National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 150 

1979 

BIOASSAY OF 

BUTYLATED HYDROXYTOLUENE (BHT) 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 128-37-0 

NCI-CG-TR-150 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

BUTYLATED HYDROXYTOLUENE (BHT)
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20205
 

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

Public Health Service
 

National Institutes of Health
 

NIH Publication No. 79-1706
 

BIOASSAY OF
 

BUTYLATED HYDROXYTOLUENE (BHT)
 

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
 

butylated hydroxytoluene (BHT) 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. A negative result, in
 

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

than control animals, does not necessarily mean that a test
 

chemical is not a carcinogen, inasmuch as the experiments are
 

conducted under a limited set of circumstances. A positive
 

result demonstrates that a test chemical is carcinogenic for
 

animals under the conditions of the test and indicates that
 

exposure to the chemical is a potential risk to man. The actual
 

determination of the risk to man from chemicals found to be
 

carcinogenic in animals requires a wider analysis.
 

CONTRIBUTORS; This bioassay of butylated hydroxytoluene (BHT)
 

was conducted at the NCI Frederick Cancer Research Center (FCRC)
 

(1), Frederick, Maryland, operated for NCI (2) by Litton
 

Bionetics, Inc.
 

The manager of the bioassay at FCRC was Dr. B. Ulland, the
 

toxicologist was Dr. E. Gordon, and Drs. R. Cardy and D. Creasia
 

compiled the data. Ms. S. Toms was responsible for management of
 

data, Mr. D. Cameron for management of histopathology, Mr. L.
 

Callahan for management of the computer branch, and Mr. R. Cypher
 

for management of the facilities. Mr. A. Butler performed the
 

computer services. Histopathologic evaluations for rats were
 

performed by Dr. J. F. Hardisty (3), and the histopathologic
 

evaluations for mice were performed by Dr. L. J. Ackerman (3).
 

The diagnoses included in this report represent the
 

interpretations of Drs. Hardisty and Ackerman.
 

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

Mason Research Institute (4). Statistical analyses were
 

ill
 
performed by Dr. J. R. Joiner (5) and Ms. P. L. Yong (5), using
 

methods selected for the bioassay program by Dr. J. J. Gart (6).
 

The chemicals used in this bioassay were analyzed at Frederick
 

Cancer Research Center by Dr. W. Zielinsky (1). The chemical
 

analyses and narrative were reviewed and approved by Dr. W.
 

Lijinsky (1).
 

This report was prepared at Tracor Jitco (5) under the direction
 

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

Dr. C. R. Angel, Acting Director of the Bioassay Program; Dr. S.
 

S. Olin, Deputy Director for Science; Dr. J. F. Robens,
 

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

Ms. L. A. Owen, Ms. M. S. King, and Mr. W. D. Reichardt, 

bioscience writers; and Dr. E. W. Gunberg, technical editor, 

assisted by Ms. Y. E. Presley. 

The following scientists at NCI were responsible for evaluating
 

the bioassay experiment, interpreting the results, and reporting
 

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

J. Fielding Douglas, Dr. Richard A. Griesemer, Dr. Thomas E.
 

Harm, Dr. William V. Hartwell, Dr. Morton H. Levitt, Dr. Harry A.
 

Milman, Dr. Thomas W. Orme, Dr. A. R. Patel, Dr. Sherman F.
 

Stinson, Dr. Jerrold M. Ward, and Dr. Carrie E. Whitmire.
 

(1)	 Frederick Cancer Research Center, P.O. Box B, Frederick,
 

Maryland.
 

(?.) Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

(3)	 Experimental Pathology Laboratories, Inc., P.O. Box 474,
 

Herndon, Virginia.
 

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

Rockville, Maryland.
 

(5)	 Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

(6)	 Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute,
 

National Institutes of Health, Bethesda, Maryland.
 

IV
 
SUMMARY

 

A bioassay of butylated hydroxytoluene (BHT) for possible 

carcinogenicity was conducted by administering the test chemical 

in feed to F344 rats and B6C3F1 mice. 

Groups of 50 rats and 50 mice of each sex were administered BHT 

at one of two doses, either 3,000 or 6,000 ppm; the rats for 105 

weeks and the mice for 107 or 108 weeks. Matched controls 

consisted of 20 untreated rats and 20 untreated mice of each 

sex. All surviving animals were killed at the end of administration of the test chemical. 

Mean body weights of the dosed rats and mice were lower than 

those of the corresponding controls and were dose related 

throughout most of the bioassay. Survival was not affected 

significantly in the dosed groups of rats or mice, and the 

survival was 60% or greater in all dosed or control groups of 

rats and mice of each sex at the end of the bioassay. Sufficient 

numbers of animals were at risk for the development of lateappearing tumors. 

