1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 106 

BIOASSAY OF 

TRICHLOROFLUOROMETHANE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 75-69-4 

NCI-CG-TR-106 

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

Public Health Service 

National Institutes of Health 
BIDASSAY OF
 

TRICHLOROFLUOROME THANE
 

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) 78-1356
 

REPORT ON THE BIOASSAY OF TRICHLOROFLUOROMETHANE
 

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 trichlorof luoromethane 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 trichlorofluoromethane was conducted
 

by Hazleton Laboratories America, Inc., Vienna, Virginia, initially
 

under direct contract to the NCI and currently under a subcontract to
 

Tracer 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. M. B. Powers (3),
 

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

(3,5) and the analytical results were reviewed by Dr. N. Zimmerman
 

(6); the technical supervisor of animal treatment and observation
 

was Ms. K. J. Petrovics (3).
 

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

represent the interpretation of these pathologists. Histopathology
 

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

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

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

111
 
R. Joiner (7), using methods selected for the Bioassay Program by Dr.
 

J. J. Gart (9).
 

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

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

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

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

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

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

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

E. Tarone (9).
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

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

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

Squire (1,10), 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.	 Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

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

Reston, Virginia.
 

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

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

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

iv
 
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.
 

10.	 No wit w h the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

v
 

SUMMARY
 

The bioassay of technical-grade trichlorofluoromethane for possible carcinogenicity was conducted using Osborne-Mendel rats and
 

B6C3F1 mice. Trichlorofluoromethane in corn oil was administered by
 

gavage, at either of two dosages, to groups of 50 male and 50 female
 

animals of each species, 5 days a week, over a period of 78 weeks.
 

The time-weighted average high and low dosages of trichlorofluoromethane in the chronic bioassay were, respectively, 977 and 488 mg/kg/
 

day for male rats, 1077 and 538 mg/kg/day for female rats, and 3925
 

and 1962 mg/kg/day for mice of both sexes. After the 78-week dosing
 

period, rats were observed for an additional period of up to 33 weeks
 

and mice were observed for an additional period of up to 13 weeks.
 

For each species, 20 animals of each sex were placed on test as
 

vehicle controls. These animals were gavaged with corn oil at the
 

same times that dosed animals were gavaged with trichlorofluoromethane. Twenty animals of each sex were placed on test as untreated
 

controls for each species. These animals were not gavaged.
 

A high rate of early deaths occurred among male and female rats
 

in this bioassay. An insufficient number of rats of either sex survived long enough to be at risk from late-developing tumors. Survival of mice was adequate for meaningful statistical analysis of
 

late-developing tumors.
 

Results of a time-adjusted statistical analysis of tumor incidence in rats indicated no significant positive associations between
 

administration of trichlorofluoromethane and tumor incidence.
 

No groups of male or female mice dosed with trichlorofluoromethane had significantly increased tumor incidences relative to their
 

respective control groups.
 

The results of the bioassay of trichlorofluoromethane in Osborne-


Mendel rats for possible carcinogenicity are not conclusive because
 

inadequate numbers of rats survived long enough to be at risk from
 

late-developing tumors. Under the conditions of this bioassay,
 

trichlorofluoromethane was not carcinogenic to male or female B6C3F1
 

mice.
 

VII
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 6
 

A. Chemicals 6
 

B. Dosage Preparation 6
 

C. Animals 8
 

D. Animal Maintenance 8
 

E. Gastric Intubation 10
 

F. Selection of Initial Dose Levels 10
 

G. Experimental Design 11
 

H. Clinical and Histopathologic Examinations 15
 

I. Data Recording and Statistical Analyses 16
 

III. CHRONIC TESTING RESULTS: RATS 22
 

A. Body Weights and Clinical Observations 22
 

B. Survival 24
 

C. Pathology 26
 

D. Statistical Analyses of Results 26
 

IV. CHRONIC TESTING RESULTS: MICE 31
 

A. Body Weights and Clinical Observations 31
 

B. Survival 31
 

C. Pathology 34
 

D. Statistical Analyses of Results 35
 

V. DISCUSSION 40
 

VI. BIBLIOGRAPHY 42
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH TRICHLOROFLUOROMETHANE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH TRICHLOROFLUOROMETHANE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH TRICHLOROFLUOROMETHANE C-l
 

IX
 
TABLE OF CONTENTS (Concluded)
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH TRICHLOROFLUOROMETHANE
 D-l 
LIST OF ILLUSTRATIONS
 

Figure Number 

1 CHEMICAL STRUCTURE OF TRICHLOROFLUOROMETHANE 

GROWTH CURVES FOR TRICHLOROFLUOROMETHANE 

CHRONIC STUDY RATS 23 

SURVIVAL COMPARISONS OF TRICHLOROFLUOROMETHANE CHRONIC STUDY RATS 25 

GROWTH CURVES FOR TRICHLOROFLUOROMETHANE 

CHRONIC STUDY MICE 32 

SURVIVAL COMPARISONS OF TRICHLOROFLUOROMETHANE CHRONIC STUDY MICE 33 

LIST OF TABLES 

Table Number 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS— 

TRICHLOROFLUOROMETHANE GAVAGE EXPERIMENT 13 

DESIGN SUMMARY FOR B6C3F1 MICE—TRICHLOROFLUOROMETHANE GAVAGE EXPERIMENT 14 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

TRICHLOROFLUOROMETHANE WHICH SURVIVED AT 

LEAST 52 WEEKS 27 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH TRICHLOROFLUOROMETHANE WHICH SURVIVED 

AT LEAST 52 WEEKS 28 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

TRICHLOROFLUOROMETHANE 36 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH TRICHLOROFLUOROMETHANE 38 

XI
 
LIST OF TABLES (Concluded) 

Table Number 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH TRICHLOROFLUOROMETHANE A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH TRICHLOROFLUOROMETHANE A-6 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH TRICHLOROFLUOROMETHANE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH TRICHLOROFLUOROMETHANE B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH TRICHLOROFLUOROMETHANE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 

TRICHLOROFLUOROMETHANE C-8 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH TRICHLOROFLUOROMETHANE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 

TRICHLOROFLUOROMETHANE D-7 

Xll
 
I. INTRODUCTION
 

Trichlorofluoromethane (NCI No. C04637), a widely used halocarbon
 

aerosol propellant and refrigerant, was selected for bioassay by the
 

National Cancer Institute because of the widespread exposure to this
 

compound resulting from the indiscriminate use of aerosol sprays, and
 

the well-documented hepatocarcinogenicity of the structurally analogous compound, carbon tetrachloride (International Agency for Research
 

on Cancer, 1972).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index (1977)
 

*
 

name for this compound is trichlorofluoromethane. It is also called
 

trichloromonofluoromethane; fluorotrichloromethane; fluorocarbon 11;
 

propellant 11; Freon 11 ; Arcton 11 , and Frigen 9®.
 

Trichlorofluoromethane is used as a low-pressure propellant and
 

solvent in a wide variety of aerosol products, such as hair sprays,
 

deodorants, and other cosmetic goods; external medicinals; house and
 

garden pesticides; cleaners and/or disinfectants; spray paints; and
 

floor and furniture polishes (Belej and Aviado, 1975; Gosselin et al.,
 

1976). The propellant in such products usually constitutes 70 percent
 

or more of the contents of the can (Crossland, 1974). As recently as
 

1976, trichlorofluoromethane was the most widely used aerosol propellant (Gosselin et al., 1976); however, the recent publication of theories which suggest that depletion of the Earth's stratospheric ozone
 

*
 

The CAS registry number is 75-69-4.
 
layer may result from the photolytic dissociation of chlorofluoromethanes (e.g., Molina and Rowland, 1974) has aroused public sentiment
 

against aerosol products which contain trichlorofluoromethane or
 

its congeners, and sales of such products have drastically declined
 

(Chemical and Engineering News, 1977).
 

Trichlorofluoromethane is also used to a substantial degree in
 

refrigeration equipment requiring a refrigerant that is effective at
 

negative pressures (Chemical Marketing Reporter, 1975; Windholz,
 

1976), and in smaller quantities as a blowing agent for polymeric
 

foams, and as an active component of liquid-type fire extinguishers
 

(Chemical Marketing Reporter, 1975; Gosselin et al., 1976).
 

In 1975, U.S. production and sales of trichlorofluoromethane were
 

269,611,000 and 253,756,000 pounds, respectively (U.S. International
 

Trade Commission, 1977). The 1972 world production rate for this
 

compound was about 0.3 megaton per year (Molina and Rowland, 1974).
 

Proposed action by three federal agencies—the Environmental Protection Agency, Food and Drug Administration, and Consumer Product Safety
 

Commission—to phase out all nonessential uses of chlorofluorocarbons
 

as propellants by April 15, 1979 (Chemical Regulation Reporter, 1977)
 

is expected to significantly reduce production and sales of trichlorofluoromethane.
 

The apparent global contamination of the biosphere by fluorocarbons (Molina and Rowland, 1974) has resulted in universal exposure to
 

low levels of trichlorofluoromethane. Measurable concentrations of
 
the compound have been found throughout the troposphere: 124 to 403
 

parts per trillion (ppt) in Washington State (Grimsrud and Rasmussen,
 

1975); 80 to 2200 ppt in the Los Angeles Basin (Simmonds et al.,
 

1974); 10 to 190 ppt in southwest Ireland (Lovelock, 1971); and 38 to
 

80 ppt over the Atlantic Ocean (from Antarctica to the United Kingdom)
 

(Lovelock et al., 1973). In the latter study, trichlorofluoromethane
 

was also detected in samples of seawater taken from corresponding locations in the Atlantic. Concentrations of the compound ranged from
 

20 to 70 ppt (Lovelock et al., 1973).
 

Users of aerosol sprays and workers at facilities which produce
 

or use trichlorofluoromethane are exposed, by both inhalation and
 

dermal contact, to concentrations of the compound that are considerably higher than ambient levels. Dermal contact is presumably
 

unavoidable when using cosmetic or medicinal sprays. Vapor concentrations may reach 400 parts per million during normal use of aerosol
 

sprays (Crossland, 1974) and vapor concentrations which are significantly higher than ambient trichlorofluoromethane levels are routinely
 

found in homes and public buildings, and in or near factories at which
 

the compound is used. Hester et al. (1974) measured fluorocarbon
 

levels in samples of air taken from homes in the Los Angeles area and
 

found concentrations of trichlorofluoromethane as high as 12 parts per
 

billion (ppb) in two of ten homes. In one instance, this was 67 times
 

higher than levels in outdoor samples collected at the same location at
 

approximately the same time. Concentrations of trichlorofluoromethane
 
found in public buildings and around factories were also high: 12
 

ppb in a drug store; 10 ppb in a supermarket; 50 ppb in a beauty shop;
 

4.1 ppb in a hospital; 8.1 ppb near the storage area of a cosmetics
 

plant (outdoors); and 42 ppb at a distance of 25 yards from a polyurethane plant (Hester et al., 1974). Elevated vapor levels in places
 

other than factories are not necessarily solely attributable to aerosol use;a substantial portion may also be due to leakage from refrigeration equipment.
 

Deliberate inhalation of aerosol propellents for the purpose of
 

inducing a euphoric mental state results in exposure to very high
 

concentrations of trichlorofluoromethane (Polkis, 1975).
 

Regulatory action to phase out chlorofluorocarbon propellant
 

uses would reduce the potential for human exposure to high concentrations of trichlorofluoromethane; however, the compound is relatively
 

stable and atmospheric concentrations would only decline slowly over
 

a period of 10 years or more (Chemical Regulation Reporter, 1977).
 

Essential items excluded from the ban would include aerosol bronchiodilators for the treatment of asthma, contraceptive vaginal foams,
 

cytology fixatives, a mine safety warning device, release agents for
 

certain plastic molds, and some flying insect sprays used on airplanes
 

and in commercial food-handling areas (Chemical Regulation Reporter,
 

1977).
 
The Freons were originally considered to have a low degree of
 

toxicity in humans; however, numerous recent accounts of spontaneous
 

deaths among persons who deliberately inhaled aerosol propellants
 

for their narcotic effect has prompted new concern over the toxic
 

effects of the fluorochloromethanes (Crossland, 1974; Polkis, 1975).
 

These deaths are thought to be an example of the "sudden death"
 

syndrome which has been associated with many deaths among workers in
 

the chemical industry since the turn of the century (Crossland, 1974).
 

These compounds apparently sensitize the heart to epinephrine, result­

(R)
 ing in severe cardiac arrhythmias and often death. Of the Freons ,
 

trichlorofluoromethane has been shown to have the highest degree of
 

cardiotoxicity in monkeys and other animals (Aviado, 1975; Gosselin
 

et al., 1976); however, similar human studies are not feasible. An
 

anesthetic or narcotic effect of Freons in humans is reported to
 

occur at levels of 4 percent by volume in air, and exposed workers
 

have experienced fainting and dizziness (Crossland, 1974).
 

