose females, 3/78
 

[4%] in pooled-control females, and 2/10 [20%] in matched-control
 

females). There was no apparent increase in the incidence of
 

this tumor in low-dose mice. Nevertheless, it seems apparent
 

that hepatchlor fed at the higher dosage concentration used in
 

this study induced a distinct increase in the incidence of
 

hepatocellular carcinoma in mice.
 

There was a low incidence of other type of neoplasms involving
 

various tissues (table B3), with no striking differences in
 

incidence between test and control groups.
 

39
 
Suppurative inflammation involving the ovaries, oviducts, and
 

endometrium, in some cases associated with cystic hyperplasia of
 

the endometrium, occurred frequently in both test and control
 

female mice. There was a low incidence of other inflammatory,
 

degenerative, and nonneoplastic proliferative lesions in test and
 

control mice of both sexes (Appendix D).
 

D- jS^at^JticjLL ^naiyses_TM9LJ^e-sulS?_(Mi££l
 

Statistical analyses of neoplasms in mice fed heptachlor are
 

given in Appendix F, tables F1-F4.
 

The small size of the control group notwithstanding, a highly
 

significant difference in hepatocellular carcinoma (table Fl) was
 

found between the high-dose male group and its control by the two
 

simple proportion analyses (P = 0.001 or P = 0.0007) and by the
 

life-table adjusted analysis (P = 0.002). The low-dose male
 

group had a somewhat smaller percentage of animals with hepatocellular carcinoma animals than the male controls, which appear
 

to have had an unusually large number of tumors. However, this
 

difference is not significant by any of the tests, although the
 

life-table adjusted test approaches a significantly negative
 

result (P = 0.05+).
 

Comparison of the incidence in treated male mice with the pooled
 

controls reinforced the positive findings (table F2). The pooled
 

40
 
controls showed a lower proportion of hepatocellular carcinomas
 

than the low-dose group, although not significantly lower.
 

Statistical tests on hepatocellular carcinoma in the high-dose
 

group yielded smaller P values using pooled controls than using
 

matched controls (table F2).
 

In female mice highly significant linear trends (P < 0.0001) were
 

found for hepatocellular carcinoma by all tests which were
 

applied (table F3). This effect reflected mainly the difference
 

between the high- and low-dose groups, since the data departed
 

significantly from a linear trend. However, when the incidence
 

in the high-dose group was compared directly to that in the
 

matched control, significance was still found by the simple
 

proportion analyses (P < 0.005). The pooled-control analyses
 

reinforced these results (table F4).
 

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

found to warrant statistical analysis.
 

41
 

V. DISCUSSION
 

Heptachlor is a member of the organochlorine group of pesticides
 

classed as neurotoxins, which in high doses produce CNS stimulation; however, neither the hyperexcitability observed in
 

bioassays of endrin nor the tremors observed with chlordane were
 

reported in the present study with heptachlor.
 

The effects of heptachlor 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 average body
 

weights of rats treated with high doses were consistently lower
 

than those of untreated controls, while body weights of low-dose
 

rats were unaffected. Adverse clinical signs in treated rats
 

included rough and discolored hair coats and, in females, vaginal
 

bleeding. Body weights of mice given either low or high doses
 

showed little or no differences from those of control mice;
 

however, adverse clinical signs of alopecia and abdominal
 

distention were noted, predominantly in high-dose females. Doses
 

were reduced during the course of the tests in an effort to
 

minimize toxic effects.
 

The effects of heptachlor on survival rates indicated that in
 

both male and female rats, mortality was affected more adversely
 

by the high dose than by the low dose, but that a statistically
 

43
 
significant dose-related trend was found only in the females.
 

Survival in male mice was unaffected by either low or high doses,
 

but survival in female mice showed a statistically significant
 

dose-related decrease, due mainly to the effect of the high dose.
 

The fact that greater toxicity was observed in the high-dose
 

females than in the high-dose males may have been due to the
 

higher total dose received by the females. A substantial
 

proportion of all groups survived, however, 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 the high dose of heptachlor (Appendix
 

F, tables Fl and F2), using the Fisher exact test either with
 

matched or pooled controls. At the low dose, the incidence of
 

proliferative lesions of the liver was significant only in male
 

mice, using pooled controls, and then only when carcinoma was
 

combined with nodular hyperplasia. Dose-related trends, however,
 

were highly significant for both males and females, using matched
 

controls for comparison; when pooled controls were used, the
 

significance of the trend in the males increased to the very high
 

levels shown by the females. Comparably high levels of significance of dose-related trends were also attained when hepatocellular carcinoma was combined with nodular hyperplasia or when the
 

data were subjected to life-table adjustment.
 

44
 
In contrast to findings with mice, no hepatocellular carcinomas
 

were induced by heptachlor in rats (Appendix E, tables El and
 

E2). Further, the incidence of proliferative lesions of the
 

liver in rats did not become statistically significant when
 

hepatocellular carcinoma was combined with neoplastic nodules or
 

when life-table adjustment was applied to the data.
 

A statistically significant dose-related trend in proliferative
 

follicular-cell lesions of the thyroid was found, however, in the
 

rats (table E3). Although the trends for follicular-cell
 

carcinoma alone were not significant in either males or females,
 

the trend for follicular-cell carcinoma combined with adenoma was
 

significant for the females. The trend for follicular-cell
 

lesions remained significant when pooled controls (table E4) were
 

used instead of matched controls and when the data were subjected
 

to life-table adjustment. The male rats showed a higher
 

incidence of follicular-cell lesions in the low-dose animals than
 

in the controls, while the incidence in the high-dose animals was
 

similar to that in the controls (tables El and E4). C-cell
 

carcinomas and/or adenomas showed a significantly negative
 

dose-related trend in female rats, using matched controls (table
 

E5). In the judgment of the pathologist, the nature, incidence,
 

and severity of the proliferative thyroid lesions were not
 

sufficient to indicate clearly a carcinogenic effect of
 

heptachlor in rats.
 

45
 
The metabolism of heptachlor in the rat consists first of
 

transformation to heptachlor epoxide, which is stored in body fat
 

(Davidow and Radomski, 1953; O'Brien, 1967). The epoxide has
 

been found in extremely low concentrations in the urine of the
 

general human population (Cueto and Biros, 1967). Dehydrochlorination of the epoxide, followed by hydroxylation and double-bond
 

rearrangement, produces a metabolite that is the principle form
 

in which heptachlor is excreted in the feces (Matsumura and
 

Nelson, 1970).
 

Unpublished reports on long-term feeding of rats with diets
 

containing heptachlor and/or heptachlor epoxide were reviewed in
 

an IARC monograph (1974). In three of five such studies, rats
 

received diets containing 12.5 ppm of the test material for at
 

least 2 years but showed no increase in tumor incidence attributable to treatment. In a fourth study, only an increase in
 

liver weight was reported for rats receiving 10 and 20 ppm
 

heptachlor. In a fifth study with rats, the incidences of tumors
 

in the animals given 0.5 to 10 ppm (65/114 [57%] in males; 92/114
 

[81%] in females) were greater than in controls (8/23 [35%] in
 

males; 13/24 [54%] in females), with most tumors located in
 

endocrine organs and with liver tumors appearing in 7 males and
 

12 females but not in the controls. In a study with C3Heb/Fe/J
 

mice, the feeding of heptachlor or heptachlor epoxide at a level
 

46
 
of 10 ppm in the diet has been reported to bring about an
 

increase in liver tumors including carcinomas (EPA, 1974).
 