Alveolar/bronchiolar carcinomas or adenomas occurred in the 

female mice at a significant incidence in the low-dose group (P = 

0.009) but not in the high-dose group, and the incidences were 

not significantly dose related (control 1/20, low-dose 16/46, 

high-dose 7/50). Thus, these lung tumors in the females cannot 

clearly be related to the administration of the BHT. No tumors 

occurred in either male or female rats at incidences that were 

significantly higher in dosed groups than in corresponding control groups. Nonneoplastic lesions that may have been related to 

the administration of the test chemical included focal alveolar 

histiocytosis at increased incidences in the dosed female rats 

and various lesions of the liver at increased incidences in the 

dosed male mice. 

It is concluded that under the conditions of this bioassay, BHT 

was not carcinogenic for F344 rats or B6C3F1 mice. 

TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 5
 

A. Chemical 5
 

B. Dietary Preparation 5
 

C. Animals 6
 

D. Animal Maintenance 7
 

E. Subchronic Studies 9
 

F. Chronic Studies 11
 

G. Clinical and Pathologic Examinations 11
 

H. Data Recording and Statistical Analyses 15
 

III. Results - Rats 21
 

A. Body Weights and Clinical Signs (Rats) 21
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 24
 

D. Statistical Analyses of Results (Rats) 25
 

IV. Results - Mice 27
 

A. Body Weights and Clinical Signs (Mice) 27
 

B. Survival (Mice) 27
 

C. Pathology (Mice) 30
 

D. Statistical Analyses of Results (Mice) 34
 

V. Discussion 37
 

VI. Bibliography 43
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Administered BHT in the Diet 47
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Administered BHT in the Diet 49
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Administered BHT
 

in the Diet 53
 

vii
 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Administered BHT in the Diet 57 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Administered BHT in the Diet 59 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Administered BHT in the Diet 63 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered BHT in the Diet.. 67 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered BHT in 

the Diet 69 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered BHT in 

the Diet 74 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered BHT in 

the Diet 79 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered BHT in 

the Diet 81 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered BHT in 

the Diet 86 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Administered BHT in the Diet 91 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Administered, BHT in the Diet 93 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Administered BHT in the Diet... 98 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered BHT in the Diet 103 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Administered BHT in the Diet 105 

viii
 
Table F2
 

Table 1
 

Table 2
 

Table 3
 

Figure 1
 

Figure 2
 

Figure 3
 

Figure 4
 

Page 

Analyses of the Incidence of Primary Tumors 

in Female Mice Administered BHT in the Diet.. 109 

TABLES 

BHT Subchronic Feeding Studies in Rats and 

Mice 10 

BHT Chronic Feeding Studies 

in Rats 12 

BHT Chronic Feeding Studies 

in Mice 13 

FIGURES 

Growth Curves for Rats Administered 

BHT in the Diet 22 

Survival Curves for Rats Administered 

BHT in the Diet ; 23 

Growth Curves for Mice Administered 

BHT in the Diet 28 

Survival Curves for Mice Administered 

BHT in the Diet 29 

ix
 

I. INTRODUCTION
 

The phenolic antioxidant
 

2,6-di-tert-butyl-p-cresol (CAS
 

128-37-0; NCI C03598), more
 

commonly known as butylated
 (CH3)3C ">

%X^C(CH3 )3
 

hydroxy toluene, or BHT, was
 OH
 

patented in 1947 (Stecher, 1968)
 

BHT
 

and received approval for use as
 

a food additive and preservative by the Food and Drug
 

Administration (FDA) in 1954 (Federal Register, 1977). Since
 

1959, BHT has been generally recognized as safe (GRAS) for use in
 

foods (Federal Register, 1977) and is one of the most commonly
 

used antioxidants in foods containing fats (Stuckey, 1972). It
 

is used alone or in combination with butylated hydroxyanisole or
 

propyl gallate (Dugan, 1963; Stuckey, 1972). Acting on an
 

evaluation of the toxicity of BHT by the Select Committee on GRAS
 

Substances (1973), the Federal Register (1977) has recently
 

proposed interim restrictions on use levels in foods until
 

additional toxicity studies have been performed. The Select
 

Committee had concluded that there was no evidence that BHT posed
 

a hazard to public health when it was used at levels then current
 

and in the manner then practiced, but that additional studies
 
would be necessary to resolve some uncertainties in the existing
 

data. In particular, the Federal Register (1977) proposed that
 

short-term metabolism studies be carried out to compare the
 

metabolism of BHT in mice with that in man, and that if similar
 

metabolisms were found, long-term feeding studies then be carried
 

out to resolve conflicting reports (Clapp et al., 1976; Brooks et
 

al., 1977) on the carcinogenicity of BHT for the lung in mice.
 

BHT prevents rancidity in foods containing fats by terminating
 

chain reactions involving free radicals that are responsible for
 

the oxidative degradation of the fats (Chapman and Kertesy, 1966;
 

Noller, 1966). Oxidation not only produces undesirable flavor
 

changes, but destroys both fat-soluble vitamins and the essential
 

fatty acids, and may generate toxic products (Dugan, 1963).
 