Trichlorofluoromethane showed no indication of carcinogenic or
 

tumorigenic activity in 45 ICR/Ha Swiss mice of both sexes following
 

neonatal injections of 0.1 ml on days 14 and 21 (Epstein et al.,
 

1967).
 
II. MATERIALS AND METHODS 

A. Chemicals
 

One batch of technical-grade trichlorofluoromethane (Figure 1)
 

was purchased from Allied Chemical Company and analyzed by Hazleton
 

Laboratories America, Inc., Vienna, Virginia. The purchased chemical
 

was initially determined to be greater than 95 percent trichlorofluoromethane, using total-area analysis gas-liquid chromatography (GLC).
 

Five small peaks were present in addition to the trichlorofluoromethane peak. When the material was assayed using the internal standard GLC method, purity was indicated to be approximately 97 percent.
 

A second purity determination was conducted approximately 2 years
 

after the first to establish the stability of the trichlorofluoromethane. GLC results were similar to those obtained in the initial analysis, indicating little or no decomposition.
 

Throughout this report the term trichlorofluoromethane is used to
 

represent this technical-grade material.
 

B. Dosage Preparation
 

®


Fresh solutions of trichlorofluoromethane in Duke's corn oil
 

(S. F. Sauer Company, Richmond, Virginia) were prepared weekly, sealed
 

and stored in dark bottles at 1°C. Each time the solution was used, a
 

slight excess was poured into a narrow-necked flask. The stock bottle
 

was immediately resealed and returned to the refrigerator. During all
 

transfers the flask was immersed in an ice bath. The concentrations
 
Cl

I 

Cl C 

Cl 

FIGURE 1 

CHEMICAL STRUCTURE OF TRICHLORO FLUOROMETHANE 
of trichlorofluoromethane in corn oil were 37.5 to 42.5 percent for
 

rats and 20 to 40 percent for mice.
 

C. An ima1s
 

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

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

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

carcinogenesis bioassays and has proved satisfactory in this capacity.
 

Rats and mice of both sexes were obtained through contracts with
 

the Division of Cancer Treatment, National Cancer Institute. The
 

Osborne-Mendel rats were procured from the Battelle Memorial Institute, Columbus, Ohio, and the B6C3F1 mice were obtained from the
 

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

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

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

dosed and control groups.
 

D.	 Animal Maintenance
 

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


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

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

conditioning system provided filtered air at a rate of 12 complete
 

changes of room air per hour. Fluorescent lighting was provided on
 

a 12-hour-daily cycle.
 

8
 
The rats were individually housed in suspended galvanized-steel
 

wire-mesh cages with perforated floors. The mice were housed by sex
 

in groups of 10 in solid-bottom polypropylene cages equipped with
 

(R)
 filter tops. Sanitized cages with fresh bedding (Sanichips , Pine-


wood Sawdust Company, Moonachie, New Jersey) were provided once each
 

week for mice. Rats received sanitized cages with no bedding with
 

the same frequency. Food hoppers were changed and heat-sterilized
 

once a week for the first 10 weeks and once a month thereafter.
 

Fresh heat-sterilized glass water bottles and sipper tubes were pro­

®
 vided three times a week. Food (Wayne Lab-Blox meal, Allied Mills,
 

Inc., Chicago, Illinois) and water were available ad_ libitum.
 

The rats dosed with trichlorofluoromethane and the untreated
 

*
 controls were housed in the same room with other rats intubated with
 

methylchloroform (71-55-6) and chloropicrin (76-06-2). The vehicle
 

control rats were housed with other rats intubated with 1,2-dichloroethane (107-06-2); 1,1-dichloroethane (75-34-3); and carbon disulfide
 

(75-15-0).
 

All mice in the trichlorofluoromethane study, including controls, were housed in the same room as other mice intubated with
 

1,1,2,2-tetrachloroethane (79-34-5); chloroform (67-66-3); allyl
 

chloride (107-05-1); chloropicrin (76-06-2); dibromochloropropane
 

* .
 CAS registry numbers are given in parentheses,
 
(96-12-8); 1,2-dibromoethane (106-93-4); 1,2-dichloroethane (107­

06-2); 1,1-dichloroethane (75-34-3); trichloroethylene (79-01-6);
 

3-sulfolene (77-79-2); iodoform (75-47-8); methylchloroform (71-55­

6); 1,1,2-trichloroethane (79-00-5); tetrachloroethylene (127-18-4);
 

carbon disulfide (75-15-0); hexachloroethane (67-72-1); and carbon
 

tetrachloride (56-23-5).
 

E. Gastric Intubation
 

Intubation was performed for five consecutive days per week on
 

a rag/kg body weight basis utilizing the most recently observed group
 

mean body weight as a guide for determining the dose. Mean body
 

weights for each group were recorded at weekly intervals for the
 

first 10 weeks and at monthly intervals thereafter. All animals of
 

one sex within a treated group received the same dose. Animals were
 

gavaged with test solutions under a hood to minimize extraneous exposure of other animals and laboratory personnel to the chemical.
 

F. Selection of Initial Dose Levels
 

In order to estimate the maximum tolerated dosages of trichlorofluoromethane for administration to treated animals in the chronic
 

studies, subchronic toxicity tests were conducted with both rats and
 

mice. Animals of each species were distributed among six groups,
 

each consisting of five males and five females. Intubation was performed 5 days per week for 6 weeks, followed by a 2-week observation
 

period to detect any delayed toxicity. Trichlorofluoromethane dissolved in corn oil was introduced by gavage to five of the six rat
 

10
 
groups and five of the six mouse groups at dosages of 1000, 1780,
 

3160, 5620, and 10,000 mg/kg/day. The sixth group of each species
 

served as a vehicle control group, receiving only corn oil.
 

A dosage inducing no mortality and resulting in a depression
 

in mean group body weight of approximately 20 percent relative to
 

controls was selected as the initial high dose. When weight gain
 

criteria were not applicable, mortality data alone were utilized.
 

At least one death was recorded for all the male rat groups receiving 1780 mg/kg/day or greater and for all the female rat groups
 

receiving 3160 mg/kg/day or greater. Mean group body weight depression was 26 percent in males receiving 1000 mg/kg/day while females
 

receiving this same dosage gained 12 percent more weight than controls. At a level of 1780 mg/kg/day mean body weight depression was
 

11 percent in females. The initial high dosages selected for male
 

and female rats in the chronic bioassay were 850 and 1500 mg/kg/day.
 

There was no mean group body weight depression when males receiving 5620 mg/kg/day or less or females receiving 3160 mg/kg/day
 

or less were compared to controls. There were deaths in male groups
 

treated with 5620 mg/kg/day or more and in female groups treated with
 

3160 mg/ kg/day or more. The initial high dosage selected for male
 

and female mice in the chronic bioassay was 3160 mg/kg/day.
 

G. Experimental Design
 

The experimental design parameters for the chronic bioassay (species, sex, group size, dosages administered, duration of treated and
 

11
 
untreated observation periods, and time-weighted average dosages) are
 

summarized in Tables 1 and 2.
 

The untreated control and treated rats were all approximately
 

6 weeks old when the bioassay was initiated and they all shared the
 

same median date of birth. The vehicle control rats were approximately 7 weeks old when they were first intubated and that was
 

approximately 7 months prior to initiation of the trichlorofluororaethane chronic bioassay. The initial dosages utilized for male
 

rats were 850 and 425 mg/kg/day and for female rats were 1500 and 750
 

mg/kg/day. Throughout this report the male rats initially receiving
 

850 mg/kg/day and the female rats initially receiving 1500 mg/kg/day
 

are referred to as the high dose groups, while the male rats initially
 

receiving 425 mg/kg/day and the female rats initially receiving 750
 

mg/kg/day are referred to as the low dose groups. After 12 weeks the
 

dosages for male rats were increased and for female rats were decreased
 

to high and low levels of 1000 and 500 mg/kg/day, respectively. These
 

dosages were maintained for the remainder of the 78-week intubation
 

period, after which there was an additional observation period of up
 

to 33 weeks.
 

The vehicle control and treated mice were all approximately 5
 

weeks old when the bioassay was initiated and they all shared the
 

same median date of birth. The untreated control rats were approximately 3 weeks old when the other animals were first intubated. The
 

initial dosages utilized for both male and female mice were 3160 and
 

12
 
TABLE 1
 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS
 

TRICHLOROFLUOROMETHANE GAVAGE EXPERIMENT
 

INITIAL TRICHLOROGROUP FLUOROMETHANE 

SIZE DOSAGE3 

MALE
 

UNTREATED CONTROL 20 0
 

VEHICLE CONTROL 20 0
 

LOW DOSE 50 425
 

500
 

0
 

HIGH DOSE 50 850
 

1000
 

0
 

FEMALE
 

UNTREATED CONTROL 20 0
 

VEHICLE CONTROL 20 0
 

LOW DOSE 50 750
 

500
 

0
 

HIGH DOSE 50 1500
 

1000
 

0
 

Q
 

Dosages, given in rag/kg body weight, were
 

tive days per week.
 

,„_ _ S (dosage
 

OBSERVATION PERIOD TIME-WEIGHTED 

TREATED UNTREATED AVERAGE 

(WEEKS) (WEEKS) DOSAGEb 

0 99 0
 

78 32 0
 

12 488
 

66
 

28
 

12 977
 

66
 

33
 

0 111 0
 

78 32 0
 

12 538
 

66
 

33
 

12 1077
 

66
 

33
 

administered by gavage 5 consecu-


X weeks received)
 

13
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

TRICHLOROFLUOROMETHANE GAVAGE EXPERIMENT
 

INITIAL TRICHLORO- OBSERVATION PERIOD TIME-WEIGHTED
 

GROUP
 

SIZE
 

MALE
 

UNTREATED CONTROL 20
 

VEHICLE CONTROL 20
 

LOW DOSE 50
 

HIGH DOSE 49
 

FEMALE
 

UNTREATED CONTROL 20
 

VEHICLE CONTROL 20
 

LOW DOSE 50
 

HIGH DOSE 50
 

a
 

Dosages, given in mg/kg
 

utive days per week.
 

FLUOROMETHANE


DOSAGE3


0
 

0
 

1580
 

2000
 

0
 

3160
 

4000
 

0
 

0
 

0
 

1580
 

2000
 

0
 

3160
 

4000
 

0
 

body weight, were
 

,„„„ - 2 (dosage
 

14
 

 TREATED UNTREATED AVERAGE
 

 (WEEKS) (WEEKS) DOSAGEb
 

0 91 0
 

78 13 0
 

7 1962
 

71
 

13
 

7 3925
 

71
 

13
 

0 91 0
 

78 13 0
 

7 1962
 

71
 

13
 

7 3925
 

71
 

13
 

administered by gavage 5 consec-


X weeks received)
 
1580 mg/kg/day. Throughout this report those mice initially receiving the former dosage are referred to as the high dose groups, while
 

those mice initially receiving the latter dosage are referred to as
 

the low dose groups. In week 8, high and low dosages were increased
 

to 4000 and 2000 mg/kg/day, respectively. These dosages were maintained for the remainder of the 78-week intubation period, after
 

which there was an additional observation period of 13 weeks.
 

The untreated controls received no trichlorofluoromethane or
 

corn oil, while the vehicle controls were intubated with corn oil.
 

H. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. Body weights, food consumption, and data concerning
 

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

and location of tissue masses were recorded at weekly intervals for
 

the first 10 weeks and at monthly intervals thereafter. From the
 

first day, all animals were inspected daily for mortality. The presence of tissue masses was determined by observation and palpation of
 

each animal.
 

A necropsy was performed on each animal regardless of whether it
 

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

bioassay. The animals were euthanized by exsanguination under sodium
 

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

15
 
of major tissues, organs, or gross lesions taken from sacrificed animals and, whenever possible, from animals found dead.
 

Slides were prepared from the following tissues: brain, pituitary,
 

adrenal, thyroid, parathyroid, trachea, esophagus, thymus, salivary
 

gland, lymph nodes (mesenteric and cervical), heart, nasal passages,
 

lung, spleen, liver, kidney, stomach, small intestine, large intestine,
 

pancreas, urinary bladder, prostate or uterus, seminal vesicles and
 

testis with epididymis or ovary, skin with mammary gland, muscle,
 

nerve, bone marrow, and tissue masses.
 

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

with hematoxylin and eosin prior to microscopic examination. An
 

occasional section was subjected to special staining techniques for
 

more definitive diagnosis.
 

A few tissues were not examined for some animals, particularly
 

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

I. Data Recording and Statistical Analyses
 

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

16
 
(Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design, clinical observations, survival, body weight, and individual pathologic results,
 

as recommended by the International Union Against Cancer (Berenblum,
 

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

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental results
 

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

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
 

17
 
that site was examined (denominator). In most instances, the denominators included only those animals for which that site was examined
 

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

(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
 

18
 
zero at the one-tailed 0.05 level of significance. Unless otherwise
 

noted, the direction of the significant trend was a positive dose relationship. This method also provides a two-tailed test of departure
 

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

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

Cochran-Armitage tests, etc.) were followed.
 

