1979 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 37 

BIOASSAY OF 

TOXAPHENE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 8001-35-2 

NCI-CG-TR-37 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

TOXAPHENE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DREW Publication No. (NIH) 79-837
 
ii

 
BIOASSAY OF
 

TOXAPHENE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORD; This report presents the results of the bioassay of
 

toxaphene conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer
 

Institute (NCI), National Institutes of Health, Bethesda,
 

Maryland. This is one of a series of experiments designed to
 

determine whether selected environmental chemicals have the
 

capacity to produce cancer in animals. Negative results, in
 

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

than control animals, do not necessarily mean that the test
 

chemical is not a carcinogen, inasmuch as the experiments are
 

conducted under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals
 

under the conditions of the test and indicate that exposure to
 

the chemical is a potential risk to man. The actual 

determination of the risk to man from chemicals found to be 

carcinogenic in animals requires a wider analysis. 

CONTRIBUTORS: This bioassay of toxaphene was conducted by Gulf
 

South Research Institute, New Iberia, Louisiana, initially under
 

direct contract to NCI (1) and currently under a subcontract to
 

Tracor Jitco, Inc., Rockville, Maryland, prime contractor for the
 

NCI Carcinogenesis Testing Program.
 

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

(1,2) and R. R. Bates (1,3); the doses were selected by Drs. T.
 

E. Shellenberger (4,5), J. H. Weisburger, and R. R. Bates.
 

Administration of the test chemical and observation of the
 

animals were supervised by Drs. T. E. Shellenberger and H. P.
 

Burchfield (4), with the technical assistance of Ms. D. H.
 

Monceaux (4) and Mr. D. Broussard (4). Histopathology was
 

performed by Drs. E. Bernal (4) and B. Buratto (4) at Gulf South
 

Research Institute, and the diagnoses included in this report
 

represent the interpretation of these pathologists.
 

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

Mason Research Institute (6). Statistical analyses were
 

performed by Dr. J. R. Joiner (7) and Ms. P. L. Yong (7), using
 

methods selected for the bioassay program by Dr. J. J. Gart (8).
 

Chemicals used in this bioassay were analyzed under the direction
 

of Dr. H. P. Burchfield, and the analytical results were reviewed
 

by Dr. S. S. Olin (7).
 

This report was prepared at Tracor Jitco (7) under the direction
 

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

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

A. Campbell, Deputy Director for Science; Dr. J. F. Robens,
 

toxicologist; Dr. R. L. Schueler, pathologist; Dr. G. L. Miller,
 

Mr. W. D. Reichardt, and Ms. L. A. Waitz, bioscience writers; and
 

Dr. E. W. Gunberg, technical editor, assisted by Ms. Y. E.
 

Presley and Ms. P. J. Graboske.
 

The following scientists at NCI were responsible for evaluating
 

the bioassay experiment, interpreting the results, and reporting
 

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

Fielding Douglas, Dr. Dawn G. Goodman (9), Dr. Richard A.
 

Griesemer, Dr. Harry A. Milman, Dr. Thomas W. Orme, Dr. Robert A.
 

Squire (10), Dr. Jerrold M. Ward, and Dr. Carrie E. Whitmire.
 

(1)	 Carcinogenesis Testing Program, Divison of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

(2)	 No wit w h the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

(3)	 No wit w h the National Institute of Environmental Health
 

Sciences, P. 0. Box 12233, Research Triangle Park, North
 

Carolina.
 

(4)	 Gulf South Research Institute, Atchafalaya Basin
 

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

(5)	 No wit w h the National Center for Toxicological Research,
 

Jefferson, Arkansas.
 

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

Rockville, Maryland.
 

iv
 
(7)	 Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

(8)	 Mathematical Statistics and Applied Mathematics Section,
 Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute,
 

National Institutes of Health, Bethesda, Maryland.
 

(9) Now with Clement Associates, Inc., 1010 Wisconsin Avenue,
 

N.W., Suite 660, Washington, D. C.
 

(10) Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

V 
vi

 
SUMMARY
 

A bioassay of technical-grade toxaphene for possible
 

carcinogenicity was conducted by administering the test chemical
 

in feed to Osborne-Mendel rats and B6C3F1 mice.
 

Groups of 50 rats of each sex were administered toxaphene at one
 

of two doses for 80 weeks, then observed for 28 or 30 weeks.
 

Time-weighted average doses for males were 556 or 1,112 ppm; for
 

females they were 540 or 1,080 ppm. Matched controls consisted
 

of groups of 10 untreated rats of each sex; pooled controls
 

consisted of the matched-control groups for toxaphene combined
 

with 45 untreated male and 45 untreated female rats from similar 

bioassays of five other test chemicals. All surviving rats were 

killed at 108-110 weeks. 

Groups of 50 mice of each sex were administered toxaphene at one
 

of two doses for 80 weeks, then observed for 10 or 11 weeks.
 

Time-weighted average doses were 99 or 198 ppm for both males and
 

females. Matched controls consisted of groups of 10 untreated
 

mice of each sex; pooled controls consisted of the matched-


control groups for toxaphene combined with 40 untreated male and
 

40 untreated female mice from similar bioassays of four other
 

test chemicals. All surviving mice were killed at 90-91 weeks.
 

Mean body weights attained by low- and high-dose female rats and
 

high-dose male mice were lower than those of matched controls,
 

but weights of other dosed groups were essentially unaffected by
 

the toxaphene. Other clinical signs of toxicity in rats included
 

generalized body tremors at week 53 in high-dose male and female
 

animals, and later, leg paralysis, ataxia, epistaxis, hematuria,
 

and vaginal bleeding> predominantly in the dosed groups of rats
 

of each sex. Abdominal distention, diarrhea, dyspnea, and rough
 

hair coats were common to both dosed rats and dosed mice. There
 

were dose-related decreases in survival rates in mice but not in
 

rats. Sufficient numbers of both rats and mice were at risk for
 

the development of late-appearing tumors.
 

In the male rats, the incidence of follicular-cell carcinomas or
 

adenomas of the thyroid was dose related (P = 0.007) using the
 

pooled controls (matched controls 1/7, pooled controls 2/44,
 

low-dose 7/41, high-dose 9/35). In the females, the incidence of
 

follicular-cell adenomas of the thyroid was dose related using
 

either the matched (P = 0.022) or pooled (P = 0.008) controls
 

(matched controls 0/6, pooled controls 1/46, low-dose 1/43,
 

vil
 
high-dose 7/42). Direct comparisons of dosed and pooled-control
 

groups but not matched controls showed significantly increased
 

incidences of follicular-cell carcinomas or adenomas in the
 

high-dose males (P = 0.008) and of follicular-cell adenomas in
 

the high-dose females (P = 0.021). Two follicular-cell tumors in
 

the high-dose males were carcinomas; all other follicular-cell
 

tumors in the rats were adenomas.
 

In the mice, the incidence of hepatocellular carcinomas was dose
 

related (P less than 0.001) for both males (matched controls
 

0/10, pooled controls 4/48, low-dose 34/49, high-dose 45/46) and
 

females (matched controls 0/9, pooled controls 0/48, low-dose
 

5/49, high-dose 34/49), using either matched or pooled controls.
 

Direct comparisons showed that the incidences of hepatocellular
 

carcinomas in low- and high-dose male mice and high-dose female
 

mice were all significantly higher (P less than 0.001) than those
 

in the respective matched or pooled controls. Statistical
 

significance was maintained when the incidence of hepatocellular
 

carcinomas was combined with that of neoplastic nodules of the
 

liver.
 

It is concluded that under the conditions of this bioassay,
 

toxaphene was carcinogenic in male and female B6C3F1 mice,
 

causing increased incidences of hepatocellular carcinomas. The
 

test results also suggest carcinogenicity of toxaphene for the
 

thyroid of male and female Osborne-Mendel rats.
 

vlli
 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 5
 

E. Subchronic Studies 7
 

F. Chronic Studies 8
 

G. Clinical and Pathologic Examinations 11
 

H. Data Recording and Statistical Analyses 13
 

III. Results - Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 23
 

D. Statistical Analyses of Results (Rats) 24
 

IV. Results - Mice 29
 

A. Body Weights and Clinical Signs (Mice) 29
 

B. Survival (Mice) 31
 

C. Pathology (Mice) 31
 

D. Statistical Analyses of Results (Mice) 35
 

V. Discussion 37
 

VI. Bibliography 41
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed Toxaphene in the Diet.f 43
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed Toxaphene in the Diet 45
 

ix
 
Page 

Table A2 Summary of the Incidence of Neoplasms in 

Female Rats Fed Toxaphene in the Diet 49 

Appendix B Summary of the Incidence of Neoplasms in 

Mice FecJ Toxaphene in the Diet 53 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Toxaphene in the Diet 55 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Toxaphene in the Diet 58 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Toxaphene in the Diet 61 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Toxaphene in 

the Diet 63 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Toxaphene in the 

Diet 67 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Toxaphene in the Diet 71 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Toxaphene in the 

Diet 73 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Toxaphene in the 

Diet 75 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Toxaphene in the Diet 79 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Toxaphene in the Diet 81 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Toxaphene in the Diet. 85 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Toxaphene in the Diet 91 

X 
Page 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Toxaphene in the Diet 93 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Toxaphene in the Diet 99 

Appendix G Analysis of Formulated Diets for Concentrations 

of Toxaphene 101 

TABLES 

Table 1 Toxaphene Chronic Feeding Studies in Rats.... 9 

Table 2 Toxaphene Chronic Feeding Studies in Mice.... 10 

FIGURES 

Figure 1 Growth Curves for Rats Fed Toxaphene 

in the Diet 20 

Figure 2 Survival Curves for Rats Fed Toxaphene 

in the Diet 22 

Figure 3 Growth Curves for Mice Fed Toxaphene 

in the Diet 30 

Figure 4 Survival Curves for Mice Fed Toxaphene in 

the Diet 32 

xi
 
xli 
I. INTRODUCTION
 

Toxaphene (CAS 8001-35-2; NCI C00259) is an organochlorine
 

insecticide that belongs to the class of compounds known as polychlorinated bicyclic terpenes with chlorinated camphenes
 

predominating (Brooks, 1975); an insecticide marketed as
 

Strobane-T® (Tenneco Chemical Co., Piscataway, N. J.) is
 

identical with toxaphene. Toxaphene is obtained from camphene by
 

photochemical chlorination, which produces a heterogeneous
 

mixture of chemicals containing 67-69% chlorine. Although the
 

exact composition of toxaphene has not been determined, it is
 

similar to that of Strobane®, an insecticide differing from
 

Strobane-T® and now discontinued. Strobane® has been shown
 

to cause hepatomas in mice (innes et al., 1969). Persons
 

involved in the manufacture or handling of toxaphene, as well as
 

human volunteers exposed twice a week at 3-week intervals to 500
 

3
 

mg/m aerosol of toxaphene 30 min/day for 10 days, showed no
 

toxic manifestations (Deichmann, 1973).
 

Toxaphene is registered for use on a wide range of fruits,
 

vegetables, nuts, field crops, animals, and agricultural premises
 

(EPA Compendium of Registered Pesticides, 1974). Tolerances for
 

residues of toxaphene have been established for many of the
 

1 
various agricultural commodities. Toxaphene was selected for
 

study in the Carcinogenesis Testing Program because it is known
 

to be related to Strobane®, a compound inducing hepatomas,
 

because it has been used extensively in agriculture, and because
 

its persistence in the ecosystem (Brooks, 1975) may lead to
 

chronic human exposure through residues in food and water.
 

2 
II. MATERIALS AND METHODS
 

A. Chemical
 

The technical-grade toxaphene used in the chronic phase of the
 

bioassay was obtained in a single batch from Hercules, Inc.,
 

Wilmington, Delaware (Lot No. X-16189-49). Analyses performed at
 

Gulf South Research Institute (melting range, elemental analysis,
 

gas-liquid chromatography, infrared spectrophotometry) confirmed 

the expected heterogeneity of this product. The chemical was 

stored at approximately 0 C. 