BHT is approved for use in enriched rice, margarine, shortening,
 

dehydrated potato products, dry breakfast cereals, chewing gum
 

base, certain food-packaging materials (Federal Register, 1977;
 

Code £f_ Federal Regulations, 1977), and animal feed (Code £f
 

Federal Regulations, 1977a). It is cleared for use by the Meat
 

Inspection Division of the U.S. Department of Agriculture in
 

rendered animal fats, fresh and dried pork sausage, and freeze-


dried meats (Furia, 1972). Among the nonfood items in which BHT
 

acts as a stabilizer are pesticides (Code of Federal Regulations,
 
1976 and 1977); gasolines, lubricants, and rubber (Dugan, 1963);
 

and oil-based lipsticks (Lauffer, 1972).
 

Although the level of BHT used in any food product has not been
 

allowed to exceed 0.02% of the weight of fat present, the total
 

amount of BHT used in foods in 1970 reached nearly 600,000
 

pounds, twice the figure reported in 1960 (Federal Register,
 

1977). By 1976, the annual production of BHT in the United
 

States had increased to 19.81 million pounds, of which. 8.86
 

million pounds were produced for use in foods and 10.95 million
 

pounds for other uses (United States International Trade
 

Commission, 1977).
 

Because humans are increasingly exposed to BHT through its wide
 

use as a food additive, the chemical was selected for
 

reevaluation of its potential carcinogenicity, using the
 

protocols of the Carcinogenesis Testing Program.
 

II. MATERIALS AND METHODS
 

A. Chemical
 

Butylated hydroxytoluene (BHT), or 2,6-di-tert-butyl-p-cresol, 

was obtained from Koppers Co., Pittsburgh, Pennsylvania, as a 

fine, white, crystalline solid. Its purity was determined to be 

99.9% by gas-liquid chromatography, with two to six contaminants 

comprising less than 0.1%. Mass spectral analysis showed a 

molecular ion at 220 m/e and a base peak at 205 m/e. The infrared spectrum was consistent with its chemical structure, and 

identical with that of a standard. The melting point was 

69.6°C (Stecher, 1968: 70°C). Elemental analysis for carbon 

and hydrogen was in agreement with theoretical. 

B. Dietary Preparation
 

Test diets containing BHT were prepared every 1 to 1-1/2 weeks in 

6-to 12-kg batches at appropriate doses. A known weight of the 

chemical was first mixed with an equal weight of autoclaved 

Wayne® Sterilizable Lab Meal containing 4% fat (Allied Mills, 

Inc., Chicago, 111.), using a mortar and pestle. The Wayne® 
Sterilizable Lab Meal contained 4% fat but no added BHT (Drews,
 

1978). The mixing was continued with second and third additions
 

of feed, and final mixing was performed with the remaining
 

quantity of feed for a minimum of 15 minutes in a
 

Patterson-Kelly® twin-shell blender with an intensifier bar.
 

The diets were stored at 7 C until used.
 

C. Animals
 

Male and female F344 (Fischer) rats and B6C3F1 mice were obtained
 

as 4-week-old weanlings, all within 3 days of the same age, from
 

the NCI Frederick Cancer Research Center (Frederick, Md.). The
 

animals were housed within the test facility for 2 weeks and were
 

then assigned four rats of the same sex to a cage and five mice
 

of the same sex to a cage. The male rats used in the chronic
 

study weighed 90 to 105 g, averaging at least 100 g; the female
 

rats, 80 to 95 g, averaging at least 90 g; the male mice, 18 to
 

22 g, averaging at least 19.5 g; and the female mice, 17 to 21 g,
 

averaging at least 18.5 g. Individual animals were identified by
 

ear punch.
 
D. Animal Maintenance
 

The animals were housed in polycarbonate cages (Lab Products,
 

Inc., Garfield, N.J.), 19 x 10-1/2 x 8 inches for the rats and
 

11-1/2 x 7-1/2 x 5 inches for the mice. The cages were suspended
 

from aluminum racks (Scientific Cages, Inc., Bryan, Tex.) and
 

were covered by nonwoven polyester-fiber 12-mil-thick filter
 

paper (Hoeltge, Inc., Cincinnati, Ohio). The bedding used was
 

Absorb-dri hardwood chips (Northeastern Products, Inc.,
 

Warrenburg, N.Y.). The feed was presterilized Wayne®
 

Sterilizable Lab Meal containing 4% fat, provided ad libitum in
 

suspended stainless steel hoppers and replenished at least three
 

times per week. Water, acidified to pH 2.5, was supplied ad
 

1ibiturn from glass bottles with sipper tubes (Lab Products, Inc.)
 

suspended through the tops of the cages.
 

