National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 100 

1978 

BIOASSAY OF 

CUPFERRON 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 135-20-6 

NCI-CG-TR-100 

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

Public Health Service 

National Institutes of Health 
BIDASSAY OF
 

CUPFERRON
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1350
 
REPORT ON THE BIOASSAY OF CUPFERRON
 

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 cupferron 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 chemicals have the
 

capacity to produce cancer in animals. Negative results, in which
 

the test animals do not have a significantly greater incidence of
 

cancer than control animals, do not necessarily mean the test chemical
 

is not a carcinogen because the experiments are conducted under a
 

limited set of circumstances. Positive results demonstrate that the
 

test chemical is carcinogenic for animals under the conditions of the
 

test and indicate a potential risk to man. The actual determination
 

of the risk to man from animal carcinogens requires a wider analysis.
 

CONTRIBUTORS: This bioassay of cupferron was conducted by Mason
 

Research Institute, Worcester, Massachusetts, initially under direct
 

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

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

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

principal investigators for the contract were Dr. E. Smith (3) and
 

Dr. A. Handler (3). Animal treatment and observation were supervised
 

by Mr. G. Wade (3) and Ms. E. Zepp (3). Chemical analysis was performed by Midwest Research Institute (4) and the analytical results
 

were reviewed by Dr. N. Zimmerman (5).
 

Histopathologic examinations were performed by Dr. R. W. Fleischman (3), Dr. A. S. Krishna Murthy (3), Dr. A. Russfield (3), and
 

Dr. D. S. Wyand (3) at the Mason Research Institute, the pathology
 

narratives were written by Dr. D. S. Wyand (3), and the diagnoses
 

included in this report represent the interpretation of these pathologists. Histopathology findings and reports were reviewed by Dr. R. L.
 

Schueler (6).
 

Compilation of individual animal survival, pathology, and summary
 

tables was performed by EG&G Mason Research Institute (7); the statistical analysis was performed by Mr. W. W. Belew (5,8) using methods
 

selected for the Carcinogenesis Testing Program by Dr. J. J. Gart (9).
 

ill
 
This report was prepared at METREK, a Division of The MITRE Corporation (5) under the direction of the NCI. Those responsible for
 

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

(5), task leader Dr. M. R. Kornreich (5,10), senior biologist
 

Ms. P. Walker (5), biochemist Dr. B. Fuller (5), and technical editor
 

Ms. P. A. Miller (5). The final report was reviewed by members of
 

the participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

Cueto, Jr. (1), Dr. J. F. Douglas (1), Dr. D. G. Goodman (1,10),
 

Dr. R. A. Griesemer (1), Dr. M. H. Levitt (l), Dr. H. A. Milman (1),
 

Dr. T. W. Orme (1), Dr. R. A. Squire (1,11), Dr. S. F. Stinson (1),
 

Dr. J. M. Ward (1), and Dr. C. E. Whitmire (1).
 

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

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

3.	 Mason Research Institute, 57 Union Street, Worcester, Massachusetts.
 

4.	 Midwest Research Institute, 425 Volker Boulevard, Kansas City,
 

Missouri.
 

5.	 Th MITR e E Corporation, METREK Division, 1820 Dolley Madison
 

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockvi1le, Maryland.
 

8.	 Now with the Solar Energy Research Institute, Cole Boulevard,
 

Golden, Colorado.
 

9.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

iv
 
10.	 Now with Clement Associates, Inc., 1010 Wisconsin Avenue, N.W.,
 

Washington, D.C.
 

11.	 Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 
SUMMARY
 

A bioassay of cupferron for possible carcinogenicity was conducted using Fischer 344 rats and B6C3F1 mice. Cupferron was administered in the feed, at either of two concentrations, to groups of 49
 

or 50 male and 50 female animals of each species. The time-weighted
 

average high and low dietary concentrations of cupferron were, respectively, 0.30 and 0.15 percent for male and female rats, and 0.4
 

and 0.2 percent for male and female mice. After a 78-week period of
 

compound administration, observation of the rats continued for an
 

additional period of up to 28 weeks and observation of the mice continued for an additional period of up to 18 weeks.
 

For each species, 50 animals of each sex were placed on test as
 

controls and fed only the basal diet.
 

Among both sexes of rats and mice there was a significant positive association between the dose of cupferron administered and
 

mortality; however, in all groups of animals sufficient numbers survived long enough to establish the carcinogenicity of this compound.
 

There were significant positive associations between the concentrations of cupferron administered to male and female rats and the
 

incidences of: squamous-cell carcinomas of the forestomach, hepatocellular carcinomas and neoplastic nodules, and hemangiosarcomas.
 

When a binomial distribution and a spontaneous incidence rate corresponding to the appropriate historical control incidence were assumed,
 

the incidences of auditory sebaceous gland neoplasms in female rats
 

and female mice were significant. There were significant positive
 

associations between the concentrations administered and: the incidences of hepatocellular carcinomas in female mice, the incidences of
 

hemangiosarcomas in both sexes of mice, and the incidence of Harderian
 

gland adenomas in both sexes of mice.
 

Under the conditions of this bioassay cupferron was carcinogenic
 

in Fischer 344 rats, causing hemangiosarcomas, hepatocellular carcinomas, and squamous-cell carcinomas of the forestomach in males and
 

females as well as carcinomas of the auditory sebaceous gland in
 

females. The chemical was also carcinogenic in B6C3F1 mice, causing
 

hemangiosarcomas in males and hepatocellular carcinomas, carcinomas
 

of the auditory sebaceous gland, a combination of hemangiosarcomas
 

and hemangiomas, and adenomas of the Harderian gland in females.
 

VII
 
TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 4
 

A. Chemicals 4
 

B. Dietary Preparation 5
 

C. Animals 5
 

D. Animal Maintenance 6
 

E. Selection of Initial Concentrations 9
 

F. Experimental Design 10
 

G. Clinical and Histopathologic Examinations 13
 

H. Data Recording and Statistical Analyses 15
 

III. CHRONIC TESTING RESULTS: RATS 20
 

A. Body Weights and Clinical Observations 20
 

B. Survival 20
 

C. Pathology 23
 

D. Statistical Analyses of Results 27
 

IV. CHRONIC TESTING RESULTS: MICE 44
 

A. Body Weights and Clinical Observations 44
 

B. Survival 44
 

C. Pathology 47
 

D. Statistical Analyses of Results 49
 

V. DISCUSSION 60
 

VI. BIBLIOGRAPHY 64
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH CUPFERRON A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH CUPFERRON B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH CUPFERRON C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH CUPFERRON D-l
 

ix
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 CHEMICAL STRUCTURE OF CUPFERRON 2 

2 GROWTH CURVES FOR CUPFERRON CHRONIC STUDY 

RATS 21 

SURVIVAL COMPARISONS OF CUPFERRON CHRONIC 

STUDY RATS 22 

GROWTH CURVES FOR CUPFERRON CHRONIC STUDY 

MICE 45 

SURVIVAL COMPARISONS OF CUPFERRON CHRONIC 

STUDY MICE 46 

LIST OF TABLES 

Table Number Page 

DESIGN SUMMARY FOR FISCHER 344 RATS— 

CUPFERRON FEEDING EXPERIMENT 11 

DESIGN SUMMARY FOR B6C3F1 MICE—CUPFERRON 

FEEDING EXPERIMENT 12 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

CUPFERRON 28 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH CUPFERRON 34 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

CUPFERRON 50 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH CUPFERRON 53 

x
 
LIST OF TABLES (Concluded) 

Table Number Page 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH CUPFERRON A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH CUPFERRON A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH CUPFERRON B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH CUPFERRON B-7 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH CUPFERRON C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH CUPFERRON C-8 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH CUPFERRON D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH CUPFERRON D-7 

XI
 
I. INTRODUCTION
 

Cupferron (Figure 1) (NCI No. C03258), an N-nitroso hydroxylamine
 

derivative used primarily as a reagent in analytical chemistry, was
 

selected for bioassay by the National Cancer Institute because of the
 

suspected carcinogenicity of nitrosamines.
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is N-hydroxy-N-nitroso-benzenamine,
 

*
 ammonium salt. It is also known as N-nitroso-N-phenyl-hydroxylamine,
 

ammonium salt; and as ammonium nitrosophenylhydroxylamine.
 

As an analytical reagent, cupferron is used to separate copper
 

and iron from other metals and to separate tin from zinc (Windholz,
 

1976; Schlecten and Thompson, 1970). It also finds application as a
 

quantitative reagent for vanadates and titanium and for the colorimetric estimation of aluminum (Windholz, 1976).
 

The chelating properties of cupferron as well as its ability to
 

inhibit respiration (and therefore oxygen consumption) in certain
 

plants have been employed in numerous experimental studies dealing
 

with elucidation of the mechanism of radiation-induced chromosomal
 

breakage in plants and subsequent repair (Kihlman, 1958; Kihlman,
 

1959; Davis, 1969; Gilot-Delhalle et al., 1973). In addition, cupferron has been found to increase the fungicidal activity of zinc
 

dimethyldithiocarbamate (Ziram), a widely used agricultural fungicide
 

(Matolcsy et al. , 1971).
 

*
 

The CAS registry number is 135-20-6.
 
FIGURE 1

 

CHEMICAL STRUCTURE OF CUPFERRON 
Production statistics for cupferron are not available. However,
 

the compound is produced in commercial quantities (greater than 1000
 

pounds or $1000 in value annually) by three U.S. companies (Stanford
 

Research Institute, 1977).
 

The potential for exposure to cupferron appears to be greatest
 

for those engaged in analytical or research studies involving its
 

use. Workers at manufacturing plants may also experience significant
 

contact with the chemical.
 
II. MATERIALS AND METHODS
 

A. Chemicals
 

Cupferron (N-nitroso-N-phenyl-hydroxylamine) was purchased by the
 

NCI for Mason Research Institute from Eastman Kodak Company, Rochester,
 

New York and chemical analysis was performed by Midwest Research Institute, Kansas City, Missouri. The experimentally determined melting
 

point of 154° to 156°C, although narrow in its range, suggested the
 

presence of minor impurities due to its difference from the literature
 

value of 163° to 164°C (Bamberger and Baudisch, 1909). The results of
 

elemental analysis, although close to theoretical, confirmed this suggestion. Thin-layer chromatography was performed utilizing two solvent
 

systems (methanol:chloroform and butanol:benzene), visualized with
 

ultraviolet light and ferricyanide-ferrichloride. The first procedure
 

indicated the presence of two impurities, while the second revealed
 

no indication of impurities. The higher polarity of the first system
 

may have contributed to this difference. High-pressure liquid chromatography indicated the presence of one less motile impurity. The
 

result of titration of the NH.+ group with eerie sulfate was approximately 98 percent of the theoretical. Infrared analysis was consistent with the structure of the compound. Ultraviolet analysis
 

revealed \ at 219 and 282 with molar extinction coefficients (O of
 

max
 

2 2
 107.5	 x 10 and 100.4 x 10 , while the published literature values
 

2
 

are X at 221 and 293 and respective e values of 91.45 x 10 and
 

max
 

2
 

107.5 x 10 . If the shifting value of the upper \ were ignored,
 
the purity approximation would be 93 percent; however, as a result of
 

the upper \ difference, the « cannot be utilized to suggest purity.
 IT13X
 

Throughout this report the term cupferron is utilized to represent this chemical.
 

B.	 Dietary Preparation
 

The basal laboratory diet for both dosed and control animals
 

consisted of Wayne Lab-Blox (Allied Mills, Inc., Chicago, Illinois).
 

Cupferron was administered to the dosed animals as a component of the
 

diet. Proper amounts of the chemical were weighed out of a working
 

stock bottle of presifted chemical under a fume hood. Ammonium carbonate in a gauze bag was suspended in the stock container to retard
 

decomposition. The compound was hand-blended in an aluminum bowl
 

with an aliquot of the ground feed. Once visual uniformity was
 

attained, the mixture was placed into a 6 kg capacity Patterson-


Kelley twin-shell stainless steel V-blender along with the remainder
 

of the meal and blended for 20 minutes. Prepared diets were placed
 

in double plastic bags and stored in the dark at 4°C. The mixture
 

was used for 1 week only.
 

C. Animals
 

Two animal species, rats and mice, were used in the carcinogenicity bioassay. Fischer 344 rats and B6C3F1 mice were obtained through
 

contracts of the Division of Cancer Treatment, National Cancer Institute. Animals were supplied by Charles River Breeding Laboratories,
 
Inc., Wilmington, Massachusetts. Dosed and control animals for
 

both species were received in separate shipments.
 

Upon arrival, a sample of animals from each shipment was examined
 

for parasites and other signs of disease. The remaining animals were
 

quarantined by species for 2 weeks prior to initiation of test.
 

Animals were assigned to groups and distributed among cages so that
 

average body weight per cage was approximately equal for a given sex
 

and species.
 

D.	 Animal Maintenance
 

All animals were housed by species in rooms having a temperature
 

range	 of 23° to 34°C. Incoming air was filtered through Tri-Dek^
 

CR)
 15/40 denier Dacron filters (Tri-Dim Filter Corp. , Hawthorne, New
 

Jersey) providing six changes of room air per hour. Fluorescent
 

lighting was provided on a 12-hour-daily cycle.
 

Rats were housed five per cage by sex. During quarantine and
 

for the first 14 months of study, they were kept in galvanized-steel
 

wire-mesh cages suspended above newspapers. Newspapers were replaced
 

daily and cages and racks washed weekly. For the remainder of the
 

study, rats were kept in suspended polycarbonate cages equipped with
 

disposable nonwoven fiber filter sheets. Clean bedding and cages
 

®


were provided twice weekly. SAN-I-CEL (Paxton Processing Company,
 

Paxton, Illinois) corncob bedding was used for the first 6 months
 

that	 rats were housed in polycarbonate cages. Aspen hardwood chip
 
bedding (American Excelsior Company, Baltimore, Maryland) was used
 

for the remainder of the study. Stainless steel cage racks were
 

cleaned once every 2 weeks, and disposable filters were replaced at
 

that time.
 

Mice were housed by sex in polycarbonate shoe box type cages.
 

During quarantine and periods of chemical administration, cages were
 

fitted with perforated stainless steel lids. During the observation
 

period following chemical administration, stainless steel wire bar
 

lids were used. Both types of lids were from Lab Products, Inc.,
 

Garfield, New Jersey. Nonwoven fiber filter bonnets were used over
 

cage lids. Dosed and control mice were housed 10 per cage for
 

the first 12 or 11 months of study, respectively, and five per cage
 

thereafter. Cages, lids, filters, and bedding were provided three
 

times per week when cage populations were ten and twice per week when
 

cage populations were reduced to five. For the first year of the
 

study, corncob bedding (SAN-I-CEL ) was used. A second corncob bedding (Bed-o-Cobs®, The Andersons Cob Division, Maumee, Ohio) was used
 

for the next 8 months, followed by Aspen bedding for the remainder
 

of the study. Reusable filter bonnets and pipe racks were sanitized
 

every 2 weeks throughout the study.
 

Water was available from 250 ml polycarbonate water bottles
 

equipped with rubber stoppers and stainless steel sipper tubes.
 

Bottles were replaced twice weekly and, for rats only, water was
 
supplied as needed between changes. Food and water were available ad
 

libitum.
 

Pelleted Wayne Lab-Blox®was supplied during the quarantine and
 

final observation periods. During chemical administration, all
 

(R)
 dosed animals were supplied with Wayne Lab-Blox meal containing
 

the appropriate concentration of cupferron. Control animals had untreated meal available. For the first 13 months of study, feed was
 

(R)
 dispensed to all rats and to dosed mice in Alpine aluminum feed cups
 

(Curtin Matheson Scientific, Inc., Woburn, Massachusetts) equipped
 

with stainless steel baffles. This same feed distribution system was
 

utilized for control mice for the first 14 months of study. Thereafter, feed was dispensed in stainless steel gangstyle feed hoppers
 

(Scientific Cages, Inc., Bryan, Texas). Pellets were fed to rats on
 

the cage floor during quarantine and final observation periods. Mice
 

were fed pellets from feeders incorporated into the cage lids during
 

the final observation period. Feed hoppers were changed twice
 

weekly. Food was replenished daily in Alpine feed cups.
 

Low dose rats were housed in rooms with other rats receiving
 

diets containing p-cresidine (120-71-8); 2,3 ,5,6-tetrachloro-4­

nitroanisole (2438-88-2); 4-chloro-o-phenylenediamine (95-83-0);
 

4-chloro-m-phenylenediamine (5131-60-2); and IH-benzotriazole (95­

14-7). High dose and control rats were housed in a room with rats
 

intubated with m-cresidine (102-50-1); and with other rats receiving
 

*
 CAS registry numbers are given in parentheses.
 
diets containing fenaminosulf (140-56-7) and 2,5-dithiobiurea (142­

46-1).
 