B. Dietary Preparation
 

All diets containing toxaphene were formulated using Wayne® Lab
 

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

added the required amount of toxaphene for each dietary
 

concentration. The test chemical was first dissolved in a small
 

amount of acetone (Mallinckrodt Inc., St. Louis, Mo.), which was
 

then added to the feed. Corn oil (LouAna®, Opelousas Refinery
 

Co., Opelousas, La.) was also added to the feed, primarily as a
 

dust suppressant, and the diets were mixed mechanically to assure
 

3 
homogeneity of the mixtures and evaporation of the acetone.
 

Final diets, including those for the control groups of animals,
 

contained corn oil equal to 2% of the final weight of feed. The
 

diets were stored at room temperature until used, but no longer
 

than 1 week.
 

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

diets, the concentration of toxaphene was determined in different
 

batches of formulated diets during the chronic study. The
 

results are summarized in Appendix G. At each dietary
 

concentration, the mean value obtained for the checked samples
 

was within 3.2% of the theoretical, and the coefficient of
 

variation was never more than 6.9%.
 

C. Animals
 

Rats and mice of each sex, obtained through contracts of the
 

Division of Cancer Treatment, National Cancer Institute, were
 

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

strain obtained from Battelle Memorial Institute, Columbus, Ohio,
 

and the mice were B6C3F1 hybrids obtained from Charles River
 

Breeding Laboratories, Inc., Wilmington, Massachusetts. On
 

arrival at the laboratory, all animals were quarantined (rats for
 

4 
7 days, mice for 13 days) and then assigned to control or dosed
 

groups.
 

D. Animal Maintenance
 

All animals were housed in rooms in which the temperature ranged
 

from 22 to 24°C, and the relative humidity from 40 to 70%. The
 

air entering each room was filtered (Air Maze Incom
 

International, Cleveland, Ohio), and room air was changed 10 to
 

12 times per hour. Fluorescent lighting provided illumination 10
 

hours per day. Food and water were provided ad libitum. Fresh
 

feed was provided daily, and excess remaining feed was discarded.
 

The rats were housed individually in hanging galvanized steel
 

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

housed in polypropylene cages (Lab Products, Inc., Garfield,
 

N.J.), five females per cage or two or three males per cage.
 

Mouse cages were covered with polyester filter bonnets (Lab
 

Products, Inc.). The rat racks and cages were sanitized every 2
 

weeks. The mouse cages were sanitized each week. These cages
 

and racks were washed in an industrial washer at 82 C with
 

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

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

5 
(Kimberly Clark Corp., Neenah, Wis.) were placed under the rat
 

cages and were changed three times per week. Absorb-dri®
 

hardwood chip bedding (Lab Products, Inc.), used in the mouse
 

cages, was provided two times per week for males and three times
 

per week for females. Filter bonnets were sanitized each week.
 

Feed jars and water bottles were changed and sanitized three
 

times per week. Sipper tubes and stoppers were sanitized two
 

times per week.
 

The filter bonnets, feed jars, water bottles, sipper tubes, and
 

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

Cage racks for each species were rotated to a new position in the
 

room once per week; at the same time, each cage was moved to a
 

different row within the same column of a rack. Rats receiving
 

toxaphene, along with their matched controls, were housed in a
 

room by themselves. Mice were maintained in a room housing mice
 

from the following studies:
 

Feed Studies
 

(CAS 57-74-9) chlordane
 

(CAS 143-50-0) chlordecone
 

6 
E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses of toxaphene, on the basis of which two concentrations (hereinafter referred to as "low" and "high" doses)
 

were determined for administration in the chronic studies. In
 

these subchronic studies, toxaphene was added to the animal feed
 

in twofold increasing concentrations, ranging from 160 to 2,560
 

ppm for rats and from 40 to 1,280 ppm for mice. The test
 

chemical was provided in feed to dosed groups of five male and
 

five female animals of each species for 6 weeks, followed by
 

observation for 2 weeks. Untreated-control groups consisted of
 

five animals of each species and each sex. A second study was
 

performed on male and female rats at doses ranging from 1,280 to
 

5,120 ppm to confirm the results and to extend the concentration
 

range of the first study.
 

At 1,280 ppm in the first and second studies, there were no
 

deaths among the rats, and mean weight gains of both males and
 

females were comparable to those of corresponding controls. At
 

2,560 ppm, two female rats died in the first study; however, the
 

mean weights of the survivors were not adversely affected.
 

During the second study, one male and one female died at 2,560
 

ppm. On the basis of these results, the low and high doses for
 

7
 
the chronic studies using rats were set at 1,280 and 2,560 ppm
 

for males, and 640 and 1,280 ppm for females.
 

Four male and two female mice died at 640 ppm, and one male and
 

one female mouse given 320 ppm died. Mean weight gains of mice
 

given 320 ppm were comparable to those of controls. On the basis
 

of these results, the low and high doses for the chronic studies
 

using mice were set at 160 and 320 ppm for males and females.
 

F. Chronic Studies
 

The test groups, doses administered, and durations of the chronic
 

feeding studies are shown in tables 1 and 2.
 

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

small, pooled-control groups also were used for statistical
 

evaluation. For the rats, matched controls from the current
 

bioassay on toxaphene were combined with matched controls from
 

studies performed on captan (CAS 133-06-02), chloramben (CAS
 

133-90-4), lindane (CAS 58-89-9), malathion (CAS 121-75-5), and
 

picloram (CAS 1918-02-1) to give pooled-control groups consisting
 

of 55 males and 55 females. For the mice, matched controls from
 

the current bioassay were combined with matched controls from
 

8 
Table 1. Toxaphene Chronic Feeding Studies in Rats
 

Sex and 

Test 

Group 

Initial 

No. of 

Animals (a) 

Toxaphene 

in Diet(b) 

(ppm) 

Time 

Dosed(c) 

(weeks) 

on Study 

Observed (d) 

(weeks) 

Time-Weighted 

Average Dose(e) 

(ppm) 

Male 

Matched-Control 10 0 108-109 

Low-Dose 50 1,280 

640 

320 

0 

2 

53 

25 

28 

556 

High -Dose 50 2,560 

1,280 

640 

0 

2 

53 

25 

28 

1,112 

Female 

Matched-Control 10 0 108-109 

Low-Dose 50 640 

320 

0 

55 

25 

30 

540 

High-Dose 50 1,280 

640 

0 

55 

25 

30 

1,080 

(a) All animals were 5 weeks of age when placed on study.
 

(b) Initial doses shown were toxic; therefore, doses were lowered after 2
 

weeks and again at 53 or 55 weeks, as shown.
 

(c) All animals were started on study on the same day.
 

(d) When diets containing toxaphene were discontinued, dosed rats and their
 

matched controls were fed control diets without corn oil for 20 weeks,
 

then control diets (2% corn oil added) for an additional 8 weeks.
 

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

E(no. of weeks receiving each dose)
 

9 
Table 2. Toxaphene Chronic Feeding Studies in Mice
 

Sex and 

Test 

Group 

Initial 

No. of 

Animals (a) 

Toxaphene 

in Diet(b) 

(ppm) 

Time 

Dosed(c) 

(weeks) 

on Study 

Observed(d) 

(weeks) 

Time-Weighted 

Average Dose(e) 

(ppm) 

Male 

Matched-Control 10 0 90-91 

Low-Dose 50 160 

80 

0 

19 

61 

11 

99 

High -Dose 

Female 

50 320 

160 

0 

19 

61 

10 

198 

Matched-Control 10 0 90-91 

Low -Dose 50 160 

80 

0 

19 

61 

11 

99 

High -Dose 50 320 

160 

0 

19 

61 

10 

198 

(a) All animals were 5 weeks of age when placed on study.
 

(b) Initial doses shown were toxic; therefore, doses were lowered at 19
 

weeks, as shown.
 

(c) All animals were started on study on the same day.
 

(d) When diets containing toxaphene were discontinued, dosed mice and their
 

matched controls were fed control diets without corn oil for 7 weeks,
 

then control diets (2% corn oil added) for an additional 3 to 4 weeks.
 

(e) Time-weighted average dose = ^(dose in ppm x no. of weeks at that dose)
 

'•(no. of weeks receiving each dose)
 

10
 
studies performed on lindane, malathion, phosphamidon (CAS
 

13171-21-6), and tetrachlorvinphos (CAS 961-11-5) to give
 

pooled-control groups consisting of 50 males and 50 females.
 

These studies on chemicals other than toxaphene were also
 

conducted at Gulf South Research Institute and were started less
 

than 5 months apart. In the studies on captan and malathion, the
 

control groups of the rats were started at different times, but
 

only those started within 5 months of the time of starting the
 

matched controls for toxaphene were used in the pooled-control
 

groups. The matched-control groups for the different test
 

chemicals were of the same strains and from the same suppliers,
 

and they were all examined by the same pathologists.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity,
 

weighed every 2 weeks for the first 12 weeks and monthly
 

thereafter, and palpated for masses at each weighing. Sick,
 

tumor-bearing, and moribund animals were observed daily.
 

Moribund animals and animals that survived to the end of the
 

bioassay were killed using ether and necropsied. Necropsies were
 

also performed on all animals found dead, unless precluded by
 

autolyses or severe cannibalization.
 

11
 
The pathologic evaluation consisted of gross and microscopic
 

examination of major tissues, major organs, and all gross
 

lesions. The following tissues were examined microscopically:
 

skin, lungs and bronchi, trachea, bone and bone marrow, spleen,
 

lymph nodes, heart, salivary gland, liver, gallbladder (mice),
 

pancreas, stomach, small intestine, large intestine, kidney,
 

urinary bladder, pituitary, adrenal, thyroid, parathyroid,
 

mammary gland, prostate or uterus, testis or ovary, and brain.
 

Occasionally, additional tissues were also examined
 

microscopically. The different tissues were preserved in 10%
 

buffered formalin, embedded in paraffin, sectioned, and stained
 

with hematoxylin and eosin. Special staining techniques were
 

utilized when indicated for more definitive diagnosis.
 

A few tissues from some animals were not examined, particularly
 

from those animals that may have died early, or been in advanced
 

states of cannibalization or autolysis. Thus, the number of
 

animals from which particular organs or tissues were examined
 

microscopically varies, and does not necessarily represent the
 

number of animals that were placed on study in each group.
 

12
 
H. Data Recording and Statistical Analyses
 

Pertinent data on this experiment have been recorded in an
 

automatic data processing system, the Carcinogenesis Bioassay
 

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

descriptive information on the chemicals, animals, experimental
 

design, clinical observations, survival, body weight, and
 

individual pathologic results, as recommended by the
 

International Union Against Cancer (Berenblum, 1969). Data
 

tables were generated for verification of data transcription and
 

for statistical review.
 

These data were analyzed using the appropriate statistical
 

techniques described in this section. Those analyses of the
 

experimental results that bear on the possibility of
 

carcinogenicity are discussed in the statistical narrative
 

sections.
 

Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically
 

censored as of the time that they died of other than natural
 

causes or were found to be missing; animals dying from natural
 

causes were not statistically censored. Statistical analyses for
 

13
 
a possible dose-related effect on survival used the method of Cox
 

(1972) for testing two groups for equality and Tarone's (1975)
 

extensions of Cox's methods for testing for a dose-related
 

trend. One-tailed P values have been reported for all tests
 

except the departure from linearity test, which is only reported
 

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic
 

examination was required to detect lesions prior to histologic
 

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

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

denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

animals. As a part of these analyses, the one-tailed Fisher
 

exact test (Cox, 1970) was used to compare the tumor incidence of
 

a control group with that of a group of dosed animals at each
 

14
 
dose level. When results for a number of dosed groups (k) are
 

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

made. The Bonferroni inequality (Miller, 1966) requires that the
 

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

cases where this correction was used, it is discussed in the
 

narrative section. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

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

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

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

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


tailed 0.05 level of significance. Unless otherwise noted, the
 

direction of the significant trend is a positive dose
 

relationship. This method also provides a two-tailed test of
 

departure from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

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

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

15
 
such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

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

methods of comparing these curves were used for two groups;
 

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

groups. The statistical tests for the incidence of tumors which
 

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

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P less than 0.05,
 

two-tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with its control was calculated
 

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

relative risk is defined as p../?., where p is the true
 

16
 
binomial probability of the incidence of a specific type of tumor
 

in a dosed group of animals and p is the true probability of
 

the spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

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

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

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

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

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

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P less than 0.025 one-tailed test when the
 

control incidence is not zero, P less than 0.050 when the control
 

incidence is zero) has occurred. When the lower limit is less
 

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

limit indicates the absence of a significant result while the
 

upper limit indicates that there is a theoretical possibility of
 

17
 
the induction of tumors by the test chemical, which could not be
 

detected under the conditions of this test.
 