The contaminated bedding was disposed of through an enclosed
 

vacuum line that led to a holding tank from which the bedding was
 

fed periodically into an incinerator. The cages were sanitized
 

twice per week and the feed hoppers twice per month at 82 to
 

88 C in a tunnel-type cagewasher (industrial Washing Corp.,
 

Mataway, N. J.), using the detergents, Clout (Pharmacal
 

Research Laboratories, Greenwich, Conn.) or Oxford D'Chlor
 

(Oxford Chemicals, Atlanta, Ga.). The bottles and sipper tubes
 
were sanitized at 82 to 88 C in a tunnel-type bottle washer
 

(Consolidated Equipment Supply Co., Mercersburg, Pa.) three times
 

per week, using a Calgen Commercial Division detergent (St.
 

Louis, Mo.). The racks for the cages were sanitized at or above
 

82 C in a rack washer (Consolidated Equipment Supply Co.) once
 

per month, using the Calgen Commercial Division detergent, and
 

the filter paper was changed at the same time.
 

The animal rooms were maintained at 22 to 24 C, and the relative
 

humidity was 45 to 55%. Incoming air was passed through a filter
 

of 65% efficiency and a bag filter of 95% efficiency at the
 

intake and expelled without recirculation through a "Z"-type
 

roughing filter of 30% efficiency and a bag system of 90 to 95%
 

efficiency at the exhaust (American Air Filters, Louisville, Ky.;
 

Mine Safety Appliances, Pittsburgh, Pa.). Room air was changed
 

15 times per hour. The air pressure was maintained negative to a
 

clean hallway and positive to a return hallway. Fluorescent
 

lighting was provided automatically on a 12-hour-per-day cycle.
 

Rats administered BHT and their corresponding controls were
 

housed in the same room as rats on feeding studies of the
 

following chemicals:
 

(CAS 88-96-0) phthalamide
 

(CAS 137-17-7) 2,4,5-trimethylaniline 
Mice administered BHT and their corresponding controls were
 

housed in the same room as mice on feeding studies of the
 

following chemicals:
 

(CAS 3165-93-3) 4-chloro-o-toluidine hydrochloride
 

(CAS 97-77-8) tetraethylthiuram disulfide
 

(CAS 148-18-5) sodium diethyldithiocarbamate
 

(CAS 636-21-5) o-toluidine hydrochloride
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses (MTD's) of BHT, on the basis of which two concentrations (referred to in this report as "low" and "high" doses)
 

were selected for administration in the chronic studies. Groups
 

of five rats and five mice of each sex were fed diets containing
 

BHT at one of several doses for 7 weeks, followed by 1 week of
 

observation, and groups of five control animals of each species
 

and sex were administered basal diet only. Each animal was
 

weighed twice per week. Table 1 shows the doses fed, the survival of animals in each dosed group at the end of the study, and
 

the mean body weights of dosed animals at week 7, expressed as
 

percentages of mean body weights of the controls. At the end of
 

the subchronic studies, all animals were killed using C0? and
 

necropsied. Histopathologic findings are shown as footnotes to
 

the table.
 
Table 1. BHT Subchronic Feeding Studies in Rats and Mice
 

Male Female 

Mean Weight Mean Weight 

at Week 7 at Week 7 

Dose Surviv­ as % of Surviv­ as % of 

( ppm) al (a) Control al (a) Control 

Rats
 

0 5/5 100 5/5 100
 

6,200 5/5 88 5/5 93
 

12,500(b) 4/5 74 5/5 84
 

25,000 5/5 38 5/5 44
 

50,000 0/5 0/5
 

Mice
 

0 5/5 100 5/5 100
 

3,100 5/5 89 5/5 88
 

6,200 5/5 94 5/5 83
 

12,500(c) 5/5 78 5/5 82
 

25,000(c) 5/5 79 4/5 74
 

50,000 4/5 73 1/5 97
 

(a) Number surviving/number in group.
 

(b) Slight increase in hematopoiesis in both sexes of rats.
 

(c) Histopathologic examination of male mice at 25,000 ppm and of
 

female mice at 12,500 ppm showed a very small amount of
 

centrilobular cytoplasmic vacuolation in the livers of the males.
 

10
 
Ten percent depression in body weight was a major criterion for
 

the estimation of MTD's. The doses required to produce this
 

response were determined by the following procedure: first,
 

least squares regressions of mean body weights versus days on
 

study were used to estimate mean body weights of each of the
 

dosed groups at day 49. Next, probits of the percent weights of
 

the dosed groups at day 49 relative to weights of corresponding
 

control groups were plotted against the logarithms of the doses,
 

and least squares regressions fitted to the data were used to
 

estimate the doses required to induce 10% depression in weight.
 

The low and high doses for the rats and mice in the chronic study
 

were set at 3,000 and 6,000 ppm, respectively.
 