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
 

19
 
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.
 

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,
 

20
 
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.
 

21
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

No distinct compound-related mean body weight depression was
 

apparent when trichlorofluoromethane-treated male and female rats
 

were compared with untreated controls, but vehicle control rats consistently gained more weight than dosed rats (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 .
 

A decline in survival was observed in the treated groups during
 

the first year of the study. Apparent compound-related deaths were
 

noted as early as week 4 in the high dose females, increasing gradually in both sexes and at both dosages as the study progressed. Survival continued to decline during the second year, but at a comparable
 

rate for all groups, including the controls.
 

Beginning with the first week of compound administration, a few
 

rats at both dose levels started to show a hunched appearance and occasional labored respiration. These signs were observed with greater
 

frequency in the treated groups than in the controls through week 30
 

but were noted at comparable rates in treated and control rats during
 

the remainder of the bioassay. Chronic respiratory disease characterized by wheezing, nasal discharge and/or labored respiration were
 

observed at a slight to moderate incidence during the first year and
 

at a high incidence in all groups during the second year. Clinical
 

22
 
750 750 

^600 

L-450 

UNTREATED CONTROL 

— 300 

VEHICLE CONTROL 

LOW DOSE 

-150 

HIGH DOSE 

1 

60 

TIME ON TEST (WEEKS) 

~T 

75 90 

~T T~ 

105 

T 

120 

750­ •750 

W 

60°­ -600 

I- 450­

X 

-450 

g 

§ 300 H 

CO 

< 

LU 

150­

FEMALE RATS 

UNTREATED CONTROL 

VEHICLE CONTROL 

LOW DOSE 

HIGH DOSE 

-300 

-150 

I 

15 30 45 60 

TIME ON TEST (WEEKS) 

T 

90 

I I 

105 120 

FIGURE 2 

GROWTH CURVES FOR TRICHLOROFLUOROMETHANE CHRONIC STUDY RATS 

23 
signs usually associated with aging were observed at a similar rate
 

in the control and treated rats. These signs included alopecia,' sores
 

on the tail and other parts of the body, discolored fur, bloating,
 

pale appearance, and palpable nodules.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and trichlorofluoromethane-dosed groups are shown in
 

Figure 3.
 

For male rats the Tarone test for positive association between
 

increased dosage and accelerated mortality was significant (P <
 

0.001) when the dosed groups were compared to the vehicle control
 

group. Accelerated mortality was observed in both the treated and
 

control groups with less than 10 percent of the animals in any group
 

surviving on test until the end of the study. By week 52 only 30
 

percent (15/50) of the high dose and 40 percent (20/50) of the low
 

dose rats were alive, while all 20 of the vehicle control and 70 percent (14/20) of the untreated control rats were still alive on test.
 

The number of male rats surviving long enough to be at risk from
 

late-developing tumors was inadequate.
 

For female rats the Tarone test also showed a significant (P <
 

0.001) positive association between increased dosage and accelerated
 

mortality when the dosed groups were compared to the vehicle control.
 

As with the males, early mortality was high as only 34 percent (17/50)
 

of the high dose and 62 percent (31/50) of the low dose compared to
 

24
 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 

ro 

VJl 
85 percent (17/20) of the vehicle control and all 20 of the untreated
 

control females were still alive on test at week 52. Survival of
 

female rats was not adequate to perform meaningful analyses of late-


developing tumors.
 

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).
 

Neoplasms present in the vehicle control and treated groups were
 

similar histologically to those present in untreated control rats or
 

those which have been observed previously in rats of similar strain,
 

age, and sex. No appreciable difference in the incidence of these
 

neoplasms was noted between the control and treated rats in this
 

study.
 

Inflammatory, degenerative, and proliferative lesions as seen
 

in the vehicle control and treated animals were usually similar, as
 

to number and kind, to those naturally occurring lesions found in
 

aged rats. Chronic murine pneumonia occurred in 88 to 100 percent of
 

the rats and appeared to be a factor in early mortality. In addition,
 

pleuritis and pericarditis were seen, primarily in dosed groups.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

rats are summarized in Tables 3 and 4. Due to the high early mortality in treated rats of both sexes, many may have died before
 

26
 
___ 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH TRICHLOROFLUOROMETHANE
 

WHICH SURVIVED AT LEAST 52 WEEKSa
 

UNTREATED VEHICLE LOW HIGH%
 

TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL DOSE DOSE
 

Pituitary: Chromophobe Adenoma 2/13(0.15) 0/20(0.00) 2/19(0.11) 0/14(0.00)
 

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

Relative Risk (Vehicle Control) Infinite
 

Lower Limit 0.324
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 64 74
 

ho Treated groups received time-weighted average doses of 488 or 977 mg/kg by gavage.
 

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

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

the corresponding control group when 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
 

vehicle control group is given beneath the incidence of tumors in the vehicle control 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.
 
TABLE 4 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN FEMALE RATS TREATED WITH TRICHLOROFLUOROMETHANE 

WHICH SURVIVED AT LEAST 52 WEEKS3 

TOPOGRAPHY : MORPHOLOGY 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Pituitary: Chromophobe Adenoma 3/20(0.15) 4/17(0.24) 1/29(0.03) 3/16(0.19) 

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

NJ 

CO 

Departure from Linear Trend 

Relative Risk (Vehicle Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

___ 

92 

P ­ 0.030 ___ 

68 

0.147 

0.003 

1.260 

111 

0.797 

0.137 

3.940 

98 

Mammary Gland: Fibroadenoma 

P Values 

Relative Risk (Vehicle Control) 

Lower Limit 

Upper Limit 

3/20(0.15) ___ 

___ 

3/17(0.18) 

N.S. ___ 

1/31(0.03) 

N.S. 

0.183 

0.004 

1.977 

2/17(0.12) 

N.S. 

0.667 

0.062 

5.067 

Weeks to First Observed Tumor 99 97 64 99 

Mammary Gland: Fibroadenoma or 

Ad eno carcinoma NOS^ 

P Values0 

Relative Risk (Vehicle Control) 

Lower Limit 

Upper Limit 

3/20(0.15) ___ 

«_ 

4/17(0.24) 

N.S. ___ 

2/31(0.06) 

N.S. 

0.274 

0.028 

1.727 

2/17(0.12) 

N.S. 

0.500 

0.051 

2.985 

Weeks to First Observed Tumor 99 97 64
 99 
TABLE 4 (CONCLUDED)
 

Treated groups received time-weighted average doses of 538 or 1077 mg/kg by gavage.
 

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

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

the corresponding control group when 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
 

vehicle control group is given beneath the incidence of tumors in the vehicle control 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 of the test for departure from linear trend is given beneath the control
 

group when P < 0.05.
 
they were at risk from late-developing tumors. To compensate for
 

this, the analyses performed were based solely upon those rats that
 

survived at least 52 weeks. The analysis is included for every type
 

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

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

None of the statistical tests for any site in rats of either
 

sex indicated a significant positive association between the administration of trichlorofluoromethane and tumor incidence. It must be
 

noted, however, that the high early mortality in both sexes precluded
 

meaningful analyses of late-developing tumors.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

risk have been estimated and entered in the tables based upon the
 

observed tumor incidence rates. In all of the intervals shown in
 

Tables 3 and 4, the value one is included; this indicates the absence
 

of statistically significant results. It should also be noted that
 

all of the confidence intervals have an upper limit greater than one,
 

indicating the theoretical possibility of tumor induction in rats by
 

trichlorofluoromethane that could not be established under the conditions of this test.
 

30
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

No distinct compound-related mean body weight depression was
 

apparent when trichlorofluoromethane-treated male and female mice
 

were compared with 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.
 

Throughout the study appearance and behavior of the treated
 

mice were generally comparable with the vehicle controls. A slight
 

decline in survival was evident for the high dose groups beginning
 

in week 18 of the study and continuing through the duration of compound administration. Clinical signs usually associated with group-


housing, particularly in the males, were observed at essentially
 

comparable frequencies in all groups and included sores on the body
 

or extremities, generalized or localized alopecia, rough or stained
 

fur, external genital irritation, abdominal distension or bloating,
 

and swollen areas. Palpable nodules and/or tissue masses were
 

observed at a slightly greater frequency in the treated male mice
 

than in the remaining groups. Isolated, apparently incidental,
 

observations included head tilt or circling in one to three high
 

dose female mice during the second year of the study.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and trichlorofluoromethane-dosed groups are shown in
 

Figure 5.
 

31
 
-50 

-40 

-30 

-20 

UNTREATED CONTROL 

VEHICLE CONTROL 

-10 

LOW DOSE 

HIGH DOSE 

1 1 

105 120 

TIME ON TEST (WEEKS) 

50- -50 

40- -40 

£30­ -30 

01 

g 

>- 20- -20 Q

m 

O UNTREATED CONTROL 

VEHICLE CONTROL 

10- -10 

LOW DOSE 

HIGH DOSE FEMALE MICE 

I I I I T I \ 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR TRICHLOROFLUOROMETHANE CHRONIC STUDY MICE 

32 
PROBABILIT

Y OF SURVIVAL PROBABILIT

Y O

F SURVIVAL 

c3 0 0 0 0 

-j 

c3 kl '-C* b> 03 b 

1 1 1 1 1 1 1 

o — 

C

c 

O 

1 i 

33 

1 ­

< 

'1

• 

 1 J 

> _, < 

! ! f F"" CTI 

o O

5 i i 

!

j 

2»1

S» _ 

m

"O 3> 

s ° s 5 I ^ o 1I i•

33 

" o z

3 r

CO OJ _ -1o J1 

0 °2

o 1 

Z o 

^ 1 

CO rf! a H ' 

0 — j"| 

H

° 1 

-1 ' 33 Ji—8

 1 

— H ^ s 0 5 •" J" I 5 I

I 

'.—>i

* 

r m I 01 o 1 s P H " ?S "i 1 Cm co ro _ i §cn H O H kl " 5 03

 m

 m

H |

H

( 

m A H co 

1 r 

X — 

^J _J

T ^ ! > cji — ~ J 

Z 

1 «•» m i r o 4 • 

X 33 

1 :^T O 1

 = 

• I— I

1 5­

CO 

H 

C 

a 

tl -< m s S > O o\ i— m m 2^

 

^ 

O 

m _,

11 1

0 

1 

!

 ' 1 ' 1 i 

0 00 O 0 

3 N3 

ji b) bo b 
For male mice the Tarone test for positive association between
 

increased dosage and accelerated mortality was not significant when
 

dosed groups were compared to the vehicle control. Adequate numbers
 

of male mice survived to be at risk from late-developing tumors, with
 

58 percent (29/50) of the high dose, 82 percent (41/50) of the low
 

dose, 75 percent (15/20) of the vehicle control, and 65 percent
 

(13/20) of the untreated control mice surviving on test at least 75
 

weeks.
 

For female mice the Tarone test showed a significant (P = 0.009)
 

positive association between increased dosage and accelerated mortality when dosed groups were compared to the vehicle control. Survival
 

was adequate, however, as 64 percent (32/50) of the high dose, 74 percent (37/50) of the low dose, 90 percent (18/20) of the vehicle control, and 85 percent (17/20) of the untreated control mice were alive
 

on test at the termination of the study.
 

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).
 

Hepatocellular carcinoma was the most commonly observed neoplasm
 

and was diagnosed in 5/19 (26 percent) vehicle control males, 12/50
 

(24 percent) low dose males, 10/47 (21 percent) high dose males, 1/19
 

(5 percent) vehicle control females, 4/50 (8 percent) low dose females, and 2/49 (4 percent) high dose females. The hepatic neoplasms
 

34
 
occurring in the vehicle control mice were not different in appearance or incidence from those noted in the trichlorofluoromethanetreated mice. No appreciable difference was noted in the incidence
 

of other neoplasms between the control and treated groups.
 

Inflammatory, degenerative, and proliferative lesions as seen
 

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

to those naturally occurring lesions found in aging B6C3F1 mice.
 

This histopathologic examination did not provide evidence for
 

the carcinogenicity of trichlorofluoromethane in B6C3F1 mice under
 

the conditions of this bioassay.
 

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 malignant tumor in either sex where at least two such
 

tumors were observed in at least one of the control or trichlorofluoromethane dosed groups and where such tumors were observed in at
 

least 5 percent of the group.
 

None of the statistical tests for any site in mice of either
 

sex indicated a significant positive association between the administration of trichlorofluoromethane and tumor incidence. Thus, at
 

the dose levels used in this experiment there was no convincing evidence that trichlorofluoromethane was a carcinogen in B6C3F1 mice.
 

To provide additional insight into the carcinogenicity of this
 

compound, 95 percent confidence intervals on the relative risk have
 

35
 
___ 

___ 

___ 

___ 

___ 

___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH TRICHLOROFLUOROMETHANE2
 

TOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Malignant
 

Lymphcmab
 

P Values0
 

Relative Risk (Vehicle Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Vehicle Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Neoplastic Nodule or Hepatocellular Carcinoma^5
 

P Values0
 

Relative Risk (Vehicle Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

UNTREATED
 

CONTROL
 

0/19(0.00)
 

3/19(0.16)
 

71
 

3/19(0.16)
 

71
 

VEHICLE
 

CONTROL
 

1/19(0.05)
 

N.S.
 