Dosed and control mice were in a room in which other mice were
 

receiving diets containing 2,5-dithiobiurea (142-46-1); 4-chloro-ophenylenediamine (95-83-0); o-anisidine hydrochloride (134-29-0);
 

p-anisidine hydrochloride (20265-97-8); and fenaminosulf (140-56-7).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

cupferron for administration to dosed animals in the chronic studies,
 

subchronic toxicity studies were conducted with both rats and mice.
 

Animals of each species were distributed among six groups, each
 

consisting of five males and five females. Cupferron was incorporated into the basal laboratory diet and supplied ad libitum
 

to five of the six rat groups in concentrations of 0.02, 0.04,
 

0.08, 0.15, and 0.30 percent and five of the six mouse groups in
 

concentrations of 0.04, 0.08, 0.15, 0.30, and 0.60 percent. The
 

sixth group of each species served as controls, receiving only the
 

basal laboratory diet. The dosed dietary preparations were administered for 8 weeks. Survivors were sacrificed at the end of the
 

test, and gross necropsies were performed.
 

The highest concentration causing no deaths, no compound-related
 

gross abnormalities, and no mean group body weight depression in
 

excess of 20 percent relative to controls was selected as the high
 

dose utilized for the chronic bioassay.
 
There were no deaths among male rats during the 8-week subchronic
 

study; however, one female in the group receiving 0.04 percent and one
 

in the group receiving 0.30 percent died. At the highest concentration mean body weight depression was approximately 38 percent among
 

male rats and approximately 20 percent among female rats. At gross
 

necropsy it was observed that rats of both sexes had black spleens
 

with dose-related, moderate to advanced enlargement of the organ.
 

The high concentration chosen for the chronic bioassay was 0.30 percent for male and female rats.
 

There were no deaths among male or female mice during the 8-week
 

subchronic study. At a concentration of 0.60 percent mean body
 

weight depression was approximately 23 percent among male and female
 

mice. At gross necropsy it was observed that mice receiving cupferron
 

had black spleens and those mice which received dosages of more than
 

0.15 percent had spleens that were also moderately enlarged. The
 

high concentration chosen for the chronic bioassay was 0.60 percent
 

for male and female mice.
 

F. Experimental Design
 

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

treated and untreated observation periods, and time-weighted average
 

concentrations) are summarized in Tables 1 and 2.
 

Rats were all approximately 6 weeks old at the time they were
 

placed on test; however, control rats were approximately 10 weeks
 

10
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

CUPFERRON FEEDING EXPERIMENT
 

INITIAL CUPFERRON OBSERVATION PERIOD 

GROUP CONCENTRATION TREATED UNTREATED 

SIZE (PERCENT) (WEEKS) (WEEKS) 

MALE 

CONTROL 50 0 0 110 

LOW DOSE 50 0.15 78 

0 26 

HIGH DOSE 49 0.30 78 

0 19 

FEMALE 

CONTROL 50 0 0 110 

LOW DOSE 50 0.15 78 

0 28 

HIGH DOSE 50 0.30 78 

0 28 

11
 
INITIAL
 

GROUP
 

SIZE
 

MALE
 

CONTROL 50
 

LOW DOSE 50
 

HIGH DOSE 50
 

FEMALE
 

CONTROL 50
 

LOW DOSE 50
 

HIGH DOSE 50
 

a
 

TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

CUPFERRON FEEDING EXPERIMENT
 

CUPFERRON OBSERVATION PERIOD TIME-WEIGHTED 

CONCENTRATION TREATED UNTREATED AVERAGE 

(PERCENT) (WEEKS) (WEEKS) CONCENTRATION3 

0 0 98 0
 

0.3 35 0.2
 

0.1 43
 

0 18
 

0.6 35 0.4
 

0.2 43
 

0 17
 

0 0 98 0
 

0.3 35 0.2
 

0.1 43
 

0 18
 

0.6 35 0.4
 

0.2 43
 

'o 17
 

S (concentration X weeks received)
 

(weeks receiving chemical)
 

12
 
older than the dosed rats. The dietary concentrations of cupferron
 

utilized were 0.30 and 0.15 percent. Throughout this report those
 

rats receiving the former concentration are referred to as the high
 

dose groups and those receiving the latter concentration are referred
 

to as the low dose groups. Dosed rats were supplied with feed
 

containing cupferron for a total of 78 weeks, followed by an observation period of up to 28 weeks.
 

All mice were approximately 6 weeks old at the time they were
 

placed on test; however, control mice were approximately 5 weeks
 

older than the dosed mice. The dietary concentrations of cupferron
 

initiallly utilized were 0.60 and 0.30 percent for the first 35
 

weeks of compound administration. Throughout this report, those mice
 

initially receiving the former concentration are referred to as the
 

high dose groups and those initially receiving the latter concentration are referred to as the low dose groups. In week 36 the concentrations for high and low dose mice were lowered to 0.2 and 0.1 percent,
 

respectively, for the remainder of the period of compound administration, due to high mortality and excess mean body weight depression.
 

Dosed mice were supplied with feed containing cupferron for a total
 

of 78 weeks, followed by an observation period of up to 18 weeks.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the experiment. Body weights were recorded twice weekly for the first
 

13
 
12 weeks of the study and at monthly intervals thereafter. From the
 

first day, all animals were inspected twice daily for mortality.
 

Food consumption, for two cages from each group, was monitored for
 

seven consecutive days once a month for the first nine months of the
 

bioassay and for three consecutive days each month thereafter. The
 

presence of tissue masses and lesions was determined by monthly
 

observation and palpation of each animal.
 

A necropsy was performed on each animal regardless of whether it
 

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

bioassay. The animals were euthanized by carbon dioxide inhalation,
 

and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examinati-on of major tissues, organs,
 

and gross lesions taken from sacrificed animals and, whenever possible,
 

from animals found dead.
 

Tissues were preserved in 10 percent buffered formalin, embedded
 

in paraffin, sectioned, and stained with hematoxylin and eosin prior
 

to microscopic examination. An occasional section was subjected to
 

special staining techniques for more definitive diagnosis.
 

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

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

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

kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid,
 

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

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

for those that died early. Also, some animals were missing, cannibalized, or judged to be in such an advanced state of autolysis as to
 

preclude histopathologic interpretation. Thus, the number of animals
 

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

animals that were placed on experiment in each group.
 

H.	 Data Recording and Statistical Analyses
 

Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data System
 

(Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design, clinical observations, survival, body weight, and individual pathologic results,
 

as recommended by the International Union Against Cancer (Berenblum,
 

1969). Data tables were generated for verification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques described in this section. Those analyses of the experimental results
 

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

15
 
missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

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

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

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

16
 
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, pp. 6-10) requires that the P-value for any comparison
 

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

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

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

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

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

when appropriate. Under the assumption of a linear trend, this test
 

determined if the slope of the dose-response curve is different from
 

zero at the one-tailed 0.05 level of significance. Unless otherwise
 

noted, the direction of the significant trend was a positive dose
 

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

found. Once this reduced set of data was obtained, the standard
 

17
 
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 animals died naturally or were sacrificed was entered as
 

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

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

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

18
 
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
 

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

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

would be within the interval calculated from the experiment. When
 

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

can be inferred that a statistically significant result (a P < 0.025
 

one-tailed test when the control incidence is not zero, P < 0.050
 

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

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

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

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

detected under the conditions of this test.
 

19
 
III. CHRONIC TESTING RESULTS: RATS
 

A.	 Body Weights and Clinical Observations
 

There were no generally consistent relationships in mean body
 

weight patterns among male rats; however, there was slight mean
 

body weight depression among dosed female rats when compared to
 

controls (Figure 2). Fluctuations in the growth curve may be due to
 

mortality; as the size of the group diminishes, the mean body weight
 

may be subject to wide variations.
 

The only clinical abnormality observed among dosed or control
 

rats was a hard, crusted subcutaneous lesion on the anterior surface
 

of a control male that subsequently died.
 

B. Survival
 

The estimated probabilities of survival for male and female
 

rats in the control and cupferron-dosed groups are shown in Figure 3.
 

For male and female rats the Tarone test indicated a significant
 

(P < 0.001) positive association between dosage and mortality. The
 

departure from linear trend for males was also significant (P = 0.016),
 

For males five rats were sacrificed from the control group in
 

week 78. In the high dose group the median survival on test was 63
 

weeks with the last rat dead by week 98. For the low dose group the
 

median survival on test was 84 weeks with the last rat dead by week
 

105. In the control group 64 percent (32/50) survived on test until
 

the termination of the study. The early mortality may have been
 

associated with tumor incidence since 78 percent (38/49) of the low
 

20
 
750­ •750 

CONTROL 

_ 600~ LOW DOSE -600 

CO 

HIGH DOSE < 

DC 

52 

(- 450­

X -450 

5 

g300- -300 

ffi 

150­ -150 

MALE RATS 

~ I I I \

 r 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

750­ •750 

CONTROL 

600­ LOW DOSE -600 

K 

O 

I- 450­ I 

O 

LU 

HIGH DOSE 

-450 

g 300­

co 

< 

LU 

S 

150­

— 300 

- 150 

FEMALE RATS 

15 30 45 60 75 

TIME ON TEST (WEEKS) 

105 120 

FIGURE 2 

GROWTH CURVES FOR CUPFERRON CHRONIC STUDY RATS 

21 
P PROBABILITY OF SURVIVA

PROBABILITY OF SURVIVAL p p p p b KJ bo * b _!_ _J_ 

£ \ 

£ I 

30 

> £ 5 8 

• C r

I iI 30 w m

£ 

33 

1 |

C/J O ,-r- O

Z O

•n -n 

25 /

/

A 

P 

H 

m 

30 ._j s 

m 

O en 

1

y I" 

r

30 

O 

,r- O 

CO 

O 

30 .•'••••r• i 

in p 

b 
dose males and 80 percent (35/44) of the high dose males examined
 

developed a hemangiosarcoma as early as week 42, while no cases of
 

this tumor were observed in the control male rats.
 

For females five rats were sacrificed from the control group in
 

week 78. In the high dose group the median survival was 68 weeks,
 

with only two rats remaining alive on test until the end of the
 

study. The median survival was 91 weeks for the low dose group, with
 

eight animals surviving on test until the end of the study. In the
 

control group 72 percent (36/50) of the female rats survived on test
 

until the end of the study. The early mortality may have resulted
 

from tumor incidence since 62 percent (28/45) of the low dose females
 

and 79 percent (37/47) of the high dose females examined developed a
 

hemangiosarcoma, while no cases of this tumor were observed in the
 

control female rats.
 

C. Pathology
 

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).
 

Dosed rats had increased incidences of hepatocellular carcinoma; carcinoma and papilloma of the forestomach; hemangiosarcoma;
 

fibromas; and tumors of the adrenal medulla and auditory sebaceous
 

glands.
 

The incidence of hepatocellular carcinoma in dosed rats was
 

significantly elevated in both males (4/43 [9 percent] high dose;
 

23
 
8/48 [17 percent] low dose) and females (10/44 [23 percent] high dose;
 

24/44 [55 percent] low dose). Neoplastic nodules, basophilic foci,
 

and clear-cell foci, while occurring in lesser numbers than carcinoma,
 

were only found in dosed rats. The histopathology of hepatocellular
 

carcinoma and altered cellular foci was similar to that described by
 

Squire and Levitt (1975). Hepatocellular carcinoma was generally
 

characterized as an expanding mass of hepatocytes that produced
 

compression of the adjacent parenchyma and lacked portal radicles.
 

Variation in cytomorphology was a common feature of the tumors.
 

Hepatocytes were basophilic, either small or large cell type, and
 

had vacuolated or granular cytoplasm and normal-sized or enlarged
 

nuclei. Mitotic activity was minimal to moderate. Cytoarchitectural
 

changes included a trabecular growth pattern, one-cell-thick or
 

multiple-cell-thick liver plates, and acinar formations. The latter
 

two arrangements of the neoplastic liver cells were often associated
 

with moderate degrees of anaplasia (e.g., cytoplasmic basophilia,
 

large cell type, nuclear enlargement, and hyperchromatism). Occasional areas of necrosis were noted. Metastases of hepatocellular
 

carcinoma were not found.
 

The squamous epithelium of the forestomach was altered in a
 

number of ways. Squatnous-cell papilloma (10/38 [26 percent] high
 

dose, 16/48 [33 percent] low dose males; 6/43 [14 percent] high dose,
 

6/43 [14 percent] low dose females) and focal and diffuse basal-cell
 

hyperplasia (16/38 [42 percent] high dose, 26/48 [54 percent] low
 

24
 
dose males; 14/43 [33 percent] high dose, 17/43 [40 percent] low dose
 

females) was significantly elevated in males and females. Carcinoma
 

in situ was noted in a few instances, but this was still considered
 

to be compound-related. These diagnoses overlap, with a single
 

animal often having two or three of the above changes. The squamouscell carcinomas of the forestomach (17/38 [45 percent] high dose,
 

19/48 [40 percent] low dose males; 22/43 [51 percent] high dose,
 

14/43 [33 percent] low dose females) originated from the prickle-cell
 

layer and were invasive. Most had locally invaded the lamina propria,
 

but some had extensively invaded through the stomach wall and had
 

invaded other visceral organs by direct extension. A few tumors
 

showed distant metastases in lymph nodes, lungs, and liver. Some
 

tumors had extensive pearl formation, while other tumors were composed
 

of invading nests and cords of more undifferentiated squamous cells.
 

Both focal and diffuse basal-cell hyperplasia were commonly seen.
 

This was characterized by downgrowths of multiple layers of basal
 

cells but without invasion of the lamina propria or overlying stratified squamous epithelium. Carcinoma in situ was judged to be present
 

when loss of polarity and cellular atypia occurred throughout the
 

squamous epithelium, but there was no invasion through the basement
 

membrane. Squamous papilloma resembled a warty or villous nodule
 

with extensive keratinization and lack of invasion.
 

Hemangiosarcomas, mostly originating in the spleen, were found
 

in very high frequency in both males (35/44 [80 percent] high dose,
 

25
 
38/49 [78 percent] low dose) and females (37/47 [79 percent] high
 

dose, 28/45 [62 percent] low dose). These were composed of variable-


sized blood spaces from capillary size to massive hematomas. There
 

were solid sarcomatous areas and villus-like structures projecting
 

into large blood spaces. The cells were pleomorphic, and mitotic
 

figures were common.
 

Subcutaneous fibromas, circumscribed tumors composed of well-


differentiated fibroblasts and mature collagen, were of moderately
 

elevated frequency in the low dose males. The incidence in the high
 

dose group was comparable to that in the controls.
 

Carcinomas of the auditory sebaceous glands occurred in 4/47
 

(9 percent) high dose and 5/45 (11 percent) low dose females and only
 

1/49 (2 percent) control females. A few were seen in male rats. A
 

rare tumor, ganglioneuroma of the adrenal medulla, was found in two
 

high dose male rats and one high dose female rat. This incidence,
 

although low, was judged to be compound-related. Histologically,
 

these tumors were composed of nerve cells in an abundant glial
 

fibrillary stroma.
 

There were numerous inflammatory and degenerative lesions noted
 

which are common in aging rats, none of which were judged to be
 

related to the administration of cupferron.
 

On the basis of the data presented by this histopathologic
 

examination, it is evident that cupferron is a carcinogenic compound
 

in Fischer 344 rats. The increased incidences of hepatocellular
 

26
 
carcinomas, squamous-cell carcinomas of the stomach, hemangiosarcomas,
 

gliomas, ganglioneuroma and tumors of auditory sebaceous glands were
 

all believed to be compound-related.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

observed in at least one of the control or cupferron-dosed groups and
 

where such tumors were observed in at least 5 percent of the group.
 

In both male and female rats increased incidences of hemangiosarcomas were observed. For both sexes the Cochran-Armitage test indicated a significant (P < 0.001) positive association between dosage
 

and tumor incidence. A significant departure from linearity was also
 

observed for both males (P < 0.001) and females (P = 0.009), principally due to the elevated incidence of this mor in the dosed rats
 

compared to the controls. For both sexes the fisher exact tests
 

supported these findings with significant (P < ~>1) r parisons
 

of both high and low dose to controls. Based on these results, the
 

administration of cupferron was associated with an increased incidence of hemangiosarcomas in both male and female rats.
 

In both male and female rats numerous stomach squamous-cell carcinomas were observed. For both sexes the Cochran-Armitage test
 

was significant (P < 0.001). In both sexes these results were
 

supported by significant (P < 0.001) Fisher exact test comparisons of
 

27
 
00 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH CUPFERRON*
 

TOPOGRAPHY : MORPHOLOGY
 

Subcutaneous Tissue: Fibroma*5
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Circulatory System: Hemangio sarcoma
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

1/50(0.02)
 

N.S.
 