F. Chronic Studies
 

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

studies are shown in tables 2 and 3.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily. Observations for sick,
 

11
 
Table 2. BHT Chronic Feeding Studies in Rats
 

Sex and Initial BHT Time on
 

Test No. of in Diet(b) Study
 

Group Animals (a) (ppm) (weeks)
 

Male
 

Matched-Control 20 0 105
 

Low-Dose 50 3,000 105
 

High-Dose 50 6,000 105
 

Female
 

Matched-Control 20 0 105
 

Low-Dose 50 3,000 105
 

High-Dose 50 6,000 105
 

(a) All animals were 6 weeks of age when placed on study.
 

(b) Test and control diets were provided ad libitum 7 days per
 

week.
 

12
 
Table 3. BHT Chronic Feeding Studies in Mice
 

Sex and Initial BHT Time on
 

Test No. of in Diet(b) Study
 

Group Animals (a) (ppm) (weeks)
 

Male
 

Matched-Control 20 0 108
 

Low-Dose 50 3,000 108
 

High-Dose 50 6,000 107
 

Female
 

Matched-Control 20 0 108
 

Low-Dose 50 3,000 108
 

High-Dose 50 6,000 107-108
 

(a) All animals were 6 weeks of age when placed on study.
 

(b) Test and control diets were provided ad libitum 7 days per
 

week.
 

13
 
tumor-bearing, and moribund animals were recorded daily.
 

Clinical examination and palpation for masses were performed each
 

month, and the animals were weighed at least once per month.
 

Moribund animals and animals that survived to the end of the
 

bioassay were killed using CCL and necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of major tissues, major organs, and all gross lesions.
 

The tissues were preserved in 10% neutral buffered formalin,
 

embedded in paraffin, sectioned, and stained with hematoxylin and
 

eosin. The following tissues were examined microscopically:
 

skin, lungs and bronchi, trachea, bone marrow (femur), spleen,
 

lymph nodes (mesenteric and submandibular), thymus, heart,
 

salivary glands (parotid, sublingual, and submaxillary), liver,
 

pancreas, esophagus, stomach (glandular and nonglandular), small
 

and large intestines, kidney, urinary bladder, pituitary, adrenal,
 

thyroid, parathyroid, testis, prostate, uterus, ovary, brain
 

(cerebrum and cerebellum), and all tissue masses. Peripheral
 

blood smears also were made for all animals, whenever possible.
 

Necropsies were also performed on all animals found dead, unless
 

precluded in whole or in part by autolysis or cannibalization.
 

Thus, the number of animals from which particular organs or
 

tissues were examined microscopically varies and does not
 

14
 
necessarily represent the number of animals that were placed on
 

study in each group.
 

H. Data Recording and Statistical Analyses
 

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

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

descriptive information on the chemicals, animals, experimental
 

design, clinical observations, survival, body weight, and
 

individual pathologic results, as recommended by the
 

International Union Against Cancer (Berenblum, 1969). Data
 

tables were generated for verification of data transcription and
 

for statistical review.
 

These data were analyzed using the appropriate statistical
 

techniques described in this section. Those analyses of the
 

experimental results that bear on the possibility of carcinogenicity are discussed in the statistical narrative section.
 

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
 

15
 
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) for testing two groups for equality and Tarone's (1975)
 

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

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

except the departure from linearity test, which is only reported
 

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic
 

examination was required to detect lesions prior to histologic
 

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

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

denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

16
 
animals. As a part of these analyses, the one-tailed Fisher
 

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

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

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

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

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

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

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

where the Fisher exact P values are shown.
 

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

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

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

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


tailed 0.05 level of significance. Unless otherwise noted, the
 

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

17
 
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 an animal died naturally or was sacrificed
 

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

methods of comparing these curves were used for two groups;
 

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

groups. The statistical tests for the incidence of tumors which
 

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

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P less than 0.05, two-


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

18
 
risk of each dosed group compared with its control was calculated
 

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

relative risk is defined as P../P where p is the true
 

binomial probability of the incidence of a specific type of tumor
 

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

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

group. The hypothesis of equality between the true proportion of
 

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

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

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

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

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

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

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

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

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

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

19
 
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.
 

20
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of dosed male and female rats were lower than
 

those of corresponding controls throughout the bioassay, and this
 

depression was dose related (figure 1). Other clinical signs
 

occurred at comparable incidences in dosed and control groups.
 

B. Survival (Rats)
 

Estimates of probabilities of survival for male and female rats
 

administered BHT in the diet at the doses of this bioassay,
 

together with those for the matched controls, are shown by the
 

Kaplan and Meier curves in figure 2. The result of the Tarone
 

test for dose-related trend in mortality is not significant in
 

either sex.
 