In the present bioassay, heptachlor was hepatocarcinogenic in
 

mice but not in rats. In rats, heptdchlor induced a statistically significant increase in proliferative lesions of thyroid
 

follicular cells. The relationship of these lesions to administration of the compound is not clear, since the spontaneous
 

incidence of the lesions has varied throughout the bioassay
 

program.
 

47
 

VI. BIBLIOGRAPHY
 

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

and Sons, Inc., New York, 1971, ch. 14.
 

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, 1968.
 

Casarett, L. J. and Doull, J. Toxicology: The Basic Science of
 

Poisons, Macmillan Publishing Co., Inc., New York, 1975.
 

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

Soc. B 34:187-220. 1972.
 

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

4 and 5.
 

Cueto, C., Jr. and Biros, F. J. Chlorinated insecticides and
 

related materials in human urine. Toxicol. Appl. Pharmacol^
 

J£:261-269, 1967.
 

Davidow, B. and Radomski, J. L. Isolation of an epoxide
 

metabolite from fat tissues of dogs fed heptachlor. J.
 

Pharmacol. Exper. Thersp. 107:259-265, 1953.
 

Division of Cancer Cause and Prevention, National Cancer
 

Institute. Bioassay of: Chlordane for Possible
 

Carcinogenicity, 1977.
 

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

pesticide chemicals in the United States (III) June 1968­

April 1970. Pest. MoniU JA 5:331-341, 1972.
 

Environmental Protection Agency. Velsicol Chemical Co. et al.
 

Cosolidated Heptachlor/Chlordane Hearing. Federal Register
 

4^:7552-7585, 1976.
 

Environmental Protection Agency. Pesticide products containing
 

heptachlor or chlordane. Federal Register 39_(229) :41298,
 

1974.
 

49
 
Environmental Protection Agency. Heptachlor; A Review of Its
 

Uses, Chemistry, Environmental Hazard and Toxicology, 1972.
 

FAO/WHO. 1966 Evaluations of Some Pesticide Residues in Food,
 

1967.
 

IARC Monographs - Heptachlor, Vol. 5, 1974, pp. 173-191.
 

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

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

1958.
 

Linhart, M. S., Cooper, J. A. Carcinogenesis bioassay data
 

system. J^ Cqmp. Biomed. Res. _7,:230-248, 1974.
 

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

extensions of Mantel-Haenszel procedures. J. Am. Statist. 

Assoc. 58;690-700, 1963. 

Matsumura, F. and Nelson, J. 0. Identification of the major
 

metabolic product of heptachlor epoxide in rat feces. Bull.
 

Environ. Contain. Tox^ 5(6) ;489-492, 1970.
 

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

Atlas of Tumor Pathology, Fascicle 4, A.F.I.P., Washington,
 

D.C., 1968.
 

Moriarty, F. ed. Organochlorine Insecticides: Persistent
 

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

O'Brien, R. D. Insecticides, Action and Metabolism, Academic
 

Press, Inc., New York, 1967.
 

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

Kaufman, D. G. Respiratory tract carcinogenesis in hamsters
 

induced by different numbers of administrations of benzo(a)
 

pyrene and ferric oxide. Cancer Res. 3_2:1073-1079, 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 hepatocellular lesions in rats.
 

Cancer Res. 35:3214, 1975.
 

50
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND OTHER
 

PROLIFERATIVE LESIONS IN RATS
 

FED HEPTACHLOR IN THE DIET
 

51
 

TABLE Al
 

SUMMARY OF THE DISPOSITION OF TISSUES
 

MALE RATS FEMALE RATS 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

Animals Initially in Study 60 10 50 50 60 10 49a 50 

Animals Necropsied 58 10 45 50 60 10 48 48 

Animals Examined 

His topatho logically 58 10 45 49 60 10 48 48 

One animal in this group was found to be a male at necropsy; data from this animal was not included
 

in this report.
 
TABLE A2
 

PROLIFERATIVE LESIONS OF THE THYROIDC
 

MALE RATS FEMALE RATS 

Pooled 

Control 

(51) 

Matched 

Control 

(8) 

Low 

Dose 

(38) 

High 

Dose 

(38) 

Pooled 

Control 

(58) 

Matched 

Control 

(9) 

Low 

Dose 

(43) 

High 

Dose 

(38) 

Follicular-cell Carcinoma 1 0 4 0 1 1 2 5 

Follicular-cell Adenoma 3 1 7 3 2 0 1 10 

C-cell Carcinoma 1 0 3 1 5 1 0 0 

C-cell Adenoma 3 0 4 1 7 2 7 3 

Follicular-cell Hyperplasia 0 0 2 2 4 1 4 4
 

C-cell Hyperplasia 29 3 13 21 30 4 21 14
 

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

OTHER NEOPLASMS OF THE ENDOCRINE SYSTEM3
 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

ADRENAL (55) (9) (42) (41) (56) (9) (45) (40) 

Pheochromo cy toma 1 1 1 0 0 0 0 1 

Cortical Carcinoma 1 1 0 0 0 0 0 0 

Cortical Adenoma 2 0 1 1 0 0 0 2 

PITUITARY (48) (7) (42) (34) (52) (6) (39) (34) 

Adenoma 14 4 8 8 23 3 9 8 

PANCREATIC ISLETS (52) (9) (40) (42) (60) (10) (41) (41) 

Carcinoma 0 0 0 0 0 0 0 1 

Adenoma 1 0 2 1 1 0 3 0 

PARATHYROID (38) (3) (22) (25) (38) (5) (32) (21) 

Adenoma 2 0 0 0 0 0 1 0 

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

NEOPLASMS OF THE DIGESTIVE SYSTEM3
 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

LIVER (58) (10) (44) (49) (59) (10) (48) (46) 

Cholangiocarcinoma 0 0 1 0 0 0 0 0 

Neoplastic Nodule 2 1 3 6 5 1 9 5 

SALIVARY GLAND (56) (9) (42) (44) (60) (10) (47) (42) 

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

Fibroma 0 0 0 0 0 0 1 0 

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

NEOPLASMS OF THE REPRODUCTIVE SYSTEM AND MAMMARY GLAND 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose ___ ___ ___ ___ 

UTERUS (56) (10) (43) (41) 

Endome trial Stromal Sarcoma 0 0 0 2 

Endome trial Stromal Polyp 6 2 7 6 

___ ___ ___ 

OVARY — .«.» (58) (9) (45) (43) 

Cys tadenocarc inoma 0 0 1 0 

Granulosa-cell Tumor 1 0 2 0 

MAMMARY GLAND 

Carcinoma 

(58)b 

0 

(io)b 

0 

(45)b 

1 

(50)b 

0 

(60)b 

2 

(10)b 

1 

(48)b 

1 

(48)b 

1 

Adenoma 0 0 0 0 1 1 4 1 

Fibrosarcoma 0 0 0 1 0 0 0 0 
TABLE A5 

NEOPLASMS OF THE REPRODUCTIVE SYSTEM AND MAMMARY GLAND 

(Continued) 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched '. Low High 