P < 0.001
 

110
 

10/50(0.20)
 

P = 0.008(N)
 

78
 

0/50(0.00)
 

P < 0.001
 

P < 0.001
 

LOW
 

DOSE
 

15/49(0.31)
 

P < 0.001
 

15.310
 

2.518
 

625.800
 

62
 

2/49(0.04)
 

P = 0.015(N)
 

0.204
 

0.003
 

0.894
 

91
 

38/49(0.78)
 

P < 0.001
 

Infinite
 

13.280
 

Infinite
 

62
 

HIGH
 

DOSE
 

5/44(0.11)
 

N.S.
 

5.682
 

0.670
 

262.000
 

60
 

2/44(0.05)
 

P = 0.024(N)
 

0.227
 

0.025
 

0.991
 

42
 

35/44(0.80)
 

P < 0.001
 

Infinite
 

13.665
 

Infinite
 

45
 
TOPOGRAPHY rMORPHOLOGY
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Q
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

N3 Liver: Hepatocellular Carcinoma or
 

Neoplastic Nodule*3
 

P Values0
 

g


Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Stomach: Squamous-Cell Carcinoma"
 

P Values0
 

g


Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED)
 

CONTROL
 

0/49(0.00)
 

N.S.
 

P = 0.016
 

0/49(0.00)
 

P = 0.048
 

P = 0.001
 

0/49(0.00)
 

P < 0.001
 

P = 0.028
 

LOW
 

DOSE
 

8/48(0.17)
 

P = 0.003
 

Infinite
 

2.334
 

Infinite
 

84
 

12/48(0.25)
 

P < 0.001
 

Infinite
 

3.747
 

Infinite
 

84
 

19/48(0.40)
 

P < 0.001
 

Infinite
 

6.245
 

Infinite
 

73
 

HIGH
 

DOSE
 

4/43(0.09)
 

P = 0.044
 

Infinite
 

1.058
 

Infinite
 

72
 

5/43(0.12)
 

P = 0.020
 

Infinite
 

1.440
 

Infinite
 

67
 

17/38(0.45)
 

P < 0.001
 

Infinite
 

7.024
 

Infinite
 

58
 
TABLE

TOPOGRAPHY : MORPHOLOGY
 

Stomach: Squamous-Cell Carcinoma or
 

Squamous-Cell Papilloma
 

P Values0
 

Departure from Linear Trend 

£
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Stomach: Carcinoma In Situ NOS
 

UJ 

O P Values0 

Departure from Linear Trend 

£
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma NOS or Chromophobe
 

Adenoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

3 (CONTINUED) 

CONTROL
 

0/49(0.00)
 

P < 0.001
 

P < 0.001
 

0/49(0.00)
 

N.S.
 

P = 0.039
 

7/45(0.16)
 

P = 0.016(N)
 

78
 

LOW
 

DOSE
 

32/48(0.67)
 

P < 0.001
 

Infinite
 

10.995
 

Infinite
 

62
 

4/48(0.08)
 

N.S.
 

Infinite
 

0.947
 

Infinite
 

71
 

3/38(0.08)
 

N.S.
 

0.508
 

0.090
 

2.048
 

73
 

HIGH
 

DOSE
 

24/38(0.63)
 

P < 0.001
 

Infinite
 

10.279
 

Infinite
 

45
 

1/38(0.03)
 

N.S.
 

Infinite
 

0.069
 

Infinite
 

50
 

0/31(0.00)
 

P = 0.021(N)
 

0.000
 

0.000
 

0.735
 
TOPOGRAPHY :MORPHOLOGY
 

Adrenal: Pheochromocytoma or
 

Pheochromocytoma, Malignant
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma"3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

CONTROL
 

3/50(0.06)
 

N.S.
 

_—
 

78
 

2/37(0.05)
 

N.S.
 

109
 

3/37(0.08)
 

N.S.
 

_—
 

109
 

LOW
 

DOSE
 

6/48(0.13)
 

N.S.
 

2.083
 

0.474
 

12.229
 

80
 

0/34(0.00)
 

N.S.
 

0.000
 

0.000
 

3.634
 

0/34(0.00)
 

N.S.
 

0.000
 

0.000
 

1.784
 

HIGH
 

DOSE
 

6/36(0.17)
 

N.S.
 

2.778
 

0.636
 

16.051
 

65
 

1/31(0.03)
 

N.S.
 

0.597
 

0.010
 

10.873
 

67
 

1/31(0.03)
 

N.S.
 

0.398
 

0.008
 

4.643
 

67
 
TABLE

TOPOGRAPHY : MORPHOLOGY
 

Testis: Interstitial-Cell Tumor
 

Q
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Cerebrum: Astrocytoma
 

P Values0
 

Relative Risk (Control)
 u> 

t-o Lower Limit 

Upper Limit 

Weeks to First Observed Tumor
 

Body Cavities: Malignant Mesothelioma
 

or Mesothelioma NOS^1
 

P Values0
 

Q
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

3 (CONTINUED) 

CONTROL
 

42/50(0.84)
 

P < O.OOl(N)
 

78
 

0/50(0.00)
 

N.S.
 

0/50(0.00)
 

P = 0.350
 

P = 0.011
 

LOW
 

DOSE
 

27/47(0.57)
 

P = 0.004(N)
 

0.684
 

0.535
 

0.911
 

67
 

2/44(0.05)
 

N.S.
 

Infinite
 

0.336
 

Infinite
 

71
 

5/49(0.10)
 

P = 0.027
 

Infinite
 

1.287
 

Infinite
 

84
 

HIGH
 

DOSE
 

8/36(0.22)
 

P < O.OOl(N)
 

0.265
 

0.145
 

0.467
 

59
 

1/38(0.03)
 

N.S.
 

Infinite
 

0.070
 

Infinite
 

58
 

1/44(0.02)
 

P = 0.468
 

Infinite
 

0.061
 

Infinite
 

72
 
TABLE 3 (CONCLUDED)
 

Treated groups received time-weighted average doses of 0.15 or 0.30 percent in feed.
 

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

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

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

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

6
The probability level of the test for departure from linear trend is given beneath the control
 

group when P < 0.05.
 
TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH CUPFERRON*
 

TOPOGRAPHY : MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Subcutaneous Tissue: Sarcoma NOS or
 

Fibrosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma°
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

2/49(0.04)
 

N.S.
 

P = 0.048
 

108
 

0/49(0.00)
 

N.S.
 

_—
 

7/49(0.14)
 

P = 0.006(N)
 

96
 

LOW
 

DOSE
 

6/45(0.13)
 

N.S.
 

3.267
 

0.621
 

31.720
 

76
 

3/45(0.07)
 

N.S.
 

Infinite
 

0.656
 

Infinite
 

76
 

3/45(0.07)
 

N.S.
 

0.467
 

0.082
 

1.904
 

77
 

HIGH
 

DOSE
 

2/47(0.04)
 

N.S.
 

1.043
 

0.078
 

13.860
 

75
 

1/47(0.02)
 

N.S.
 

Infinite
 

0.056
 

Infinite
 

67
 

0/47(0.00)
 

P = 0.008(N)
 

0.000
 

0.000
 

0.536
 
TOPOGRAPHY: MORPHOLOGY
 

Circulatory System: Hemangiosarcoma
 

P Values0
 

Q
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

OJ P Values0 

Ul £ 

Departure from Linear Trend 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma or
 

Neoplastic Nodule"
 

P Values0
 

g


Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED) 

CONTROL
 

0/49(0.00)
 

P < 0.001
 

P = 0.009
 

1/48(0.02)
 

P = 0.012
 

P < 0.001
 

98
 

1/48(0.02)
 

P = 0.004
 

P < 0.001
 

98
 

LOW
 

DOSE
 

28/45(0.62)
 

P < 0.001
 

Infinite
 

10.180
 

Infinite
 

62
 

24/44(0.55)
 

P < 0.001
 

26.180
 

4.657
 

1024.000
 

79
 

26/44(0.59)
 

P < 0.001
 

28.360
 

5.104
 

1100.000
 

79
 

HIGH
 

DOSE
 

37/47(0.79)
 

P < 0.001
 

Infinite
 

13.240
 

Infinite
 

43
 

10/44(0.23)
 

P = 0.002
 

10.910
 

1.657
 

459.900
 

70
 

12/44(0.23)
 

P < 0.001
 

13.090
 

2.074
 

542.600
 

70
 
___ 

___ 

TOPOGRAPHY : MORPHOLOGY
 

Stomach: Carcinoma In Situ NOS
 

P Values0
 

e
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Stomach: Squamous-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Stomach: Squamous-Cell Carcinoma or
 

Squamous-Cell Papilloma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED)
 

CONTROL
 

0/49(0.00)
 

N.S.
 

P = 0.011
 

0/49(0.00)
 

P < 0.001
 

0/49(0.00)
 

P < 0.001
 

LOW
 

DOSE
 

3/43(0.07)
 

N.S.
 

Infinite
 

0.686
 

Infinite
 

81
 

14/43(0.33)
 

P < 0.001
 

Infinite
 

4.986
 

Infinite
 

76
 

19/43(0.44)
 

P < 0.001
 

Infinite
 

6.990
 

Infinite
 

76
 

HIGH
 

DOSE
 

0/43(0.00)
 

N.S.
 

22/43(0.51)
 

P < 0.001
 

Infinite
 

8.205
 

Infinite
 

60
 

24/43(0.56)
 

P < 0.001
 

Infinite
 

9.022
 

Infinite
 

60
 
___ 

___ 

___ 

TABLE 4 (CONTINUED)
 

TOPOGRAPHY: MORPHOLOGY
 

Pituitary: Adenoma NOS or Chromophobe
 

Adenoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma or Pheochromocytoma, Malignant
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

17/39(0.44)
 

P < O.OOl(N)
 

78
 

3/49(0.06)
 

N.S.
 

110
 

12/49(0.24)
 

P = 0.003(N)
 

103
 

LOW
 

DOSE
 

4/35(0.11)
 

P = 0.002(N)
 

0.262
 

0.072
 

0.710
 

76
 

7/41(0.17)
 

N.S.
 

2.789
 

0.684
 

15.720
 

97
 

10/45(0.22)
 

N.S.
 

0.907
 

0.390
 

2.056
 

93
 

HIGH
 

DOSE
 

0/34(0.00)
 

P < O.OOl(N)
 

0.000
 

0.000
 

0.208
 

6/42(0.14)
 

N.S.
 

2.333
 

0.533
 

13.603
 

79
 

1/47(0.02)
 

P = 0.002(N)
 

0.087
 

0.002
 

0.550
 

106
 
TABLE

TOPOGRAPHY :MORPHOLOGY
 

Nervous System/Brain: Astrocytoma, Oligodendroglioma, or Glioma NOS^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Zymbal's Gland: Ceruminous Carcinoma
 

u> P Values0 

oo 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Zymbal's Gland: Squamous-Cell Carcinoma or
 

Ceruminous Carcinoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

4 (CONTINUED) 

CONTROL
 

1/49(0.02)
 

N.S.
 

98
 

1/49(0.02)
 

N.S.
 

80
 

1/49(0.02)
 

N.S.
 

80
 

LOW
 

DOSE
 

5/43(0.12)
 

N.S.
 

5.698
 

0.673
 

262.570
 

88
 

4/45(0.09)
 

N.S.
 

4.356
 

0.453
 

209.400
 

67
 

5/45(0.11)
 

N.S.
 

5.444
 

0.642
 

251.270
 

76
 

HIGH
 

DOSE
 

3/42(0.07)
 

N.S.
 

3.500
 

0.294
 

179.151
 

52
 

2/47(0.04)
 

N.S.
 

2.085
 

0.112
 

120.400
 

54
 

4/47(0.09)
 

N.S.
 

4.170
 

0.433
 

200.705
 

54
 
TABLE 4 (CONCLUDED)
 

Treated groups received time-weighted average doses of 0.15 or 0.30 percent in feed.
 

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

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

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

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

6

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

group when P < 0.05.
 
both high and low dose to control group. Based on these results, the
 

administration of cupferron was associated with an increased incidence
 

of squamous-cell carcinomas of the stomach in both male and female
 

rats.
 

In both male and female rats numerous stomach squamous-cell carcinomas were observed. For both sexes the Cochran-Armitage test
 

was significant (P < 0.001). In both sexes these results were
 

supported by significant (P < 0.001) Fisher exact test comparisons of
 

both high and low dose to control group. Based on these results, the
 

administration of cupferron was associated with an increased incidence
 

of squamous-cell carcinomas of the stomach in both male and female
 

rats.
 

In both male and female rats increased incidences of liver neoplasms were observed. When incidences were combined so that the numerator represented rats with either a hepatocellular carcinoma or a
 

neoplastic nodule, for male rats the Cochran-Armitage test indicated
 

a significant (P = 0.048) positive association between dose and incidence. The Fisher exact: test supported these findings with significant comparisons of both high dose (P = 0.020) and low dose (P <
 

0.001) to control. Similarly, for females the Cochran-Armitage test
 

on the combined incidence of hepatocellular carcinomas and neoplastic
 

nodules of the liver was significant (P = 0.012) and both the high
 

dose (P = 0.002) and low dose (P < 0.001) Fisher exact test comparisons were significant. Based on these results, the administration of
 

40
 
cupferron was associated with the increased incidence of liver
 

neoplasms in both male and female rats.
 

For male rats the Fisher exact test indicated a significantly
 

(P < 0.001) higher incidence of fibromas of the subcutaneous tissue
 

in the low dose than in the control. The Cochran-Armitage test and
 

the comparison of high dose to control, however, were not significant.
 

In historical control data compiled by this laboratory for the NCI
 

Carcinogenesis Testing Program, 11/250 (4 percent) of the untreated
 

male Fischer 344 rats had this tumor.
 

The combined incidence of squamous-cell carcinomas or ceruminous
 

carcinomas of the Zymbal's gland in female rats, although increased
 

in the dosed groups compared to the controls, did not yield significant results using the Fisher exact or Cochran-Armitage tests.
 

They were, however, rare tumors occurring in only 0.8 percent (2/249)
 

of the untreated historical control female Fischer 344 rats. The
 

incidence rates observed under the conditions of this experiment
 

were 11 percent (5/45) for the low dose group and 9 percent (4/47)
 

for the high dose group—compared to 2 percent (1/49) in the control
 

group. When a binomial distribution with a spontaneous incidence
 

rate of 2/249 was assumed, the probability of observing 5 or more
 

animals with such a tumor out of 45 animals (as in the low dose
 

female group) was P < 0.0001 and the probability of observing 4
 

animals with such a tumor out of 47 animals (as in the high dose
 

female group) was P < 0.0006, both significant results.
 

41
 
For male rats the low dose to control Fisher exact comparison
 

of the combined incidences of mesothelioma NOS and malignant mesothelioma of the body cavities had a probability level of P = 0.027,
 

a marginal result which was not significant under the Bonferroni
 

criterion.
 

A number of possible negative associations between dose and incidence were observed: leukemia/malignant lymphomas, interstitial-


cell tumors of the testis, and pituitary adenomas in the males, plus
 

leukemia/malignant lymphomas, mammary fibroadenomas, and pituitary
 

adenomas in the females. Since all of these tumors are relatively
 

late-developing tumors it is possible that these results merely reflected the higher early morality noted in dosed rats of both sexes.
 

A rare tumor—a ganglioneuroma of the adrenal gland or adrenal
 

medulla—was observed in 2/36 high dose males. In historical data,
 

none of the 250 untreated males had this tumor. When a binomial
 

distribution with a spontaneous incidence rate of 1/251 was assumed
 

(the most conservative approach), the probability of observing 2 or
 

more rats with such a tumor out of 36 males was P < 0.009, a significant result. In other historical data of interest 32/250 (13
 

percent) of the males and 16/249 (6 percent) of the females had an
 

adrenal pheochromocytoma or a malignant pheochromocytoma; none of
 

the 249 historical control females had a glioma of the brain or an
 

adrenal ganglioneuroma.
 

Thus, the statistical results indicated that the administration of cupferron was associated with the increased incidence of
 

42
 
hemangiosarcomas, squamous-cell carcinomas of the stomach, and liver
 

neoplasms in both male and female rats—and possibly was associated
 

with the increased incidence of ganglioneuromas of the adrenal gland
 

or adrenal medulla in males and of carcinomas of the Zymbal's gland
 

in females.
 

43
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Distinct compound-related mean body weight depression was evident in both male and female dosed mice when compared to controls
 

(Figure 4). Fluctuations in the growth curve may be due to mortality; as the size of the group diminishes, the mean body weight may
 

be subject to wide variations.
 

No clinical abnormalities were reported for dosed or control
 

mice of either sex.
 

B. Survival
 

The estimated probabilities of survival for male and female
 

mice in the control and cupferron-dosed groups are shown in Figure 5.
 