In male rats, 36/50 (72%) of the high-dose group, 39/50 (78%) of
 

the low-dose group, and 13/20 (65%) of the control group lived to
 

the end of the bioassay. In females, 39/50 (78%) of the high21
 
bUO • 

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Q 

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Z 200 • 

01 

MALE RATS 

S MATCHED CONTROL a 100] LOW DOSE i 0 

A HIGH DOSE 

( 10 20 30 40 50 60 70 80 90 100 11 

TIME ON STUDY (WEEKS) 

500­

400­

D 2 a 1- D 

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100. I 0 LOW DOSE 

A HIGH DOSE 

0 • 

100 

TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered BHT in the Diet 

22 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 

TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered BHT in the Diet 

23 
dose group, 37/50 (74%) of the low-dose group, and 13/20 (65%) of
 

the control group lived to the end of the bioassay.
 

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

development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

are summarized in Appendix C, tables Cl and C2.
 

A variety of neoplasms commonly seen in aged F344 rats occurred
 

with approximately equal frequency in dosed and control rats. In
 

the male rats, interstitial-cell tumors of the testes and
 

pheochromocytomas of the adrenal were the most frequently
 

observed neoplasms. In the female rats, fibroadenomas of the
 

mammary gland and endometrial stromal polyps of the uterus were
 

observed frequently.
 

Several inflammatory, degenerative, and proliferative lesions
 

commonly seen in aged F344 rats occurred with approximately equal
 

frequency in dosed and control animals. Focal alveolar
 

24
 
histiocytosis in the lung was observed in both dosed and control
 

animals, but this lesion was most often observed in the high-dose
 

female rats. This lesion consisted of focal aggregates of large
 

mononuclear cells within the alveolar lumen. These cells
 

contained abundant foamy vacuolated cytoplasm. This lesion
 

occurred in all dosed and control groups, as shown in the
 

following table:
 

MALES FEMALES
 

Low High Low High
 

Control Dose Dose Control Dose Dose
 

Number of Animals
 

with Tissues
 

Examined 20 49 49 18 48 49
 

Focal
 

Alveolar
 

Histiocytosis 1(5%) 4(8%) 7(14%) 2(11%) 12(25%) 21(43%)
 

Based on the histopathologic examination, the administration of
 

BHT at the doses used in this bioassay did not induce either
 

neoplastic or nonneoplastic lesions in the F344 rat, with the
 

possible exception of focal alveolar histiocytosis in the females.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

25
 
least two animals of one group and at an incidence of at least 5%
 

in one or more than one group.
 

In each sex, the results of the Cochran-Armitage test for
 

dose-related trend in the incidence of tumors and the results of
 

the Fisher exact test comparing the incidence of tumors in each
 

dosed group with that in the control group are not significant in
 

the positive direction. However, significant results in the
 

negative direction are observed in the incidence of adenomas of
 

the pituitary in female rats.
 

In each of the 95% confidence intervals for relative risk, shown
 

in the tables, the value of one or less than one is included;
 

this indicates the absence of significant positive results. It
 

should also be noted that each of the intervals, except that for
 

the incidence of adenomas of the pituitary in high-dose female
 

rats, has an upper limit greater than one, indicating the
 

theoretical possibility of the induction of tumors by BHT, which
 

could not be detected under the conditions of this test.
 

26
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of dosed male and female mice were lower than
 

those of corresponding controls throughout the bioassay, and were
 

dose related (figure 3). Tissue masses occurred at comparable
 

incidences in dosed and control groups.
 

B. Survival (Mice)
 

Estimates of the probabilities of survival for male and female
 

mice administered BHT in the diet at the doses of this bioassay,
 

together with those for the matched controls, are shown by the
 

Kaplan and Meier curves in figure 4. In male mice, the result of
 

the Tarone test for dose-related trend in mortality is significant (P = 0.005), but in the negative direction. In females, the
 

result of the Tarone test is not significant.
 

In male mice, 46/50 (92%) of the high-dose group, 43/50 (86%) of
 

the low-dose group, and 12/20 (60%) of the control group lived to
 

the end of the bioassay. In female mice, 45/50 (90%) of the
 

27
 
50- n OD D 

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2 

I 30-

UJ 

s 

a^R 

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3P 

a 

0 X

?Li 

Z 20­

UJ 

2 

MALE MICE 

D MATCHED CONTROL 

10 • 

O LOW DOSE 

A HIGH DOSE 

0 • 

c 10 20 30 40 !K> 60 70 80 90 100 11 

TIME ON STUDY (WEEKS) 

50^ 

a n a i 

a ( n a a a n 40^ a n 0 i a ° oo ° 0

a o o 0 0 

O A o o o A 

I DO A A A A A A 30- oa A A u! A an a o o ^O A >- n AA a a a 

O «L3«i fitiii 00 Cj

Z 20- rn 

2 iRB 

s £_i 

FEMALE MICE 

D MATCHED CONTROL 

10 ­ O LOW DOSE 

A HIGH DOSE 

0 ­

0 10 20 30 40 50 60 70 80 90 100 11 

TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Administered BHT in the Diet 

28 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 

TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves for Mice Administered BHT in the Diet 

29 
high-dose group, 41/50 (82%) of the low-dose group, and 17/20 

(85%) of the control group lived to the end of the bioassay. 