18
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

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

lower than those of the matched controls throughout most of the
 

bioassay, suggesting a dose-related effect of toxaphene on body
 

weights (figure 1). Weights of low- and high-dose male rats were
 

essentially unaffected by the toxaphene. Fluctuation in the
 

growth curve may be due to mortality; as the size of a group
 

diminishes, the mean body weight may be subject to variation.
 

During the first 16 weeks of the bioassay, the dosed animals were
 

generally comparable to the controls in appearance and behavior,
 

with the exception of the high-dose males, which appeared
 

hyperactive during week 2; doses for male rats were lowered at
 

that time. At week 53, the concentration of toxaphene in the
 

feed was reduced because a majority of the high-dose males and
 

females developed generalized body tremors. From week 52 to week
 

80, clinical signs including alopecia, diarrhea, dyspnea, pale
 

mucous membranes, rough hair coats, dermatitis, ataxia, leg
 

paralysis, epistaxis, hematuria, abdominal distention, and
 

vaginal bleeding were noted predominantly in the dosed groups.
 

19
 
Figure 1. Growth Curves for Rats Fed Toxaphene in the Diet
 

20
 
During the second half of the year, these adverse signs were
 

noted with increasing frequency in both control and dosed
 

groups. Two females, one high-dose and one low-dose, had
 

impaired equilibrium. A purulent vaginal discharge was noted in
 

one high-dose female.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats fed toxaphene in the diet at
 

the doses of this bioassay, together with those of the controls,
 

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

dose-related trend in mortality is not significant in either sex.
 

In male rats, 45/50 (90%) of the high-dose group, 47/50 (94%) of
 

the low-dose group, and all 10 animals of the control group lived
 

at least as long as week 52 on study. In females, 48/50 (96%) of
 

the high-dose group, 46/50 (92%) of the low-dose group, and all
 

10 animals of the control group lived beyond week 52 on study.
 

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

the development of tumors.
 

21
 
Figure 2. Survival Curves for Rats Fed Toxaphene in the Diet
 

22 
C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

are summarized in Appendix C, tables Cl and C2.
 

The majority of neoplastic and nonneoplastic lesions observed in
 

the rats were not unusual findings, and the incidences of lesions
 

in animals of the dosed groups were generally similar to those of
 

the matched-control groups. Certain lesions (follicular-cell
 

tumors of the thyroid; follicular and C-cell hyperplasias of the
 

thyroid; endometrial stromal polyps of the uterus; chromophobe
 

adenomas of the pituitary gland; tumors of the mammary gland;
 

cyst of the bile duct; and testicular atrophy) appeared with a
 

higher incidence in the dosed rats; however, they are frequently
 

observed in this strain of rat independent of any chemical
 

administration. The follicular-cell neoplasms, primarily
 

adenomas, were seen in 1/7 control males, 7/41 low-dose males,
 

9/35 high-dose males; 0/6 control females, 1/43 low-dose females,
 

and 7/42 high-dose females. Thyroid follicular-cell hyperplasias
 

were observed only in dosed rats (males: low-dose 3/41,
 

high-dose 3/35; females: low-dose 5/43, high-dose 3/42). These
 

tumors and lesions occurred at relatively low incidences, and, in
 

view of the low numbers of animals used in the matched controls,
 

23
 
it could not be concluded from comparisons of incidences in dosed
 

and matched-control groups that the increased incidences of
 

neoplasms or proliferative lesions were a result of the
 

administration of toxaphene.
 

Based on the histopathologic examination, there was no conclusive
 

evidence that toxaphene was carcinogenic in Osborne-Mendel rats
 

under the conditions of this bioassay, although thyroid
 

follicular-cell neoplasms may have been associated with
 

administration of the test chemical.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

In male rats, the Fisher exact test shows that the incidence of
 

follicular-cell carcinomas or adenomas of the thyroid was
 

significantly higher (P = 0.008) in the high-dose group than in
 

the pooled-control group, and the Cochran-Armitage test for
 

linear trend, using the pooled controls, had a probability level
 

24
 
of P = 0.007. All of these tumors were adenomas except for two
 

carcinomas in the high-dose group.
 

In the female rats, follicular-cell adenomas of the thyroid
 

appeared in the high-dose group in significant proportions (P =
 

0.021), when comparisons were made with the pooled controls.
 

When compared with the historical controls (data obtained to date
 

on 20 studies at this laboratory in this bioassay program), the
 

incidence in the high-dose group (7/42 or 17%) was higher than
 

that reported for follicular-cell adenomas in the female
 

historical controls (4/184 or 2.2%). Furthermore* the incidences
 

in neither the matched nor the pooled controls were different
 

from that in the historical controls. The Cochran-Armitage test
 

for significant linear trend applied to the proportions in
 

pooled-control (1/46), low-dose (1/43), and high-dose (7/42)
 

females showed a probability level of P = 0.008; the
 

Cochran-Armitage test applied to the data using the matched
 

controls (P = 0.022) supported the findings using the pooled
 

controls.
 

Also in the female rats, the Cochran-Armitage test results on the
 

incidence of animals with chromophobe adenomas, chromophobe
 

carcinomas, or adenomas of the pituitary are significant (P =
 

0.012 using the pooled controls and P = 0.046 using the matched
 

25
 
controls). The Fisher exact test shows that the incidence in the
 

high-dose group is significantly higher than that in the
 

pooled-control group (P = 0.013). The historical-control data
 

obtained to date on 20 similar studies at this laboratory show an
 

incidence of 58/185 (31.4%), although there are incidences as
 

high as 6/10 (60%), 5/10 (50%), 3/6 (50%), and 4/9 (44%).
 

Considering these high spontaneous incidences seen in control
 

groups, the conclusion cannot be made that there is an
 

association between administration of the chemical and the
 

increased incidence of tumors of the pituitary in female
 

Osborne-Mendel rats under the conditions of this bioassay.
 

In male rats, the Fisher exact test on the incidence of
 

neoplastic nodules of the liver between the low-dose and pooled-


control groups indicates a P value of 0.034, which is above the
 

0.025 level required for significance when the Bonferroni
 

inequality criterion is used for multiple comparison. The
 

high-dose group does not show a significantly higher incidence
 

than that in either control group. The results of the
 

Cochran-Armitage test are not significant.
 

Significant results in the negative direction are seen in the
 

incidence of adrenal tumors in male rats.
 

26
 
In summary, the statistical results suggest that the incidence of
 

thyroid tumors in each sex of rats is associated with the
 

administration of toxaphene.
 

27
 
28

 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

The mean body weights attained by the high-dose male mice were
 

generally lower than those of the matched controls, while weights
 

of low-dose males and both low- and high-dose females were
 

essentially unaffected by the toxaphene (figure 3). Fluctuation
 

in the growth curve may be due to mortality; as the size of a
 

group diminishes, the mean body weight may be subject to
 

variation.
 

Several animals died before week 19 of the bioassay, and doses
 

were lowered at that time. Following this, the dosed mice were
 

generally comparable to the controls in appearance and behavior
 

during the first year of the study. A few animals, both control
 

and dosed, had alopecia.
 

During the second year of the bioassay, abdominal distention was
 

noted in all dosed groups, but predominantly in the high-dose
 

males. Other clinical signs included alopecia, diarrhea, rough
 

hair coats, and dyspnea. From weeks 60 to 76, the low-dose males
 

appeared hyperexcitable.
 

29
 
Figure 3. Growth Curves for Mice Fed Toxaphene in the Diet
 

30
 
B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice fed toxaphene in the diet at
 

the doses of this bioassay, together with those of the controls,
 

are shown in figure 4. In each sex, the result of the Tarone
 

test for dose-related trend in mortality is significant (P less
 

than 0.001 in males and P = 0.017 in females).
 

In male mice, 46/50 (92%) of the high-dose group, 49/50 (98%) of
 

the low-dose group, and all 10 animals of the control group lived
 

beyond week 52 on study. In females, 46/50 (92%) of the
 

high-dose group, 46/50 (92%) of the low-dose group, and 9/10
 

(90%) of the control group lived beyond week 52 on study.
 

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

development of tumors.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

31
 
Figure 4. Survival Curves for Mice Fed Toxaphene in the Diet
 

32
 
With the exception of the incidences of hepatocellular carcinomas
 

and neoplastic nodules of the liver, the incidences of lesions in
 

the control mice were comparable to those in the dosed animals.
 

Hepatocellular carcinomas occurred only in the dosed mice —
 

males: low-dose 34/49 (69%), high-dose 45/46 (98%); females:
 

low-dose 5/49 (10%), high-dose 34/49 (69%). In male mice without
 

hepatocellular carcinoma, neoplastic nodules of the liver
 

occurred in 2/10 (20%) matched-control males, 6/49 (12%) low-dose
 

males, 0/9 (0%) matched-control females, 13/49 (27%) low-dose
 

females, and 6/49 (12%) high-dose females.
 

The lesions diagnosed as hepatocellular carcinomas varied in
 

gross appearance from small, single growths to large,
 

tnultinodular growths, randomly positioned throughout the liver.
 

These growths were generally tan to dark brown in color and
 

variegated in pattern, with tiny scattered areas of hemorrhage
 

and necrosis. Histologically they were quite variable. They
 

appeared as circumscribed masses of obviously malignant cells
 

encroaching on the periphery. Liver cell plates were thickened
 

and branched haphazardly; occasionally nests were formed, as were
 

pseudorosettes. For the most part the cells were more basophilic
 

than normal, and the cytoplasmic and nuclear borders were
 

commonly enlarged and/or irregular in outline. Markedly enlarged
 

33
 
hyperchromatic nuclei were frequently seen. Areas of infraction
 

and hemorrhage were not uncommon. Mitoses were also seen with
 

some abnormal forms. Metastases were not found to be associated ,
 

with the hepatocellular carcinomas.
 

The diagnosis "neoplastic nodule" is used to describe one or more
 

than one clearly delineated nodular growth within the liver
 

parenchyma. Grossly, these nodules varied in size up to 0.5 cm in
 

diameter. Most commonly they were pale tan and homogeneous on
 

the cut surface. Histologically they were expansive growths
 

compressing the surrounding tissue. The sinusoidal pattern was
 

mostly haphazard, the cords being of variable thickness. A
 

tinctorial change was commonly evident between the proliferative
 

mass and the surrounding liver. The cells were regular and the
 

nuclei were essentially within normal limits. Vascular
 

structures within these lesions were not remarkable. Bile ducts
 

were not evident. The diagnosis of neoplastic nodules vs. small
 

carcinomas was based on the more anaplastic cellular features of
 

the latter. Occasionally, cellular atypia was relatively marked,
 

suggesting malignant transformation.
 

Most of the other neoplastic and nonneoplastic lesions observed
 

in the mice were not unusual findings, and the incidences of
 

lesions in animals of the control groups were similar to those of
 

34
 
the dosed groups. These lesions are, therefore, considered to
 

have occurred spontaneously.
 

Based on the histopathologic examination, toxaphene was
 

carcinogenic in B6c3:?l mice, causing increased incidences of
 

hepatocellular carcinomas and neoplastic nodules under the
 

conditions of this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group. Only tumors of the liver occurred
 

at this incidence; however, in this report, the incidence of
 

neoplastic nodules is not listed separately but is combined with
 

the incidence of hepatocellular carcinomas.
 