Control Control Dose Dose Control Control 'Dose Dose 

MAMMARY GLAND (Continued) (58)b (io)b (45)b (50)b (60)b (io)b (48)b (48)b 

Sarcoma, N.O.S. 0 0 0 0 0 0 1 0 

Fibroadenoma 0 0 0 0 8 1 10 5 

Fibroma 1 0 3 0 1 0 3 1 

Lipoma 0 0 1 0 0 0 0 0 

wumbers 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. 
TABLE A6
 

NEOPLASMS OF THE CARDIOVASCULAR SYSTEM3
 

MALE RATS FEMALE RATS 

Pooled 

Control 

(58)b 

Matched 

Control 

Cio)t> 

Low 

Dose 

(45)b 

High 

Dose 

(50)b 

Pooled 

Control 

(60)b 

Matched 

Control 

(10)b 

Low 

Dose 

(48)b 

High 

Dose 

(48)b 

Hetnang iosar coma 3 0 0 1 0 0 1 0 

HEART 

Sarcoma, N.O.S. 

(58) 

2 

(10) 

0 

(44) 

0 

(49) 

0 

(57) 

1 

(10) 

0 

(48) 

2 

(44) 

0 

Fibroma 1 1 0 0 0 0 0 0 

lumbers 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.
 
TABLE A7
 

NEOPLASMS OF THE HEMATOPOIETIC SYSTEM3
 

MALE RATS FEMALE RATS 

Pooled 

Control 

(58)b 

Matched 

Control 

(10)b 

Low 

Dose 

(45)b 

High 

Dose 

(50)b 

Pooled 

Control 

(60)b 

Matched 

Control 

(10)b 

Low 

Dose 

(48)b 

High 

Dose 

(48)b 

Monocytic Leukemia 0 0 0 0 1 1 0 0 

Thymoma 0 0 0 1 0 0 0 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.
 
TABLE A3
 

MISCELLANEOUS NEOPLASMS
 

MALE RATS FEMALE RATS 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

SUBCUTIS (58)b U0)b (45)b (50)b (60)b (10)b (48)b (48)b 

Malignant Fibrous 

Histiocytoma 1 0 1 0 0 0 0 0 

Fibrosarcoma 0 0 1 0 0 0 0 1 

Leiomyoma 0 0 1 0 0 0 0 0 

Fibroma 0 0 0 1 1 1 0 0 

LUNG (58) (10) (44) (47) (58) (10) (47) (46) 

Adenoma 0 0 1 0 0 0 0 0 

BRAIN 

Meningioma 

(57) 

0 

(10) 

0 

(43) 

0 

(47) 

0 

(59) 

0 

(10) 

0 

(48) 

1 

(46) 

0 

a

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 HEPTACHLOR IN THE DIET
 

63
 

TABLE Bl 

SUMMARY OF THE DISPOSITION OF TISSUES 

MALE MICE FEMALE MICE 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

Animals Initially in Study 100 20 50 48 80 10 50 50 

Animals Necropsied 92 20 47 47 79 10 48 45 

Animals Examined 

His topatho logically 92 19 46 47 79 10 48 43 
TABLE B2
 

PROLIFERATIVE LESIONS OF THE LIVER
 

MALE MICE FEMALE MICE 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

LIVER (92) (19) (46) (47) (78) (10) (47) (42) 

Hepitocellular Carcinoma 17 5 11 34 3 2 3 30 

Nodular Hyperplasia 3 1 9 6 2 0 3 0 

Diffuse Hyperplasia 3 0 1 3 1 0 0 0 

Hepatocytomegaly 0 0 1 0 1 0 0 1 

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

MISCELLANEOUS NEOPLASMS2 

MALE MICE FEMALE MICE 

Pooled Matched Low High Pooled Matched Low High 

Control Control Dose Dose Control Control Dose Dose 

HEMATOPOIETIC SYSTEM 

Malignant Lymphoma 

(92)b 

5 

(20)b 

1 

(47)b 

0 

(47)b 

0 

(79)b 

8 

(io)b 

2 

(48)b 

4 

(45)b 

1 

LUNG (91) (19) (46) (47) (79) (10) (47) (42) 

Alveolar/Bronchiolar Adenoma 6 1 1 7 0 0 1 1 __ __ __ 

UTERUS — (78) (10) (47) (37) 

Adenocarcinoma 0 0 1 0 — — — —— __ __ 

OVARY — — (73) (8) (45) (35) 

Luteoma 1 1 0 0 — — — — 

THYROID (79) (14) (32) (36) (72) (8) (33) (22) 

Follicular-cell Adenoma 1 0 0 0 1 0 1 0 
TABLE B3 

MISCELLANEOUS NEOPLASMS 

(Continued) 

MALE MICE FEMALE MICE 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

PITUITARY 

Adenoma 

(70) 

0 

(12) 

0 

(16) 

0 

(19) 

0 

(58) 

0 

(5) 

0 

(20) 

1 

(12) 

0 

ADRENAL CORTEX 

Carcinoma 

(88) (17) 

0 

(41) 

0 

(44) 

0 

(78) (9) 

0 

(39) 

1 

(36) 

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.
 

°For purposes of this summary, the following lesions are grouped under "Malignant Lymphoma".
 Lymphosarcoma (of any site or multiple sites), lymphocytic lymphosarcomas, and reticulum cell sarcoma.
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS FED HE?TACHLOR
 

IN THE DIET
 

69
 

__ 

TABLE C1 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED HEPTACHLOR IN THE DIET 

ANIMAL S INITIALL Y IN STUD Y 

ANIMAL S NJSCROPSIED 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY 

ANIMAL S HU H NON-TUMO R PATHOLOGY 

INTtGUMfcNTAR Y SYSTE M 

NON E 

RESPIHATOiY SYSTEM * 

TRACHE A 

INFLAMMATIO N CHRONI C 

LUN G 

EMPHYSEM A 

EDEH A 

INFLAMMATIO N 

INFLAMMATIO N FOCAL

 

INFLAMMATIO N CHRONIC 

ALVUOLA R MACROPHAGE S 

LUHG/ALVEOL I 

INFLAMMATIO N FOCAL 

CIHCULATOR Y SYSTEM 

MYOCARDIU M 

INFLAMMATIO N FOCAL 

lUFLAMilATIOt l IN iEiiSTIIIAL 

FIBHOSIS 

FIBEOSIS FOCA L 

CALCIFICATION 

DIGESTIV E SYSTEM 

SALIVAR Y GLAN D 

tii&lillOH^iiUCIS _ 

'SYSTE M PEaCENIAGE S ARE BASE D ON THE

CONTROL 

10 

10 (100X) 

10 

9 (90X ) 

2 (20%) 

2 

2 

5 (50X) 

5 

1 

H 

9 (90X) 

1 

1 

 NUMBE R 3F

71 

LOM DOSE HI3B DOSE 

50 50 

1(5 (100J) 50 (100X) 

U5 50 

14 j (96X) 4i» (88X) 

1o (36 X) 18 (36*) 

1 

1 

15 18 

1 

1 

2 

1 

1 

15 18 

1 

1 

11 (31X} 16 (32X) 

I K 16 

1 2 

It 15 

1 

1 

39 (87X) 34 (68X) 

 A.NISALS_N_£C jjOP SIED 
TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONT.) 