The Tarone test indicated a significant positive association between
 

dosage and mortality for both male (P < 0.001) and female (P = 0.009)
 

mice.
 

For males, five mice were sacrificed from the high dose group in
 

week 78 and five from the control group in week 80. Adequate numbers
 

of males were at risk from late-developing tumors as 62 percent
 

(31/50) of the high dose, 88 percent (44/50) of the low dose, and 98
 

percent (49/50) of the control mice survived on test at least 75
 

weeks.
 

For females five mice were sacrificed from the high dose group
 

in week 78 and five from the control group in week 80. With 58
 

percent (29/50) of the high dose, 68 percent (34/50) of the low dose
 

44
 
50- -50 

40 — — 40 

< 

tr 

— 30 

I 

13 

>­

Q 

O 

 20­ — 20 

CQ 

< 

LU 

10 — LOW DOSE —10 

MALE MICE _ ^^_ HIGH DOSE 

I \ ' 1 < I \ I ' 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

50- -50 

40- -40 

< 

£C 

-30 

O 

O 

CO 

CONTROL 

10 — LOW DOSE —10 

FEMALE MICE HIGH DOSE 

I ' I > I I 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR CUPFERRON CHRONIC STUDY MICE 

45 
PROBABILIT

Y OF SURVIVAL 

|­

m 

O 

m 

c

•33 <I

8 

1 30 

\ m V) O O o m CO 30 Ul m 2) m O 

O 

I 3D 

O 

O 

C/l 

O 

O 

m 

sH 

PROBABILITY OF SURVIV

O kj * 0> 

O O ° O 

1 1 1 1 1 1 1 

> r~ 

m 

S O 

in 

m

en 

s­

- 

=! £-

5 m 

O

z 

m 

% 8­ 

§ 

m 

m ^ 

C/5 ^j 

Ol J 

1 

1 

rf 

sj

T r 

I f ,./ 

| 

s-

x f— n

1 i i 

-

° s * 0 g 0 S m *" 

r

O o — 

1 1 1 1 1 1 1 

c3 O O O 

c3 Kj ^ Ol 
and 80 percent (40/50) of the controls surviving on test until the
 

termination of the study, adequate numbers of female mice were at
 

risk from late-developing tumors.
 

C. Pathology
 

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).
 

Dosed mice had increased but relatively low incidences of
 

hepatocellular neoplasms, hemangiomas and hemangiosarcomas, Harderian
 

gland adenomas, and tumors of the Zymbal's gland.
 

The incidence of hepatocellular carcinoma was considerably
 

higher in dosed female mice (13/45 [29 percent] high dose, 9/46 [20
 

percent] low dose) than in the controls (2/49 [4 percent]). This
 

increase was not seen in dosed male mice. The hepatocellular carcinomas were cellular, pleomorphic masses of hepatocytes devoid of
 

any architectural arrangement and infiltrating the adjoining parenchyma.
 

In each sex, a few hepatocellular carcinomas had metastasized to the
 

lungs. Hepatocellular adenomas were generally spherical noninfiltrating expanding nodules of well-differentiated hepatocytes which
 

moderately compressed adjacent hepatic cords. In some the cells were
 

smaller than normal and hyperbasophilic while others had cells
 

larger than normal with eosinophilic cytoplasmic vacuolation.
 

Hepatocytes were arranged in thickened trabecular and sheet-like
 

growth patterns. Mitotic figures and small bile ducts were rare.
 

47
 
The incidence of hemangiosarcomas was elevated in the high dose
 

male mice (7/40 [18 percent]), low dose female mice (5/47 [11 percent]), and high dose female mice (6/46 [13 percent]) with 1/50 (2
 

percent) in control males and 1/50 (2 percent) in control females.
 

The number of hemangiomas was elevated in low dose females (5/47 [11
 

percent]; controls 0/50). The majority of hemangiosarcomas were
 

found in the spleen, and although histologically malignant, there was
 

only one instance of pulmonary metastasis. The hemangiosarcomas were
 

characterized by irregular blood spaces with ingrowths of pleomorphic
 

lining cells. Some tumors had large blood-filled spaces, while
 

others had extensive areas of solid spindle-shaped sarcomatous cells.
 

Mitotic figures were numerous. Hemangiomas tended to have blood
 

spaces bordered by several layers of cells with uniform size and
 

shape of the lining epithelium. Mitotic figures were rare in hemangiomas. These tumors were also more circumscribed than their malignant
 

counterparts.
 

Neoplasms of the Zytnbal's gland were found in 3/47 (6 percent)
 

high dose females and in 1/45 (2 percent) high dose males; none were
 

detected in controls.
 

The number of adenomas of the Harderian gland was elevated in
 

the low dose (3/45 [7 percent]) and high dose (4/40 [10 percent])
 

males and the low dose (2/47 [4 percent]) and high dose (6/46 [13
 

percent]) females as compared with controls (female 0/50; male 0/50).
 

These were well-differentiated, circumscribed expanding tumors with
 

48
 
a papillary glandular structure composed of epithelial cells closely
 

resembling the normal glandular epithelium. No mitoses were seen.
 

There were numerous inflammatory and degenerative lesions
 

noted, as expected, in aging mice. None were judged to be related to
 

the administration of cupferron.
 

The results of this histopathologic examination indicate that
 

cupferron induced hemangiomas, hepatocellular carcinomas, and Zymbal's
 

gland carcinomas in female mice, and adenomas of the Harderian gland
 

and hemangiosarcomas in both males and females. Therefore, the
 

compound is considered to be carcinogenic in B6C3F1 mice.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

mice are summarized in Tables 5 and 6. The analysis is included for
 

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

observed in at least one of the control or cupferron-dosed groups and
 

where such tumors were observed in at least 5 percent of the group.
 

In both male and female mice numerous hemangiosarcomas were
 

observed. The Cochran-Armitage test indicated a significant positive
 

association between dose and tumor incidence for both males (P =
 

0.008) and females (P = 0.038). For males the Fisher exact test
 

supported these findings with a significant (P = 0.013) comparison of
 

the high dose group to the control. For females when incidences were
 

combined so that the numerator represented mice with either a hemangiosarcoma or a hemangioma, the Fisher exact test comparison of low
 

49
 
TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH CUPFERRONJ
 

LOW
 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE
 

Skin and Subcutaneous Tissue: Squamous-


Cell Carcinomab 0/50(0.00) 3/45(0.07)
 

P Values N.S. N.S.
 

Relative Risk (Control) Infinite
 

Lower Limit 0.669
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 95
 Ln
 

Subcutaneous Tissue: Sarcoma NOS 0/50(0.00) 1/45(0.02)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) Infinite
 

Lower Limit 0.060
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 96
 

Lung: Alveolar/Bronchiolar Carcinoma 3/47(0.06) 0/44(0.00)
 

P Values0 P = 0.047(N) N.S.
 

Relative Risk (Control) 0.000
 

Lower Limit 0.000
 

Upper Limit 1.769
 

Weeks to First Observed Tumor 98
 

HIGH
 

DOSE
 

2/40(0.05)
 

N.S.
 

Infinite
 

0.370
 

Infinite
 

0/40(0.00)
 

N.S.
 

0/40(0.00)
 

N.S.
 

0.000
 

0.000
 

1.941
 

78 
_ __ 

_ __ 

TABLE

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/ Bronchiolar Adenoma or
 

Alveolar/Bronchiolar Carcinoma*3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Circulatory System: Hemangiosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

5 (CONTINUED) 

CONTROL
 

7/47(0.15)
 

N.S.
 

98
 

1/50(0.02)
 

P = 0.008
 

—
 

98
 

15/49(0.31)
 

N.S.
 

94
 

LOW
 

DOSE
 

8/44(0.18)
 

N.S.
 

1.221
 

0.422
 

3.620
 

79
 

3/45(0.07)
 

N.S.
 

3.333
 

0.279
 

171.000
 

87
 

9/44(0.20)
 

N.S.
 

0.668
 

0.287
 

1.454
 

79
 

HIGH
 

DOSE
 

5/40(0.13)
 

N.S.
 

0.839
 

0.226
 

2.819
 

87
 

7/40(0.18)
 

P = 0.013
 

8.750
 

1.194
 

382.700
 

70
 

8/40(0.20)
 

N.S.
 

0.653
 

0.267
 

1.459
 

86
 
TABLE 5 (CONCLUDED) 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Harderian Gland:

P Values0 

 Adenoma NOS 0/50(0.00) 

P = 0.028 

3/45(0.07) 

N.S. 

4/40(0.10) 

P = 0.036 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Infinite 

0.669 

Infinite 

Infinite 

1.161 

Infinite 

Weeks to First Observed Tumor 95 95 

Treated groups received time-weighted average doses of 0.2 or 0.4 percent in feed. 

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

Q

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

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability 

level for the Fisher exact test for the comparison of a treated group with the control group is 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group. 

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

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

group when P < 0.05.
 
___ 

___ 

TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

a
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH CUPFERRON
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Adenoma or
 Ul Alveolar/Bronchiolar Carcinoma^5
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

3/50(0.06)
 

N.S.
 

80
 

4/50(0.08)
 

N.S.
 

80
 

6/50(0.12)
 

N.S.
 

98
 

LOW
 

DOSE
 

3/45(0.07)
 

N.S.
 

1.111
 

0.156
 

7.893
 

84
 

11/45(0.24)
 

P = 0.027
 

3.056
 

0.983
 

12.240
 

84
 

7/47(0.15)
 

N.S.
 

1.241
 

0.385
 

4.146
 

87
 

HIGH
 

DOSE
 

1/46(0.02)
 

N.S.
 

0.362
 

0.007
 

4.318
 

95
 

9/46(0.20)
 

N.S.
 

2.446
 

0.737
 

10.160
 

79
 

4/46(0.09)
 

N.S.
 

0.725
 

0.160
 

2.852
 

94
 
___ 

___ 

__ _ 

TOPOGRAPHY :MORPHOLOGY
 

Circulatory System: Hemangiosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Circulatory System: Hemangiosarcoma
 

or Hemangioma"3
 

P Values0
 

g
 Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 6 (CONTINUED) 

CONTROL
 

1/50(0.02)
 

P - 0.038
 

98
 

1/50(0.02)
 

N.S.
 

P = 0.017
 

98
 

2/49(0.04)
 

P = 0.001
 

98
 

LOW
 

DOSE
 

5/47(0.11)
 

N.S.
 

5.319
 

0.627
 

245.700
 

84
 

10/47(0.21)
 

P = 0.003
 

10.638
 

1.610
 

449.503
 

84
 

9/46(0.20)
 

P = 0.019
 

4.794
 

1.063
 

43.590
 

79
 

HIGH
 

DOSE
 

6/46(0.13)
 

P = 0.044
 

6.522
 

0.836
 

292.800
 

64
 

6/46(0.13)
 

P = 0.044
 

6.522
 

0.836
 

292.687
 

64
 

13/45(0.29)
 

P = 0.001
 

7.078
 

1.730
 

61.400
 

74
 
TOPOGRAPHY : MORPHOLOGY
 

Liver: Hepatocellular Carcinoma or
 

Hepatocellular Adenoma"3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Ovary: Tubular-Cell Adenoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Harderian Gland: Adenoma NOS
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 6 (CONTINUED) 

CONTROL
 

2/49(0.04)
 

P < 0.001
 

98
 

0/48(0.00)
 

N.S.
 

0/50(0.00)
 

P = 0.006
 

_—
 

LOW
 

DOSE
 

12/46(0.26)
 

P = 0.002
 

6.391
 

1.533
 

56.028
 

79
 

1/37(0.03)
 

N.S.
 

Infinite
 

0.070
 

Infinite
 

96
 

2/47(0.04)
 

N.S.
 

Infinite
 

0.315
 

Infinite
 

96
 

HIGH
 

DOSE
 

16/45(0.36)
 

P < 0.001
 

8.711
 

2.222
 

73.866
 

74
 

3/41(0.07)
 

N.S.
 

Infinite
 

0.706
 

Infinite
 

95
 

6/46(0.13)
 

P = 0.010
 

Infinite
 

1.740
 

Infinite
 

87
 
___ 

___ 

TABLE 6 (CONCLUDED) 

LOW HIGH
 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE
 

Ovary: Granulosa-Cell Tumor 0/48(0.00) 2/37(0.05) 1/41(0.02)
 

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

Relative Risk (Control) Infinite Infinite
 

Lower Limit 0.385 0.063
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 96 95
 

Zymbal's Gland: Squamous-Cell Carcinoma
 

or Sebaceous Adenocarcinoma" 0/50(0.00) 0/47(0.00) 3/46(0.07)
 

Ui
 P Values0 P = 0.033 N.S. N.S.
 

Relative Risk (Control) Infinite
 

Lower Limit 0.654
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 87
 

Treated groups received time-weighted average doses of 0.2 or 0.4 percent in feed.
 

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

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

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

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

e

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

group when P < 0.05.
 
dose to control was significant (P = 0.003). The comparison of high
 

dose to control had a probability level of P = 0.044, a marginal result which was not significant under the Bonferroni criterion. Based
 

on these results, administration of cupferron was associated with an
 

increased incidence of hemangiosarcomas in male and either hemangiosarcomas or hemangiomas in female mice.
 

For female mice the incidence of hepatocellular carcinomas was
 

increased in the dosed groups compared to the control group. The
 

Cochran-Armitage test showed a significant (P = 0.001) positive
 

association between dose and tumor incidence compared to the control.
 

This was supported by significant Fisher exact test results comparing the incidences in the high dose (P = 0.001) and the low dose
 

(P = 0.019) to that in the control. Based upon these results the
 

administration of cupferron was associated with an increased incidence
 

of hepatocellular carcinomas in female mice.
 

In both male and female mice increased incidences of Harderian
 

gland adenomas NOS were also observed in both dosed groups. For
 

females the Cochran-Armitage test showed a significant (P = 0.006)
 

positive association between chemical administration and tumor incidence. This was supported by a significant (P = 0.010) Fisher exact
 

test comparison of the high dose group to the control. For male mice
 

the Cochran-Armitage test was significant (P = 0.028), but the Fisher
 

exact tests were not significant under the Bonferroni criterion.
 

Based on these results the administration of cupferron was associated
 

57
 
with an increased incidence of Harderian gland adenomas in female
 

mice.
 

In female mice an increase in the combined incidence of squamouscell carcinomas or sebaceous adenocarcinomas of the Zymbal's gland
 

was observed in the high dose group. The Cochran-Armitage test
 

showed a significant (P = 0.033) positive association between dosage
 

and tumor incidence, but Fis-her exact tests were not significant.
 

These rare tumors did not occur in any of the untreated 275 historical control B6C3F1 female mice observed by Mason Research Institute
 

for the NCI Carcinogenesis Testing Program. When a binominal distribution with a 1/276 probability of spontaneous incidence was assumed
 

(the most conservative approach), the probability of observing 3 mice
 

with such tumors out of 46 female mice was P < 0.0007, a significant
 

result.
 

For male mice the Cochran-Armitage test showed a significant
 

(P = 0.047) negative trend for alveolar/bronchiolar carcinoma, but
 

the Fisher exact tests were not significant. For alveolar/bronchiolar
 

neoplasms in the females the comparison of low dose to control was
 

P = 0.027, a marginal result which was not significant under the
 

Bonferroni criterion.
 

Thus, the statistical results indicated that the administration
 

of cupferron was associated with an increased incidence of hemangiosarcomas in male mice and of hepatocellular carcinomas, of Harderian
 

58
 
gland adenomas, of hemangiosarcomas or hemangiomas, and possibly of
 

carcinomas of the Zymbal's gland in female mice.
 

59
 
V. DISCUSSION
 

Among both sexes of rats and mice there was a significant positive association between the dosage of cupferron administered and
 

mortality which could be attributed to tumor formation.
 

For male and female rats there was a significant positive association between dosage and the incidences of circulatory hemangiosarcomas (i.e., 0/50, 38/49 [78 percent], and 35/44 [80 percent] in the
 

control, low dose, and high dose males, respectively, and 0/49, 28/45
 

[62 percent], and 37/47 [79 percent] in the control, low dose, and
 

high dose females). For both sexes the high dose to control and low
 

dose to control Fisher exact comparisons were also significant.
 

Squamous-cell carcinomas of the forestomach were observed at increased incidences in the dosed rats (i.e., 0/49, 19/48 [40 percent],
 

and 17/38 [45 percent] in the control, low dose, and high dose males,
 

respectively, and 0/49, 14/43 [33 percent], and 22/43 [51 percent] in
 

the control, low dose, and high dose females). The Cochran-Armitage
 

tests for both sexes were significant and all the Fisher exact comparisons supported these findings. In addition to the squamous-cell
 

carcinomas of the forestomach, squamous-cell papillomas and basal-cell
 

hyperplasias of the forestomach were observed at increased incidences
 

in the dosed rats when compared to controls, further strengthening
 

the association between abnormalities of the squamous epithelium of
 

the forestomach and administration of cupferron.
 