The proportions of hepatocellular carcinomas in the low- and
 

high-dose groups of male mice and in the high-dose group of
 

female mice were significant (P less than 0.001) when compared
 

with either the matched- or pooled-control groups by the Fisher
 

35
 
exact test. Also, the results of the Cochran-Armitage test for 

dose-related trend, using either of the control groups, had 

values of P less than 0.001 in both male and female groups, but 

with significant departures from linearity due to the steep rise 

in incidence in the dosed groups compared with that in the 

controls. Although the matched controls of the present studies 

show no hepatocellular carcinomas, the historical records taken 

from 20 similar studies at this laboratory showed 45/245 (18.4%) 

hepatocellular carcinomas in male B6C3F1 mice and 6/224 (2.7%) in 

females. 

When the incidence of hepatocellular carcinomas was combined with 

that of neoplastic nodules, the results of the Fisher exact test 

for comparisons of incidences for low- and high-dose males and 

high-dose females with those of the respective matched and pooled 

controls indicated that in each of these tests P was less than 

0.001. 

36 
V. DISCUSSION
 

Mean body weights attained by low- and high-dose female rats and
 

high-dose male mice were lower than those of matched controls,
 

but weights of other dosed groups were essentially unaffected by
 

the toxaphene. Other clinical signs of toxicity included
 

generalized body tremors in high-dose male and female rats at
 

week 53, after which the concentrations of toxaphene were
 

reduced. Later, leg paralysis, ataxia, epistaxis, hematuria, and
 

vaginal bleeding were observed in a few animals, predominantly in
 

the dosed groups. Abdominal distention, diarrhea, dyspnea, and
 

rough hair coats were observed predominantly in the dosed groups
 

of both rats and mice; the abdominal distention was noted
 

particularly among the high-dose male mice. Several high-dose
 

male mice died during later weeks of the study, and the survival
 

rates showed a significant dose-related trend in male mice.
 

High-dose female mice had a significant decrease in survival.
 

Sufficient numbers of both rats and mice were at risk for the
 

development of late-appearing tumors.
 

In the male rats, the incidence of follicular-cell carcinomas or
 

adenomas of the thyroid was dose related (P = 0.007) using the
 

pooled controls (matched controls 1/7, pooled controls 2/44,
 

37
 
low-dose 7/41, high-dose 9/35). In the females, the incidence of
 

follicular-cell adenomas of the thyroid was dose-related using
 

either the matched (P = 0.022) or pooled (P = 0.008) controls
 

(matched controls 0/6, pooled controls 1/46, low-dose 1/43,
 

high-dose 7/42). Direct comparisons of dosed and pooled-control
 

groups showed significantly increased incidences of the follicular-cell carcinomas or adenomas in the high-dose males
 

(P = 0.008) and of the follicular-cell adenomas in the high-dose
 

females (P = 0.021). Two follicular-cell tumors in the high-dose
 

males were carcinomas; all other follicular-cell tumors in the
 

rats were adenomas.
 

In the female rats, the incidence of tumors of the pituitary
 

(adenomas, chromophobe adenomas, and chromophobe carcinomas) was
 

dose related using either matched (P = 0.046) or pooled (P =
 

0.012) controls, and, in a direct comparison, the incidence of
 

pituitary tumors in the high-dose group was significantly higher
 

(P = 0.013) than that in the pooled-control group (matched
 

controls 3/8, pooled controls 17/51, low-dose 15/41, high-dose
 

23/39). One pituitary tumor, in a high-dose female, was a
 

carcinoma; all other pituitary tumors in the rats were adenomas.
 

The historical-control data obtained to date on 20 similar
 

studies at this laboratory show an incidence of pituitary tumors
 

of 58/185 (31.4 %), although there are incidences as high as 6/10
 

38
 
(60%), 5/10 (50%), 3/6 (50%), and 4/9 (44%).- Considering these
 

high spontaneous incidences observed in control groups, the
 

conclusion cannot be made that the tumors in this study are
 

associated with the administration of the test chemical.
 

In the mice, the incidence of hepatocellular carcinomas was dose
 

related (P less than 0.001) for both males (matched controls
 

0/10, pooled controls 4/48, low-dose 34/49, high-dose 45/46) and
 

females (matched controls 0/9, pooled controls 0/48, low-dose
 

5/49, high-dose 34/49), using either matched or pooled controls.
 

Direct comparisons showed that the incidences of hepatocellular
 

carcinomas in low- and high-dose male mice and high-dose female
 

mice were all significantly higher (P less than 0.001) than those
 

in the respective matched or pooled controls. Statistical
 

significance was maintained when the incidence of hepatocellular
 

carcinomas was combined with that of neoplastic nodules of the
 

liver.
 

Both the FDA in 1949 and Kettering Laboratories in 1952 (Lehman,
 

1965) conducted 2-year feeding studies of toxaphene in rats
 

(strain not specified). Concentrations of toxaphene used in the
 

two investigations were 25, 100, 400, and 1,600 ppm and 10, 100,
 

1,000, and 1,500 ppm, respectively. Histologic changes in the
 

liver were noted in rats given more than 25 ppm in the FDA study
 

39
 
and more than 100 ppm in the Kettering study, but no increase in
 

the incidence of tumors was noted. Doses used in the present
 

study fall within these ranges. Sherman rats fed 50 or 200 ppm
 

toxaphene in the diet for 9 months did not show clinical signs,
 

but on histopathologic examination, mild liver changes were found
 

in some of the dosed animals (Ortega et al., 1957). In other
 

studies, hybrid mice were administered the related chemical,
 

Strobane®, at 4.64 mg/kg by stomach tube for 3 weeks, then at a
 

concentration of 11 ppm in the diet for 75 weeks (innes et al.,
 

1969). A significantly elevated incidence of hepatomas was
 

reported in male C57BL/6 x C3H/Anf hybrid mice and of lymphomas
 

in male C57BL/6 x AKR hybrid mice.
 

It is concluded that under the conditions of this bioassay,
 

toxaphene was carcinogenic in male and female B6C3F1 mice,
 

causing increased incidences of hepatocellular carcinomas. The
 

test results also suggest carcinogenicity of toxaphene for the
 

thyroid of male and female Osborne-Mendel rats.
 

40
 
VI. BIBLIOGRAPHY
 

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

Wiley & Sons, Inc., New York, 1971.
 

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

Panel on Carcinogenicity of the Cancer Research Commission
 

of UICC, Vol. 2^, International Union Against Cancer, Geneva,
 

1969.
 

Brooks, G. T., Polychloroterpene insecticides (toxaphene). In:
 

Chlorinated Insecticides, Vo 1. 1^, Biological and Environmental
 

Aspects, Chemical Rubber Company, Cleveland, Ohio, 1975, pp.
 

205-210.
 

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

Soc. B. 34;187-220, 1972.
 

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

1970, pp. 48-58.
 

Deichmann, W. B., The chronic toxicity of organochlorine
 

pesticides in man. In: Pesticides and the Environment Vol. II,
 

W. B. Deichmann, ed., Intercontinental Medical Book
 

Corporation, New York, 1973.
 

Environmental Protection Agency, EPA Compendium of Registered
 

Pesticides, U. S. Government Printing Office, Washington,
 

D.C., III-T-17.1 to 17.13, 1974.
 

Gart, J. J., The comparison of proportions: a review of
 

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Stat. Inst. 39:148-169, 1971.
 

Innes, J. R., Ulland, B. M., Valeric, M. G., Petrucelli, L.,
 

Fishbein, L., Hart, E. R., and Pallotta, A. J., Bioassay of
 

pesticides and industrial chemicals for tumorigenicity in
 

mice: A preliminary note. J. Natl Cancer Inst.
 

42:1101-1114, 1969.
 

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

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

1958.
 

Lehman, A. J., Chlorinated organics. In: Summaries of Pesticide
 

Toxicity, Assoc. Food and Drug Officials, p. 37, 1965.
 

41
 
Linhart, M. S., Cooper, J. A., Martin, R. L., Page, N. P., and
 

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

and Biomed. Res. 7^:230-248, 1974.
 

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

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

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

changes in the liver of rats after feeding low levels of
 

various insecticides. Arch. Path. 64;614-622, 1957.
 

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

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

induced by different numbers of administrations of
 

benzo(a)pyrene and ferric oxide. Cancer Res. 32;1073-1081,
 

1972.
 

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

Biometrika 62;679-682, 1975.
 

42
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS FED TOXAPHENE IN THE DIET
 

43
 
44

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

FED TOXAPHENE IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS HISSING
 

ANIMALS NKCBOPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

*SKIN
 

SCUAMOUS CELL PAPILLOSA
 

*SOBCOT TISSUE
 

FIBBOMA
 

FIBROUS H1STIOCYTOMA
 

FIBROUS H1STIOCYTOMA, MALIGNANT
 

HESPlBATOflY SYSTEM
 

tLUNG
 

ALVEOLAR/BKONCHIOLAfi ADENOMA
 

HEMATOPOIETIC SYSTEM
 

*MULTIPL£ OBGANS
 

MALIGNANT LYMPHOMA, NOS
 

MALIG.LYMPHOMA, UISTIOCYTJC TYPE
 

tSPLEEN
 

HEMANGIOMA
 

»THYMUS
 

CARCINOMA, NOS
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

*LIVEH
 

NEOPLASTIV NQPULE
 

MATCHED
 

CONTROL
 

10
 

10
 

9
 

(10)
 

(10)
 

(9)
 

(10)
 

(9)
 

(9)
1 nui
 

LOW DOSE
 

50
 

50
 

U7
 

(50)
 

(50)
 

1 (2X)
 

1 (2%)
 

2 (H%)
 

(US)
 

(50)
 

(45)
 

3 (7X)
 

(«4)
 

6 P1**!
 

HIGH DOSE
 

50
 

1
 

45
 

45
 

(45)
 

1 (2X)
 

C»5)
 

1 (2X)
 

(43)
 

1 <2X)
 

(45)
 

1 (2X)
 

1 (2«)
 

(42)
 

3 (7X)
 

(1)


1 (100X)
 

(45)
 

4 (9X>
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

45
 
__ 

TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

NitITCHED 

CO NTROL LOW DOSE HIGH DOSE 

FIBROUS HISTIOCYTOMA, HBIASTATIC 1 (2X) 

tBILE DUCT 

PAPILLARY ADENOMA 

HAHARTONA 

(9) (44) 1 (45) 

1 

1 

(2X) 

(2%) 

URINAR Y SYSTEH 

*KIi)NEY (9) (45; 1 (45) 

TUBULAR-CiiLL ADENOM A 2 C*X) 1 (2X) 

MIXED TUSJB. HALIGNAHI 1 (2*) 1 (2X) 

f HAHARTOHA 1 (2%) 

ENDOCRINE SYSTEM 

tPITOITABY (7) (42) i (31)

CARCINOMA, NOS 1 (3X) 

ADENOMA, NOS 1 (14%) 1 (2%) 

CHHOMOPHOflE ADENOM A 2 (29S) 12 (29%) 4 (13X) 

•ADRENAL (9) (41] 1 (37) 

ADENOMA, iilOS 2 (22X) 

CORTICAL ADENOMA 2 (22%) 4 (10X) 3 (8X) 

CORTICAL CARCINOMA 1 (2X) 

PHEOCHROHOCYTOMA 1 (1U) 1 (3X) 

ITHYROID (7) (41] I (35) 

FOLLICOLArf-CELL ADENOHA 1 (14%) 7 (17X) 7 (20X) 

FOLLICULAfi-CELL CARCINOMA 2 (6X) 

C-CELL ADuNOMA 1 (2%) 

C-CELL CARCINOMA 1 (2X) 

tPARATHYHOID (5) (26] I (21) 

ADENOMA, NOS 1 (4X) 

•PANCREATIC ISLETS (9) {42] I (30) 

ISLET-CELL ADENOMA 1 (2*) 1 (3X) 

REPRODUCTIVE SYSTEM 

*MAMHARY GLAND (10) (50] I (45) 

CARCINOMA. NOS 1_J2*1 

* NUBBEB OF ASIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

t This is considered to be a benign form of the malignant mixed tumor
 

of the kidney and consists of lipocytes, tubular structures, and
 

fibroblasts in varying proportions.
 