—_
 

75
 

5/19(0.26)
 

N.S.
 

87
 

5/19(0.26)
 

N.S.
 

87
 

LOW
 

DOSE
 

4/50(0.08)
 

N.S.
 

1.520
 

0.168
 

73.309
 

72
 

12/50(0.24)
 

N.S.
 

0.912
 

0.359
 

2.959
 

79
 

15/50(0.30)
 

N.S.
 

1.140
 

0.476
 

3.571
 

79
 

HIGH
 

DOSE
 

4/47(0.09)
 

N.S.
 

1.617
 

0.178
 

77.876
 

88
 

10/47(0.21)
 

N.S.
 

0.809
 

0.302
 

2.699
 

88
 

10/47(0.21)
 

N.S.
 

0.809
 

0.302
 

2.699
 

88
 
___ 

00 

TABLE 5 (CONCLUDED) 

UNTREATED VEHICLE LOW HIGH
 

TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL DOSE DOSE
 

Lung: Alveolar/Bronchiolar Adenoma
 

or Alveolar/Bronchiolar Carcinoma*5 0/19(0.00) 2/19(0.11) 0/50(0.00) 1/47(0.02)
 

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

Relative Risk (Vehicle Control) 0.000 0.202
 

Lower Limit 0.000 0.004
 

Upper Limit 1.278 3.710
 

Weeks to First Observed Tumor 91 80
 

Treated groups received time-weighted average doses of 1962 or 3925 mg/kg by gavage.
 

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

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

the corresponding 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
 

vehicle control group is given beneath the incidence of tumors in the vehicle control 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.
 
TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH TRICHLOROFLUOROMETHANE3
 

UNTREATED VEHICLE LOW HIGH
 

TOPOGRAPHY : MORPHOLOGY CONTROL CONTROL DOSE DOSE
 

Liver: Hepatocellular Carcinoma 1/19(0.05) 1/19(0.05) 4/50(0.08) 2/49(0.04)
 

c
 

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

_—
 

Lower Limit 0.168 0.044
 

Upper Limit 73.309 44.838
 

Relative Risk (Vehicle Control)d 1.520 0.776
 

Weeks to First Observed Tumor 91 91 84 74
 

w Treated groups received time-weighted average doses of 1962 or 3925 mg/kg by gavage.
 CO b
 

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

£

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

the corresponding control group when 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
 

vehicle control group is given beneath the incidence of tumors in the vehicle control 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.
 
been estimated and entered in the tables based upon the observed
 

tumor incidence rates. In all of the intervals shown in Tables 5 and
 

6, the value one is included; this indicates the absence of statistically significant results. It should also be noted that all of the
 

confidence intervals have an upper limit greater than one, indicating
 

the theoretical possibility of tumor induction in mice by trichlorofluoromethane that could not be established under the conditions of
 

this test.
 

39
 
V. DISCUSSION
 

Due to a high rate of early deaths among rats of both sexes, the
 

number of rats at risk from late-developing tumors was not adequate
 

for conclusions to be based on results of this bioassay. Survival of
 

mice was adequate for meaningful statistical analysis of tumor incidence.
 

In an attempt to compensate for the large number of early deaths
 

in the rat bioassay, statistical analyses of tumor incidence in rats
 

were based on those animals surviving at least 52 weeks. None of
 

these statistical tests indicated a significant positive association
 

between administration of trichlorofluoromethane and tumor incidence.
 

No unusual tumors were observed during histopathologic examinations
 

of dosed or control rats in this bioassay. The fact that vehicle
 

control rats were placed on test approximately seven months before
 

dosed rats had no apparent effect on the results of this study.
 

No significant increase in tumor incidence occurred among mice
 

dosed with trichlorofluoromethane. No unusual tumors were observed
 

during the histopathologic examinations of mice in this bioassay.
 

Negative results for carcinogenesis were obtained by Epstein
 

et al. (1967) in a one-year bioassay of Swiss mice (iCR/Ha) in which
 

trichlorofluoromethane solutions (10 percent by volume in tricaprylin)
 

were injected subcutaneously into the neck of neonatal mice (0.1 ml
 

into 1- and 7-day old mice, then 0.2 ml into 14- and 21-day old mice).
 

40
 
The results of the bioassay of trichlorofluoromethane in Osbornc-


Mendel rats for possible carcinogenicity are not conclusive because
 

inadequate numbers of rats survived long enough to be at risk from
 

late-developing tumors. Under the conditions of this bioassay, trichlorofluoromethane was not carcinogenic to B6C3F1 mice.
 

41
 
VI. BIBLIOGRAPHY
 

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

J. Wiley & Sons, New York, 1971.
 

Aviado, D.M., "Toxicity of Aerosol Propellants in the Respiratory and
 

Circulatory Systems. IX. Summary of the Most Toxic: Trichlorofluoromethane (FC 11)." Toxicology 3:311-319, 1975.
 

Belej, M.A. and D.M. Aviado, "Cardiopulmonary Toxicity of Propellants
 

for Aerosols." Journal of Clinical Pharmacology 15(1):105-115,
 

1975.
 

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, B.C., 1977.
 

Chemical and Engineering News, "Aerosol Producers Have Another Bad
 

Year." 55_(21):6, 1977.
 

Chemical Marketing Reporter, "Chemical Profile: Fluorocarbons."
 

208_(10), 1975.
 

Chemical Regulation Reporter, "Federal Agencies to Phase Out Nonessential Propellant Use By 1979." J_(9) :298-299, 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.
 

Crossland, J., "Aerosols." Environment ^6(6): 16-20, 25 ,26 , 1974.
 

Epstein, S.S., S. Joshi, J. Andrea, P. Clapp, H. Falk, and N. Mantel,
 

"Synergistic Toxicity and Carcinogenicity of 'Freons' and Piperonyl Butoxide." Nature 214:526-528, 1967.
 

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.
 

42
 
Gosselin, R.E., H.C. Hodge, R.P. Smith, and M.N. Gleason, Clinical
 

Toxicology of Commercial Products, 4th edition. The Williams
 

and Wilkins Company, Baltimore, Maryland, 1976.
 

Grimsrud, E.P. and R.A. Rasmussen, "The Analysis of Chlorofluoromethanes in the Troposphere by Gas Chromatography-Mass Spectrometry." Atmospheric Environment 9:1010-1013, 1975.
 

Hester, N.E., E.R. Stephens, and O.C. Taylor, "Fluorocarbons in the
 

Los Angeles Basin." Journal of the Air Pollution Control Association 24(6):591-595. 1974.
 

International Agency for Research on Cancer, "IARC Monographs on the
 

Evaluation of Carcinogenic Risk of Chemicals to Man, Volume 1.
 

IARC, Lyon, France, 1972.
 

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

Observations." Journal of the American Statistical Association
 

53_:457-481, 1958.
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

Lovelock, J.E., "Atmospheric Fluorine Compounds as Indicators of Air
 

Movements." Nature 230:379, 1971.
 

Lovelock, J.E., R.J. Maggs, and R.J. Wade, "Halogenated Hydrocarbons
 

In and Over the Atlantic." Nature 241:194-196, 1973.
 

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

Co., New York, 1966.
 

Molina, M.J. and F.S. Rowland, "Stratospheric Sink for Chlorofluoromethanes: Chlorine Atom-Catalysed Destruction of Ozone."
 

Nature 249:810-812, 1974.
 

Polkis, A., "Determination of Flurocarbon 11 and Fluorocarbon 12 in
 

Post-Mortem Tissues: A Case Report." Forensic Science 5:53­

59, 1975.
 

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

in Male Rats Given Subcutaneous Carbon Tetrachloride." Journal
 

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

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.
 

43
 
Simmonds, P.G., S.L., Kerrin, J.E. Lovelock, and F.H. Shair, "Distribution of Atmospheric Halocarbons in the Air over the Los
 

Angeles Basin." Atmospheric Environment 8:209-216, 1974.
 

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

62:679-682, 1975.
 

U.S.	 International Trade Commission, Synthetic Organic Chemicals:
 

United States Production and Sales, 1975. USITC Publication
 

804, Washington, D.C., 1977.
 

Windholz, M., editor, The Merck Index; An Encyclopedia of Chemicals
 

and Drugs, Ninth edition. Merck and Co., Inc., Rahway, New
 

Jersey, 1976.
 

44
 


TABLE A1

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED 

WITH TRICHLOROFLUOROMETHANE 

CONTROyUNTH) CONTgO^VBH ) LOW DOSE HIG H DOSE 01-172M 01-173M 

ANIMAL S

ANIMAL S

ANIMAL S

 INITIALL Y I N STUD Y 2 0

 NECROPSIED 20

 EXAMINE D HISTOPATHOLOGICALLY *¥ 20

 2 0 

 20 

 20 

50 

50 

50 

50 

5C 

50 

INTEGUMENTAR Y SYSTEM 

*SUBCDT TISSUE

SEBACEOUS ADENOM A 

FIBROM A 

 (20) (20) (50) 

1 (2%) 

1 <2X ) 

(50) 

BESPIRATOR Y SYSTEM 

#LUN G

ADINOSQUAMOU S CARCINOM A

 (20) (20) 

1 (5X) 

(50) (50) 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGANS

MALIG.LYMPHOMA , HISTIOCYTIC TYPE

 (20)

 1 (5X) 

 (20) (50) (50) 

#SPLEE N

HEMANGIOSABCOMA

MAIIG.LYMPHOMA , HISTIOCYTIC TYPE 

 (20)

 1 (5%)

 ! 

! (5X) 

(49) 

1 (2«) 

(50) 

ICEEVICAL LYMP H NODE

ADENOSQUAMOU S CARCINOMA , HEIASTA

 (19) (20) 

1 (5X) 

(46) (19) 

tLUN G

MALIG.LYMPHOMA , HISTIOCYTIC TYPE

 (20)

 1 (5%) 

 (20) (50) (50) 

CIRCULATOR Y SYSTEM 

NONE 

DIGESTIVE SYSTEM 

__NQNE 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D

* NUMBE R OF ANIHALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

A-3 
TABLE AI (CONTINUED) 

CONTROL (UNTR) CONTROL! VEH) LON DOSE HIGH DOSE 

01-171H 01-0918 01-172H 01-17311 

UilNARY STSTEH 

•KIDNE Y

ADENOSQUAHOUS CARCINOMA,

MIXED TtmOR, MALIGNANT

HAHA8TOKA +

 HETASTA

 (20) (20)

 1

 2

 1

 (5X) 

 (10X) 

 (5X) 

 (50) (£0) 

ENDOCRINE STSTER 

•PITUITARY (19) (20) (US) (U9) 

CHROHOPHOBE ADENOMA 2 (11X) 2 (UX) 

IADREIAL (20) (20) (50) (50) 

PHIOCHROHOCTTOHA 1 (5X) 

•THTROIE (20) (20) (48) (SO) 

rOHICOUR-CELL ADENOMA 1 (5X) 2 («X) 

IKPIIODDCtlVE SYSTEM 

NONB 

HERVOOS STST£M 

IBRAIN (20) (20) (49) (SO) 

HeUROriBHOS»HCOH» 1 (2X) 

SPECIAL SENSE ORGAM S 

NONE 

•OSCOtOSKELETAL STSTEH 

NONE 

BOOT CAVITIES 

• ABDOMINAL CAVITY (20) (20) (50) (SO) 

SPIUBLH/GIAST-CELL CARCINOMA 1 (5X) 

ALL OTHER SYSTEMS 

I NUMBER Or ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF ANIMALS NECROPSIEC 

+ THIS IS CONSIDERED TO BE A BENIGN FORM OF THE MALIGNANT MIXED TUMOR OF THE KIDNEY AND CONSISTS OF PROLIFERATIVE 

LIPOCYTES, TUBULAR STRUCTURES, FIBROBLASTS, AND VASCULAR SPACES IN VARYING PROPORTIONS. 