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

development of late-appearing tumors. 

C. Pathology (Mice) 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

The liver was the most common organ to have proliferative
 

lesions. The incidences of the lesions are summarized as follows:
 

MALES FEMALES 

Low High Low High 

Control Dose Dose Control Dose Dose 

Number of Animals with 

Tissues Examined 20 48 49 20 46 49 

LIVER 

Hepatocytomegaly 0(0%) 9(19%) 20(41%) 0(0%) 1(2%) 1(2%)
 

Hepatocellular Adenoma 2(10%) 11(23%) 7(14%) 0(0%) 3(7%) 2(4%)
 

Hepatocellular
 

Care inoma 9(45%) 12(25%) 6(12%) 1(5%) 1(2%) 3(6%)
 

Angiosarcoma 1(5%) 0(0%) 1(2%) 1(5%) 1(2%) 1(2%)
 

Peliosis 0(0%) 34(71%) 43(88%) 0(0%) 0(0%) 0(0%)
 

Hepatocellular Degeneration and Necrosis 2(10%) 34(71%) 45(92%) 0(0%) 0(0%) 0(0%)
 

Cytoplasmic Vacuolation 3(15%) 20(42%) 22(45%) 0(0%) 0(0%) 0(0%)
 

30
 
Focal hepatocytomegaly was characterized by well-demarcated areas
 

of slightly enlarged hepatocytes. Typically, the cytoplasm of
 

the hepatocytes was more eosinophilic and mildly to severely
 

vacuolated. The edges of these foci were continuous with the
 

surrounding hepatocytes, and there was little or no compression
 

of the adjacent hepatic parenchyma. Multifocal hepatocytomegaly
 

was used to describe less well-demarcated areas of hepatocytic
 

enlargement and cellular change. The hepatocytes within these
 

areas usually were vacuolated or had a slightly more eosinophilic
 

staining quality than the surrounding liver parenchyma. The term
 

"hepatocellular adenoma" was used to describe focal areas of
 

hepatocellular proliferation which compressed the adjacent
 

hepatic parenchyma. Within these foci, there was increased
 

cellular pleomorphism, and mitotic figures were sometimes present. Typically, the cytoplasm of the cells was vacuolated, and
 

it stained slightly more basophilic than the surrounding hepatocytes. Hepatocellular carcinomas were characterized by poorly
 

circumscribed areas of proliferating hepatocytes. As a rule, the
 

cells were basophilic and extremely variable in size, and the
 

cytoplasm varied from being finely vacuolated to containing
 

large, clear vacuoles or large eosinophilic-staining bodies.
 

Nuclear atypia and mitotic figures were common. These growths
 

compressed the adjacent liver parenchyma, but usually had areas
 

of invasion into the adjacent liver lobules. Metastatic nodules
 

31
 
of cells having similar morphologic characteristics were found in
 

the lungs of three control and three low-dose male mice.
 

Angiosarcomas were characterized by large, cavernous blood-filled
 

spaces lined by proliferating spindle cells that invaded the
 

adjacent liver parenchyma.
 

Tn addition to proliferative lesions of the liver, there was a
 

high incidence of other liver lesions in most of the dosed male
 

mice. These were peliosis, hepatocellular degeneration and
 

necrosis, and varying degrees of hepatocellular vacuolation.
 

Peliosis was characterized by areas of sinusoidal dilatation and
 

spaces containing erythrocytes. These blood-filled spaces were
 

surrounded by cellular material resembling hepatocytic cytoplasm
 

and contained free hepatocytic nuclei. Many of these areas
 

resembled foci of intrahepatocytic hemorrhage. These areas were
 

scattered throughout the sections of liver and were primarily
 

located in the midzonal portion of the lobules. Surrounding
 

these areas of peliosis, there were areas of hepatocellular
 

degeneration and necrosis. These hepatocytes showed varying
 

degrees of swelling, hyalinization, and fine to coarse cytoplasmic vacuolation. Admixed with these areas of degenerating
 

hepatocytes were single or multiple enlarged hepatocytes.
 