46
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

tPROSTATE
 

SARCOMA, H OS
 

NERVOUS SYSTEM
 

»BRAIN
 

ASTHOCYTOaA
 

HENINGIOMA
 

»CKANIAL NERVE
 

NEUBILEHOflA
 

SPECIAL SENSE OHGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

*SKULL
 

OSTEOBLASTOHA
 

*SK£LETAL MUSCLE
 

SARCOMA, HOS
 

BODY CAVITIES
 

*BODY CAVITIES
 

HESOrHELIOHA, NOS
 

ALL OTHER SYSTEMS
 

•MULTIPLE	 ORGANS
 

UNpIfFBBEiJTIATED CASCINOflA
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

(9) (37) (35) 

1 <3X) 

(9) (44) 

1 (2X) 

(43) 

1 (2%) 

(10) (50) («5) 

1 (2*) 

(10) (50) (»5) 

1 (2%) 

(10) (50) (45) 

1 (2») 

(10) (50) (45) 

1 (2X) 

(10) (50) 

1 (2%} 

(45) 

* NUMBER OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF AillHALS N£CBOPSI£D
 

47
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUD!
 

NATURAL DtATHa
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIHAL HISSING
 

d INCLUDES AUIOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TOHORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUHOHS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALiGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS HITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR SETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED
 

CONTROL
 

10
 

2
 

2
 

6
 

7
 

10
 

7
 

9
 

1
 

1
 

LOW DOSE
 

50
 

9
 

13
 

28
 

33
 

49
 

27
 

35
 

7
 

8
 

1
 

1
 

6
 

6
 

HIGH DOSE
 

50
 

14
 

15
 

1
 

19
 

1
 

24
 

42
 

20
 

26
 

10
 

11
 

5
 

5
 

* PRIMARY TOHORS: ALL TUBOH5 EXCEPT SECONDAHY TOHOHS
 

» SECONDARY TUHOBS: METASTATIC TOBOES 08 TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

48
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

FEDTOXAPHENEINTHEDIET 

MATCHED 

CONTROL LOW DOSE 

ANIMALS INITIALLY IN STUDY 10 50 

ANIHALS N3CBOPSIED 10 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 10 49 

INTEGUHBHTARY SYSTEM 

*SOBCUT TISSUE (10) (50) 

FIBROUS H1STIOCYTOHA, HALIGKANT 1 (2X) 

LIPOMA 

BESPIBATOBY SYSTEH 

#LONG (9) (46) 

TEBATOHA, flETASTATIC 

HEH4TOPOIETIC SYSTEfl 

ISPLEEM (7) (47) 

HEMANGIOMA 1 (2X) 

CIBCOLiTORY SYSTEM 

HONE 

DIGESTIVE SYSIEH 

tLIVEB (10) (42) 

NEOPLASTIC NODULE 1 (10*) 4 (10X) 

HEPATOCfiLLULAfi CABCIHOHA 1 <2X) 

tBILE DUCT (10) (42) 

HAMABTOMA 

•PANCREAS (9) (46) 

LIPOHA ^1

 JiSl 

* NUHBEB OF ANIMALS HITH TISSUE EXAHINED MICROSCOPICALLY
 

* NUHBhR OF AalHALS NECBOPSIED
 

HIGH DOSE 

50 

49 

49 

(49) 

3 (6X) 

1 (2*) 

(48) 

1 (2X) 

(48) 

1 (2X) 

(40) 

4 (10X) 

(40)

1 (3X) 

(45) 

49
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

URINARY SYSTEM 

•KIDNEY (8) (49) TUBULAR-CALL ADENOMA 

ENDOCRINE SYSTEM 

•PITUITARY (8) (41) 

ADENOMA , NOS 1 (13X) 

CHHOMOPHOhE ADENOMA 2 (25X) 15 (37X) 

CHROHOPHOriE CARCINOMA 

•ADRENAL (8) (U4) 

CORTICAL ADENOMA 3 (7X) 

CORTICAL l-ARCINOMA 

•THYROID (6) («3) 

FOLLICULAK-CELL ADENOMA 1 (2X) 

C-CELL CARCINOMA 1 (17X) 

REPRODUCTIVE bYSTEM 

*MAHMAR Y GLAriD (10) (50) 

ADENOMA , SOS 2 (4X) 

ADENOCARCINOHA, NOS 1 (2X) 

PAPILLARY ADENOCARCINOHA 

FIBROMA 1 (2X) 

FIBROADENONA 1 (10*) 10 (20X) 

TERATOMA, MALIGNAN T 

•UTERUS (9) (41) 

CARCINOMA . NOS 1 (2X) 

PAPILLARY ADENOM A 

ENDOMBTHIAL STROHAL POLYP 9 (22X) 

•OVARY (8) (40) 

CARCINOMA, NOS 

GRANULOSA-CELL TUMOR 1 (3X) 

NERVOOS SYSTEd 

NONE 

* NOBBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

(48) 

1 (2X) 

(39) 

4 (10X) 

18 (46X) 

1 (3X) 

(43) 

4 (9X) 

2 (5X) 

(42) 

7 (17X) 

(<»9) 

1 (2X) 

1 (2X) 

1 (2X) 

2 (4X) 

10 (20X) 

1 (2X) 

(45) 

1 (2X) 

5 (11X) 

(36) 

1 (3X) 

50
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE ORGAN S 

NONE 

flUSCULOSKilLETAL

NON£ 

 SYSTEM 

BODY CAVITIES 

NONil ' 

ALL OTHER

NONE 

 SYSl'EMS 

ANIHAL DISPOSITION SUHHABI 

ANIMALS INITIALLY IN STUDY 

NATUBAL DEATHS 

MORIBUND SACBIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE 

ANIHAL HISSING 

10 

2 

4 

4 

50 

4 

18 

28 

50 

4 

11 

35 

3 INCLUDES AUXOLYZED ANIMALS 

I NUHBBB OF AUIHALS WITH TISSUE EZAHIMED

* NUHBEB OP ANIHALS NEC80PSIED 

 MICROSCOPICALLY 

51
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

•
 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

TUMOR SUMMARY
 

TOTAL ANIMALS BITH PRIMARY TO NOBS* 6 31 40
 

TOTAL PRIdARY TUMORS 6 52 70
 

TOTAL ANIMALS WITH BENIGN TUMOBS 4 28 36
 

TOTAL BENIGN TUBORS 4 43 56
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 1 4 10
 

TOTAL MALIGNANT TUMORS 1 4 10
 

TOTAL ANIMALS HITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 1 5 4
 

TOTAL UNCERTAIN TUMOBS 1 5 4
 

TOTAL ANIMA1.S HITH TUMORS UNCERTAINPRIMARY OR UETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUHOBS: ALL TUMORS EXCEPT SECONDARY TOHOfiS
 

« SECONDARY TJHOBS: METASTATIC TUHOBS OB TUHOBS INVASIVE INTO AN ADJACENT ORGAN
 

52
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE FED TOXAPHENE IN THE DIET
 

53
 
54

 
TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED TOXAPHENE IN THE DIET 

MATCHED 

CONTROL LOW OOSE HIGH DOSE 

ANIMALS INITIALL Y IN STODY 10 50 50 

ANIMAL S NECBOPSIED 10 50 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 10 50 46 

INTEGUMENTAR Y SYSTEM 

NON E 

BESPIBATOBY SiSTEM 

*LUNG (10) (49) (46) 

ALVEOLAB/tiRONCHIOLAB ADENOM A 1 (10X) I (2X) 2 <<•*) 

HfcMATOPOlETIC SYSTEM 

'MULTIPLE OBuAN S (50) (50) (50)

 

GHANULOCYI'IC LEUKEMIA 2 (4X)

 

CIHCOLATOB? SYSTEM 

NONE 

DIGESTIVE SYSTEM 

*LIVEB (10) (49) (46) 

NoOPLASTIC NODULE 2 (20%) 6 (12X) 

HEPATOCELLULAB CARCINOMA 3U (69%) 45 (98X) 

ANGIOSARCOMA 1 (2X) 

UBINABY SYSTEM 

NON E 

ENCOCBIHE SYSTEM 

NONE 

* NUMBER OF ANIMALS SITH TISSUE EXAMINED HICBOSCOPICALLI
 

* NUMBEB OF ANIMALS NECBOPSIED
 

55
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

BEPHODOCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*£YE/LACBI«Ai, GLAND (10) (50) (50)
 

PAPILLARY CYSTADENOMA, NOS 1 (2*)
 

MUSCOLOSKELETAL SYSTEM
 

NOME
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 10 50 50
 

NATURAL DEATHS 3 8
 

MORIBUND SACRIFICE 1 6 21
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 9 41 21
 

ANIMAL HISSING
 

d INCLUDES AUIOLYZED ANIMALS
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

56
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

— -----' ­ ­

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUHO8 SUMMAR Y 

TOTAL ANIBAi-S HITH PRIMARY TUHORS* 2 14 45 

TOTA1 PBIflARY TUMORS 3 45 47 

TOTAL ANIMALS WITH BENIGN TOflOBS 1 2 2 

TOTAL BENIGN TUHORS 1 2 2 

TOTAL ANIMALS WITH HALIGNABT TOMOHS 37 45 

TOTAL BAL1GNANT TUMORS 37 45 

TOTAL ANIMALS HITH SECONDAR Y TUBORS* 

TOTAL SECONDARY TOBORS 

TOTAL ANIMALS WITH TUBORS UNCEBTAINBENIGN OR MALIGNAN T 2 6 

TOTAL UNCERTAI N TUMORS 2 6 

TOTAL ANIBAi,S HITH TOBOfiS UNCERTAINPRIBARY OR MET ASTATIC 

TOTAL UNCiiRTAIN TOBOBS 

* PBiaARI TUHORS: ALL TOHOBS EXC£PT SECONDARY TUHORS
 

I SECONDARY TUMORS: HETASTAIIC TOHORS OR TUBORS INVASIVE INTO AN ADJACENT ORGAN
 

57
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

FED TOXAPHENE IN THE DIET 

ANIHALS INITIALLY IN STODT
 

ANIMALS M EC R OP SI ED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

ILUNG
 

ALVEOLAR/UfiONCHIOLAR ADBNOHA
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE OJWANS
 

MALIGNANT LYMPHOMA, NOS
 

CIBCOLATORY SltSTEH
 

NONE
 

DIGESTIVE SISIE8
 

ISALIVARY GLAND
 

ADENOMA, NOS
 

*L1VEB
 

NEOPLASTIC NODULE
 

HEPATOCELLULAR CARCINOMA
 

URINARY SYSTEd
 

NONE
 

ENDOCRINE SYSIEH
 

NONE
 

MATCHED
 

CONTROL
 

10
 

10
 

10
 

(10)
 

1 (10X)
 

(10)
 

(8)
 

(9)
 

LOW DOSE
 

50
 

50
 

49
 

(49)
 

(50)
 

1 (2%)
 

(46)
 

1 UX)
 

(49)
 

13 (21%)
 

5 (10X)
 

HIGH DOSE
 

50
 

50
 

49
 

(49)
 

(50)
 

(47)
 

(49)
 

6 (12%)
 

34 (69*)
 

* NUMBER 0? ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIHALS NECBOPSIED
 

58
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

REPRODUCTIVE SYSTEM
 

*BAMBARY GLAMD
 

ADENOMA, NOS
 

*UTEHUS
 

LEIOMYOBA
 

ENDOMBTRIAL STROBAL POLYP
 

NERVOUS SYSTES
 

HOME
 

SPECIAL SENSt ORGANS
 

*HAHDEBIAN Gi-AND
 

PAPILLARY CYSTADEHOHA, DOS
 

MUSCULOSKELETAL SYSTEH
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEBS
 

NONE
 

AN1HAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IH STUDY
 

NATURAL DiATHd
 

BORIBUSD SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIBAL MISSING
 

d INCLUDES AUTOLYZED ANIMALS
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED


* NUKBER OF ANIMALS NECROPSIED
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

(10) (50) (50) 

1 (2X) 

(6) (48) 

1 (2X) 

(44) 

1 (2*) 

(10) (50) (50) 

1 121) 

10
 50 50
 

1 1 6
 

2 7
 

9 «7 37
 

 MICROSCOPICALLY
 

59
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

TUMOR SUMMARY
 

TOTAL ANIMAi.3 WITH PRIMARY TUHOBS*
 

TOTAL PRIdABY TUHOBS
 

TOTAL ANIMALS WITH BENIGN TOHOBS
 

TOTAL BENIGN TUHOBS
 

TOTAL ANIMALS HITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS KITH SECONDARY TUMOBS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMOBS UNCEBTAINBENIGH OB MALIGNANT
 

TOTAL UNCERTAIN TUMOBS
 

TOTAL ANIMALS WITH TUHOBS UNCERTAINPBINABY OR dETASTATIC
 

TOTAL UNCERTAIN TUMOBS
 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

1 22 10
 

1 23 41
 

1 4 1
 

1 4 1
 

6 34
 

6 34
 

13 6
 

13 6
 

* PRIMAHT TOHUBS: ALL TUflOBS EXCEPT SECONDARY TOMOBS
 

I SECONDARY TJMOKS: HE1ASTATIC TUMORS OB TDHOBS INVASIVE INTO AH ADJACEHT OBGAN
 

60
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS FED TOXAPHENE
 

IN THE DIET
 

61
 
62

 
TABLE C1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

FED TOXAPHENE IN THE DIET 

1 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS MISSIdG 

ANI3ALS NECBOtfSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

10 

10 

9 

50 

50 

47 

50 

1 

45 

45 

INTEGUMENTABY SYSTEM 

•SKIN 

tPIDEBflAL INCLUSION CYST 

(10) (50) 

1 (2X) 

(45) 

1 (2X) 

RESPIBATORY SYSTEM 

*LUNG 

INFLAMMATION, CHRONIC 

HYPERPLASiA, ALVEOLAE EPITHELIUB 

<9) (US) (43) 

1 <2X) 

1 (2%) 

HEMATOPOIETIC SYSTEM
 

•SPLEEN
 

HEMORRHAGE
 

FIBROSIS, FOCAL
 

PtRIARTERITIS
 

HYPERPLASiA. RETICULOM CELL
 

fLYMPH NODE
 

INFLAMMATION, FOCAL
 

CIRCOLATOBY SYSTEM
 

tHEART
 

FIBBOSIS, DIFFUSE
 

*MYOCABDIUM
 

INFLAHHATXON, CHRONIC
 

FIBBOSIS, FOCAL
 

IENDOCARDIUH
 

flBROSI?
 

» NUMBE R OF AtilH&LS 8ITU TISSUE

* NUMBER OF AhlHALS NECHOPSIED 

(9) (45) 

1 (2*) 

1 (2X) 

1 <2X) 

(42) 

1 (2X) 

(8) (43) (35) 

1 (3X) 

(9) (47) (42) 

1 (2X) 

(9)

1 (11X) 

1 (1») 

(47) (42) 

(9) (47) 

1 (2*1 

(42) 

 EXAMINED HICBOSCOPICALLI 

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

11

 -- mi J.- .. IT- - - — _i«_i_i_i__n.|-T-r-"r- .._._... . L ^—J

*-"J*JJ""~— -1-""— TM" —TM "• "•"""-— 

*AORTA 

ARTERIOSCLEROSIS, NOS 

HEDIAL CALCIFICATION 

DIGESTIVE SYSTEM 

tLIVER 

DEGENERATION , BALLOONING 

NECROSIS, FOCAL 

METAMORPHOSI S FATTY 

FOCAL CELLULAR CHANGE 

ANGIECTASJ.S 

#LIVER/CENTR1LOBULAR 

BETAMOBPHOSIS FATTY 

*BILE DUCT 

HYPEBPLASiA , NOS 

HYPEBPLASIA , FOCAL 

• PANCREATIC ACINUS 

ATROPHY , SOS 

*STO»ACH 

ULCEB, NOS 

ULCER, CHHONIC 

CALCIFICATION, METASTATIC 

ACANTHOSI 3 

URINAR Y SYSTErt 

#KIDNE Y 

CYST, NOS 

MULTIPL E cYSTS 

INFLAMMATION , CHRONIC 

DEGENERATION , CYSTIC 

ENDOCRIN E SYSTEM 

*PITUITAR Y 

DEGENERATION , CYSTIC

 

* NUMBE R OF AttlMAL S WIT H TISSUE

* NUMBE R OF AinIMAL S NECROPSIED 

MATCHED 

CONTROL LOW DOSE 

(10) (50) 

1 (10%) 

1 (2%) 

(9) (44) 

1 (2X) 

2 (5X) 

1 (11%) 

1 (2*) 

(9) (44) 

1 (11%) 

(9) (44) 

1 (IIS ) 3 (7% ) 

1 (11*) 

(9) (42) 

(9) (43) 

1 (2X) 

1 (11*)

1 (11%) 

(9) (45) 

1 (11*) 1 (2%) 

4 (44*) 9 (20%) 

1 (2%) 

(7) (42) 

2 J5S1 — — —. 

 EXAMINE D MICROSCOPICALLY 

HIGH DOSE 

(45) 

(45)

1 (2*) 

1 (2%) 

4 (9*) 

(45) 

(45) 

1 (2X) 

(30 ) 

1 (31) 

(40) 

1 (3%) 

(45) 

20 (44%) 

(31) 

2 i^Y 

64 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

HYPERPLASlA, NOS	 1 (14X)
 

ANGIECTASiS	 1 (2X)
 

•ADRENAL	 19) (41) (37)
 

METAHORPHOSIS FATTY 1 (11X)
 

ANGIECTASIS 2 (5X)
 

*ADRENAL CORTEX (9) (41) (37)
 

HYPERPLAS1A, NOS 1 (11%)
 

•THYHOID	 (7) (U1) (35)
 

FOLLICULAa CYST, NOS 2 (5X) 1 (3X)
 

HYPEBPLASiA, C-CELL 2 (5X) 1 (3X)
 

HYPEBPLASiA, FGLLICULAR-CELL 3 (7S) 3 (9X)
 

tPABATHYBOID (5) (26) (21)
 

HYPEHPLASIA, HOS 1 (20X)
 

BEPBODOCTIVE SYSTEB
 

tPKOSTATE (9) (37) (35)
 

INFLAflHATiON, NOS 2 (6X)
 

INFLAHHAT10M, ACUTE 1 (3X) 1 (3%)
 

INFLAMBAT10N, CHBONIC 1 (3X)
 

tTESTIS (9) (i>6) (43)
 

ATEOPHY, NOS 2 (22%) 11 (24%) 10 (23%)
 

ATROPHY, fOCAL 1 (2%)
 

NERVOUS SYSTEM
 

NONi
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

*FEMUH (10) (50) (45)
 

FIBBOOS OaTEODYSTBOPHY . ^ 1 J1QX1 T , , ., ,. ,.
 

* NUHBEB OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY
 * NOHBEB OF ANIMALS NBC80PSIBD
 

65
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

* ABDOMINAL CAVITY (10) (50) (45)

 

NECROSIS, FAT 1 (10%) 

*fl£SENTERI (10) (50) (U5) 

THROHBOSIS, NOS 1 (10X) 

PERIARTERITIS 1 (2X) 2 (4X) 

ALL OTHER SI SI EMS 

ADIPOSE TISSUE 

INFLAMMATION, CHRONIC 1 

SPECIAL MORPHOLOGY SOHHAR Y 

NO LESION REPORTED 6 5 

ACCIDENTAL DEATH 1 

NECROPSY PERF/BO BISTO PEBFOHHED 1 1 

AOTO/NECROPSY/BISTO PERF 1 

AUTO/NBCROPSY/NO HISTO 1 2 

AUTOLYSIS/NO NECROPSY 3 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS BECROPSIED
 

66
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

FED TOXAPHENE IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROfSIED
 

ANIMALS EXAMINED HISTO PATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

tLONG
 

INFLAMMATION, CHRONIC
 

HYPtffiPLASIA, ALVEOLAE EPITHELIUM
 

HfiMATOPOIBTIC SYSTEM
 

ISPLEEN
 

HBMORRHAGJi
 

CIRCULiTOBY SiSTEM
 

*£NDOCABDIOM
 

FIBROSIS
 

DIGESTIVE SYSTEM
 

tLIVER
 

METAMORPHOSIS FATTY
 

BASOPHILIl. CYTO CHANGE
 

CLEAR-CELL CHANGE
 

tBILE DUCT
 

HIPERPLASiA, NOS
 

HYPERPLASIA, CISTIC
 

tSTOMACH
 

DLCER. NOi
 

MATCHED
 

CONTROL
 

10
 

10
 

10
 

(9)
 

(7)
 

(9)
 

(10)
 

1 (10X)
 

(10)
 

2 (20%)
 

(9)
 

LOW DOSE	 HIGH DOSE
 

50 50
 

50 49
 

49 49
 

(»6) (48)
 

1 (2%)
 

1 (2X)
 

(47)	 (48)
 

1 12%)
 

(49)	 (47)
 

1 (2t)
 

(42)	 (40)
 

2 (5*) 2 (5X)
 

1 (3X)
 

1 (2*)
 

(42)	 (40)
 

1	 (2X) 1 (3X)
 

3 (8X)
 

(47)	 (49)
 

1 (2%)
 

«	 NUMBER OF ANIMALS WITH TISSOE EXA HIRED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NEC80PSIED
 

67
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

TCHED 

HIGH DOSE 

(48) 

2 (4X) 

(48) 

(39) 

(43) 

1 (2X) 

(<»3) 

1 (2«) 

(42) 

2 (5*) 

2 (5X) 

3 (7X) 

(49) 

(45) 

ULCER, CHBONIC 

UBINAH Y SYSTEfl 

*KIDNE Y 

CAST, NOS 

IMFLAHHAT10N, CHRONIC 

fKIDNEY/TUBUi.E 

CAST, SOS 

ENDOCRIN E SYSIEN 

iPITUITAHY 

CYTOPLASMIC VACUOLIZATION 

HYPERPLASiA, FOCAL 

1 ADRENA L 

ACCESSOR! STRUCTURE 

ANGIECTASiS 

IADRENA L CORTEX 

HYPERPLASiA, FOCAL 

•THYROID 

FOLLICULAo CYST, NOS 

HYPfiRPLASIA, C-CELL 

HYPERPLASIA, FOLLICOLAB-CELL 

REPRODUCTIVE SYSTEH 

*VAGINA 

INFLAMMATION , SUPPUBATIVE 

POLYP, INFLAMMATORY 

tUTEBUS/ENDOaETRIUH 

CYST, ^OS 

INFLAMMAXxON , NOS 

NERVOU S SYSTEd 

NON E 

COI NTROL LOW DOSE 

1 (11*) 

(8) (U9) 

1 (13X) 

1 (13%) 8 (16*) 

(8) (49) 

1 (13X) 

(8) (41) 

1 (13X) 

1 (13X) 

(8) (44) 

1 (13X) 

2 (25*) 3 (7*) 

(8) (44) 

(6) (43) 

4 (9X) 

5 (12*) 

(10) (50) 

1 (10X) 

1 (10*) 

(9) (41) 

1 (11*) 2 (5X) 

1 (2X) 

*	 NUMBER OF ANIMALS UITH TISSUE EXAHIHKO MICROSCOPICALLY
 

* NUMBER OF ANIMALS NKCROPSIED
 

68
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

SPECIAL SENS£ ORGANS
 

HOME
 

MUSCOLOSKELETAL SYSTEM
 

•PHALANGES
 

GRANULOaA, NOS
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL HOBPHOLOGY SOHBARY
 