A-4 
TABLE A1 (CONCLUDED) 

CONTROL (UNTB) CONTROLJVEH, 1

8U9I5 B^M°3MSE 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALLY I N STDDY NATURA L DEATHi 

MORIBUN D SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE 

ANIMA L MISSING 

2

?9 

1 

2

% 5

29

1 

5

«9 

i INCLUDES AUTOLYZED ANIMAL S 

TUMO R SUflMAF Y 

TOTA L ANIMAL S WIT H PRIMAR Y TOTAL PEIMAS Y TUMORS TUMORS * 6 6 5

7 >t 7 

TOTAL ANIMAL S WIT H BENIG N TOTAL BFNIG N TUMORS TUMORS 3 3 2

2 « 6 

TOTAL ANIMAL S WIT H MALIGNAN T TUHOBS TOTA L MALIGNAN T TUMORS 3 3 5

5 1 1 

TOTAL ANIMAL S WIT H SECONDAB Y TUMORS* TOTAL SECONDARY TUMORS 1 2 

TOTAL ANIMALS WITH TUMORS UNCERTAIN ­ BENIG N O P MALIGNAN T 

TOTAL UNCERTAI N TUMORS 

TOTAL ANIMAL S WITH TUMORS PBIMARY OR METASTATIC 

TOTAL UNCERTAI N TUMOBS 

 UNCERTAIN ­

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDAR Y TUMORS * SECONDAR Y TUMORS : METASTATIC TDMOBS OR TUMORS INVASIV E INTO AN ADJACENT ORGA N 

A-5 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED 

WITH TRICHLOROFLUOROMETHANE 

CONTROLJUNTB) CONTROL^VEH) LOU DOSE 01-174F HIG H DOSE 01-175F 

ANIMALS INITIALLY IN STUDY 20 20 50 5C 

ANIMAL S NECBOPSIED 20 20 50 50 

ANIMAL S EXAMINE D HI STOPATHOLOGICALL Y "* 2 0 20 1(9 5C 

INTEGUMENTAR Y SYSTE M 

*SKIN (20) (20) (50) (50) 

SQUAMOH S CELL CARCINOM A 1 (2%) 

*SUBCHT TISSUE (20) (20) (50) (50) FIBROSARCOMA 1 <2X) 

RESPIRATOR Y SYSTEM 

ITRACHEA (20) (16) (49) (48) HEMANGIOSARCOMA , METASTATIC 1 <2X ) 

HEMATOPOIETIC SYSTEM 

tSPLEEN (20) (20) (49) (50) HENANGIOSAECOMA 1 (5JS) 

MALIG.LYMPHOHA , HISTIOCYTIC TYPE 1 (5X) 

tLUN G (20) (20) (49) (50) MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (5X) 

CIRCULATOR Y SYSTEM 

NONE 

DIGESTIVE SYSTEM 

iESOPHAGUS (20) (17) (49) (46) HEMANGIOSARCOMA , METASTATIC 1 (2*) 

UBINAR Y SYSTEM 

*KIDNE Y (20) (20) (119) (50) HAMBXSM + 

* NUMBE R OF ANIMAL S »ITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

+ THIS IS CONSIDERED TO BE A BENIGN FORM OF THE MALIGNANT MIXED TUMOR OF THE KIDNEY AND CONSISTS OF PROLIFERATIVE 

LIPOCYTES, TUBULAR STRUCTURES, FIBROBLASTS, AND VASCULAR SPACES IN VARYING PROPORTIONS. 

A-6 
TABLE A2 (CONTINUED) 

CONTROL (II NTR) 01-171F CONTROL (VEH ) 01-091F LOW DOSE 01-174F HIGH DOSE 

01-175F 

ENDOCRIN E SYSTEM 

tPITUITAR Y 

CHROMOPHOBE ADENOM A 

(20)

3 (15X)

 (20)

 1 (20X)

 (48) 

1 (2X) 

(15) 

3 (7X) 

*ADRENA L 

CORTICA L CARCINOM A 

PHEOCHROMOCYTOMA 

(20)

2 (10X) 

 (20) (49) (49) 

1 (2X) 

#THYROIE 

FOLLICULAR-CELL CARCINOM A 

(20)

2 (10X) 

 (20) (48) (16) 

REPRODUCTIV E SYSTEM 

*MAMMAB Y GLAN D 

ADENOCARCINOMA ,

FIBROADENOM A 

 NOS 

(20)

3 (15X)

 (20)

1 (5X)

 3 (15X)

 (50) 

1 (2X) 

1 (2X) 

(50) 

#UTEBUS 

ADENOCABCINOMA, NOS 

ENDOMETBIAL STROMA L POLYP 

tOVAR Y 

ADENOCABCINOMA , NOS, METASTATIC 

GRANOLOSA-CELL TUMOR 

GRANULOSA-CELL CARCINOM A 

#OVABY/FOLLICLE 

CYSTADENOMA, NOS 

(19)

(20)

1 (5X) 

(20)

 (19)

 (20)

1 (5«) 

 (20)

 (49) 

1 (2X) 

 (49) 

1 <2X ) 

 (49) 

(19) 

1 <2X ) 

(49) 

(49) 

1 (2X) 

NERVOU S SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NON E 

HOSCULOSKELFTAL SYSTEM 

*MOSCLE OF THORA X 

HEMANGigSARCOaA 

(20) (20) (50) 

TJ21 L 

(50) 

* SDMBE R

* NUMBER

 OF ANIMAL S

 OF ANIMALS

 KITH TISSUE

 NECROPSIED 

 EXAMINE D MICROSCOPICALLY 

A-7 
TABLE A2 (CONCLUDED) 

J»BH) 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEHS 

NONE __ 

ANIMAL tISPCSITIOM SO (IMA El 

ANIMAL S INITIALLY IN STUDY NATDBAL DEATH}

MOBIBDNC SACBIFICE

SCHIEULED SACBIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACBIFICE

ANIMAL MISSING 

» INCLUDES AUTOLYZED ANIMALS 

20

 18

 2 

20

 12

8 

SO

 46

 2

2 

5C 

15 

3 

2 

1DHOB SD8HAFY 

TOTAT AL ANIMALAN S WITH PRIMARY TOTAL PBIRABY TUMORS 

TUMOBS* To 

6. 

TOTAL UNCEBTAIN TDHOBS 

TOTAL nNCEBTAIN TO HOES 

INTO AN ADJACER ORGAN 

A-8 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH TRICHLOROFLUOROMETHANE
 

TABLE Bl 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED 

WITH TRICHLOROFLUOROMETHANE 

ANIMALS INITIALLY IN STUDY 20 20 50 19 

ANIMAL S MISSING 1 

ANIMAL S NECFOPSIED 19 19 50 17 

ANIMAL S EXAMINED HISTOPATHOLOGICAllI** 18 19 50 17 

INTEGUMENTAR Y SYSTEH 

*SUBCUT TISSUE (19) (19) (50) (17) 

FIBROSARCOHA 1 (5X) 1 (2X) 2 (IX) 

FIBROUS HISTI3CYTOMA, MALIGNAN T 1 (2X) 

RESPIRATORY SYSTEM 

tLUNG (19) (19) (50) (17) 

HEPATOCELLULAB CARCINOMA, METAST 1 (2X) 1 (2X) 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (5X) 1 (2X) 

ALVEOLAR/BRONCHIOLAH CARCINOMA 1 (5X) 

HEMATOPOIETIC SYSTEM 

*NERV E TRACT (19) (19) (50) (1(7) 

MALIG.LYMPHOMA, ONDIFFEB-TYPE 1 (2X) 

*MULTIPLE ORGANS (19) (19) (50) (17) 

MALIG.LYMPHOMA . LYMPHOCYTIC TYPE 3 (6X) 

MALIG.LYMPHOMA , HISTIOCYTIC TYPE 1 (5X) 2 (IX) 

*SUBCDT TISSUE (19) (19) (50) (17)

 

MALIG.LYMPHOMA . HISTIOCYTIC TYPE 1 (2X)

 

tMESENTIRIC L. NODE (17) (19) (U7) 

HEHANGIOMA 1 (2X)

 

tKIDNIY (19) (19) (19) 

MAIIG.LYMPHOMA. HISTIOCYTIC TYPE 1 (2X) 

CIRCULATORY SYSTEM 

__NONE 

* NUMBE R OF ANIMAL S KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
TABLE Bl (CONTINUED) 

CONTROL (UNTR) 02-M18T CONTBOLIVEH) 02-M171 LOtl DOSE 02-R17 2 HIG H DOSE 02-M173 

DIGESTIVE SYSTEM 

#LIVER

NEOPIASTIC NODUL E

HEPATOCELLULAB CARCINOM A

HEMANGIOSARCOMA

 (19)

 3 (16*)

 (19)

 5 (26X)

 (50)

 3

 12

 (6X) 

 (2UX)

 (<»7) 

 10

 1

 (21*) 

 (2*) 

UEINA8 Y SYSTEH 

NONE 

ENDOCR IN E SYSTEM 

IPITUITARY

CHROBOPHOBE ADENOMA

 (16) (16) (t5) (<i7) 

1 (2%) 

REPRODUCTIV F SYSTE M 

tTESTIS

INTERSTITIAL-CELL TOMOR

 (19) (19) (50)

 1 (2% ) 

 (U7) 

NERVOU S SYSTEM 

NONE_ _ 

SPECIAL SENSE ORGAN S 

*HARDERIA N GLAN D

ADENCMA , NOS

 (19) (19) (50)

 1 (2X) 

 (47) 

MUSCULOSKELETA L SYSTE M 

NON E 

BODY CAVITIE S 

_NON E 

A LL OTHER SYSTEMS 

NON E 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

B-4
 
TABLE Bl (CONCLUDED) 

CONTHOLflJNTR) CONTROL(VBH) LOU DOSE HIGH DOSE 

02-M181 02-M171 02-M172 02-B173 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 2 NATURAL DEATHS °

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 13 30 21 

ANIMAL MISSING 1 

9 INCLUDES AUTOLYZED ANIMALS 

TUMOR SUHMABY 

TOTAL ANIMALS WITH PRIMARY IOMORS* 

TOTAL PRIM AH Y TUMORS 

TOTAL ANIMALS WITH BENIGN TONORS 

TOTAL BENIGN TOMORS 

TOTAL ANIMALS WITH MALIGNANT TUMORS 

TOTAL MALIGNANT TUMORS 1517 

TOTAL ANIMALS WITH SECONDARY TUMORS* 

TOTAL SECONDARY TUMORS 

TOTAL ANIMALS WITH TOMORS UNCERTAIN­ BENIG N OB MALIGNANT

 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMOBS UNCERTAIN­ PRIMAR Y OR BETASTATIC

 

TOTAL UNCERTAIN TUHOBS 

* PRIMARY TDBORS: ALL TUMORS EXCEPT SECONDARY TUMORS * SECONDARY TUMOBS: METASTATIC TUHOHS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

B-5 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

TREATED WITH TRICHLOROFLUOROMETHANE 

CONTROL (DUTB) CONTROL (VEH) LOW DOSE HIGH DOSE 02-F181 02-F171 02-F174 02-F175 

ANIMAL S INITIALLY IN STUDY 2 0 20 

ANIMAL S NECROPSIED 19 19 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY ** 18 19 

INTEGUMENTAR Y SYSTEM 

NON E 

RESPIRATOR Y SYSTEM 

ILUN G (19) (19) 

ALVECLAR/BRONCHIOLA R ADENOM A 1 (5X) 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGAN S (19) (19) 

MA1IG. LYMPHOMA . ONDIFFEB-TYPE 

MALIG. LYMPHOM A , LYI1PHOCYTIC TYPE 

MALIG. LYMPHOH A i HISTIOCYTIC TYPE 

tSPLEEN (19) (19) 

HEHANGIOSARCOMA 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVEB (19) (19) 

HEPATOCELLULAR CARCINOM A 1 (5X) 1 (5X) 

#STOMACH (19) (19) 

SQUAMOU S CELL PAPILLOMA 

*RECTDM (19) (19) 

LEIOMYOSAHCOMA 

* NDMBE B OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

» NUMBE R OF ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

50 

50 

50 

(50) 

(50) 

1 (2%) 

1 (2X) 

(50) 

1 (2X) 

(50) 

4 (8X) 

(50) 

1 (2X) 

(50) 

_1_J2XJ 

50 

49 

49 

(49)
 

2 <4X)
 

(49) 

1 <2X) 

(49) 

(49) 

2 (4X) 

(49) 

<2X) 

(49) 

B-6 
UBINAR Y STSTEH 

•URETER 

LEIOH TO SARCOMA, NETASTATIC 

ENDOCRINE STSTEH 

IPITUITARY 

CHROBOPHOBE ADENOM A 

IADHENAL 

PREOCHROMOCTTOMA 

tTHYROIB 

FOLLICULAR-CELL ADENOHA 

REPRODUCTIVE SISTEH 

*?AGINA 

LEIOHTOSARCOHA, HETASTATIC 

tOTEROS 

LEIOHTOSARCOHA , METASTATIC 

ENDOMETBIAL STROHAL POLTP 

tOVARI 

GRANOLOSA-CELL TO H08 

NERVOUS STSTEH 

•CRAIIAL NtRTE 

HEOROFIBROHA 

SPECIAL SEISE ORGANS 

*BARDE8IAN GLAND 

ADE1CHA. EOS 

HUSCULOSKELETAL STSTEH 

NONE 

t IDMBEB OF A1IHALS HITB TISSUE

* IOBBEB OF A HI HALS VECBOPSIED 

TABLE B2 (CONTINUED) 

CONTROL (ONTB) CONTROL (VEH) 02-F18T 02-F171

(19) (19)

(16) (18)

1 (6X)

(18) (19)

(15) (15)

(19) (19)

(17) (19)

1 (6X)

(18) (19)

1 (5X) 

(19) (19)

(19) (19)

 EIAIIIED HICBOSCOPICALLI 

 LOW DOSE HIGH DOSE 02-F174 02-F175 

 (50) (19) 

1 (2%) 

 (50) (19) 

1 (2X) 

 (50) (49) 

1 (2X) 

 (50) (49) 

1 (2X) 

 (50) (49) 

1 (2X) 

 (50)

 (19) 

1 (2«) 

1 (2X) 

 (50) (49) 

 (SO) (49) 

1 (2X) 

 (50) (49) 

1 (2X) 2 <4X) 

B-7 
TABLE B2 (CONCLUDED) 

CONTROLJDNTB, CONTROL|VBH> tO^DOS^ HJG.H, 

BODY CAVITIES 

HOME 

ALL OTHER SYSTEMS 

NOME 

ANIMA L EISPCSITION SDMHABY 

AIIHALS IMITIALLY IM STODT 20 20 SO 50 

NATURAL DEATH* 3 2 12 17 

HOBIBUND SACRIFICE 1 1 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 17 18 37 32 

AilHAL HISSING 

i INCLUDES ADTOLYZED ANIRALS 

TOHOB SORHARY 

T°HOI1S 22 t0?OTl

Aj"igNi!»S|55olsE"IG"

1 

, 

TOTAL AMIBALS HITR TOBORS UNCERTAIN­ BENIGN OR HALI6NANT 

TOTAL UNCERTAIN TUHOBS 

TOTAL UNCERTAIN TUMORS 

INTO AN ADJACENT OBGAN 

B-8 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS TREATED WITH TRICHLOROFLUOROMETHANE
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH TRICHLOROFLUOROMETHANE

 

c:ONTBOL (UNTR) 01-171M CONTROL (VEH) 01-0911!