LIVEB 

CONGESTION 

HEMORRHAGE 

PEBIABTEBITIS 

flETAMORPHOSIS FAT1Y 

HEPATOCYTOHEGALY 

ANGIECTASIS 

BILK DUCT 

DILATATION 

INFLAMMATION 

FIBBOSIS 

HYPBBPLASIA 

PANCBEAS 

FIBBOSIS 

PJUIABTBBITIS 

PANCREATIC ACINUS 

A1BOPHY 

STOMACH 

ULCEB 

NEC80SIS FOCAL 

GASTBIC HOCOSA 

CALCIFICATION 

CALCIFICATION DISTBOPUIC 

SHALL INTESTINE 

HEHOBBHAGE 

INFLAMMATIO N 

UBINAB Y SYSTEM * 

KIDNE Y 

INFLAMMATION CHBONIC 

METAMORPHOSIS FATTY 

KIDNEY/PELVIS 

HYPEBPLASIA EPITHELIAL 

UBINABY BLADDEB 

INFLAMMATION CUBONIC 

aiEMiUSii-fiEilflSiiii. 

*SY5T£U PEBCENTAGES ABE BASED

ZOITBOL 

7 

1 

4 

U 

B 

a 

2 

5 

1 

1 

2 

2 

2 

1 

1 

1 

1 

8 (80*) 

8 

8 

1 

1 

1 

 ON THE JOBBER OF

LOV DOSE 

20 

3 

2 

18 

« 

32 

2 

18 

1 

26 

6 

5 

1 

1 

1 

1 

1 

1 

1 

1 

39 (87*) 

39 

39 

1 

5 

5 

5 

5 

 4Kil4t5_Jig£IS£5iSB 

HIGH DOSE 

21 

1 

U 

17 

8 

25 

21 

8 

19 

5 

3 

2 

3 

3 

1 

1 

39 (78*) 

39 

39 

5 

5 

2 

2 

72 
TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONT.) 

CONTBOL 

EH DOC BIKE SYSTEH * 7 (70X) 

PITUITABY 5

 

cist 1

 

CONGESTION 

HYPEBPLASIA CHBOMOPHOBE-CELL 

ANGIBCTASIS 14 

ADRENA L 

CYTOHEGALY 

AOB£NAL COBTEX 1

 

CYTOMEGAL Y 1

 

THYBOID 3

 

ULTIHOBRANCHIAL CIST 

CYSIIC FOLLICLES 

HEHOBBUAGE 

HYPBBPLASIA C-CELL 3 

HYPEBPLASIA FOLLICULA B-CELL 

PAEAIHYBOI D 1

 

HYPEEPLASI A 1 

FANCBEATIC ISLETS 

HYPEBPLASI A 

UEHATOPOIETIC SYSTEM 1 (10%) 

SPLEEN 1

 

CONGESTION 

UYPBBEHIA 

INFLAHHATION CHBONIC 

INFLAHHATION FOCAL GBANULOHAIOO s

 

HEUOSIDSBOSIS 1 

LYHPHOI D HYPSBPLASI A 

HEMATOP01ESIS 

CEBVICAL LYHPH NODE 

DILATATION 

liaEH2I£_flIE£aELA5IA 

• SYSTEH PEBCENTAGES ABE BASED ON THE NUMBE R OF

LOM DOSE HI3H DOSE 

27 (BOX) 29 (58X) 

6 6

 

2 2 

1 

1 t 

3 3 

1 

1 

5 7 

5 7 

17 26 

2 

3 1 

1 

13 21

 

2 1

 

2 1

 

2 1 

1 

1 

6 (13XJ U <8X) 

5 It 

2 

1 

2 

1 

3 

1 

2 

2 

1 

1. - — — _ _ _—-. 

 A.£m4S._N.gC.B,QeS.i.£Q 

73 

TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONT.)

 

CONIBOI. LOH DOSE 

ALL OTHER SYSIiMS * 9 (20*) 

ABDOMINAL CAVITY 1

 

NECHOSIS FAT 1

 

P£BITONEUM 1 

INFLAMMATIO N 1

 

NO ASSOCIATED OBGA H 7

 

NO LESION BEPORTED 2

 

AUTO/NECfiOESY PEfiP/HISIO PBKP 

AUTOLYSIS/NO NECROPSY PERFOBBiD 5

 

ADIPOSE TISSUE 

INFLAMMATIO N GBAJIULOM ATOUS 

DESilNTEfiY 1

 

PBBIABTEBI1IS 1 

*SYSTEB PEBCENTAGES ARi BASiiD O» THE NUBBiSH OF 

HIG H COSE 

10 (20X) 

6 

5 

1 

1 

1 

3 

3 

75 

TABLE C2 FEMALE RATS: NONNEOPLASTIC

;O«TBOL 

LZVttB (CCHT.) 

IHFIAHS AIICH SUPPUBATIVE 

FIBBOSIS 

HtCaOSIS 

«ETA«Ot<PHOSIS FATTY 

HiPAIOCYTOBtGALY 2 

ATBOfHY 

HYPiBPLASIA FOCAL 

AUGIECTASIS 1 

BILE DUCT 6 

DILAiATION 1 

IfcFiAMHATIOli 5 

FIBBOSIS 1 

HYPEBPLASIA 4 

PANCBEAS 

FIBBUSIS 

PtRIAHIEBIIIS 

PAHCHtATIC ACINUS 

AIBOPHI 

STOMACH 

ULCii FOCAL 

CA1CIFICATIOH 

UvIUABY SYSltM * 8 (SOS) 

KIDNc Y a 

IMFLAKBAIIOM CHBOMIC 8 

netAnofipHosis FATIJ 

KIOK£Y/P£LVIS 

HYP£BPi.ASIA aPIIHELiAi. 

1 

1 

UEIHA8! BLAKDEB 1 

INFLAHaATICU CUBOHIC 

ai£l££U5iA_t£ilfliiiIAi ._ _ J._ 

•SYSTift FcBCEHTAGES ABE BASED ON THE HUH BEE OF

 LESIONS (CONT.) 

LOU DOSE 

1 

2 

16 

23 

21 

1 

13 

6 

17 

1 

1 

3 

3 

1 

1 

1 

3U (69S) 

30 

31 

7 

7 

3 

1 

3 

 AJJIJA,1;£_1!£C£O£SI ED 

HIGH DOSE 

1 

1 

4 

15 

1 

1 

21 

34 

2 

21 

6 

24 

6 

4 

2 

5 

5 

31 (65%) 

31 

31 

1 

5 

5 

1 

1 

77 
6 

TABLE C2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONT.) 