NO LESION REPORTED
 

AOTO/HECROPSY/HISTO PEHF
 

AUTO/HECHOPSY/NO HISTO
 

AOTOLYSIS/NO NECROPSY
 

* NUflBEB OP ANIHALS HITH TISSUE

*	 NDHBEB OP ASIHALS N£CBOPSI£D
 

MATCHED
 

CONTROL LOW DOSE HIGH OOSE
 

(10)	 (50) (49)
 

1 (10X)
 

11	 7
 

1
 

1
 

1
 

 EXAMINED HICBOSCOPICALLY 

69
 
70

 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE FED TOXAPHENE
 

IN THE DIET
 

71
 
72

 
TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

FEDTOXAPHENEINTHE DIET 

ANIMAL S INITIALLY IN STUDY 

A HI HALS HBCROt>SIED 

ANIMALS EXAMINE D HXSTO PATHOLOGICALLY 

INTEGUMENTAR Y SYSTEM 

*SKIN 

INFLAMMATION ACUTE AND CHRONIC 

flESPIRATORY SYSTEM 

NONE 

HEMATOPOIETIC SYSTEM 

tLYMP H NODE 

INFLAMMATION, CHRONIC 

tMESENTEBIC L. NODE 

HYPEBPLAS1A, NOS 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

tLIVEH 

FOCAL CELLULAR CHANGE 

UfllNAH Y SYSTEM 

NONE 

iNDOCRINE SYSIEH 

NONE 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

10 50 50 

10 50 50 

10 50 46 

(10) (50) (50) 

1 (2%) 

(7) (36) 

1 (3%) 

(39) 

(7) (36) (39) 

1 (3*) 

(10)

1 (10X) 

(U9) (U6 ) 

* NUMBER OF ANIMALS BITH TISSUE EXAMINED MICBOSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

73
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

REPRODUCTIVE SYSTEM 

tTESTIS (10) (49) (45)

 

ATROPHY, NOS 1 (2X)

 

NEBVOUS SYSTEd 

NONE 

SPECIAL SENSE ORGAN S 

NON E 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUNMAB Y 

NO LESION REPORTED 7 6 1

 

AUTO/BECROPSY/HO HISTO 4

 

t NUHBEB OF A8IHALS WITH TISSUE EXAHIBED MICROSCOPICALLY
 

* NUMBER OP AKIMILS NBCBOPSIED
 

74
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

FED TOXAPHENE IN THE DIET 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECBOPSIED 

ANIMALS EXAMINE D HISTO PATHOLOGICALLY 

INTEGUMENTAR Y SYSTEH 

NONf c 

BESPIRATOBY S1STEM 

*LUNG 

HVPEEPLAS1A, ALVEOLAK

HEHATOPOIETIC SYSTEH 

tSPLEEN 

HYPEHPLAS1A, LYHPHOID 

#HESENTERIC L. NODE 

INFLAMMATION , NOS 

CIRCULATOBY SKSTEfl 

NONE 

DIGESTIVE SYSIEB 

*LIV£B 

INFLAMMATION, CHfiONIC 

BETAMOBPHOSIS FATTY 

tSHALL INTESTINE 

HYPEBPLASIA, LYBPHOID 

UBINAR Y SYSTEH 

IKIDNEY/TUBULE 

CAST. SOS 

 £PlTHELIUfl 

MATCHED 

CONTROL LOW OOSE 

10 50 

10 50 

10 49 

(10) (<»9) 

1 (2X) 

(10) (48) 

2 (<*X) 

(7) (44) 

(9) (49) 

1 (2X) 

(9) (49) 

(10) (47) 

* NUHBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF AAIMALS NECBOPSIED
 

75
 

HIGH OOSE 

50 

50 

49 

(49) 

(44) 

1 <2X) 

(40) 

1 (3«) 

(49) 

1 <2X) 

(45) 

1 (2%) 

(48) 

112%} 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

ENDOCRINE SYSTEM 

4THYROID 

HYPJBHPLASiA, FOLLICOLAR-CELL 

BEPRODUCTIVE SYSTEM 

*MABHARY CLAUD 

HYPERPLAS1A, NOS 

•UTERUS 

INFLABBATJ.ON, C HBO NIC 

DEGENERATION, CYSTIC 

*JTt8US/ENDOrtETKIUM 

DILATATION, NOS 

INFLAMHATiON, FOCAL 

DEGENERATION, CYSTIC 

UyPEBPLASIA. NOS 

HYPERPLASJ.A, CYSTIC 

IOVABY/OVIDUCT 

INFLABHATION, DOS 

INFLAMMATION, CHRONIC 

tQVABY 

DISTENTION 

CYST, HOS 

FOLLICULAfc CYST, NOS 

INFLAHHATiON, SUPPORATIV E 

1NFLAHHATION ACUTE AND CHRONIC 

INFLAMMATION, CHRONIC 

NERVOOS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

HUSCOLOSKELETaL SYSTEM 

NONE 

MATCHED 

CONTROL 

(7) 

(10) 

(6) 

(6) 

1 (17X) 

(6) 

1 (17*) 

(7) 

1 (14X) 

1 (1<4X) 

LOW DOSE 

(«5) 

(50) 

(48) 

1 (2X) 1 (2X) 

(48) 

1 (2X) 

4 (8X) 

1 (2X) 

(48) 

1 (2X) 

(<»8) 

1 (2X) 

1 (2X) 

2 (4X) 

5 (10X) 

HIGH DOSE 

(38) 

1 (3X) 

(50)

1 (2X) 

(44) 

(44) 

1 (2X) 

1 (2X) 

1 (2X) 

(«4) 

(45) 

1 (2X) 

* DUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

76
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

*«ESENT£BY (10) (50) (50) 

HECBOSIS, FAT 1 (2X) 

ALL OTHER SYSiEBS 

NONE 

SPECIAL MORPHOLOGY SUHMAHY 

MO LESION REPORTED 6 17 6 

AUTO/NECBOPSY/HISTO PERF 2 

AUTO/NECHJPSY/NO HISTO 1 1 

I NUMBER OF AMIMALS ilTH TISSUB EXAMINED MICROSCOPICALLY 

* NUNflEB OF ANIMALS NECBOPSIED 

77
 
78

 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED TOXAPHENE IN THE DIET
 

79
 
80

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

Fed Toxaphene in the Diet (a)
 

Topography: Morphology 

Matched 

Control 

Pooled 

Control 

Liver: Neoplastic Nodule (b) 1/9 (11) 1/52 (2) 

P Values (c,d) N.S. N.S. 

Relative Risk (Matched

Lower Limit 

Upper Limit 

Control) (f) 

Relative Risk (Pooled Co

Lower Limit 

Upper Limit 

ntrol) (f) 

Weeks to First Observed Tumor 109 — 

Pituitary: Chromophobe Adenoma, 

Carcinoma, NOS, or Adenoma, NOS (b) 3/7 (43) 8/46 (17) 

P Values (c,d) N.S. N.S. 

Relative Risk (Matched Control) (f) 

Lower Limit 

Upper Limit 

Relative Risk (Pooled Control) (f) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 102 — 

Low 

Dose 

6/44 (14) 

P = 0.034** 

1.227 

0.190 

55.074 

7.091 

0.908 

317.788 

108 

13/42 (31) 

N.S. 

0.722 

0.315 

3.371 

1.780 

0.763 

4.429 

85 

High 

Dose 

4/45 (9) 

N.S. 

0.800 

0.099 

38.517 

4.622 

0.479 

222.281 

94 

5/31 (16) 

N.S. 

0.376 

0.118 

2.069 

0.927 

0.260 

2.878 

95 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Toxaphene in the Diet (a)
 

(continued) 

Topography: Morphology
 

Adrenal: Adenoma, NOS, Cortical
 

Adenoma, or Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Spleen: Hemangioma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled 

Control 

Low 

Dose 

High 

Dose 

5/52 (10) 5/41 (12) 3/37 (8) 

N.S. P = 0,043 (N)* P = 0.020 (N)* 

0.274 

0.088 

1.189 

0.182 

0.039 

0.931 

1.268 

0.311 

5.126 

0.843 

0.138 

4.032 

85 85 

0/49 (0) 3/45 (7) 3/42 (7) 

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

Infinite 

0.136 

Infinite 

Infinite 

0.145 

Infinite 

Infinite 

0.656 

Infinite 

Infinite 

0.703 

Infinite 

— 83 85 —
 

Matched
 

Control
 

4/9 (44)
 

P - 0.019 (N)
 

_
 

0/9 (0)
 

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

Fed Toxaphene in the Diet (a)
 

(continued)
 

Topography: Morphology 

Matched 

Control 

Pooled 

Control 

Low 

Dose 

High 

Dose 

Thyroid: Follicular-cell 

Carcinoma or Adenoma (b) 1/7 (14) 2/44 (5) 7/41 (17) 9/35 (26) 

P Values (c,d) N.S. P ­ 0.007 N.S. P = 0.008** 

Relative Risk (Matched Control) (f) 

Lower Limit 

Upper Limit 

1.195 

0.211 

53.423 

1.800 

0.346 

76.080 

Relative Risk (Pooled Control) (f) 

Lower Limit 

Upper Limit 

3.756 

0.769 

35.292 

5.657 

1.272 

50.584 

Weeks to First Observed Tumor 109 — 104 56 

(a) Dosed groups received time-weighted average doses of 556 or 1,112 ppm. 

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

(c) Beneath the incidence of tumors in a control group is the probability level for the CochranArmitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath 

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

the comparisons of that dosed group with the matched-control group (*) or with the pooledcontrol group (**) when P less than 0.05 for either control group; otherwise, not significant 

(N.S.) is indicated. 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

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

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the specified
 

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

Matched
 

Topography: Morphology Control
 

Integumentary System: Malignant
 

Fibrous Histiocytoma of the
 

Subcutaneous Tissue (b) 0/10 (0)
 

P Values (c,d) N.S.
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

Mammary Gland: Fibroadenoma (b) 1/10 (10)
 

P Values (c,d) N.S.
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 87
 —
 

Fed Toxaphene in the Diet (a)
 

Low
 

Dose
 

1/50 (2)
 

N.S.
 

Infinite
 

0.012
 

Infinite
 

Infinite
 

0.059
 

Infinite
 

105
 

10/50 (20)
 

N.S.
 

2.000
 

0.357
 

84.786
 

1.833
 

0.652
 

5.693
 

19
 

Pooled
 

Control
 

0/55 (0)
 

N.S.
 

6/55 (11)
 

N.S.
 

High
 

Dose
 

3/49 (6)
 

N.S.
 

Infinite
 

0.136
 

Infinite
 

Infinite
 

0.674
 

Infinite
 

83
 

10/49 (20)
 

N.S.
 

2.041
 

0.363
 

86.461
 

1.871
 

0.667
 

5.801
 

67
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Liver: Hepatocellular Carcinoma
 

or Neoplastic Nodule (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromphobe Adenoma,
 

Carcinoma, or Adenoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/10 (10)
 

N.S.
 

109
 

3/8 (38)
 

P = 0.046
 

85
 

Pooled
 

Control
 

1/55 (2)
 

N.S.
 

—
 

17/51 (33)
 

P = 0.012
 

Low
 

Dose
 

5/42 (12)
 

N.S.
 

1.190
 

0.165
 

54.892
 

6.548
 

0.770
 

301.508
 

108
 

15/41 (37)
 

N.S.
 

0.976
 

0.410
 

4.521
 

1.098
 

0.583
 

2.021
 

75
 

High
 

Dose
 

4/40 (10)
 

N.S.
 

1.000
 

0.121
 

46.976
 

5.500
 

0.570
 

263.516
 

109
 

23/39 (59)
 

P = 0.013**
 

1.573
 

0.706
 

6.767
 

1.769
 

1.062
 

2.897
 

—
 79 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Thyroid: Follicular-cell Adenoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Cortical Adenoma or
 

Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

0/6 (0)
 

P = 0.022
 

—
 

0/8 (0)
 

N.S.
 