60
 
Hepatocellular carcinomas, relatively uncommon tumors in Fischer
 

344 rats (i.e, 0/250 and 1/249 observed in the Mason Research Institute
 

historical control male and female rats, respectively), were detected
 

in 0/49, 8/48 (17 percent), and 4/43 (9 percent) of the control, low
 

dose, and high dose males, respectively, and in 1/48 (2 percent),
 

24/44 (55 percent), and 10/44 (23 percent) of the control, low dose,
 

and high dose females. When the rats having hepatocellular carcinomas
 

were combined with rats having neoplastic nodules and the resulting
 

incidences (i.e., 0/49, 12/48 [25 percent], and 5/43 [12 percent]
 

control, low dose, and high dose males, respectively, and 1/48 [2 percent], 26/44 [59 percent], and 12/44 [23 percent] control, low dose,
 

and high dose females) analyzed, the Cochran-Armitage tests for both
 

sexes provided significant positive associations between dosage and
 

incidence. High dose to control and low dose to control Fisher exact
 

comparisons for each sex supported the relationship.
 

When female rats with squamous-cell carcinoma of the auditory
 

sebaceous gland (i.e., 0/49, 1/45 [2 percent], and 2/47 [4 percent]
 

for the control, low dose, and high dose, respectively) were combined
 

with those having ceruminous carcinoma (i.e., 1/49 [2 percent], 4/45
 

[9 percent], and 2/47 [4 percent] for the control, low dose, and high
 

dose, respectively), no statistically significant results were provided using either the Cochran-Armitage test or the Fisher exact test.
 

However, when a binominal distribution and a spontaneous incidence
 

rate corresponding to the appropriate historical control incidence
 

61
 
were assumed, the incidences of neoplasms of the auditory sebaceous
 

gland observed in this bioassay were significant.
 

In addition, several uncommon tumors were observed in male
 

and female rats, further strengthening the evidence for carcinogenic
 

action of cupferron in rats.
 

For female mice hepatocellular carcinomas were observed in
 

2/49 (4 percent), 9/46 (20 percent), and 13/45 (29 percent) of the
 

control, low dose, and high dose groups, respectively. Statistical
 

analysis indicated a significant positive association between dosage
 

and tumor incidence and both the high dose to control and low dose to
 

control Fisher exact comparisons reinforced the finding.
 

Hemangiosarcomas occurred in mice of both sexes. For males and
 

females there was a statistically significant positive association
 

between the cupferron dosages administered and tumor incidence. For
 

males these results were supported by the high dose to control Fisher
 

exact test. For females only the low dose to control Fisher exact
 

comparison was significant and that occurred only when the female
 

rats with circulatory hemangiosarcomas and/or hemangiomas were
 

combined and the resulting tumor incidences analyzed.
 

Adenomas of the Harderian gland were observed in dosed male
 

and female mice. For both sexes the Cochran-Armitage test was significantly positive. The high dose to control Fisher exact comparison supported this finding for the females. The same was not true,
 

however, for males.
 

62
 
When female mice with squamous-cell carcinoma of the auditory
 

sebaceous gland (i.e., 0/50, 0/47, 2/46 [4 percent] for the control,
 

low dose, and high dose groups, respectively) were combined with
 

those having sebaceous adenocarcinoma (i.e., 0/50, 0/47, and 1/46
 

[2 percent] for the control, low dose, and high dose groups, respectively), no statistically significant results were provided using
 

either the Cochran-Armitage test or the Fisher exact test. However,
 

when a binominal distribution and a spontaneous incidence rate
 

corresponding to the appropriate historical control incidence were
 

assumed, the incidences of auditory neoplasms observed in this bioassay were significant.
 

Due to the extensive carcinogenic effect on multiple tissues,
 

it must be considered that dose-related trends and incidences of
 

tumors at a particular site may have been modulated by competing or
 

enhancing effects from several types and sites of tumors in both rats
 

and mice.
 

Under the conditions of this bioassay cupferron was carcinogenic
 

in Fischer 344 rats, causing hemangiosarcomas, hepatocellular carcinomas, and squamous-cell carcinomas of the forestomach in males and
 

females as well as carcinomas of the auditory sebaceous gland in
 

females. The chemical was also carcinogenic in B6C3F1 mice, causing
 

hemangiosarcomas in males and hepatocellular carcinomas, carcinomas
 

of the auditory sebaceous gland, a combination of hemangiosarcomas
 

and hemangiomas, and adenomas of the Harderian gland in females.
 

63
 
VI. BIBLIOGRAPHY
 

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

J. Wiley & Sons, New York, 1971.
 

Bamberger, E. and 0. Baudisch, "Oxydation Normaler Diazotate mit
 

Hydroperoxyd." Berichte der Deutschen Chemischen Gesellschaft
 

45_: 35 68-3582, 1909.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS)
 

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

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

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

Co., Ltd., London, 1970.
 

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

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

Davis, H.L., "The Effect of Cupferron on Recovery from Radiation Damage in Tradescantia Microspores." Radiation Botany 10:191-198,
 

1969.
 

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

Tests, Confidence Limits, and Adjustments for Stratification."
 

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

Gilot-Delhalle, J., R. Thakare, and J. Moutschen, "Fast Rejoining
 

Processes in Nigella Damascena Chromosomes Revealed by Fractionated 60CO Y-Ray Exposures." Radiation Botany 13:229-242, 1973.
 

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

Observations." Journal of the American Statistical Association
 

_53_:457-481, 1958.
 

Kihlman, B.A. "The Effect of Oxygen, Nitric Oxide, and Respiratory
 

Inhibitors on the Production of Chromosome Aberrations by X-Rays."
 

Experimental Cell Research 14:639-642, 1958.
 

Kihlman, B.A., "Studies on the Production of Chromosomal Aberrations
 

by Visible Light: The Effects of Cupferron, Nitric Oxide, and
 

Wavelength." Experimental Cell Research 17:590-593, 1959.
 

64
 
Linhart, M.S., J.A. Cooper, R.L. Martin, N.P. Page, and J.A. Peters,
 

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

Matolcsy, G., M. Hamran, and B. Bordas, "Increased Antifungal Action
 

of Zinc Dimethyldithiocarbamate in the Presence of Complex Forming Compounds." Pesticides Science 2:229-231, 1971.
 

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

Co., New York, 1966.
 

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

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

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

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

Schlecten, A.W., and A.P. Thompson, "Zinc and Zinc Alloys." Encyclopedia of Chemical Technology, 2nd edition, Volume 22. R.E. Kirk
 

and D.F. Othmer, editors. John Wiley and Sons, Inc., New York,
 

1970; as cited in Urso, 1977.
 

Squire, R.A. and M.H. Levitt, "Report of a Workshop on Classification
 

of Specific Hepatocellular Lesions in Rats." Cancer Research
 

15:3214-3223, 1975.
 

Stanford Research Institute. 1977 Directory of Chemical Producers,
 

U.S.A. Menlo Park, California, 1977.
 

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

6^:679-682, 1975.
 

Urso, S., Research Analyst, Chemical-Environmental Program, Chemical
 

Industries Center, Stanford Research Institute. Menlo Park,
 

California. Personal communication, June 8, 1977.
 

Windholtz, M., editor, The Merck Index: An Encyclopedia of Chemicals
 

and Drugs, 9th edition. Merck and Company, Inc., Rahway, New
 

Jersey, 1976.
 

65
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH CUPFERRON
 
TABLE A1

 

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

CONTROL (UNTR) LOW DOSE KIGH DOSE
 

01-0160 01-0180 01-0185
 

ANIMALS INITIALLY III STUDY 50 50 3'50
 

ANIMALS NECROPSIED 50 49 44
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 50 49 44
 

INTIGDM2NTARY SYSTEM 

*SKIN 

SEBACEOUS ADENOCARCINOMA 

FIBR0.1A 

FIBHOSAHCOMA 

HEMANGIOSARCOMA 

(50)

1

1

 (2X) 

 (2X) 

 (49) 

1 <2X) 

1 (2X) 

(44) 

1

1

 (2%) 

 (2*) 

*SUBCDT TISSUE 

SARCOMA, NOS 

FIBROMA 

FIBROSARCOMA 

(50)

1

1

1

 (2X) 

 (2X)

 (2K)

 (49) 

 15

 1

 (31X) 

 (2X) 

(44) 

1

5

1

 (2S) 

 (11X) 

(2%) 

RESPIRATORY SYSTEM
 

*LUNG (49) (47) (43)
 

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

HEMANGIOSARCOMA, METASTATIC 2 (4X)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (50) (49) (44)
 

MALIGNANT LYMPHOMA, NOS 1 (2X)
 

LEUKEMIA, NOS 1 (2X)
 MYELOMONOCYTIC LEUKEMIA 9 (18X) 2 (4X) 1 (2X)
 

JSPLEEN (50) (46) (43)
 

HEMANGIOSARCOMA 38 (83X) 35 (81X)
 

tTRACHEAL LYMPH NODE (49) (43) (39)
 

C-CELL CARCINOMA, METASTATIC 1 (3«)
 

#B£DIASTINAL L.NODE (49) (43) (39)
 

* tiU«BER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED **EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

9 50 ANIMALS WERE INITIALLY IN THE STUDY, BUT ONE ANIMAL WAS FOUND TO BE A FEMALE IN A MALE GROUP.
 

A-3
 
TABLE Al (CONTINUED) 

CONTROL (UNTR ) LOU DOSE 

01-0160 01-0180 

CIRCULATOR Y SYSTEM 

*HEAR T (48) (U7)
 

SARCCBA, NOS, METASTATIC 1 (2%)
 

DIGESTIVE SYSTEM
 

tLIVER (49) (48)
 

NEOPLASTIC NODULS 4 (8X)
 HEPATOCSLLULAH CARCINOMA 8 (17%)
 

SARCOMA, NOS 1 (2%)
 

FIBHOSARCOMA, METASTATIC
 

•STOMACH	 (49) <«8)
 

CARCINOMA-IN-SITU, HOS 4 (8X)
 

SQUAMOUS CELL PAPILLOBA 16 (33X)
 

SQUAMOUS CELL CARCINOMA 19 (40%)
 

URINAR Y SYSTEM
 

NJN E
 

ENDOCRINE SYSTEM
 

tPITUITARY (45) (38)
 

ADENOMA, NOS 5 (11X) 3 (8X)
 

CHBOMOPHOBE ADENOMA 2 (UX)
 

#ADRENAL (50) (48)
 

EHEOCHROMOCYTOMA 3 (6X) 4 (8X)
 

PHEOCHROMOCYTOMA, MALIGNANT 2 (4X)
 

HEHANGIOSABCOHA 1 (2X)
 

jANGLIONEUROMA
 

#ADRENAL MEDULLA (50) (48)
 

GANGLIONEUROMA
 

*THYPOID (37) (34)
 

FOLLICULAR-CBLL CARCINOMA 1 (3X)
 C-CELL ADENOMA 1 (3X)
 C-CELL CARCINOMA 2 (5X)
 

#PANCREATIC ISLETS (47) (41)
 

ISLETrCELL_ADENQMA
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NBCROPSIED
 

A-4
 

HIGH DOSE 

01-0185 

(43)
 

(43)
 

1 <2X)
 4 (9X)
 

(38)
 

1 (3X)
 

10 (26X)
 

17 (45X)
 

(31)
 

(36)
 

5 (14X)
 

1 (3X)
 

1 (3X)
 

(36)
 

1 (3X)
 

(31)
 

(3X)
 

(31)
 
TABLE A i (CONTINUED) 

CONTROL (UNTR )

01-0160

 LOU DOSE

 01-0180

 HIGH DOSE 

 01-0185 

REPRODUCTIV E SYSTEM 

*PREPUTIA L GLAN D

CARCINOMA,NO S

tTESTIS ­

INTERSTITIAL-CELL TUMOR

 (50)

 2 (4X) 

 (50)

 42 (84X)

 (49)

 (47)

 27 (57%)

 (44) 

 (36) 

8 (22%) 

NEBVOOS SYSTEM 

#CEHEBBUM

ASTROCYTOMA

• CEREBRAL CORTEX

GLIOMA, NOS

#CEREBELLUM

FIBFOSARCOHA. INVASIVE

 (50)

 (50)

 1 (2%) 

 (50)

 (UK)

 2 (5%)

 (144)

 (44)

 (38) 

1 (33) 

 (38) 

 (38) 

1 (3«) 

SPECIAL SENSE ORGANS 

*EAR CANAL

FIBROSARCOMA

*ZYMBAL'S GLAND

CERUMINOUS CARCINOMA

 (50)

 (50)

 (49)

 (49)

 2 (4!5)

 (44) 

1 (2X) 

 (44) 

1 (2S) 

MUSCULOSKEIETAL SYSTEM 

*SKELETAL MUSCLE

HEMANGIOSARCOMA, INVASIVE

 (50) (49) (44) 

1 (2)t) 

BODY CAVITIES 

*BODY CAVITIES

MESOTHELIOMA, NOS

MISOTHELIOMA, MALIGNANT

• ABDOMINAL CAVITY

SAECCMA, NOS

FIBROSARCOMA

 (50)

 (50)

 (49)

 2 (4X)

 3 (6%) 

 (49)

 1 (2%) 

1 (2%) 

 (44) 

1 (2X) 

 (44) 

ALL OTHER SYSTEMS 

__NJNE . 

i NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECP.OPSIED 

A-5 
TABLE Al (CONCLUDED) 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATURAL DEATHS


MORIBUND SACRIFICE


SCHEDULED SACRIFICE


ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIMAL MISSING
 

ANIMAL DELETED (WRONG SEX)


a> INCLUDES AUTOLYZED ANISAL5
 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*


TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TBMORS UNCERTAINBENIGN OH MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR)

01-0160

 50


 5


 8


 5
 

 32
 

 49


 76


 46


 56


 17


 20


 1


 1


 LOW DOSE HIGH DOSE 

 01-0180 01-0185 

 50 50
 

 32 31
 

 18 18
 

1
 

 45 41
 

 158 101
 

 37 21
 

 65 31
 

 44 39
 

 87 68
 

3 3
 

4 3
 

6 2
 

6 2
 

* PRIMARY IUKORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TUtlOES: METftSTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-6
 
TABLE A2 

SUMMARY OF THE INCIDENT OF NEOPLASMS IN FEMALE RATS TREATED WITH CUPFERRON 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

02-0160 02-0180 02-0185
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

ANIMALS MISSING 1
 ANIMALS NECROPSIED 19 45 147
 

ANIMALS EXAMINED HISTO PATHOLOGICALLY ** 49 14 44
 

INTEGUMENTARY SYSTEM 

*SKIN 

SQOAMOUS CELL CARCINOMA 

SEBACEOUS ADENOCAHCINOMA 

(49) (45) (47) 

1 (2%) 

1 (2%) 

*SUBCUT TISSUE 

SARCOMA, NOS 

FIBROMA 

FISBOSARCOMA 

HIMANGIOSARCOBA 

(49) 

2 (4X) 

(45) 

2 (4*) 

6 (13%) 

1 <2X) 

1 (2*) 

(47) 

1

2

 (2%) 

 (4%) 

RESPIBATORY SYSTEM
 

#LUNG (149) (44) (41)
 

SQUAMOUS CELL CARCINOMA, METASTA 2 (5%)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (2%)
 

ALVE01AB/BRONCHIOLAR CARCINOMA 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (49) (45) (47) 

MALIGNANT LYMPHOMA, NOS 1 (2%) 

LEUKEMIA,NOS 1 (2%) 

HYELCMONOCYTIC LEUKEMIA 6 (12%) 2 (4% ) 

*SPLEEN (47) (44) (44) 

SQUAMOUS CELL CARCINOMA, INVASIV 2 (5X) 

HEMANGIOSARCOKA 28 (64X) 37 (84%) 

tLYBPH NODE (44) (38) (40) 

SQUAMOUS CELL CARCINOMA, METASTA 1 (3%) 

tPANCREATIC L.NODS (44) (38) (40) 

S2UAMOUS_CELL_CARCINOMfi,._METASTA 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D HICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7 
TABLE A2 (CONTINUED)
 

CONTROL (UNTR) LOH DOSE
 

02-0160 02-0180
 

CIRCULATORY SYSTEM
 

NJN E
 

DIGESTIVE SYSTEM 

*LIVER 

SQUAMOUS CELL CARCINOMA, INVASI

SQUAMOUS CELL CARCINOMA, KETAST

NEOPLASTIC NODULE 

HEPATOCELLULAR CARCINOMA 

HEMANGIOSABCOMA, BETASTATIC 

V 

A 

(48) 

1 (2%) 

(44) 

1

2

24

 (2*) 

 (5X) 