 LOR DOSE 01-172M 

ANIMAL S INITIALLY IN STUDY 20 20 50 

ANIMALS NECBOPSIED 20 20 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 20 20 50 

INTEGUMENTAR Y SYSTEM 

*SKIN (20) (20) (50) 

EPIDERMAL INCLUSION CYST 

INFLAMMATION , NO S 1 (5X) 

ULCER, ACUTE 1 (2*) 

HYPERKEBATOSIS 1 (5X) 

ACANTHOSIS 1 (5X) 

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

ABSCESS, NOS 1 (5X) 

NECROSIS, FOCAL 1 (2X) 

RESPIEA10RY SYSTEM 

tTRACHEA (20) (20) (49) 

INFLAMMATION , CHRONIC 1 (2X) 

*LUNG (20) (20) (50) 

INFLAMBATION, ACUTE 

PNEUMONIA. CHRONIC MORINE 20 (100X) 19 (95X) 48 (96X) 

HEMATOPOIETIC SYSTEM 

#SPLEEN (20) (20) (49) 

HEMCSIDEROSIS 

HEHATOPOIESIS 2 (10X) 2 (4*) 

*CERVICAL LYMPH NODE (19) (20) (46) 

INFLAMMATION, NOS 1 (2X) 

*HEDIASTINAL L. NODE (19) (20) (46) 

HYPZRPLASIA. LYHPHOID 

*BESENTIRIC L. NODE (19) (20) (46) 

. _ PERIART_EBITIS_ ._ . . .. _ _ 1 (5X) 

* NDBBER OF ANIHALS HITH TISSDE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIHALS NECROPSIEC 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

HIGH DOSE 01-173H 

50 

5C 

50 

(50) 

1 (2X) 

1 (2X) 

(50) 

(50) 

(50) 

1 (2X) 

49 (98X) 

(50) 

3 (6X) 

(49) 

1 (2X) 

(49) 

1 (2X) 

(49) 

C-3
 
TABLE Cl (CONTINUED) 

CONTROL (UNTR) 01-171K CONTROL (VEH) 01-091H LOB DOSE 01-172M H:G H DOSE 

0 1-173M 

*THYMD S (18) (17) (i»6) <U5) 

INFLAMMATION , NOS 1 (6X) 

CIECULATOEY SYSTEM 

#MYOCAREIU M (20) (20) (50) (50) 

MINERALIZATIO N 1 (2X) 

INFLAMHATION, SDPPUBATIV E 1 <2X ) 

INFLAMMATION , CHRONIC 1 (2X) 

FIBEOSIS 

DEGENERATION , NO S 

1 <2X) 

1 (2X) 

2 (UX) 

1 (2X) 

DIGESTIVE SYSTEM 

ISALIVAB Y GLAND (17) (17) (18) (22) 

INFLAMMATION , NOS 1 (6X) 

INFLAMMATION, ACOTE/CHRONIC 1 (5X) 

tLIVER (20) (19) (50) (50) 

HEMOBRHAG E 

INFLAMMATION , CHRONIC FOCAL 1 (2X) 

1 (2X) 

GRANULOMA , NOS 

PERIABTEBITIS 

PELIOSIS HEPATIS 

NECROSIS, FOCAL 1 (5X) 

1 (2X) 

1 <2X) 

1 (2X) 

METAMORPHOSIS FATTY 1 (5X) 

iHEPATIC LOBULE (20) (19) (50) (50) 

CYT010GIC DEGENEBATIO N 1 (2X) 

*LIVER/CENTBILOBULAH (20) (19) (50) (50) 

NECBOSIS, NOS 1 (5X) 2 (UX) 

NECBOSIS, COAGULATIVE 1 (2X) 

METAMOBPHOSIS FATTY 1 (5X) 

•BILE DUCT 

INFLAMMATION, FOCAL 

(20) (20) (50) (50) 

1 (2X) 

#PANCBEAS 

PESIAETEBITIS 

(20) (19)

1 <5X) 

 (K9) (50) 

1 (2X) 

tESOPHAGUS (20) (19) («9) (17) 

FBACTURE, NOS 1 (2X) 3 <6X) 

# NUMBE R OF ANIMAL S HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMALS NECEOPSIED 

C-4 
ULCEF, NOS 

INFLAMMATIOS, SUPPU8ATIVE 

INFLAMMATION , ACUTE 

*STOMACH 

ULCER, NOS 

INFLAMMATION , FOCAL 

ULCEE, FOCAL 

ULCER. ACUTE 

INFLAMMATION , CHRONIC FOCAL 

CALCIUM DEPDSIT 

HYPERKERATOSIS 

ACANTHOSIS 

ILARGE INTESTINE 

NEMATODIASIS 

URINAR Y SYSTEM 

IKIDNEY 

MINERALIZATION HEMORBHAG E

 

PYELONEPHBITIS, NOS 

PYELONEPHRITIS, ACUTE 

ABSCESS, NOS 

INFLAMMATION , CHRONIC 

*RENA L PAPILLA 

MINERALIZATIO N 

HYPERPLASIA, EPITHELIAL 

*KIDNEY/PELVI S 

MINERALIZATION 

INFLAMMATION, NOS 

• URINAR Y BLADDER 

CALCULUS, NOS 

ULCER, NOS 

INFLAMMATION , ACDTE 

ULCER, ACUTE 

INFLAMMATION , CHRONIC 

HYPERPLASIA , EPITHELIAL

 

ENDOCRIN E SYSTEM 

tPITUITARY 

INFLAMMATION, . NOS 

# KUMBE E OF ANIMALS WITH TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

TABLE Cl (CONTINUED) 

CONTROL (ONTR) COSTROL(VEH) 01-171S 01-0918 

(20) (19) 

1 (5*) 

1 (5X) 1 (5X) 

1 (5X> 

1 (5X) 

1 (5X) 

1 (5X) 

(20) (18) 

(20) (20) 

2 (10X) 

1 (5X) 

8 (UOX) 7 (35X) 

(20) (20) 

(20) (20) 

1 (5X) 

(19) (19) 

1 (5%) 

1 <5X) 

(19) (20) 

1 (5*) 

 EXAMINED MICROSCOPICALLY 

LOW DOSE 

01-172M 

1 <2X) 

1 (2%) 

(U9) 

1 (2X) 

1 (2X) 

K (8X) 

1 <2X ) 

(18) 

3 (6X) 

(50) 

1 <2X) 

1 (2*) 

1 (2X) 

12 (2KX ) 

(50) 

2 (UX ) 

(50) 

1 (2X) 

(48) 

1 (2X) 

1 (2X) 

1 (2X) 

(K5) 

HIG H DOSE 

01-173H 

1 (2X) 

(50) 

6 (12X) 

1 (2X) 

(50) 

3 (6X) 

(50) 

1 (2X) 

1 <2X) 

1 (2X) 

11 (22X) 

(50) 

1 (2X) 

(5C) 

(50) 

1 (2X) 

1 (2X) 

1 (2X) 

1 (2X) 

(U9) 

C-5 
HYEERPLASIA , CH ROHOPHOBE-CELL 

tADRENA L CORTEX 

DEGENERATION, NOS 

HYPERPLASIA, FOCAL 

ANGIECTASIS 

*ADRENA L MEDULLA 

HYPFBPLASIA, NOS 

HYPERPLASIA, FOCAL 

MHYHOI D 

CYST, NOS 

INFLAMMATION , NOS 

REPRODUCTIVE SYSTEM 

IPROSTATE 

INFLAMMATION , NOS 

INFLAMMATION , ACUTE 

INFLAMMATION , ACUTE FOCAL 

INFLAMMATION , CHRONIC 

*SEMINA L VESICLE

 

INFLAMMATION , NOS 

INFLAMMATION , ACUTE 

ITESTIS 

MINERALIZATIO N 

ATROPHY, NOS 

HYPOSPEPMATO GENES IS 

*EPIDICYMIS 

STEATITIS 

NECROSIS, FAT 

NERVOUS SYSTEM
 

NONE_
 

SPECIAL SENSE OEGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

__NOJ<E
 

TABLE Cl (CONTINUED) 

CONTROL (0 NTS) 01-171IJ

 

1 (5%) 

(20) 

9 (145%) 

1 (5«) 

3 (15%) 

(20) 

1 (5%) 

1 (5%) 

(20) 

(19) 

1 (5%) 

(20) 

(20) 

5 (25%) 

6 (30%) 

(20) 

CONTROLJVEH, 

(20) 

(20) 

(20) 

1 (5%) 

(2) 

1 (50%) 

(20) 

1 (5%) 

(20) 

3 (15%) 

(20) 

1 (5%) 

LOB DOSE 01-172H 

2 («%) 

(50) 

11 (22%) 

3 (6%) 

(50) 

1 (2%) 

(U8) 

(41) 

7 (16%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

5 (10%) 

7 (14%) 

(50) 

HIGH DOSE 01-173M 

1 (2%) 

(50) 

3 (6%) 

2 (4%) 

(50) 

(50) 

1 (2%) 

(44) 

4 (9%) 

5 (11%) 

2 (5%) 

(50) 

(50) 

1 (2%) 

3 (6%) 

2 (4%) 

(50) 

1 (2%) 

* NUMBE R OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIHAL S NEC80PSIED 

C-6 
TABLE Cl (CONCLUDED) 

CONTROL (DNTR) CONTROL 

91 LOU DOSE 01-1728 

BODY CAVITIES 

•PLEURA 

INFLAMMATION, ACUTE 

INFLAMMATION. ACOTE/CHRONIC 

INFLAMMATION, PYOGRANULOSATOOS 

(20) (20) (50) 

1 (2X) 

(50) 

2 (1%) 

1 (2X) 

3 (6*) 

•PERICARDIUM 

INFLAMMATION, 

INFLAMMATION , 

INFLAMMATION, 

INFLAMMATION, 

ACUTE 

ACUTE/CHRONIC 

CHRONIC 

PYOGRANULOMATOUS 

(20) (20) (50)

1 <2X) 

 (50) 

1 <2X) 

1 (2X) 

1 (2X) 

• EPICARDIUM 

INFLAMMATION, 

INFLAMMATION , 

INFLAMMATION, 

INFLAMMATION, 

ACUTE 

ACUTE/CHRONIC 

CHRONIC 

PYOGRANULOHATOUS 

(20) (20) (50)

1 (2X)

 (50) 

2 (4X) 

1 (2X) 

2 (IX) 

1 (2X) 

• MESENTERY 

PERIAR1ERITIS 

(20) (20) 

1 (5X) 

(50) 150) 

1 (2X) 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

NONE 

t NUMBI H OF ANIMALS

* NUMBE R OF ANIMALS

 WITH TISSUE EXAMINE D

 NECROPSIED 

 MICROSCOPICALLY 

C-7 

TABLE C2 (CONTINUED) 

CONTBOL ID NIB) CONTBOL(TEH) 01-171F 01-091F 

INILAHMATION, NOS 

ILIVER (20) (19) 

INFLAMMATION, NOS 1 (5X)

 

GRANULOMA, NOS 1 (SX) 

METAMORPHOSIS FATTY 1 (5X) 

ANGIECTASIS 

•LIVER/CENTBILOBOLAR (20) (19)

 

NECBOSIS, ISCHEMIC 

•PANCREAS (20) (18)

 

CYST, NOS 1 (6X)

 

INFLAHMATION, ACUTE NECBOTIZING 

INFLAMMATION, ACUTE/CHRONIC 

PERIAR1ERITIS 

ATROPHY, KOS 1 (6X) 

• ESOPHAGUS (20) (")

 

FRACTURE, NOS 

ISTOMACH (20) (20)

 

ULCER. NOS 1 (5X) 

INFLAMMATION. FOCAL 4 (20X) 

ULCER, FOCAL 2 (10X) 1 (5X)

 

INFLAMMATION , CHRONIC 

HY PERK ERATO SIS 

ACANTHOSIS 

•GASTRIC SUBMUCOSA (20) (20) 

HYPERPLASIA. LYMPHOID 

tSHALI INTESTINE (20) (19) 

ULCIR, ACUTE 

•LARGE INTESTINE (20) (20) 

NEHATODIASIS 

URINARY SYSTEM 

• KIDNE Y (20) (20)

 

MINERALIZATION 1 (SX) 

CYST, NOS 1 (5X)

 

PYELONEPHRITIS, ACUTE 

INFLAMMATION, ACUTE FOCAL, .._ ^ _ 

* NUMBER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY * DUMBER OF ANIMALS NECROPSIED 

LOU DOSE 

01-174F 

1 (3X) 

(49) 

1 (2X) 

(49)

1 (2X) 

(49) 

1 (2X) 

1 (2X) 

1 (2X) 

(U9) 

2 (4X) 

(18) 

7 (15X) 

1 (2X) 

1 (2X) 

(48) 

1 (2X) 

(48) 

1 (2X) 

(49) 

3 (6X) 

(49) 

4 (8X) 

1 (2*) 

HIGH DOSE 

01-175F 

(5C) 

1 (2X) 

(50) 

(50) 

(46) 

(50) 

2 (4X) 

(50) 

(50) 

(49) 

6 (12X) 

(50) 

1 12X). 