COHTBOi. LOU DOSE HIGH DOSE 

&NDOCHIN E SYSTEM * 5 (50X) 29 (59 X) 27 (56X) 

PITUITAR Y 1 3 8 

HYPEBPLASIA 1 1 

HYPEHPLASIA CHBOHOPHOBE-CELL 2 

ANGIECTASIS 1 3 

ADRENA L 1 3

6 

HEIUOBPUOSIS FATTY 2

 

CYTOHEGALY 1 1

 

ADRENA L CORTEX 1 9 

CYTOBBGAtY 1 9 8 

THYROID U 23 15 

ULTIBOBEANCHIA L CYST 2 

CYSt'IC FOLLICLES 1 1 

3

3 

INFLAHHATION ACUTE 1 

HYPEHPLASIA C-CELL a 21 11) 

HYPEflPLASIA FOiLICULAB-CELL i it 4 

HEMATOiOIETIC SYSTEM 2 <20X) 7 (1UXJ 8 (17X) 

SPLEEN 2 7 7

 

INFLAHHATIOM FOCAL GRAtlULOHATOUS 1

 

HEflOSIDiEOSIS 1 3 

ANGItCTASIS 

HEflATOPOIESIS 1 

1 

2 6 

CEBVICAL LYHPH NODE 

LYMPHOID HYPEBPLASIA 

1 

1 

-

PELVIC LYHPH NODi 

INFLAMMATIO N CUBONIC 

 M£ific.SASI3 _ __ 

1 

1 

1 

*SYS!EM PEBCENTAGES ABE BASED ON THE NUHBEB 3F iH.I«lIt5._{15CBQPSI£D 

78 

1 
TABLE C2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONT.) 

CONTBOL LOU DOSE 

BEPBODUCTIVE SYSTEM * 2 («) 

U1EBUS 1 

HEHOBHHAGE 

ULCER 1 

INFLAMMATIO N FOCAL 1 

INFLAHHATION SUPPOBATIVE 

UTEBUS/ENDOflETaiUH 1 

CYST 

HJPEBPLASIA CYSTIC 1 

NBBVOUS SYSTEM 1 (10*) 

BBAI N 1 

HEMORRHAG E 1 

SOSCDLOSKBLETAL SYSTEM 

NON E 

SPECIAL SiNSE OSGANS 

HONE 

ALL OTHER SYSTEMS 3 (6*) 

NO ASSOCIATED OBGAN 2 

NO LESION BEPOHTED 1 

iOTOLYSIS/NO NECfiOPSY PBHFOEMBD 1 

flESEblTBBY 1 

P.£fiIAai£fiilI5_ _ 1 

*SYST£H PERCENTAGES ARE BASED ON THE NUHBER OF AJIUA.L.S._i!E.Cg2PS.iE.p. 

HIGH DOSE 

"» <8X) 

2 

1 

1 

1 

2 

1 

1 

8 <17X) 

8 

6 

2 

79
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE FED HEPTACHLOR
 

IN THE DIET
 

81
 

TABLE D1

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE MICE FED HEPTACHLOR IN THE DIET 

HIGH DOSE LOW DOSE LOU DOSE HIGH DOSE 

CONTROL CONTROL 

ANI4AI.S INI II ALL If IN STUDY 10 10 5« 49 

ANIMALS NECBOPSIED 10(100*) 10(100%) 47(100%) 47{ 100%) 

ANIMAL S EXAMINE D HIStOPATHOLOGICALLY 9 10 46 47 

ANIMALS WITH NON-TUHOB PATHOLOGY 3 (30%) 3 (30%) 21 (t5%) 10 (21%) 

ISTEGUMENTAB Y SYSTEM * 1 (10X) 1 (2%) 

SKIN 1 1 

ULCBS 1 

INFLAMMATIO N GiAN ULOMATOUS 1

 

BESPIBA10RY SYSTEM 1 (10X) 1 (10%) 4 (9%) 1 (2%) 

LUNG/fiBONCHUS 1

 

INFLAHMATION FOCAL 1

 

LUNG/BRONCHIOLE 1

 

INFLAMMATION SUPPUBATIVE 1 

LUNG 1 1 2 1 

INFLAMMATIO N FOCAL 1 

ALVEOLAR MACBOPHAGES 2 

HYPiBPLASIA ALVEO LAB- CELL 1 1 

LONG/ALVEOLI 1 1

 

INFLAMMATION 1 

INFLAMMATIO N SUPPUBATIV E 1 

CIHCULAlOfi Y SYSIliB 

NUN E 

DIGESTIVE SYSTEM 1 (10X) 1 (10%) 13 (28%) 3 (17%) 

LIVE3 1 1 13 8

 

INFLAMMATIO N FOCAL 1 _ £i£iN£fi_A.2ION_

 1 

•SYSTEM PiBCtNTAGES ABE BASED ON THE NUMBER OF A1II.MALS_NE.CBO£SIED 

83 
TABLE D1 MALE MICE: NONNEOPLASTIC LESIONS (CONT.) 

HIGH DOSE LOW DOSE LOU DOSE HIGH 03 SS 

CONTROL CONTROL 

LIVSS (COMT.) 

SEfAHOHPHOSZS F1IIY 1 

HEPiTOCJTOHKGtLJ 1 

HYPEBPLASIA NOUULAB 1 9 6 

UlPEJiPLiSZi DIFFUSE 1 3 

DBIMABY SYSTEM * 6 (131) 

KIDM2Y 6

 

LKMPHOCYIIC IIFUfl IHFILTBATE «

 

IHFLAHaATIOH CttSOMIC 2

 

EKOOCBIHi SYSTiH 1 (M) 2 J»») 

THYROI D 1 2 

HYPiEPLASIA C-CELL 1 

HYPEBPLASIA FOiLICUUI-CCLI. 2 

HoSATOIOItrlC SVSTEB 1 (2») 

HSStHlEHIC LYBPHIiODE 1 

COKGESriON t

 

EDtiMA 1 

HIPtSPLASIA BETICULUfl-CBLL 1 

KBPHOilUCIIII. SYSI£H 1 (2t) 

I2SIIS 1 

ATiOPHY 1

 

kKHVOUS SYSTtl) t (»0») 

BBAIN 1

 

C06P08A ANYUCt^ 1

 

HU3CUJ.OSK«.LET*i SKS1EH 

IHHt. ...rjr^.>. j—ui^^t..^. >««««­

•Stall* PfcBtfcNTiGiS ARE BASED OK IHi. DUHBEB 3F [lECBQPS^ED 

84 
TABLE 01 MALE MICE: NONNEOPLASTIC LESIONS (GONT.) 

HIGH DOSE 

CONTROL 

LOW DOSE 

CONTROL 

LOU DOSE HIGH D3SE 

SPECIAL SENSE ORGAN S 

NONE 

ALL OTHiS SYSTEMS * 

NO ASSOCIATED ORGA N 

NO LESION BEPOBTED 

AUIOLlfSIS/NECEOPSY P.ERF/NO HISIO 

AUTOLYSIS/110 NtCBOtSK PiSBFOHHSD 

5 (50*) 

5 

4 

1 

3 (30X) 

3 

3 

20 (43X) 

20 

1S 

1 

3 

6 (13%) 