—
 

Pooled
 

Control
 

1/46 (2)
 

P = 0.008
 

—
 

3/50 (6)
 

N.S.
 

—
 

Low
 

Dose
 

1/43 (2)
 

N.S.
 

Infinite
 

0.009
 

Infinite
 

1.070
 

0.014
 

82.048
 

102
 

3/44 (7)
 

N.S.
 

Infinite
 

0.126
 

Infinite
 

1.136
 

0.160
 

8.065
 

104
 

High
 

Dose
 

7/42 (17)
 

P = 0.021**
 

Infinite
 

0.340
 

Infinite
 

7.667
 

1.049
 

335.959
 

105
 

6/43 (14)
 

N.S.
 

Infinite
 

0.344
 

Infinite
 

2.326
 

0.530
 

13.580
 

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

Fed Toxaphene in the Diet (a)
 

(continued)
 

Topography: Morphology 

Matched 

Control 

Pooled 

Control 

Low 

Dose 

High 

Dose 

Uterus: Endometrial Stromal 

Polyp (b) 0/9 (0) 5/53 (9) 9/41 (22) 5/45 (11) 

P Values (c,d) N.S. N.S. N.S. N.S. 

Relative Risk (Matched Control) (f) 

Lower Limit 

Upper Limit 

Infinite 

0.657 

Infinite 

Infinite 

0.286 

Infinite 

Relative Risk (Pooled Control) (f) 

Lower Limit 

Upper Limit 

2.327 

0.760 

8.142 

1.178 

0.288 

4.787 

Weeks to First Observed Tumor — — 87 109 

(a) Dosed groups received time-weighted average doses of 540 or 1,080 ppm.
 

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

(c) Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparisons of that dosed group with the matched-control group (*) or with the pooled-


control group (**) when P less than 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

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

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the specified
 

control group.
 
90

 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE FED TOXAPHENE IN THE DIET
 

91
 
92

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

Fed Toxaphene in the Diet (a)
 

Topography: Morphology
 

Liver: Hepatocellular Carcinoma (b)
 

P Values (c,d)
 

Departure from Linear Trend (e)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

0/10 (0)
 

P less than
 

0.001
 

P = 0.028
 

—
 

Pooled
 

Control
 

4/48 (8)
 

P less than
 

0.001
 

P = 0.044
 

—
 

Low
 

Dose
 

34/49 (69)
 

P less than
 

0.001*
 

P less than
 

0.001**
 

Infinite
 

2.610
 

Infinite
 

8.327
 

3.379
 

27.877
 

73
 

High
 

Dose
 

45/46 (98)
 

P less than
 

0.001*
 

P less than
 

0.001**
 

Infinite
 

4.108
 

Infinite
 

11.739
 

5.764
 

15.812
 

59
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

Topography: Morphology 

Matched 

Control 

Pooled 

Control 

Low 

Dose 

High 

Dose 

Liver: Hepatocellular Carcinoma 

or Neoplastic Nodule (b) 2/10 (20) 7/48 (15) 40/49 (82) 45/46 (98) 

P Values (c,d) P less than 

0.001 

P less than 

0.001 

P less than 

0.001* 

P less than 

0.001* 

P less than 

0.001** 

P less than 

0.001** 

Departure from Linear Trend (e) P ­ 0.007 P ­ 0.002 

Relative Risk (Matched Control) (f) 

Lower Limit 

Upper Limit 

4.082 

1.430 

28.580 

4.891 

1.883 

9.530 

Relative Risk (Pooled Control) (f) 

Lower Limit 

Upper Limit 

5.598 

2.949 

11.332 

6.708 

4.049 

7.956 

Weeks to First Observed Tumor 90 — 73 59 

(a) Dosed groups received time-weighted average doses of 99 or 198 ppm.
 

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

(c) Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise not significant (N.S.) is indicated. Beneath the
 

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

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


control group (**) when P less than 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

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

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the specified
 

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

Fed Toxaphene in the Diet (a)
 

Topography: Morphology
 

Liver: Hepatocellular Carcinoma (b)
 

P Values (c,d)
 

Departure from Linear Trend (e)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

0/9 (0)
 

P less than
 

0.001
 

P = 0.022
 

—
 

Pooled
 

Control
 

0/48 (0)
 

P less than
 

0.001
 

P = 0.002
 

—
 

Low
 

Dose
 

5/49 (10)
 

P = 0.030**
 

Infinite
 

0.262
 

Infinite
 

Infinite
 

1.237
 

Infinite
 

89
 

High
 

Dose
 

34/49 (69)
 

P less than
 

0.001*
 

P less than
 

0.001**
 

Infinite
 

2.384
 

Infinite
 

Infinite
 

11.267
 

Infinite
 

72
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

Topography: Morphology 

Matched 

Control 

Pooled 

Control 

Low 

Dose 

High 

Dose 

Liver: Hepatocellular Carcinoma 

or Neoplastic Nodule (b) 0/9 (0) 0/48 (0) 18/49 (37) 40/49 (82) 

P Values (c,d) P less than 

0.001 

P less than 

0.001 

P = 0.026* P less than 

0.001* 

P less than 

0.001** 

P less than 

0.001** 

Relative Risk (Matched Control) (f) 

Lower Limit 

Upper Limit 

Infinite 

1.200 

Infinite 

Infinite 

2.854 

Infinite 

Relative Risk (Pooled Control) (f) 

Lower Limit 

Upper Limit 

Infinite 

5.649 

Infinite 

Infinite 

13.554 

Infinite 

Weeks to First Observed Tumor — — 89 72 

(a) Dosed groups received time-weighted average doses of 99 or 198 ppm.
 

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

(c) Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise not significant (N.S.) is indicated. Beneath the
 

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

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


control group (**) when P less than 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Toxaphene in the Diet (a)
 

(continued)
 

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

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the specified
 

control group.
 
APPENDIX G
 

ANALYSIS OF FORMULATED DIETS FOR
 

CONCENTRATIONS OF TOXAPHENE
 

99
 
100

 
APPENDIX G
 

Analysis of Formulated Diets for
 

Concentrations of Toxaphene
 

A 2 g sample of formulated diet was shaken at ambient temperature
 

with 50 ml hexane for 2 hours, then filtered through Celite with
 

hexane washes, and reduced in volume to 10 ml.
 

The toxaphene then was dehydrohalogenated by the following
 

procedure. A 1 ml aliquot was added to 0.5 ml of alcoholic KOH
 

(2.5 g KOH/10 ml 95% ethanol), and the mixture was heated at
 

75-80 C for 15 minutes. The mixture was allowed to cool, 1 ml
 

hexane and 8 ml saturated aqueous Na.SO, were added, and the
 

mixture was shaken for 2 minutes. The hexane layer was drawn
 

off, another 1 ml of hexane was added, and the mixture was again
 

shaken and the hexane layer removed. This extraction was
 

repeated one more time, and the combined hexane extracts were
 

brought up to 10 ml volume with hexane and dried over anhydrous
 

Na2S04.
 

The resultant solution was analyzed quantitatively by gas-liquid
 

chromatography (electron capture detector, 5% QF-1 on Chromosorb
 

101
 
W column). Recoveries were checked with spiked samples carried
 

through the entire procedure.
 

Theoretical
 

Concentrations
 

in Diet (ppm)
 

80
 

160
 

320
 

640
 

1,280
 

No. of Sample Coefficient of Range (ppm) 

Samples Analytical Variation (%) 

Mean (ppm) 

4 81.8 1.8% 81-84 

8 155.0 3.3% 150-164 

3 330.0 1.0% 327-333 

11 640.0 6.9% 592-714 

12 1,286.0 2.9% 1,200-1,340 

102
 
Review of the Bioassay of Toxaphene* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

August 31, 1978
 

The Clearinghouse on Environmental Carcinogens was established in
 

May, 1976, in compliance with DHEW Committee Regulations and the Provisions of the Federal Advisory Committee Act. The purpose of the
 

Clearinghouse is to advise the Director of the National Cancer Institute
 

(NCI) on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed. The
 

members of the Clearinghouse have been drawn from academia, industry,
 

organized labor, public interest groups, State health officials, and
 

quasi-public health and research organizations. Members have been
 

selected on the basis of their experience in carcinogenesis or related
 

fields and, collectively, provide expertise in chemistry, biochemistry,
 

biostatistics, toxicology, pathology, and epidemiology. Representatives
 

of various Governmental agencies participate as ad hoc members. The
 

Data Evaluation/Risk Assessment Subgroup of the Clearinghouse is charged
 

with the responsibility of providing a peer review of reports prepared
 

on NCI-sponsored bioassays of chemicals studied for carcinogenicity. It
 

is in this context that the below critique is given on the bioassay of
 

Toxaphene for carcinogenicity.
 

A toxicologist with Hercules presented a public statement regarding
 

the bioassay of Toxaphene. He noted that Hercules is the sole U.S.
 

manufacturer of Toxaphene, the largest selling insecticide in the world.
 

He said that Hercules disagreed with the conclusion in the report that
 

"The test results also suggest carcinogenicity of Toxaphene for the
 

thyroid of male and female Osborne-Mendel rats." Based on an evaluation
 

of the histopathology and statistics by consultants to Hercules, the
 

representative said that the follicular-cell thyroid tumor incidence in
 

treated and pooled control male rats was virtually identical. Furthermore,
 

the incidence of follicular-cell thyroid tumors in females was not
 

statistically significant. He said that classic thyroid carcinogens
 

induce mainly bilateral tumors, whereas those observed in control and
 

treated animals were essentially unilateral. He objected to the use
 

of the term "carcinogen" in reference to benign tumors. He noted a
 

discrepancy in the historical control tissue count (for thyroids)
 

between that given in the report and the number found in the Hercules
 

evaluation. The Hercules representative recommended that the report
 

include the range of thyroid tumors observed in each control group.
 

103
 
The primary reviewer indicated that his -emarks were predicated
 

solely on the information in the report and n>t from other sources. He
 

concluded that the bioassay demonstrated that the test compound induced
 

a neoplastic response in the rat thyroid and :he mouse liver. He also
 

pointed out an increased number of liver carcinomas observed in low dose
 

treated male rats. He suggested that this fiiding should receive greater
 

emphasis. He further noted that the test com sound came from a single
 

batch of Toxaphene. Since Toxaphene is a mixzure, he wondered if the
 

carcinogenic component(s) would be common to all batches. Although he
 

agreed with the conclusion in the report, the primary reviewer cautioned
 

against estimating the potential hazard of ToKaphene for man, particularly
 

since no evidence exists that polychlorinated alkanes or terpenes are
 

human carcinogens. He recommended that the report on the bioassay of
 

Toxaphene be accepted as written. He also recommended that the test
 

batch of Toxaphene be analyzed to determine its quantitative similarity
 

to other batches of Toxaphene in general use.
 

Although the staff viewed the thyroid tumors as "suspicious," a
 

Program pathologist said that the histopathology was confirmed at
 

several review levels. Despite the question about the thyroid tumors,
 

he noted that the liver tumors in mice were clearly treatment-related.
 

He acknowledged that any necessary modification to the report would be
 

made after examining the concerns expressed by Hercules.
 

A motion was approved unanimously that the report on the bioassay
 

of Toxaphene be accepted as written.
 

Members present were:
 

Arnold L. Brown (Chairman), University of Wisconsin School of Medicine
 

Joseph Highland, Environmental Defense Fund
 

Michael Shimkin, University of California at San Dip^o
 

Louise Strong, University of Texas Health Sciences Center
 

*	 Subsequent to this review, changes may have been made in the
 

bioassay report either as a result of the review or other
 

reasons. Thus, certain comments and criticisms reflected in
 

the review may no longer be appropriate.
 

104
 

MJ.S. GOVERNMENT PRINTING OFFICE: 1978 281-217/3310 1-3 

DHEW Publication No. (NIH) 79-837