C-9 
TABLE C2 (CONTINUED) 

COHTROl(OllTB) 01-171? CONTROL(VEH ) 01-091F 

INFLAMMATION , CHRONIC 

INFABCT, FOCAL 

1 (20X) 5 (25X) 

*KIDNEY/CORTEX (20) (20) CYS1, NOS 

IRENA L PAPILLA (20) (20) HYPEBPLASIA, EPITHELIAL 

HYPEPPLASIA , FOCAL 

• KIDNEY/PELVI S (20) (20) MINERALIZATIO N 

IURINAB Y BLADDER (20) (18) INFLAMMATION , ACUT E 

HYPERPLASIA , EPITHELIA L 

ENDOCRIN E SYSTEM 

IPITUITAR Y (20) (20) CYST, NOS 

HYPEBPLASIA, CHBOMOPHOBE-CELL 1 <5X) 

fADBENA L CORTEX (20) (20) DEGENERATION , NOS 2 (10*) ANGIECTASIS 8 (40X) 2 (10X) 

#ADRENA l MEDULL A (20) (20) HYPERPLASIA, FOCAL 

tTHYRCI D (20) (20) INFLAMHATION , G RA NULOMATOU S 

HYPERPLASIA , C-CELL 

REPRODUCTIVE SYSTEM 

*MAMMAR Y GLAN D (20) (20) INFLAMMATION , ACUTE 

tUTEBUS (19) (19) HYCROMFTB A 

tUTEBUS/SNDOMETRIU M (19) (19) CYST, NOS 1 15XJ. 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECBOPSIED 

LOW DOSE 01-17UF 

5 (10X) 

1 (2X) 

(49) 

1 (2X) 

(i»9) 

8 (16X) 

1 <2X) 

(H9) 

8 (16X) 

(47) 

1 (2X) 

1 (2%) 

(48) 

1 (2X) 

(49) 

3 (6X) 

9 (18X) 

(49) 

1 (2X) 

(48) 

1 (2X) 

(50) 

1 (2X) 

(49) 

1 (2X) 

(49) 

HIGH DOSE 01-175F 

1 (2X) 

(50) 

(50) 

(50) 

9 (18X) 

(45) 

(45) 

(49) 

4 (8X) 

13 (27X) 

(49) 

(46) 

1 (2X> 

(50) 

(49) 

4 (8X) 

(49) 

C-10 
TABLE C2 (CONTINUED) 

CONTROL (ONTR) 01-171F CONTROL (VEH) 01-091F LOW DOSE 01-17UF HIGH DOSE 01-175P 

INFLAMMATION, ACUTE

tOVAR Y

MULTIPLE CYSTS

INFLAMMATION , ACUTE

 2 (11S)

 (20)

 1 (5X) 

 (20)

 2 (UX) 

 (1*9)

 2 («% ) 

 (H9) 

NERVOU S SYSTEM 

NON E 

SPECIAL SENSE ORGAN S 

NONE 

MUSCULOSKELETAL SYSTEM 

NON E 

BODY CAVITIES 

*PLEURA

INFLAMMATION ,

INFLAMMATION ,

INFLAMMATION,

*PERICARDIUM

INFLAMMATION,

ABSCESS, N3S 

INFLAMMATION ,

INFLAMMATION ,

*MESENTER Y

PERIAR1ERITIS 

 ACUTE 

 ACUTE/CHRONIC 

 PYOGB ANULOM ATOUS 

 ACUTE 

 ACUTE/CHRONIC 

 CHRONIC 

 (20) 

 (20) 

 (20) 

(20) 

(20) 

(20) 

(50) 

1 (2X) 

1 (2%) 

(50) 

1 (2X) 

(50) 

(50) 

1 <2X) 

1 (2X) 

(SO) 

1 (2X) 

2 (i»X) 

3 (6X) 

(50) 

1 (2%) 

ALL OTHER SYSTEMS 

DIAPHRAGM 

NECROSIS, FAT 

ADIPOSE TISSUE 

ISFLAMMATIONi_CHHONIC_FOCAL 

* NUMBER OF ANIHALS WITH TISSUE

* NUMBER OF ANIMALS NECROPSIED 

 EXAMINED CICROSCOPICALLY 

C-ll 
TABLE C2 (CONCLUDED) 

CONTROL (0 NTH) 01-171F CONTROL(VEH ) 01-091F LOU DOSE 01-17HF HIG H DOSE C1-175F 

SPECIAL HORPHOLOGY SUMMAR Y 

NO LESION REPORTE D 

AOTC/NECROPSY/NO HISTO 

* NUMBE R OF ANIMALS WITB TISSUE EXAHINE D MICROSCOPICALLY 

* NUMBE R OF ANIMALS NECROPSIED 

C-12 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE TREATED WITH TRICHLOROFLUOROMETHANE
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH TIRCHLOROFLUOROMETHANE 

CONTBOLJDNTH, CgB^OLJVEH) LOW DOSE 02-M172 

ANIMALS INITIALLY IN STUDY 20 20 50 

ANIMALS HISSING 

ANIMAL S NECROPSIED 19 19 50 

ANIMALS EXAMINED HI STOPATHOLOGICALL Y ** 18 19 50 

INTEGUMENTARY SYSTEM 

*SKIN (19) (19) (50) 

EPIDERMAL INCLUSION CYST 1 (5X) 

INFLAMMATION, ACUTE 

PARASITISM 33 (66X) 

*SUBCUT TISSUE (19) (19) (50) 

EDEMA , NOS 1 <2X) 

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

RESPIRATORY SYSTEM 

#LUNG (19) (19) (50) 

INFLAMMATION , ACUTE 2 (IX) 

INFLAMMATION, ACUTE FOCAL 

INFLAMMATION, ACUTE/CHRONIC 1 (2X) 

NECROSIS, NOS 1 (2X) 

HYPERPLASIA, ALVEOLAR EPITHELIUM 1 (5X) 

HEMATOPOIETIC SYSTEM 

tSPLEEN

AMYLOIDOSIS

 (19) 

7 (37X) 

(19) (50) 

7 (1KX) 

HEMATOPOIESIS 1 (8X) 

*HEMOLYMP H NODES (19) (19) (50) 

ANGIECTASIS 

HYPEBPLASIA, LYMPHOID 

1

1

 (2X) 

 (2X) 

tLYMP H NODE (17) (19) (17) 

HYPERPLASIA. LYMPHOID 1 (5X) 

*CERVICAL LYMPH NODE (17) (19) (17) 

HYPEEPLASIA. LYMPHOID . . . ._ 8 J17%1_ 

* NUMBF R OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

**EXCHJDES PARTIALLY AUTOLYZED ANIMALS
 

HIGH DOSE C2-M173 

149 

1 

U7 

(17) 

1 (2X) 

2U (51X) 

(17) 

(17) 

1 (2X) 

(17) 

6 (13X) 

1 (2X) 

(17) 

(17) 

1 (2X) 

(17) 

D-3
 
• HESEN7ERIC L. NODE 

CONGESTION, NOS 

INFLAM RATION. NOS 

INFLAHHATION. ACUTE 

INFLAMMATION , GRAROLOHATODS 

ANGIECTASIS 

ERYTHROPHAGOCYTOSIS 

HYPERPLASIA, LYHPROID 

CIRCOLATOR Y SYSTEM 

•HEART 

INFLAMMATION, CHRONIC FOCAL 

AHYLOIDOSIS 

CALCIOM DEP3SIT 

DIGFSTIVE SYSTEM 

• SALIVAR Y GLAND 

ABSCESS, NOS 

INFLAMMATION , CHRONIC FOCAL 

• LIVEB 

THROMBOSIS, NOS 

INFLAMMATION, NOS 

INFLAHHATION. FOCAL 

I NFL AH NATION, A COTE 

NECROSIS, ISCHEHIC 

AHYLOIDOSIS 

HYPERPLASTIC NODDLE 

HYPEBPLASIA, FOCAL 

•LIVER/PERIPORTAL 

DEGENERATION, NOS 

NECROSIS, NOS 

*BILE DOCT 

INFLAHHATION, CHRONIC 

HYPERPLASIA. NOS

 

•PANCREAS 

INFLAHHATION, NOS 

AMYLOIDOSIS 

f NUMBER OF ANIHALS KITH TISSUE

* NUMBE R OF ANIHALS NECBOPSIED 

TABLE DI (CONTINUED) 

CONTROL (UNTa) CONTROL (VEH) 

02-M18T 02-MT71 

(17) (19) 

1 <5X) 

1 (6X) 

1 (5X) 

(18) (19) 

1 (6X) 

(19) (19) 

8 (42X) 

(19) (19) 

1 (5X) 

1 (SX) 

1 (SX) 

2 (11X) 

(19) (19) 

(19) (19) 

2 (11X) 

(18) (18) 

1 (6X) 

 EXAMINED MICROSCOPICALLY 

>fl.m 

(17) 

2 (»X) 

1 (2X) 

2 (IX) 

10 (21X) 

(50) 

(49) 

2 («X) 

11 (29X) 

(50) 

1 (2X) 

1 (2X) 

« (8X) 

1 (2X) 

1 (2X) 

(50) 

1 (2X) 

1 (2X> 

(50) 

1 (2X) 

(50) 

, 1 (2X1, 

02-K 

C«l 

1 

9 

3 

(47) 

1 

1 

(06) 

7 

(«7)

1 

3 

(U7) 

(147) 

1 

(47) 

OF 

(2X) 

(19X) 

<6X) 

<2X ) 

(2X) 

(15X) 

(2X) 

(6X) 

(2X) 

D-4 
TABLE Dl (CONTINUED) 

COJTgOLJOm, CONTROLJVEH) 

•STOMACH (19) (18)

 

MINERALIZATION 1 (SX)

 

ULCER, 80S 

INFLAMMATION, CHRONIC FOCAL 

PARASITISM 1 (5*)

 

•SMALL INTESTINE (18) (19)

 

AMYLOIDOSIS 

•LABGE INTESTINE (15) (19)

 

PARASITISM 

•COLOH (15) (19)

 

NEMATODIASIS 1 (7X)

 

URINARY SYSTEM 

•KIDNEY (19) (19) 

MINERALIZATION 2 (11X) 

HYDRONEPHBOSIS 2 (11X) 

MULTIPLE CYSTS 

INFLAMMATION, ACUTE 

INFLAMMATION. CHRONIC 9 (47X) 1 (5X) 

INFLAMMATION, CHROMIC FOCAL 13 (68X) 

INFLAMMATION. CHRONIC DIFFUSE 

AMYLOIDOSIS 4 (21X) 

• RENA L PAPILLA (19) (19)

 

INFLAMMATION, NEC BOTH ING 

ENDOCRINE SYSTEM 

•ADRENAL (17) (19)

 

AMYLCIDOSIS 

•THYROIC (15) (17) 

FOLLICULAB ZYST. NOS 3 (18*) 

INFLAMMATION, CHBONIC 1 (6<) 

INFLAMMATION, CHRONIC FOCAL 1 (6S) 

REPRODUCTIVE SYSTEM 

•PROSTATE (16) (19)

 

INFLAMMATION. ACUTE 

* NUMBE R OF ANIMALS ilTH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMALS KECROPSIED 