(55%) 

tPANCREAS 

HEMANGIOSASCOMA, INVASIVE 

(48) (37) 

1 (3%) 

tPERIPANCRSATIC TISSU 

SQOAMOUS CELL CARCINOMA, METASTA 

("8) (37) 

1 (3X) 

tSTOMACH 

CARCINOMA-IN-SITU, NOS 

SQUAMOUS CELL PAPILLOP1A 

SQUAMOUS CELL CARCINOMA 

(49) (43) 

3

6

14

 <7S) 

 (14X) 

 (33%) 

ICOLON 

ADENOMATOUS POLYP, NOS 

(49) (41) 

1 (2%) 

URINAR Y SYSTEM
 

KKIDNEY (48) (44)
 

HEMANGIOSARCOMA 1 (2X)
 

ENDOCRINE SYSTEM
 

*PITUITARY (39) (35)
 

ADENOMA, NOS 15 (38%) 4 (11%)
 

CHROMOPHOB2 ADENOMA 2 (5%)
 

•ADRENAL	 (49) (41)
 

SG.UAMOUS CELL CARCINOMA, INVASIV
 

CORTICAL ADENOMA 2 (5%)


PHEfiCHJiOMQCYTOMA 3 _(6£l t_-UO»l
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-8
 

HIGH DOSE
 

02-0185
 

(44)
 

3 (7X)
 

2 (5%)
 

10 (23X)
 

1 (2%)
 

(38)
 

(38)
 

(43)
 

6 (14%)
 

22 (51%)
 

(40)
 

(44)
 

(34)
 

(42)
 

1 (2%)
 

1 I2%1
 
TABLE A2 (CONTINUED) 

CONTROL (UNTR) LOU DOSE HIG H DOSE 

02-0160 02-0180 02-0185 

PHECCH80MOCYTCMA, MALIGNANT 3 (7%) 5 (12%)
 

GANGLIONEUROMA 1 (2%)
 

#THYHOID (45) (31) (35)
 

FOLLICULAR-CELL CARCINOMA 1 (3S)
 

C-CELL CARCINOMA 2 (U*) 1 (3%)
 

PAPILLARY CYSTADENOMA, NOS 1 (3*)
 

REPRODUCTIVE SYSTEM
 

*MAMdAR Y GLAND (49) (145) (17) 

ADENOCARCINOHA, SOS 1 (2%) 

FIBROADENOMA 12 (21%) 10 (22%) 1 (2%) 

*CLITORAL GLAND (19) (1*5) (17)

 

CARCINOMA,NOS 1 (2%) 

ADENOMA, NOS 1 (2%)

 

tUTERUS (U6 ) (12) (10) 

ENDOMETRIAL STROMAL POLYP 5 (11*) 3 (7X)

 

#OVAR Y (17) (12) (10) 

HEMANGIOSARCOM A 1 (2%) 

NERVOU S SYSTE M 

•CEREBRUM	 (49) (13) (12)
 

ASTROCYTOMA 2 (5%) 2 (5%)
 

OLIGODJENDROGLIOMA 1 (2%)
 

#BRAIN (19) (13) (42)
 

GLIOMA, NOS 1 (2%)
 

ASTPOCYTOMA 1 (2%) 1 (2%) 1 (2%)
 

SPECIAL SENSE ORGANS
 

*HARDERIAN GLAND (49) (15) (47)
 

ADENOCARCINOMA, NOS 1 (2%)
 

*ZYMBAL'S GLAND (49) (15) (47)
 

SUUAMOUS CELL CARCINOMA i (2%) 2 (4%)
 CERUMINOUS CARCINOMA 1 (2%) 1 (9%) 2 (1%)
 

HUSCULOSKEIETAL SYSTEM
 

NJNE
 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

« NUMBER OF ANIMALS NECROPSIED
 

A-9
 
TABLE A2 (CONTINUED) 

CONTROL (UNTR ) LOW DOSE HIGH DOSE 

02-0160 02-0180 02-0185 

BODY CAVITIES
 

*A8DOMINAL CAVITY (49) (15) (U7)
 

SARCOMA, DOS 1 (2X)
 

ALL OTHER SYSTEMS
 

DIAPHRAGM
 

SQUAMOOS CELL CARCINOMA, INVASIV . 1
 

OMENTUM
 

SQUAMOUS CELL CARCINOMA 1
 

HEMANGIOSARCOHA 1
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

NATURAL DEATHS 2 27 29
 

MORIBUND SACRIFICE 6 15 19
 

SCHEDULED SACRIFICE 5
 ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 36 8 2
 ANIMAL MISSING 1
 

a INCLUDES AUSOLIZED.AfilMALS _ .
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-IO
 
TABLE A2 (CONCLUDED)
 

TUMO R SUMMAR Y 

TOTAL ANIMALS KITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIrtALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*


TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR MSTASTATIC
 TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR)


02-0160


 31


 52


 27


 HO


 10
 12


 LOW DOSE HIGH DOSE 

 02-0180 02-0185 

 t2 12 

 136 101 

 24 12 

 37 12 

 41 12 97 87 

2 4 

3 12 

2 2 

2 2 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

A-ll
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH CUPFERRON
 
TABLE Bl 

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

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

05-0160 05-0185 05-0190
 

ANIMALS INITIALLY IN STUDY 50 50 50
 ANIMALS MISSING 1 2
 

ANIMALS NECBOPSI2D 50 U5 40
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 49 44 to
 

INTEGUMENTARY SYSTEM
 

»SKIN (50) (15) («0)
 

SQUAMODS CELL CARCINOMA 1 (2%) 1 (3*)
 

*SU3CUT TISSUE (50) (45) (40)
 

SQUAMOUS CELL CARCINOMA 2 (»%) 1 (3%)
 

SARCOMA, NOS 1 (2%)
 

HEMANGIOSARCOMA 2 (14%) 2 (5%)
 

RESPIRATORY SYSTEM 

*LUNG 

HEPATOC2LLUIAR CARCINOMA, METUST 

ALVEOLAF./BRONCHIOLAR ADENOMA 

ALVEOLAR/BRONCHIOLAH CARCINOMA 

HEMAN3IOMA 

(47) 

2

I*

3

 (4%) 

 (9%) 

 (6%) 

(44) 

8 (18%) 

(»0) 

2

5

1

 (5%) 

 (13%) 

 (3%) 

HEMATOPOIE1IC SYSTEM
 

*MOLTIPLE ORGANS (50) (15) (10)
 

MALIGNANT LYMPHOMA, NOS 1 (2%) 3 (7%) 2 (5«)
 

GRANULOCYTIC LEUKEMIA 1 (2%)
 

*SPLEEN (U9) (i»U) (38)
 

HEMANGIOSARCOMA 1 (2%) 1 (2%) u (11S)
 MALIGNANT LYMPHOMA, NOS 1 (2%) 1 (3%)
 

#MESENTERIC L. NOD5 0»0) (38) (33)
 

MALIGNANT LYMPHOMA, NOS 1 (3%)
 

*PEYERS PATCH (49) (U1) (37)
 MALIGNANT LYMPHOMA, NOS 1 (2%)
 

CIRCULATORY SYSTEM
 

__N2»E
 

t NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R 01 ANIMAL S NECHOPSIE D 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
TABLE Bl (CONTINUED) 

CONTROL (UNTR ) LOW DOSE HIGH DOSE 

05-0160 05-0185 05-0190 

DIGESTIVE SYSTEM
 

•LIVER	 (19) (11) (10)
 

HEPATOCSLLULAR ADENOMA 1 (3%)
 HEPATOCELLULAE CARCINOMA 15 (315!) 9 (20*) 8 (20%)
 

SARCOMA, NOS 1 <2X)
 

•STOMACH	 (19) (U2) (39)
 

SQOAMOUS CELL PAPILLOMA 2 (5X)
 

ADENOMATOUS POLYP, NOS 1 (2X)
 

•JEJUNUM	 (19) (11) (37)
 

CYSTADENOCARCINOMA, NOS 1 (3S)
 

UBINARY SYSTEM
 

iKIDNEY (19) (4U) (10)
 

PAPILLARY CYSTADENOMA, NOS 1 (3%)
 

ENDOCRINE SYSTEM
 

• THYROID (12) (39) (33)
 

FOLLICULAR-CELL CARCINOMA 1 (3%)
 

REPRODUCTIVE SYSTEM
 

*PHEPUTIAL GLAND (50) (15) (10)
 

CARCINOMA,NOS 1 (2%)
 

SQUAKOUS CELL CARCINOMA 1 (2%)
 

•TESTIS	 (19) (11) (10)
 

INTERSTITIAL-CELL TUMOR 1 (254)
 

NERVOUS SYSTEM
 

*BRAIN/MENINGES (48) (K2) (39)
 

FIBROSARCOMA 1 (2*)
 

SPECIAL SENSE ORGANS
 

*HARDE8IAN GLAND	 (50) (15) (40)
 

t NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

» NUMBER OF ANIMALS NiJCROPSIED
 

B-4
 
*ZYMBAL'S GLAND


SQUAMOUS CELL CARCINOMA


MOSCULOSKELETAL SYSTEM
 

*BONE/UPPER EXTREMITY


OSTEOSARCOBA


BODY CAVITIES
 

*BODY CAVITIES


HEMANGIOSARCOMA


ALL OTHER SYSTEMS
 

NOSE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATURAL DEATHS


MORIBUND SACRIFICE


SCHEDULED SACRIFICE


ACCIDENTALLY KILLED


TERMINAL SACRIFICE


ANIMAL MISSING


S_INCLUDES_AUTOLYZED_ANI|?ALS 

TABLE Bl (CONTINUED) 

CONTROL (ONTR) LOW DOSE HIGH DOSE 

05-0160 05-0185 05-0190 

 (50) (t5) (MO) 

1 (3%) 

 (50) (U5) (UO) 

1 (2X) 

 (50) (15) (10) 

1 (3X) 

 50 50 50 

3 11 19 

8 t 

5 5 

1 

 U2 29 20 

1 2 

» NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

B-5
 
TABLE Bl (CONCLUDED)
 

CONTBOL(UNTH) LOW DOSE HIGH DOSE
 05-0160 05-0185 05-0190
 

TUMOB SUMMABY
 

TOTAL ANIMALS WITH PRIMARY TUMOBS* 20 25 26
 TOTAL PRIMARY TUMORS 26 41 35
 

TOTAL ANIMALS WITH BENIGN TUMORS 6 12 10
 TOTAL BENIGN TUMORS 6 13 12
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 18 21 23
 TCTA1 MALIGNANT TUMOBS 20 28 23
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 2 2
 

TOTAL SECONDARY TUMORS 2 2
 

TOTAL ANIMALS WITH TUMORS•UNCERTAINBESIGN OR MALIGNANT
 TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCSRTAINPBIMARY 08 METASTATIC
 TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 « SECONDARY TUMORS: METASTATIC TUMORS OH TUKORS INVASIVE INTO AN ADJACENT ORGAN
 

B-6
 
TABLE B2 

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

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

06-0160 06-0185 06-0190
 

ANIMALS INITIALLY IN STUDY 50 50 50
 ANIMALS MISSING 1
 ANIMALS NECROPSIED 50 17 U6
 ANIMALS iXAMINSD HISTO PATHOLOGICALLY ** 50 16 16
 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (« 7)
 SQUAMOUS C2LL CARCINOMA 1 (2X)
 HEMANGIOSARCOMA 1 12%)
 

*SUBCUT TISSUE (50) (1*7) 46)
 FIBROSARCOMA 1 (2%) 1 (2*)
 HEMANGIOMA 2 (IX)
 HEMANGIOSAHCOMA 1 (25!) 2 (IX)
 

RESPIRATORY SYSTEM
 

*EXTERNAL NOSE (50) (17) («6)
 

SARCOMA, NOS 1 (2X)
 

*LU!JG (50) (t5) (16)
 

HEPATOCKLLULAR CARCINOMA, HETAST 1 (2%) 1 (2%)
 ALVEOLAR/BRONCHIOLAR ADENOMA 1 (2*) 8 (18%) 8 (17X)
 

ALVEOLAR/BRONCHIOLAB CARCINOMA 3 (6%) 3 (7X) 1 (2X)
 HEMANGIOSARCOMA, METASTATIC 1 <2X)
 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGANS

MALIGNANT LYMPHOMA, NOS

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 

 (50) 

3 (6X) 3

3

 (6X) 

 (6X) 

(16) 

1

2

 (2X) 

 (IX) 

#SPLEEN

TRANSITIONAL-CELL CARCINOMA, MET 

HEMANGIOMA 

HEMANGIOSARCOHA

MALI5.LYMPHOMA, HISTIOCYTIC TYPE

 (U9) 

1 (2X) 

1 (2X) 

(11) 

1

1

1

 (2X) 

 (2*) 

 (2X) 

(12) 

«

1

 (10X) 

 (2X) 

#MANDIBOLA R L. NODi

«ALISNANT_LYMPiiQM4i_NOS 

 (10) (12) (10) 

* NUMBE R OP ANIMAL S WITH TISSUE EXAMIN

* NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

E D MICROSCOPICALLY 

B-7 
TABLE B2 (CONTINUED) 

CONTROL (UNTR)

06-0160

 LOS DOSE

 06-0185

 HIGH DOSE 

 06-0190 

iPULMONARY LYMPH NODE

TRANSITIONAL-CELL CARCINOMA, MET

IPEYEPS PATCH

MALIGNANT LYMPHOHA, NOS

IKIDNEY

MALIG.LYMPHOMA. HISTIOCYTIC TYPE

 (<»0)

 (19)

 1 (2%) 

 (19)

 («2)

 1 (2X) 

 (11)

 (15)

 1 (2%) 

 (10) 

 (11) 

 (13) 

CiacULATOHY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

#LIVER

TRANSITIONAL-CELL CARCINOMA, MET

HEPATOCELLOLAE ADENOMA

HEPATOCELLULAR CARCINOMA

SARCOMA, NOS, METASTATIC

ISTCMACH

SQUAMOUS CELL PAPILLOMA

 (19)

 2 (1%)

 (19)

 (16)

 1 (2%) 

3 (7X)

 9 (20%)

 1 (2%) 

 (13)

 (15) 

3 (7X) 

 13 (29JS) 

 (12) 

2 (5X) 

URINAR Y SYSTEM 

#KIDNEY/CORTEX

TUBULAR-CELL ADiNOMA

tURINARY BLADDER

TRANSITIONAL-CELL CARCINOHA

 (19)

 (50)

 (15)

 1 (2)5) 

 (11)

 2 (5S) 

 (13) 

 (U5) 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

#UTERUS (19) (11)

HEMMSIQMA 

* NUMBEE OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

 (13) 

B-8 
TABLE B2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE

06-0160 06-0185

tUTIRINE SEROSA (49) (41)


HEMANGIOMA 1 (2%)
 

fOVHRY (18) (37)


GRANU10SA-CELL TUMOR 2 (5%)


TUBULAR ADENOMA 1 (3%)


NERVOU S SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*HARD£RIAN GLAND (50) (HI)


SQDAMOUS CELL CARCINOMA


ADL'NOMA, NOS 2 (H%)


*ZYMBAL'S GLAND (50) (17)


SQUAMOUS CELL CARCINOMA


SEBACEOUS ADENOCARCINOMA


HUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

•ABDOMINAL	 CAVITY (50) (47)


HEilANGIOSARCOMA 1 (2X)


ALL OTHER SYSTEMS
 

THIGH
 

H jiMA NGIQ SARCOMA 1
 

t NUIBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

B-9
 

 HIGH DOSE 

 06-0190 

 (13)
 

 (11)
 

1 (2*)
 

3 (7*)
 

 (16)
 

1 (2X)
 

6 (13X)
 

 (16)
 

2 (4%)
 

1 (2%)
 

 («6)
 

1 (2X)
 
TABLE B2 (CONCLUDED)
 

ANIMA L EISFOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 NATURAL DSATH3
 MORIBUND SACRIFICE
 SCHEDULED SACRIFICE
 ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*


TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OP. METASTATIC
 TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR)
 

06-0160
 

50
 

3
 

2
 

5
 

i»0
 

12
 

15
 

1
 

1
 

11
 

1U
 

1


 1


LOW DOSE HIGH DOSE 

06-0185 06-0190 

50 50 

8 12 6 14 

5 

1 

34 29 

1 

33 31 

50 52 

17 16 

20 22 

22 21 

28 29 

2 2 

4 2 

2 1 

2 1 

* PRIMARY TUMORS: ALL TUMORS EXCtPT SECONDARY TUMORS
 t SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-IO
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH CUPFERRON
 
TABLE Cl

 

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

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

01-0160 01-0180 01-0185
 

ANIMALS INITIALLY IN STUDY 50 50 850
 

ANIMALS NECROPSIED 50 49 44
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 50 49 44
 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (49) (44)
 

INFLAMMATION, SUPPURATIVE 1 (2X)
 

ULCER, ACUTE 1 (2*)
 

*SUBCUT TISSUE (50) (49) (44)
 EPIDERMAL INCLUSION CYST 1 <2X)
 

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

RESPIRATORY SYSTEM
 

*LUNG (49) (47) (43)
 

CONGESTION, CHRONIC PASSIVE 1 (2%)
 

INFLAMMATION, INTERSTITIAL 4 (8X)
 

FIBROSIS, DIFFUSE 1 (2X)
 

HYPERPLASIA, NOS 1 (2X)
 

HYPERPLASIA, ADENOMATOUS 1 (2%)
 

HYPERPLASIA, ALVEOLAR EPITHELIUM 1 (2%)
 

tLUNG/ALVEOLI (49) (47) (43)
 

HEMORRHAGE 1 (2X)
 

HEHATOPOIEIIC SYSTEM
 

#SPLEEN (50) (46) (43)
 

CONGESTION, NOS 1 (2*)
 CONGESTION, ACUTE 1 (2*)
 

CONGESTION, PASSIVE 4 (9X) 3 (7X)
 

CONGESTION, ACUTE PASSIVE 1 (2%)
 

HEMATOMA, NOS 1 (2X)
 FIBROSIS 1 (2X) 2 (4X)
 

HEMOSIDEROSIS 2 (4JS) 3 (7%) 4 (9»)
 

_ METAPJ,ASJAt OSSJQ^S __	 1 12J1
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECPOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

31 50 ANIMALS WERE INITIALLY IN THE STUDY, BUT ONE ANIMAL WAS FOUND TO BE A FEMALE IN A MALE GROUP. 