6 

5 

1 

»SYiTEH PERCENTAGES ABE BASED OH TUB MONBEB 3F 4JLIttiI.S_ftfi£6a£51BD 

85 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE MICE FED HEPTACHLOR IN THE DIET 

COSTBOL LOti DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 10 50 50 

ANIMALS NECBOPSIED 10(10QX) as(ioox) U5(100X) 

ANIMALS KXAMINtLI HISTOFATHO LOGICALLY 10 48 43 

ANIMALS WITH NON-TUMOR PATHOLOGY 6 (60*) 25 (52*) 12 (27X) 

INIEGUMENTABY SYSTEM 

NONE 

BESflBATOBY SYSTEM * 1 (2X) 

LUNG 1 

INFLAMMATION CHBOUIC 1 

CIBCULATCBY SYS1£M 1 (10X) 

ABTEBY 1

 

INFLAMMATION 1

 

DIGESTIVE SYSTEM 1 (10X) 6 (13*) 4 (9X) 

LIVEB 1 3 2

 

HBMOBBHAGE 1

NECBOSIS 1 

HEPATOCYTOH£GALY 1 

HYPESPLASIA NODULAR 3 

NODULA R REGENERATION 1 

LIVEB/CAUDAT£ LOBE 1 

NECBOSIS 1 

BILE DUCT 1 

HYP2BPLASIA 1 

PANCBEA S 3

 

INFLAMMATIO N CHBONIC 1

 

- UfJJflaillSN. S.£MUi2«llIOUS i 

•SISTEW PERCENTAGES ABE BASED ON THE NUNBE B OF iSi54J;S_NEC£gP^lED 

86 
TABLE 02 FEMALE MICE: NONNEOPLASTIC LESIONS (CONT.) 

-OMT80L L OW DOSE 

URINAR Y SYSTEM * 1 (10X| 7 (15*) 

KIDNE Y 1 7

 

INFLAMMATIO N 

LYMPHOCYTI C IKFLA M INFILTRAT E 6

 

INFLAMMATIO N CHBONIC 1 1

 

ENDOCRIN E SYSTkM J (6«) 

THYBOID 3

 

INFIAHHATIO N CHRONIC 1 

HYPEBFLASI A C-CELL 1

 

HYPEBPLASIA FOLLJCU LA fi-CELL 1

 

HESATOfOIETIC SYS1EM 

SPLEEN 

LYMPHOID HYPE8PLASI A 

LYMP H NODE 

HYPiBPLiSIA REIICUtUH-CELL 

B8PBODUCIIVE SYSTEM 3 (30%) 13 (38%) 

UTEBU S 

INFLAHNATIO N SUPPURATIV E 

INFLAMMATIO N ACUTE 

UTEBUS/ENDOMETRIOH 2 10

 

IHFLAHMATIO N SUPPUBATIV E 5

 

ABSCESS 1 

HYPEBPLASI A CYSTIC 2 u

 

DtCIDUJ 1

 

OVABY/OVIDUC T 

INFLAMHATIO N SUBACUTE 

OVAB Y 1 13

 

FOLLICULA S CYST 1

 

IUFLAMMATIO N 2

 

INFLAMMATIO N SUPPUBATIV E 1 9

 

INFLAftMAIIO N ACUl'E SUPPUBATIV L 

_ ifiEWflflAII2fi SUfiAiUli 1 

*SYSTEM PKBCENTAGES ABE BASED ON THE NUMBER OF AJISAJtS._lJEC.B.p.P.S.IE.p.
 

HIG H DOSE 

2 (U« ) 

2 

1 

1 

i («%) 

2 

2 

2 (<•*) 

2 

2 

1 

\ 

6 (13%) 

2 

1 

1 

2 

1 

1 

1 

1 

3 

2 

1 

n-»aa_L^_Lii_ i i_ «. 

87
 
TABLE D2 FEMALE MICE: NONNEOPLASTIC LESIONS (CONT.) 

CONTRO L ^OVi ^CSE. 

NEB70U S SYSTE M * 

BSAIN/HENINGES 

INFLAMMATIO N 

2 (20%) 1 (2%) 

1 

1 

BSAI N 

CORPOftA AMYI.ACBA 

2 

2 

MUSCULOSKiLETAL SYSTEM 

BONE 

FIBBCSI3 

SKELETAL MUSCLE 

INFLAMMATIO N ACUTE 

SPECIAL SENSE ORGANS 

nous 

ALL OTHEB SYSTEMS 

NO ASSOCIATED ORGA N 

NO LESION BEPOBTfcD 

AUTOLYSIS/V£CBOPS¥ PEEF/NO HISIO 

AUIOLISIS/NO NECBOP5Y P^BFOBHEO 

2 (20X) 

2 

2 

19 (10%) 

19 

17 

2 

*SYSTtM PEBCEN1AGES ABE BASED ON THE NUBBBB OF A^S MBCHQPSIBD 

nI5 H JOSE 

2 («*) 

1 

1 

1 

1 

1U (31%) 

1U 

7 

2 

5 

88
 
APPENDIX E
 

STATISTICAL ANALYSES OF NEOPLASMS IN RATS
 

FED HEPTACHLOR IN THE DIET
 

89
 

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

in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Test for Tests After
 

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

Trend Adjustment
 £ b
 

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

Hepatooellular Carcinoma
 

Male 0/10 0/44 0/49
 

0% 0% 0%
 

Female 0/10 0/48 0/46
 

0% 0% 0% -"—
 

Hepatocellular Carcinoma <and/or Neoplastic Nodule
 

Male 1/10 3/44 6/49
 

10% 7% 12% 0.37 0.20
 

Female 1/10 9/48 5/46
 

10% 19% 11% 0.38C 0.41°
 

^Males: 38.9 ppm; females: 25.7 ppm.
 

Males: 77.9 ppm; females: 51.3 ppm.
 

P value given in the direction of a negative linear trend.
 
Table E2. Statistical Analysis of Neoplasms of the Liver in Rats Fed Heptachlor
 

in the Diet (using pooled control)
 

No. Rats with Lesion/
 

No. Rats with Liver 'Tissue Examined
 

Sex Control Low Dosea
 

Hepatooellular Carcinoma
 

Male 1/58 0/44
 

2% 0%
 

Female 0/59 0/48
 

0% 0%
 

Hepatoeellular Carcinoma <and/or Neoplastic
 

Male 3/58 3/44
 

5% 7%
 

Female 5/59 9/48
 

8% 19%
 

rMales: 38.9 ppm; females: 25.7 ppm.
 

Males: 77.9 ppm; females: 51.3 ppm.
 