•urn 

(50) 

1 (2X) 

(50) 

(46) 

7 (15X) 

(46) 

(49) 

1 (2X) 

1 (2X) 

2 (4X) 

6 (12*) 

27 (55*) 

3 (6*) 

1 (2X) 

0»9) 

1 <2X) 

(50) 

2 (4X) 

(«4) 

(50) 

TWIT 

J»6) 

1 (2X) 

(47) 

1 (2X) 

(41) 

4 (10X) 

(41) 

(47) 

1 (2X) 

9 (19X) 

19 (40X) 

1 (2X) 

(47) 

(47) 

1 <2X) 

(44) 

(47) 

1 (2*) 

D-5 
TABLEDl (CONCLUDED) 

CONTROL [UNTR) CONTROL JVEH, LOU DOSE HIGH DOSE 02-M181

 02-H172 02-B173 

INFLAMMATION . CHRONIC 

INFLAMMATION , CHRONIC FOCAL 

tIESTIS 

HINEEALIZATIO N 

ATROPHY, NOS 

*EPIDIDYMIS 

SPERMATOCELE 

NECROSIS, FAT 

*SCROTUM

FOREIGN BODY, NOS

NERVOU S SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

HOSCULOSKELETA L SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

ADIPOSE TISSUE 

NECROSIS, FOCAL

SPECIAL MORPHOLOG Y SOMHA8Y 

N O LESION REPORTED

ANIBAL MISSING/NO NECROPSY

AUTO/NECROPSY/HISTO PEEF

AOTC/NECROPSY/NO HISTO

AtlTOLIS IS/NO NECROPSY

I NUBBE B OF ANIMAL S WITH TISSDE

* NUMBE R OF ANIMAL S NECROPSIED 

1 (5X) 1 (2X) 

1 (5X) 

(19) (19) (50) (17) 

1 (2%) 

2 (IX) 

(19) (19) (50) 

<2X) 

1 (5X) 

 (19) (19) (50) (M7) 

1 (5X) 

1 

3 2 1 6 

1 

2 

1 

1 J i__ 

 EXAMINED MICROSCOPICALLY 

D-6 
TABLE D2 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH TRICHLOROFLUOROMETHANE

 

CONTROL (UNTR) CONTROL fVEH) LOW DOSE HIGH DOSE 02-F181 02-F17t 02-F17U C2-F175 

ANIMALS INITIALLY IS STUDY 20 20 50 50 

ANIMALS NECROPSIED 19 19 50 U9 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 18 19 50 49 

INTEGUMENTAR Y SYSTEM 

*SKIN (19) (19) (50)

 

EPIDERMAL INCLUSION CYST <5X)

 

ULCER. FOCAL 1 (5X)

 

PARASITISM 11 (58X) 38 (7SX) 31 (63X)

 

*SUBCOT TISSUE (19) (19) (50) (U9)

 

STEATITIS 2 (UX) 

RESPIRATORY SYSTEM 

tLUNG (19) (19) (50) (t9)

 

INFLAMMATION , ACUT E 1 (2X)

 

INFLAMMATION , ACUTE/CHRONIC 1 (2X)

 

PNEUMONIA. CHRONIC MDHIN E 2 (11X) 2 (11X) 6 (12X) U (8*) 

INFLAMMATION , CHRONIC FOCAL 1 (2X1

 

HYPEFPLASIA. ALVEOLA R EPITHELIUM 1 (2X)

 

HEMATOPOIETIC SYSTEM 

•SPLEEN (19) (19) (50) (U9)

 

AMYLOICOSIS (5X) 1 (2%) 

HYSERPLASIA , LYHPHOID <5X> 1 (2X)

 

HEMATOPOIESIS 1 (2X) t (8X)

 

ILYMPH NODE (19) (19) (50) |t9)

 

HYPERPLASIA, LYKPHOID 1 (2X>

 

*CERVICAL LYMPH NODE (19) (19) (50) (149) 

HYPEBPLASIA, LYMPHOID 1 (5X) 2 (KX) 1 (8X) 

#MESENTIRIC L. NODE (19) (19) (50) (19) 

LYHPHANGIECTASIS 1 (2X) 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-7 
TABLE D2 (CONTINUED) 

CONTROL JUNTR) CONTROL(VEH) 02-F171 

INILAMMATION , NOS 2 (11X) 

INFLAMMATION. ACUTE 

HYPERPLASIA, LYMPHOID 3 (16X) 

CIRCULATORY SYSTEM 

•MYOCARDIUM (19) (19)

 

MINERALIZATION 

DIGESTIVE SYSTEM 

•SALIVARY GLAND (17) (19) 

INFLAMMATION, CHRONIC FOCAL 13 (68X) 

•LIVER (19) (19)

 

INFLAMMATION, ACUTE 

INFLAMMATION, ACUTE FOCAL 

INFLAMMATION , FOCAL GRANULOHATOU 1 (5X)

 

NECROSIS, F3CAL 

NECROSIS, ISCHEMIC 1 (5X)

 

•BILE DUCT (19) (19) 

INFLAMMATION . CHRONIC 10 (S3X) 

•PANCREAS (19) (19)

 

DILATATION/DUCTS 

INFLAMMATION , NOS 2 (11X) 

ATROPHY. NOS 

• ESOPHAGUS (19) (17)

 

INFLAMMATION , ACUTE/CHRONIC 

HYPERKEPATOSIS 

•STOMACH (19) (19)

 

ULCER. FOCAL 

HYPE8KERATOSIS 

• LARGE INTESTINE (18) (19)

 

PARASITISM 

URINAR Y SYSTEM 

• KIDNE Y (19) (19)

 

CYST, NOS 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

Will 

1 (2X) 

(50) 

1 (2X) 

(50) 

22 (U4X) 

(50) 

1 (2X) 

3 (6X) 

(50) 

9 (18X) 

(50) 

1 (2X) 

(50) 

1 (2X) 

(50) 

(50) 

2 <«X) 

(50) 

1 (2X) 

•HMW 

1 (2*) 

3 (6X) 

<*•> 

(19) 

la (29*) 

(19) 

1 (2*) 

(19) 

a (8*) 

(K9) 

2 (1*) 

•' 2 CM) 

(U9)

2 (a*» 2 (IX) 

(U9) 

8 (16X) 

(•9) 

0-8 
TABLE D2 (CONTINUED) 

CONTROL (U NTS) CONTBOLJVEH ) 02-F18T 

PYELONEPHRITIS, DOS 1 (5X) 

INFLAMHATION . CHRONIC 1 (5X) 

INFLAMMATION. CHBONIC FOCAL 12 (63X) 

ENDOCRIN E SYSTEM 

*ADRENA L (18) (19) ANGIECTASIS

 

*ADEENAI CORTEX (18) (19) CYST, NOS

 

tTHYROI D (15) (15) F01IICDLAR lYST, NOS 1 (7X)

 

HYFERPLASIA . FO LLICOLAR -CELL 

REPRODUCTIV E SYSTEM 

IUTERU S (17) (19)

 HYEBOMETRA 1 (6*)

 INFLAMMATION . NO S 2 (12*)

 INFLAMMATION, ACUTE

 

»UTERUS/ENDOMETBIUM (") (19) CYST, NOS 

HYPEBPLASIA. CYSIIC 5 (29*) 17 (89*) 

tOVARY/OVIDUCT (17) (19)

 RETENTIO N FLUID 1 <5X)

 INFLAMMATION, NOS 1 <6X)

 

tOVAR Y (18) (19)

 CYST, NOS 3 (17*)

 FOLLICULAR CYST, NOS 1 (5X)

 PAROVARIA N CYST 1 (5*)

 HEMORPHASIC CYST

 

INFLAMMATION , NO S 3 (17*) 

NEBVOU S SYSTEM 

tBRAI N (19) (19)

 

COMPRESSION _ . . ._ ._ 

# NUMBER OF ANIMALS WITH TISSUE EXABINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

LOW DOSE 

02-F174 

1 (2*> 28 (56X) 

(50) 

(50) 

1 (2X) 

(50) 

1 (2X) 

(50) 

1 (2X) 

(50) 

1 (2X) 

3 1 (62X) 

(50) 

(50) 

8 (16X) 

2 («*) 

(50) 

1 12*) 

HIGHpD07SE 

1 (2*) 

2H (U9X) 

(49) 

1 (2X) 

(19) 

(1(9) 

(U9) 

(49) 

28 (57*) 

(149) 

(19) 

8 (16X) 

1 (2*) 

3 (6X) 

(U9) 

D-9 
TABLE D2 (CONCLUDED) 

CONTROL (UNTR) CONTROL (VEH) LOR DOSE HIGH DOSE 02-F181 02-F171 02-F174 02-F175 

SPECIAL SENSE ORGANS 

*EYE (19) (19) (50) (K9 ) 

MICBOPHTHALMIA 1 (2*) 

ATBOPHY, NOS 1 (5U) 

PHTHISIS BULBI 2 (11%) 

MUSCULOSKELETAL SYSTEM 

*SKELITAL MUSCLE (19) (19) (50) 1<19) 

INFLAHHATION , ACOTE FOCAL 2 (<4]S) 

BODY CAVITIES 

*PEBITONEUM (19) (19) (50) (H9) 

INFLAMMATIOH , NOS 1 (5X) 

*EFICABEIUH (19 ) (19) (50) («9) 

INFIABMATION , CHRONIC 1 (2X) 

ALL OTHER SYSTEMS 

NONE 

SPECIAL HORPHOLOGY SUMMAR Y 

N O LESION REPORTED 5 2 6 

AOTC/NECROPSY/HISTO PERF 2 

AUTO/NECBOPSY/NO HISTO 1 

ADTOLYSIS/NO NECROPSY 1 1 1 

* NUMBE R OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

D-10 
Review of the Bloassay of Trichlorofluoromethane*
 

for Carclnogenicity by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

March 6, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academia, industry, organized labor, public interest
 

groups, State health officials, and quasi-public health and
 

research organizations. Members have been selected on the
 

basis of their experience in carcinogenesis or related fields
 

and, collectively, provide expertise in chemistry, biochemistry,
 biostatistics, toxicology, pathology, and epidemiology.
 Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse is charged with the responsibility of
 

providing a peer review of reports prepared on NCI-sponsored
 

bioassays of chemicals studied for carcinogenicity. It is in
 

this context that the below critique is given on the bioassay
 

of Trichlorofluoromethane for carcinogenicity.
 

The primary reviewer agreed that the rat portion of
 

the study was too inadequate, due to the high early mortality,
 to evaluate the carcinogenicity of Trichlorofluoromethane
 

in this species. Under the conditions of test, he said
 

that Trichlorofluoromethane was not carcinogenic in either
 

sex of treated mice. After a brief description of the
 

experimental design, the primary reviewer added that no
 

unusual tumors or increases in overall tumor incidence were
 

observed among the treated mice. In his critique, he said
 

that a number of other chemicals were studied in the same
 

room during the bioassay of Trichlorofluoromethane and that
 

the vehicle control group was initiated several months prior
 

to the treated animals being placed on test.
 

The secondary reviewer noted that occupational exposure
 

to Trichlorofluoromethane could be in the same range as the
 

low dosages administered in the bioassay. He pointed out
 

the high incidence of lymphoid hyperplasia among the low
 

dose male mice and said that these lesions may be under-
 diagnosed. He recommended that they be reevaluated to
 

45
 
determine if some are malignant lymphomas. He criticized
 

the report for not sufficiently noting the lymphoid hyper­ plasias in the treated male mice. A Program pathologist
 

said the slides of the lymphoid hyperplasias will be
 

reevaluated in view of the secondary reviewer's comments.
 

Several members agreed that it was more likely that the
 

lymphoid hyperplasias were over-diagnosed rather than
 

under-diagnosed. It was noted that abnormal lymph nodes
 

are commonly found among older animals. A staff pathologist
 

pointed out that the mice had amyloidosis, which may have
 

contributed to the lymphoid hyperplasia. It was agreed
 

that the Subgroup would be notified if the results of the
 

reevaluation differed from the diagnoses given in the report,
 

The primary reviewer moved that the report on the
 

bioassay of Trichlorofluoromethane be accepted as written.
 

The motion was seconded. A Subgroup member offered an
 

amendment, which was accepted, that the report be represented to the Subgroup if the conclusion is changed based
 

on the reevaluation of the lymphoid hyperplasia. A vote on
 

the amended motion was approved unanimously.
 

Members present were:
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Arnold Brown, Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

E. Cuyler Hammond, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Henry Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Michael Shimkin, University of California at San Diego
 

John Weisburger, American Health Foundation
 

Sidney Wolfe, Health Research Group
 

Subsequent to this review, changes may have been
 

made in the bioassay report either as a result of
 

the review or other reasons. Thus, certain comments
 

and criticisms reflected in the review may no longer
 

be appropriate.
 

OU.S. GOVERNMENT PRINTING OFFICE: 1978 260-899/3169 1-3 46 

DHEW Publication No. (NIH) 78-1356