C-3 
TABLE Cl (CONTINUED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

01-0160 01-0180 01-0185
 

ERYTHROPOIESIS 1 (2X) 2 (5S)
 

fMANDIBULAR i. NODr) (U9) (13) (39)
 

HYPERPLASIA, PLASMA CELL 1 (2*) 2 (5X)
 

#MEDIASTINAL L.NODE C»9) (13) (39)
 

CONGESTION, NOS 2 (5X) 1 (3%)
 

HEMORRHAGE 2 (5X)
 

HEMOSIDEROSIS 12 (28*) 1 (10X)
 

• PANCREATIC L.NODii (U9) (13) (39)
 

CONGESTION, NOS 1 (2X) 2 (5X)
 

HEMOSIDEROSIS it (9X) 9 (23X)
 

•MESENTERIC L. NODn (U9) (13) (39)
 

HEMOSIDiiKOSIS 4 (9X) 5 (13X)
 

HYPEEPLASIA. PLASMA CELL 1 (215)
 

IRENAL LYMPH NODU («9) (13) (39)
 

HEMOSIDEROSIS 1 (3X)
 

CIRCULATORY SYSTEM
 

tMYOCARDIUM (18) (17) (13)
 

INFLAMMATION, INTERSTITIAL 1 (2X)
 

FIBROSIS 2 (UX)
 

DEGENERATION, NOS 1 (2K)
 

*BLOOD VESSEL (50) (19) (11)
 

THROMBOSIS, NOS 1 (2%)
 

DIGESTIVE SYSTEM
 

ISALIVARY GLAND (50) (10) (10)
 

HYPERPLASIA, INTRADUCTAL 1 (2%)
 

DYSPLASIA, NOS 1 (3%)
 

IlIVER (19) (18) (13)
 

INFLAMMATION, CHRONIC FOCAL 1 (2%)
 

INFLAMMATION. CHRONIC DIFFUSE 1 (2%)
 

NECROSIS, FOCAL 3 (6%) 1 (2X>
 

BASOPHILIC CYTO CHANGE 1 (2X)
 

CLEAK-CELL CHANGE 1 (2S)
 

HIPiEEiASIS^.FQCA!, J_I251
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

« NUMBER OF ANIMALS NECROPSIED
 

C-4
 
TABLE Cl (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

01-0160 01-0180 01-0185
 

ANGIECTASIS	 3 (6X)
 

ILIVER/CENTRILOBULAR (t9) (U8) C*3)
 

CONGESTION, PASSIVE 1 (2X)
 NECROSIS, NOS 2 <UX)
 

NECROSIS, DIFFUSE 5 (10X) 10 (23X)
 

*LIVER/KUPFFER CELL (U9) (U8) (K3)
 HEMOSIDEROSIS 15 (31X) 28 (65X)
 

*BILE DUCT (50) (19) (It)
 HYPERPLASIA, NOS 2 (H%)
 

tPANCREAS (17) <«1 > (3D
 INFLAMMATION. NOS 2 (US)
 INFLAMMATION, CHBONIC 2 (6X)
 

INFLAMMATION, CHRONIC FOCAL 1 (2X)
 

iSTOMACH	 (U9) (U8) (38)
 

HYPERPLASIA, BASAL CELL 26 (5I4X) 16 (42S)
 HYPF.RKERATOSIS 1 (2X)
 ACASTHOSIS 1 <2X)
 

#GASTRIC MUCOSA (U9) (18) (38)
 

EROSION 2 C*X)
 NECROSIS, FOCAL 2 (<4X)
 

#PEYERS PATCH (U9) (145) (37)
 

HYPERPLASIA, NOS 1 (2X)
 

URINARY SYSTEM
 

ItKIDNEY (50) (U8) (39)
 

HYDfiONEPHROSIS 1 (2X)
 

CONGESTION, NOS 1 (2%)
 GLOMERULONEPHRITIS, NOS H (8*)
 PYELONEPHHITIS. ACUTE 1 (2*)
 NEPHROSIS, NOS 35 (VOX) 5 (10X) 2 (5X)
 

HEMOSIDEEOSIS 1 (2%)
 

#KIDNEY/TUBULE (50) («8) (39)
 CALCIFICATION, NOS 1 (3S)
 HEMOSIEEBOSIS 35 (73*) 38 (97X)
 

•URINARY	 BLADDER (50) (tt) .(38)
 

INFLAMMATION.. ACUTi NECROTIZING . ._ 1 ^asi_ .
 * N01BER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-5
 
TABLECl (CONTINUED) 

ENDOCRINE SYSTEM
 

tPITUITARY
 

CONGESTION, NOS
 

IADRENAL MEDULLA
 

HYPZRPLASIA, FOCAL
 

tTHYROID
 

CYSTIC FOLLICLES
 

HYPEBPLASIA, C-CELL
 

REPRODUCTIVE SYSTEM
 

tPROSTATE
 

INFLAMMATION, NOS
 

INFLAMMATION, SUPPURATIVS
 

INFLAMMATION, ACUTE NECEOTIZING
 

*TESTIS
 

PERIVASCULITIS
 

CALCIFICATION, NOS
 

CALCIFICATION, FOCAL
 

ATROPHY, NOS
 

HYPEEPLASIA, INTERSTITIAL CELL
 

NERVOUS SYSTEM
 

#CEREBP.UM
 

ABSCESS, NOS
 

SPECIAL SENSE ORGANS
 

NONE
 

HUSCULOSKELETA L SYSTE M 

N JNE 

BODY CAVITIES
 

*PL3UIU 

____ £IBEQSISX_DIFFUSE 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

01-0160 01-0180 01-0185 

(45) (38) (31) 

1 (2%) 

(50) (18) (36) 

1 (3*) 

(37) (34) (31) 

1 (3%) 

2 (5%) 

(48) (46) (36) 

3 (6%) 

1 (2%) 

1 (2X) 

(50) (47) (36) 

1 (2%) 

3 (6X) 

1 11 (28) 

 (22%) 12 (26%) 3 (8%) 

4 (8S) 1 (3%) 

(50) (44) (38) 

1 (2X) 

(50) (49) (44) 

I NUMBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-6
 
TABLE Cl (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

01-0160 01-0180 01-0185
 

ALL OTHER SYSTEMS
 

ADIPOSE TISSUE
 NECROSIS, FAT 1
 

CMENTUM
 

HEMOSIDiEOSIS 1
 

SP5CIAL MORPHOLOGY SUMMAR Y
 

NO LESION REPORTED 1
 

AUTO/NECROPSY/HISTO PERF 1
 

AUTOLYSIS/NO NECROPSY 1 5
 

« NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMALS NilCROPSIED
 

C-7
 
TABLE C2

 

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

CCNTHOL(ONTR) 

02-0160 

LOU DOSE 

02-0180 

HIGH DOSE 

02-0185 

ANIMALS INITIALLY IN STUDY 

ANIMALS MISSING 

ANIMALS NECEOPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

50 

1 

49 

** 49 

50 

t5 

44 

50 

1*7 

14 !4 

INTEGUMENTARY SYSTEM
 

•SKIN
 

SEBACEOUS CYST
 

HESPIEATOBY SYSTEM
 

*L'JNG
 

THROMBOSIS, NOS
 

CONGESTION, NOS
 

CONGESTION, ACUTE PASSIVE
 

HEMORRHAGE
 

CALCIFICATION, NOS
 

HEMATOPOIETIC SYSTEM
 

*SPLEEN
 

CONTRACTURE
 

CONGESTION, NOS
 

CONGESTION, PASSIVE
 

HEMOSIDEROSIS
 

ATROPHY, NOS
 

HEMATOPOIESIS
 

E<?YTHHOPOIESIS
 

#SPLENIC CAPSULE
 

HYPZRPLASIA, NOS
 

*LYMPH NODJ2
 

HEMOSIDEROSIS
 

#MANDIEULAR L. NODr.
 

HYPERPLASIA, PLASMA CELL
 

tMEDIASTINAL L.NODE


HEMORRHAGE


t NUMBE R OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIMAL S NECROPSIE D 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

(45)	 (47)
 

1 (2%)
 

(19)	 (44) (U1)
 

1 (2X)
 

1 (2%)
 

1	 (2X)
 

1 (2%)
 

1 (2X)
 

(47)	 (tH) (4«) 1 (2%) 

1 (2%) 

1 (2%) 3 (7% ) 

3	 (6%) 10 (23S) 3 (7%) 

3 (7%) 

2 (5X) 1 (2%) 

2 (5X) 

(47) (14) (U4 ) 

1 (2%) 

(44)	 (38) (40) 

1 (3%) 

<t«) (38) (40) 

1 (3%) 

 (14)	 (38) (40) 

6_J.16%1_
 

 EXAMINE D MICROSCOPICALLY 
TABLE C2 (CONTINUED) 

CONTROL (UNTR) LOU DOSE HIGH DOSE
 

02-0160 02-0180 02-0185
 

HEMOSIDBROSIS 5 (13X) 2 (5X)
 

HYPERPLASIA, PLASMA CELL 1 (3%)
 

#PASCREATIC L.NODZ (44) (38) (40)
 

HEMORRHAGE 1 (3%)
 

HEMOSIESROSIS 6 (16X) 7 (18%)
 

#MESENTERIC L. NODi (44) (38) (40)
 

CONGESTION, NOS 1 (3%)
 

HEHOSIDEKOSIS 2 (5X) 7 (18X)
 

*RENHL LYMPH NOD3 (44) (38) (40)
 

HEHOSIDEROSIS 1 (3%) 1 (3%)
 

CIRCULATORY SYSTEM
 

tMYOCARDIUH (48) (44) (41)
 

FIBHOSIS 1 (2%)
 

CALCIFICATION, NOS 1 (2%)
 

CALCIFICATION, FOCAL 1 (2%) 1 (2%)
 

'AORTA (49) (45) (47)
 

MEDIAL CALCIFICATION 1 (2X)
 

DIGESTIVE SYSTEM 

ULIVER 

HEMORRHAGE 

INFLAMMATION, ACUTE/CHRONIC 

INFLAMMATION, CHRONIC FOCAL 

NECROSIS, NOS 

NECROSIS, FOCAL 

METAMORPHOSIS FATTY 

BASOPHILIC CYTO CHASG2 

CLEAR-CELL CHAN3E 

HYPEBPLASIA, FOCAL 

ANSIECTASIS 

(48) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

(44) 

1

2

1

1

 (2%) 

 (5%) 

 (2%) 

 (2%) 

(44) 

1 (2%) 

2 (5%) 

2 (5%) 

KLIVER/CENTRILOBULAR 

NECROSIS, DIFFUSE 

(48) (44) 

3 (7%) 

(44) 

2 (5X) 

tLIVER/KUPFFER CELL 

HEMOSIDEROSIS 

(48) (44) 

9 (20X) 

(44) 

33 (75K) 

*3ILE DUCT 

HYPEFPLASIA^ NOS 

(49) 

2_J4%1. 

(45) (47) 

NUMBER OF ANIMALS WITH TISSUE

NUMBER OP ANIMALS NECF.OPSIED 

EXAMINED MICROSCOPICALLY 

C-9
 
TABLE C2 (CONTINUED) 

CONTROL (UNTR) 

02-0160 

LOW DOSE 

02-0180 

HYPEBPLASIA, FOCAL 1 (2%) 

fPANCREAS 

HEHORRHAGIC CYST 

INFIAKHATION. CHRONIC 

(18) (37) 

1 (3%) 

KSTOMACH 

EPIDERMAL- INCLUSION CYST 

INFLAMMATION. NCS 

HYPERPLASIA. BASSL CELL 

(<*9) 

1 (2%) 

(13) 

1

17

 (2%) 

 (1*0%) 

tGASTRIC MtlCOSA 

VESICLE 

EROSION 

CALCIFICATION, NOS 

(49) (1*3) 

2 (5X) 

#GASTRIC SUBMUCOSA 

EDEMA, NOS 

(149) 

1 (2%) 

(1*3) 

•PEYERS PATCH 

HYPERPLASIA, NOS 

(U9) 

2 (US) 

(1*3) 

#COLON 

PARASITISM 

(1*9) 

1 (2%) 

(11) 

URINAR Y SYSTEM
 

#KIDNEY	 (48) (KK)
 

EMBRYONAL DUCT CYST 1 (2%)
 

GLOMEHULONEPHRITIS, NOS it (8%)
 

NEPHROSIS, NOS 29 (60%) 2 (5«)
 

NECROSIS, MEDULLARY 1 (2%)
 

•KIDNEY/CORTEX	 (US) (Kl*)
 

CYST, NOS
 

METAMORPHOSIS FATTY 1 (2%)
 

ItKIDNEY/TUBULE (H8) (1*1*)
 

NEPHROSIS, NOS 1 (2%)
 

CALCIFICATION, NOS 1 (2%)
 

HEMOSIDE80SIS 1*2 (95%)
 

tKIDNEY/PELVIS (U8) (1*1*)
 

CALCIFICATION, NOS
 

#0. ELADDER/SUBMUCOSA (1*9) (1*0)
 

HEMORRHAGE
 

« NUBBEE OF ANIMALS «ITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS N2CP.OPSIED
 

C-IO
 

HIGH DOSE
 

02-0185
 

(38)
 

1 (3%)
 

(1*3)
 

11* (33%)
 

(«3)
 

1 (2%)
 

2 (5%)
 

(«3)
 

(«0)
 

(«0)
 

(KU)
 

2 (5%)
 

C*t)


2 (5%)
 

(KU)
 

2 (5%)
 

UO (91%)
 

(!*«)
 

1 (2%)
 

(37)
 

1 _[3%1
 
ENDOCRINE SYSTEM
 

IADRENAL CORTSX
 

HEMOSIDEROSIS
 

#THYROID
 

HYPERPLASIA, C-CELL
 

IPARATHYROID
 

HYPERPLASIA, NOS
 

REPRODUCTIVE SYSTEM
 

•MAMMARY	 GLAND
 

DILATATION/DUCTS
 

•MAMMARY	 DUCT
 

HYPERPLASIA, CYSTIC
 

»DTERUS
 

HYDROMETRA
 

HEMATOflA, NOS
 

POLYP, INFLAMMATORY
 

#UTERUS/ENDOMETRIUM
 

HYPERPLASIA, CYSTIC
 

IOVARY
 

INFLAMMATION, CHRONIC
 

NERVOUS SYSTEM
 

#BRATN
 

PERIVASCULITIS
 

tPONS
 

GLIOSIS
 

SPECIAL SENSE ORGANS
 

»LENS CAPSULE
 

CALCIFICATION, NOS
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

TABLE C2 (CONTINUED) 

CONTROL (UNTR) 

02-0160 

LOW DOSE

02-0180

 HIGH DOSE 

 02-0185 

(49) 

(45) 

2 (4X) 

(27) 

(41)

1

(31)

1

(17)

1

 (2X) 

 (3X) 

 (6X) 

 (42) 

 (35) 

 (23) 

(49) 

1 (2%) 

(49) 

1 (2X) 

(46) 

1 (2X) 

1 (2X) 

1 (2X) 

(46) 

(47) 

1 (2X) 

(45)

(45)

(42)

(42)

1

(42)

 (2X) 