High Doseb
 

0/49
 

0%
 

0/46
 

0%
 

Nodule
 

6/49
 

12%
 

5/46
 

11%
 

Exact Test for
 

Dose-related
 

Trend
 

(P)
 

—
 

——
 

0.13
 

0.37
 

Tests After
 

Life- table
 

Adjustment
 

(P)
 

0.14
 

0.20
 
Table E3. Statistical Analysis of Neoplasms of Thyroid Follicular Cells in Rats
 

Fed Heptachlor in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Test for Tests After 

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

Sex Control 

b 

Low Dose 

c 

High Dose 

Trend 

(P) 

Adjustment 

(P) 

Carcinoma 

Male	 0/8(0/8) 4/38(4/37) 0/38(0/33)
 d e H f
 

0% (0%) 11% (11%) 0% (0%) 0.23a

'e(0.26)°
 

Female	 1/9(1/7) 2/43(2/38) 5/38(5/29)
 

11% (14%) 5% (5%) 13% (17%) 0.31(0.26) 0.19
 

Carcinoma and/or Adenoma
 

Male	 1/8 9/38 3/38
 d	 d
 

13% 24% 8%	 0.16d 0.15d
 

Female	 1/9 3/43 14/38
 

11% 7% 37% 0.002 0.001
 

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: 38.0 ppm; females: 25.7 ppm.
 

^Males: 77.0 ppm; females: 51.3 ppm.
 

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

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

Validity of test is dubious because of the small number of tumorous animals observed,
 
Table E4. Statistical Analysis of Neoplasms of Thyroid Follicular Cells in Rats
 

Fed Heptachlor in the Diet (using pooled control)
 

Sex 

No. 

No. Rats 

Rats with 

Control 

with Lesion/ 

Thyroid Evprn-ftipd 

Low Dosea High DoseD 

Exact Test for 

Dose-related 

Trend 

(P) 

Tests After 

Life- table 

Adjustment 

(P) 

Carai-noma 

Male 1/51 

2% 

4/38 

11% 

0/38 

0% 

e d 

0.51C'a r d 

0.30C'U 

Female 1/58 

2% 

2/43 

5% 

5/38f 

13% 0.02 0.001 

Ccofoi-noma and/or Adenoma 

Male 4/51 

8% 

9/38S 

24% 

3/38 

8% 0.48e 0.36 

Female 3/58 

5% 

3/43 

7% 

14/38h 

37% <o.oie <o.oie 

?Males: 38.9 ppm; females: 25.7 ppm
 

Males: 77.9 ppm; females: 51.3 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).
 

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

jfSignificantly higher than its pooled control in unadjusted exact test (P < 0.05)
 

Significantly higher than its pooled control in both tests (P < 0.01).
 
Table E5. Statistical Analysis of Neoplasms of Thyroid C-cells in Rats Fed
 

Heptachlor in the Diet (using matched control)
 

No. No. Rats Rats with 

with Lesion/ a 

Thyroid Examined 

Exact Test for 

Dose-related 

Tests After 

Life-table 

Sex Control 

b 

Low Dose 

c 

High Dose 

Trend 

(P) 

Adjustment 

(P) 

Carcinoma 

Male 0/8(0/8) 

0% (0%) 

3/38(3/36) 

8% (8%) 

1/38(1/32) 

3% (3%) 

d d 0.37 (0.56) 

f 

Female 1/9 

11% 

0/43 

0% 

0/38 

0% d e 0.10 'e f 

Carcinoma and/or Adenoma
 

Male	 0/8 7/38 2/38
 d e	 d ee
 0%	 18% 5% 0.34 >S 0.34a

'


Female	 3/9 7/43 3/38 A /j


33% 16% 8% 0.046 0.03a
 

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

.before first tumor was found in any of the groups being compared to one another.
 Males: 38.9 ppm; females: 25.7 ppm.
 ^Males: 77.9 ppm; females: 51.3 ppm.
 f value given in the direction of negative linear trend.
 ?Data depart significantly from a linear trend (P < 0.05)
 

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

Heptachlor 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 Dose3 High Dose0 (P) (P) 

Carcinoma 

Male	 1/51 3/38 1/38
 

2% 8% 3% 0.49 0.48
 

Female	 5/58 0/436 0/386
 

9% 0% 0% 0.11° 0.01°
 

Carcinoma and/or Adenoma
 

Male	 4/51 7/38 2/38
 

8% 18% 5% 0.48c»d 0.28°
 

Female	 11/58 7/43 3/38
 

19% 16% 8% 0.10C 0.07°
 

fMales: 38.9 ppm; females: 25.7 ppm.
 

Males: 77.9 ppm; females: 51.3 ppm.
 

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

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

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

Table E7. Statistical Analysis of Neoplasms of the Pituitary Gland in Rats
 

Fed Heptachlor in the Diet (using matched control)
 

No. Rats with Lesion Exact Test for Tests After
 

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

Trend Adjustment
 

Sex Control Low Dosea High Doseb (P) (P)
 

Chromophobe Adenocarcinoma oic Carcinoma
 

Male	 0/7 0/42 0/34
 

0% 0% 0%
 

Female	 0/6 0/39 0/34 __ —
 

0% 0% 0%
 

Chromophobe Adenocaroinoma or Carcinoma and/or Chromeiphobe Adenoma
 

Male	 4/7 8/42 8/34 0.21C 0.14C
 

57% 19% 24%
 

Female	 3/6 9/39 8/34
 

50% 23% 24% 0.25° 0.16C
 

fMales: 38.0 ppm; females: 25.7 ppm.
 

Males: 77.0 ppm; females: 51.3 ppm.
 

P Value given in the direction of a negative linear trend.
 
Table E8. Statistical Analysis of Neoplasms of the Pituitary Gland in Rats
 

Fed Heptachlor in the Diet (using pooled control)
 

No. Rats with Lesion/
 Exact Test for Tests After
 

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

Trend Adjustment
 

Sex Control Low Dosea High Doseb (P) (P)
 

Chromophobe Adenoaarainoma or Carcinoma and/or Adenoma
 

Male	 16/48 8/42e 8/34e
 

33% 19% 24% 0.16° o.ooic

'd
 

Female	 23/52 9/39f 8/34f
 

44% 23% 24% 0.02° 0.02C
 

?Males: 38.9 ppm; females: 25.7 ppm.
 

Males: 77.9 ppm; females: 51.3 ppm.
 

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

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 lower than its pooled control in both tests (P < 0.05).
 
Table E9. Statistical Analysis of Neoplasms of the Mammary Gland in Female
 

Rats Fed Heptachlor in the Diet (using matched control)
 

No. Rats with Lesion/ Exact Test for Tests Alter
 

No. Rats Necropsieda Dose-related Lite-table
 

Control Low Dose High Dose Trend Adjustment
 

(0 ppm) (25.7 ppm) (51.3 ppm) (P) (P)
 

Adenooaroinoma or Papillary Adenoaarcinoma
 

1/10(1/10) 1/48(1/46) 1/48(1/39)
 c
 10% (10%) 2% (2%) 2% (3%) 0.17D

(0.34)D
 

Sarcoma or> Fibrosarooma
 

0/10 1/48 0/48
 

0% 2% 0% 0.30b c
 

Adenoaareinoma or Papillary Adenooaroinoma and/or Saraoma or Fibrosarooma
 

1/10 2/48 1/48
 

10% 4% 2% 0.23 c
 

Adenooaroinoma, or Papillary Adenoaarainoma and/or Saraoma or Fibrosarooma and/or
 

Fibroma or Adenoma or Fibroadenoma
 

2/10 18/48 8/48 V, •K
 

20% 38% 17% 0.09b 0.22b
 

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

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

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

°Validity of test is dubious because of the small number of tumorous animals observed.
 
Table E10. Statistical Analysis of Neoplasms of the Mammary Gland in Female
 

Rats Fed Heptachlor in the Diet (using pooled control)
 

No. Rats with Lesion/ Exact Test for
 Tests After
 

No. Rats Necropsied Dose-related Life-table
 

Control Low Dose High Dose Trend Adjustment
 

(0 ppm) (25.7 ppm) (51.3 ppm)
 (P) (P)
 

Adenocarcinoma or Papillary Adenocarcinoma
 

2/60 1/48 1/48
 

3% 2% 2% 0.46a e
 

Sarcoma or Fibrosarcoma
 

0/60 1/48 0/48
 e
 

0% 2% 0% 0.62
 

Adenocarcinoma or Papillary Adenocarcinoma and/or Sarcoma or Fibrosarcoma
 

2/60 2/48 1/48
 

3% 4% 2% 0.48a 0.44a
 

Adenooarcinoma or Papillary Adenocarcinoma and/or Sarcoma or Fibrosarcoma and or Fibroma
 

10/60 18/48d 8/48
 V, f»
 

17% 38% 17% 0.47b 0.22
 

«a
 

,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).
 

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

Validity of test is dubious because of the small number of tumorous animals observed.
 
Table Ell. Statistical Analysis of Neoplasms of the Uterus in Female Rats Fed
 

Heptachlor in the Diet (using matched control)
 

No Exact Test for Tests After . Rats with Lesion/ 

No. Rat's wi t~Vi llfpfiiR F.vamlnpfl Dose-related Life-table 

Control Low Dose High Dose Trend Adjustment 

(0 ppm) (25.7 ppm) (51.3 ppm) (P) (P) 

Endome trial Stromal Sarcoma 

0/10 0/43 2/41 

0% 0% 5% 0.19 a 

Endometrial Stromal Sarooma and/ or Endometr-Lal Stromal Polyp 

2/10 7/43 8/41 

20% 16% 20% 0.52 0.39 

validity of test is dubious because of the small number of tumorous animals observed.
 
Table E12. Statistical Analysis of Neoplasms of the Uterus Fed Heptachlor
 

in the Diet (using pooled control)
 

No

Control 

1Q ppml 

No. Rats with Lesion/ 

. Rats with Uterus Examined 

Low Dose 

(25.7 ppm) 

High Dose 

(51.3 ppm) 

Exact Test for 

Dose-related 

Trend 

(P) 

Tests After 

Life-table 

Adjustment 

(P) 

Endometrial Stromal Sarcoma 

0/56 

0% 

0/43 

0% 

2/41a 

5% 0.08 

b 

Endomt, trial Stromal Sarcoma and/or Endometrial Stromal Polyp
 

6/56 7/43 8/41
 

11% 16% 20% 0.14 0.09
 

^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 tumorous animals observed,
 
APPENDIX F
 

STATISTICAL ANALYSES OF NEOPLASMS IN MICE
 

FED HEPTACHLOR IN THE DIET
 

103
 

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

Heptachlor in the Diet (using matched control)
 

No. M:Lee 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 

!Qj!pmJ £6vl_Ei)m) I13..JLBBSX- (P) (Pi (P) 

Hepatooellulap Carcinoma 

__ 1/9(1/9) 34/47(34/46) 

11% (11%) 72% (74%) 0.001(0.0007) 0.002 — __ 4/10(4/8) 

40% (50%) 

11/46(11/43) 

24% (26%) — 

v> v> 

0.25D(0.17) 

V. 

0.05+ 

Combined 0.001C(0.0025)b 0.003 

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.
 

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

°Two slopes are significantly different (P < 0.05).
 
__ 

Table F2. Statistical Analysis of Neoplasms of the Liver in Male Mice Fed
 

Heptachlor in the Diet (using pooled control)
 

No. Mice with Lesion/
 

No. Mice with lAvei: Ti,ssue Examined
 

Control Low Dose High Dose
 

(0 j>pm) (6.1 ppm) (13.8 ppm)
 

Hepatoaellular Carcinoma
 

17/92 34/47
 —
 18% 72%
 —
 

17/92 11/46
 

18% 24%
 —
 

17/92 11/46 34/47
 

18% 24% 72%
 

Fisher
 

Exact
 

Test
 

(P)
 

< 0.0001
 

0.30
 

Exact Test for
 

Dose-related
 

Trend
 

(P)
 

<o.oooia
 

Tests After
 

Life-table
 

Adjustment
 

(P)
 

< 0.0001
 

0.26
 

•i
 

< 0.0001
 

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

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

Heptachlor in the Diet (using matched control)
 

No. Mice with Lesion Exact Test for Tests After 

JlQJ__J£Lce_jHijJi.JJLy.£r lisaue__Examinadf_ Dose-related Lite-table 

Control Low Dose High Dose Trend Adjustment 

(0 ppm) J!_E2SLL (18 ppm) (P) (P) 

Hepatocellular' Carcinoma 

2/10(2/10) 3/47(3/43) 30/42'b

(30/38)b

 

20% (20%) 6% (7%) 71% (79%) < 0. 0001 (< 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.
 

Significantly higher than matched control (P < 0.01).
 

°Data depart significantly from linear trend (P < 0.01).
 
Table F4. Statistical Analysis of Neoplasms of the Liver in Female Mice
 

Fed Heptachlor in the Diet (using pooled control)
 

No. Mice with Lesion/ Exact Test for
 

No. Mice with Liver Tissue Examined Dose-related
 

Control Low Dose High Dose Trend
 

(0 ppm) (9 ppm) (18 ppm) (P)
 

Hepatoeellular Carcinoma
 

3/78 3/47 30/42t>
 

4% 6% 71% <o.oooia
 

depart significantly from a linear trend (P < 0.01).
 'Significantly higher than .pooled controls (P < 0.01).
 

Tests After
 

Lite-table
 

Adjustment
 

(P)
 

<o.oooia
 
APPENDIX G
 

ANALYSIS OF FORMULATED DIETS
 

FOR CONCENTRATION OF HEPTACHLOR
 

109
 

APPENDIX G
 

Analysis of Formulated Diets for
 

Concentration of Heptchlor
 

A 100 g sample of the diet mixture was shaken with 125 mis hexane
 

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

with hexane washes, and reduced in volume to 10 mis. After
 

appropriate dilutions, the solution was quantitatively analyzed
 

for heptachlor 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 No. of Sample Coefficient of Range (ppm) 

Dietary Samples Analytical Variation (%) 

Level (ppm) Mean (ppm) 

5 17 4.92 7.5 4.0-5.4 

10 18 9.83 3.7 9.2-10.6 

20 20 20.2 5.9 17.5-23.4 

40 12 39.2 5.7 35.2-42.1 

80 10 80.6 6.4 71.0-90.7 

120 2 110.0 0 110.0 

160 5 151.2 6.9 137.0-162.0 

111 

*U.S. GOVERNMENT PRINTING OFFICE: 1977 Z41-161/3051 1-3 






DHEW Publication No. (NIH) 77-809