 (47) 

 (47) 

 (40) 

 (40) 

 (40) 

(49) 

(49) 

(43)

1

(43)

1

 (2X) 

 (2X) 

 (42) 

 (42) 

(49) (45) (47) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-ll
 
TABLE C2 (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 02-0160 02-0180 02-0185
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 2
 ANIMAL MISSING/NO NECROPSY 1
 

NECROPSY PEEF/NO HISTO PERFORMED 2
 

AUTO/NECROPSY/NO HISTO 1 1
 

AUTOLYSIS/NO NECROPSY 5 3
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIEB
 

C-I2
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH CUPFERRON
 
TABLE Dl

 

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

CONTROL (UNTR) 

05-0160 

LOW DOSE 

05-0185 

HIGH DOSE 

05-0190 

ANIMALS INITIALLY

ANIMALS HISSING 

ANIMALS NECHOPSIE

ANIMALS EXAMINED

 IN STUDY 50 

D 50 

 HISTOPATHOLOGICALLY** 19 

50 

1 

45 

44 

50 

2 

DO 

to 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (45) («0)
 INFLAMMATION, CHSONIC 1 <2X)
 INFLAMMATION, CHRONIC DIFFUSa 1 (3*)
 

*SU3CUT TISSUE (50) (45) (40)
 HEMORRHAGE 1 (2S)
 HEMATOMA, NOS 1 (2X)
 INFLAMMATION, ACUTE FOCAL 1 (2%)
 ABSCESS, NOS 1 (2S)
 

RESPIRATORY SYSTEM
 

*LUNG («7) (44) (40)
 

ABSCESS, NOS 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

*DONE MARBO H (49) (38) (35)
 

HYPERPLASIA, HEMATOPOIETIC 2 (5S)
 

tSPLiEN (49) (44) (38)
 

RUPTURE 1 (3K)
 

HEMORRHAGE 1 (3X)
 

NECROSIS, FOCAL 1 (3X)
 

HYPERP1ASIA, NOS 1 (3X)
 

HEMATOFOIESIS 3 (7X) 1 (3X)
 

ERYTHROPOIESIS 2 (4)S) 2 (5S)
 

GRANULOPOIZSIS 1 (2*)
 

ItMEDIASTINAL L.NODci (40) (38) (33)
 

HEMORRHAGE 1 (3*)
 

#MESENTERIC L. NODE (40) (38) (33)
 

LIMPHANGIECTASIS
 

* NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

» NUMBE R OF ANIMALS NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-3 
HYPEBPLASIA, LYMPHOID
 

IRENAL LYMPH NODE
 

HYPERPLASIA, NOS
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tSALIVARY GLAND
 

INFLAMMATION, CHRONIC
 

FIBROSIS. FOCAL
 

#LIVEB
 

CYST. NOS
 

HEMORRHAGE
 

HEMATOMA, NOS
 

HEMOERHAGIC CYST
 

SCAR
 

NECROSIS, FOCAL
 

SECROSIS, DIFFUSE
 

METAMORPHOSIS FATTY
 

ANGIECTASIS
 

CLIVER/KUPFFER CELL
 

HYPERPLASIA, HOS
 

*BILE DUCT
 

INFLAMMATION, CHRONIC
 

#PANCREAS
 

CYSTIC DOCTS
 

INFLAMMATION, ACUTE/CHRONIC
 

PEBIVASCOLITIS
 

NECROSIS, FAT
 

tPANCREATIC ACINUS
 

ATROPHY, NOS
 

• STOMACH


INFLAMMATION, ACUTE/CHRONIC


HYP£SPLASIAJl_BASAL_CEtL


TABLE Dl (CONTINUED) 

CONTROL (UNTR ) LOW DOSE HIGH DOSE 

05-0160 05-0185 05-0190 

2 (5X) 

(HO) (38) (33) 

2 (5%) 

(47) (40) 

1 (3X) 

1 (3X) 

(44) (40) 

2 (5X) 

1 (3*) 

1 (3X) 

1 (3X) 

1 (3%) 

1 (3*) 

1 (3X) 

1 (2%) 

1 (2%) 

(U9) (44) (40) 

1 (2«) 

(50) (45) (10) 

1 (2X) 

(46) (38) (36) 

1 (2%) 2 (6*) 

1 (2%) 

1 (2%) 

(46) (38) (36) 

2 (6X) 

 (49) (42) (39) 

1 (2%) 

; 1_I2S1_ 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-4
 
TABLE Dl (CONTINUED) 

CONTROL (UNTR)

05-0160

 LOW DOSE

 05-0185

 HIGH DOSE 

 05-0190 

IPEYERS PATCH

INFLAMMATION, ACUTE

HYPERPLASIA, LYMPHOID

 (49)

 1 (2%) 

1 (2%) 

 (141) (37) 

URINARY SYSTEM 

#KIDNEY

HYDRONEPHROSIS

INFLAMMATION, SOPPURATIVE

INFLAMMATION, CHRONIC

INFLAMMATION, CHRONIC FOCAL 

INFARCT, NOS 

*KIDNEY/CORTEX

SCAR 

KKIDNEY/GLOMERULUS

AMYLOIDOSIS 

*U. BLADDiR/SUBMUCOSA

INFLAMMATION, CHRONIC FOCAL 

 (1(9)

 2 (US) 

1 (2%) 

 (49) 

 (49) 

 (49) 

 {Itt)

 1 (2X) 

(44) 

1 (2X) 

(44) 

1 (2%) 

(43) 

1 (2%) 

 (40) 

1 (3%) 

1 (3X) 

(HO) 

(40) 

(39) 

ENDOCRINE SYSTEM 

#THYROID

HYPERPLASIA, FOCAL

 (42)

 1 (2%) 

 (39) (33) 

REPRODUCTIVE SYSTSM 

*PHE?UTIAL GLAND

DIL.MATION, NOS

tTESTIS

HEMORRHAGE

NECPOSIS, NOS

t"2SIIS/TUBULE

NECROSIS, FOCAL

 (50)

 1 (2?!) 

 (49)

 (49)

 1 (2%) 

 (45)

 (44)

 1 (21,) 

1 (2%)

 (44)

 (40) 

 (40) 

1 (3%) 

 (40) 

NERVOUS SYSTEM 

NONE 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

D-5 
TABLE Dl (CONCLUDED) 

CONTROL (UNTH ) LOW DOSE HIGH DOSE 

05-0160 05-0185 05-0190 

SPECIAL SENSE ORGAN S 

*EYE (50) (45) (40) 

PUS 1 (3%) 

*EYE/COP.NEA (50) (45) (HO)
 

ULCER. NOS 1 (3%)
 

MUSCULOSKELEIAL SYSTEM
 

*POPLITEAL MUSCLE (50) (1(5) (40)
 

HEMORRHAGE 1 (2%)
 

BODY CAVITIES
 

*ABDOMINA1 CAVITY (50) (45) (40)
 

ADHESION, NOS 1 (2%)
 

*MESENTERY (50) (45) (40)
 

STEATITIS 1 (2%)
 

ABSCESS, NOS 1 (2%)
 

ALL OTHER SYSTEMS
 

ADIPOSE TISSUE
 

STEATITIS 1
 

NECROSIS, FAT 2
 

SPECIAL MORPHOLOGY SUMMAR Y
 

NO LESION REPORTED 17 12 8
 

ANIMAL MISSING/SO NECROPSY 1 2
 

ACCIDENTAL DEATH 1
 

AUTO/NECROPSY/HISTO PERF 1
 

AUTO/NECROPSY/Nu HISTO 1 1
 

AUTOLYSIS/NO NECROPSY 3 8
 

« NUMBE R OF ANIMALS WITH TISSUE EXAMINEE MICROSCOPICALLY
 

« NUMBER OF ANIMALS NECROPSIED
 

D-6
 
TABLE D2 

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

CONTROL (UNTE) LOW DOSE HIGH DOSE
 

06-0160 06-0185 06-0190
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

ANIMALS MISSING 1
 

ANIMALS NECROPSIED 50 47 46
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 50 46 46
 

INTiGOMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETIC SYSTEM
 

IBONE MARROW (49) (34) (37)
 

HYPERPIASIA, HEMATOPOIETIC 1 (3%)
 

*SPLE£N (49) (44) (42)
 

THRCM30SIS, NOS 1 (2%)
 

HEMAIOflA, NOS 2 (5%)
 

HYPERPL&SIA, LYMPHOID 1 (2%) 1 (2%)
 

HEMATOPOIESIS 1 (2%)
 

ERYTHBOPOIESIS 1 (21) 1 (2%)
 

tMANQIBULAR L. NODE (40) (42) (40)
 

HYPF.RPLASIA, PLASMA CELL 1 (3X) 1 (2%)
 

fMEDIASTINAL L.NODE (40) (42) (40)
 

'IYPERFLASIA, NOS 1 (3^)
 

#LUMBAR LYMPH NODE (40) (42) (40)
 

HYPZRPLASIA, NOS 1 (3%)
 

*«ji5£STERIC L, MODE (40) (42) (40)
 

HYPERPLASIA, NOS 1 (3*)
 

HYPERPLASIA, LYMPHOID 1 (2%)
 

tRENA L LYMPH NODE (40) (42) (40)
 

HYPERPLASIA,. NOS _J 1321 _
 

« NUMBE R O F ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY * NUMBE R OF ANIMAL S NECROPSIE D 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-7 
TABLE D2 (CONTINUED)
 

HYPEBPLASIA, PLASMA CELL
 

CIRCULATORY SYSTEM
 

(MYOCARDIUM
 

INFLAMMATION, ACUTE DIFFUSE
 

DIGESTIVE SYSTEM
 

ILIVER
 

NECROSIS, NOS
 

NECROSIS, FOCAL
 

INFARCT, NOS
 

MEGALOCYTOSIS
 

HYPEBPLASTIC NODULE
 

ANGIECTA5IS
 

HEHATOFOIESIS
 

*LIVEE/CAUUATE LOBti
 

NECROSIS, NOS
 

*LIVER/KUPFFaR CELL
 

HYPERPLASIA, NOS
 

*BILE DUCT
 

INFLAMMATION, CHRONIC FOCAL
 

tSMMACH
 

INFLAMMATION, ACUTE FOCAL
 

INFLAMMATION, CHRONIC
 

tPEYERS PATCH
 

HYPERPLASIA, LYMPHOID
 

tCOLON
 SEMATODIASIS
 

URINASY SYSTEM
 

itKIDNEY


LYMPHOCYTIC INFLAMMATORY INFILTR


INFLAMMATION, CHRONIC


IC_FQCAL


CONTROL (UNTR) LOW DOSE HIGH DOSE 

06-0160 06-0185 06-0190 

1 (3«) 

(50) (15) (46) 

1 US) 

(It9) (t6) (45) 

1 (2X) 1 (2%) 1 (2X) 

1 (2%) 2 (4%) 

1 (2%) 1 (2%) 

1 (2%) 

2 (US) 

1 (2%) 

(49) (46) (45) 

1 (2X) 

(49) (46) (15) 

1 (2%) 

(50) (47) (46) 

2 («%) 

(U9) (43) (42) 

1 (2%) 

1 (2%) 

(t9) (44) (44) 

1 (2%) 

(50) (43) (44) 

1 (2«) 

 (49) (45) (43) 

1 (2%) 

2 (4%) 

 1_12S1 

I NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NIJ.1BER OF ANIMALS NECROPSIED
 

0-8
 
TABLE D2 (CONTINUED)
 

CONTROL (UNTR)

06-0160

 LOW DOSE

 06-0185

'5LOMISULOSCLEROSIS, NO

HYPEHPLASIA. TUBULAP

S

CELL 

1(2%) 

#KIDNEY/TUBULE 

CALCIFICATION, NOS 

(U9) (U5) 

1 (2X) 

IURINARY BLADDER 

INFLAMMATION, ACUTE F

INFLAMMATION, CHRONIC

OCAL 

 FOCAL 

(50) 

1 (2%) 

to. 3LADD£H/SUBMUCOSA 

INFLAMMATION, CHRONIC 

INFLAMMATION, CHRONIC

EEHIVASCULITIS 

 FOCAL 

(50) 

1 (2X) 

16 (32%) 

1 (2%) 

*U.BLADCER/MU3CULARIS 

CALCIUM DEPOSIT 

(50) (It) 

ENDOCRINE SYSTEM
 

tTHYROID C*1) (37)
 

HYPiRPLASIA, C-CELL 2 (5S)
 

HYPERPLASIA, FOLLICULAR-CELL 1(2%)
 

REPRODUCTIVE SYSISM
 

(UTERUS (U9) 

HYDROMETRA 5 (10%) 1 (2*) HEMATOHA, NOS 1 <2X) 

HEMA10MA, ORGANIZED 1 (2S) 

HEMOERHAGIC CYST 1 (2*) 

NECSOSI5, FAT 1 (2X ) 

CALCIFICATION, NOS 1 (2S) 

•UTERUS/ENDOMETRIUM	 (19) (11) 

INFLAMMATION, SUPPURATIVi 2 (US) 

HYPEKPLftSIA, CYSTIC 32 (65*) 10 (2«S) 

»OVAR Y (18) (37) 

CYST, NOS 6 (US) 5 (14X) 

THROMBOSIS, NOS 

HEMA10BA, NOS
 

4EMOREHAGIC CYST
 

_ 1-J2*1 

* NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

« NUMBE R OF ANIMALS NECPOPSIED
 

D-9
 

 HIGH DOSE
 

 06-0190
 

1 (2X)
 

1 (2X)
 

(K3)
 

(US)
 

1 (2%)
 

(15)
 

(31)
 

(13)
 

(13) 

2 (5%) 

(11) 

5 (12*) 

1 (2*) 

1 (2X) 

1 (2X) 
INFLAMMATION, CHRONIC


HECEOSIS, NOS


HYPEEPLASIA, ADENCMATOUS


NERVODS SYSTEM
 

#BRAIN/M2NINGES


INFLAMMATION, NOS


*ERAI N

HrlMORRHAG E

SPECIAL SENSE ORGANS
 

N JNE
 

BUSCULOSKEL2TAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

'MULTIPLE ORGANS


AMYLOIDOSIS


OMEMTUM
 

PERIVASCULITIS


SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED


ANIMAL MISSING/NO NECROPSY


AUTO/NECEOPSY/HISTO PERF


AUTO/NECROPSY/NO HISTO


AUTOLYSIS/NO NECROPSY


*	 NUMBER OP ANIMALS WITH TISSUE


*	 NUMBER OP ANIMALS N3CROPSIED
 

TABLE D2 (CONCLUDED) 

CONTROL (UNTP.) LOW DOSE HIGH DOSE 

06-0160 06-0185 06-0190 

1 (254)
 

1 (2%)
 

2 (5%) t (10%)
 

 (U9) («1) (115)
 

1 (2X)
 

 (119) (tl ) (1)5) 

1 (2X) 

 (50)	 (147) (1*6)
 

1 (2%)
 

1
 

2 5 10
 

1
 

2
 

1
 

2 U
 

 EXAMINED MICROSCOPICALLY
 

D-IO
 
Review of the Bioassay of Cupferron* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

June 29, 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 carcinogenic­ ity. It is in this context that the below critique is given
 

on the bioassay of Cupferron for carcinogenicity.
 

The reviewer agreed with the conclusion in the report
 

that Cupferron was carcinogenic in rats and mice, under the
 

conditions of test. After a brief description of the experimental design, he said that the hemosiderosis detected in
 

several organs should be further evaluated. He added that
 

the hemosiderosis was not reported in the same target organs
 

for the chronic phase as in the subchronic study. He also
 

commented on the wide temperature variation in the animal
 

rooms, on the fact that mice were housed in a room in which
 

other chemicals were under study, and on the lack of data on
 

the stability and concentration of Cupferron in the diet.
 

The reviewer questioned the basis for selecting the chronic
 

dose levels since it did not correspond with the criteria
 

described in the protocol. He opined that the studies'
 

shortcomings cast doubt on its validity. The reviewer said
 

that the data do not allow an assessment of human risk.
 

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A Program staff member said that the bioassay was too
 

limited to allow an interpretation of the hemosiderosis
 

problem. He noted that it did not appear to interfer with
 

the survival of the treated animals. It was moved that the
 

report on the bioassay of Cupferron be accepted as written.
 The motion was approved without objection.
 

Clearinghouse Members present:
 

Arnold L. Brown (Chairman), Mayo Clinic
 

Paul Nettesheim, National Institute of Environmental
 

Health Sciences
 

Verne Ray, Pfizer Medical Research Laboratory
 

Verald K. Rowe, Dow Chemical U.S.A.
 Michael B. Shimkin, University of California at San Diego
 

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.
 

i. GOVERNMENT PRINTIN G OFFICE i 1978-260-899/3196
 

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DHEW Publication No. (NIH) 78-1350