32
 
Other common neoplasms in mice of this study were pulmonary
 

alveolar/bronchiolar adenomas and carcinomas. The incidence of
 

these lung neoplasms is summarized as follows:
 

MALES FEMALES 

Low High Low High 

Control Dose Dose Control Dose Dose 

Number of Animals 

with Tissues 

Examined 20 50 49 20 46 50 

Alveolar/Bronchiolar
 

Carcinoma 5(25%) 12(24%) 7(14%) 1(5%) 4(9%) 4(8%)
 

Adenoma 2(10%) 9(18%) 10(20%) 0(0%) 12(26%) 3(6%)
 

The alveolar/bronchiolar adenomas were characterized by
 

circumscribed masses of well-differentiated cuboidal epithelial
 

cells resting on a thin, fibrovascular stroma. These masses
 

often compressed the surrounding pulmonary parenchyma, and on
 

occasion protruded into the lumen of a bronchiole or elevated the
 

pleura. The alveolar/bronchiolar carcinomas were usually large
 

in size and less circumscribed than the adenomas; they usually
 

invaded the surrounding lung parenchyma. The cells stained more
 

basophilic, were piled up on one another, and showed cellular
 

pleomorphism. In several of the mice with alveolar/bronchiolar
 

adenocarcinomas, the pulmonary parenchyma adjacent to the tumor
 

contained intra-alveolar mononuclear or multinucleated cells
 

containing richly eosinophilic-staining cytoplasmic material.
 

33
 
Adenomas of the eye/lacrimal gland occurred in four high-dose
 

male mice and in two low-dose females but not in corresponding
 

controls. The significance of these findings is difficult to
 

evaluate, however, since only animals with grossly apparent
 

lesions at necropsy were examined microscopically.
 

Several inflammatory and neoplastic and nonneoplastic
 

proliferative lesions commonly seen in aged B6C3F1 mice were
 

observed, and the incidences were about the same in the control
 

and dosed groups of mice.
 

Based on the histopathologic examination, under the conditions of
 

this bioassay, the administration of BHT was associated with a
 

high incidence of nonneoplastic hepatocellular changes in dosed
 

male B6C3F1 mice compared with controls. Also, there was an
 

increased incidence of lung tumors in the female mice.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

34
 
In male mice, four adenomas of the eye/lacrimal gland are observed in the high-dose group, but none in the other two groups.
 

The result of the Cochran-Armitage test for positive dose-related
 

trend is significant (P = 0.039), but the results of the Fisher
 

exact test are not significant. The historical records of this
 

laboratory show an incidence of 5/422 (1.2%) as compared with
 

0/20 in the control group, 0/50 in the low-dose group, and 4/50
 

(8%) in the high-dose group of this study.
 

The incidence of alveolar/bronchiolar carcinomas or adenomas in
 

low-dose female mice is significantly higher (P = 0.009) than
 

that in the control group, but the incidence in the high-dose
 

group is not significant. Historical records at this laboratory
 

indicate that female control mice had an incidence of alveolar/
 

bronchiolar carcinomas or adenomas of 21/440 (4.7%), compared
 

with 1/20 (5%) in the female controls in this study, 16/46 (35%)
 

in the low-dose group, and 7/50 (14%) in the high-dose group.
 

The result of the Cochran-Armitage test also is not significant.
 

Significant results in the negative direction are observed in the
 

incidence of tumors of the liver in male mice and in the incidence
 

of sarcomas of multiple organs in female mice.
 

In each of the 95% confidence intervals for relative risk, shown
 

35
 
in the tables, the value of one or less than one is included;
 

this indicates the absence of significant postive results. It
 

should also be noted that most of the intervals have an upper
 

limit greater than one, indicating the theoretical possibility of
 

the induction of tumors by BHT, which could not be detected under
 

the conditions of this test.
 

36
 
V. DISCUSSION
 

Mean body weights of the dosed rats and mice were lower than
 

those of the corresponding controls and were dose related
 

throughout most of the bioassay. Survival was not affected
 

adversely in any of the dosed groups of rats or mice and was 60%
 

or greater in all dosed or control groups of rats and mice of
 

each sex at the end of the bioassay. Sufficient numbers of
 

animals were at risk for the development of late-appearing tumors.
 

No neoplastic lesions occurred in the rats or mice at incidences
 

that could clearly be related to administration of the BHT.
 

Nonneoplastic lesions that may have been related to the test
 

chemical consisted of focal alveolar histiocytosis at increased
 

incidences in the lungs of dosed female rats and various lesions
 

of the liver, including peliosis, hepatocellular degeneration and
 

necrosis, cytoplasmic vacuolation, and hepatocytomegaly at
 

increased incidences in the dosed male mice. Four high-dose male
 

mice were observed to have adenomas of the lacrimal gland;
 

however, these tumors cannot clearly be related to administration
 

of the test compound, since all glands were not examined in the
 

same manner. Alveolar/bronchiolar carcinomas or adenomas
 

occurred at a significant incidence (P = 0.009) in the low-dose
 

37
 
female mice; however, the incidence of the tumor in the high-dose
 

group was not significant, and the overall incidences were not
 

significantly dose related (control 1/20, low-dose 16/46, high-


dose 7/50). Historical records at this laboratory indicate that
 

female control mice had an incidence of alveolar/bronchiolar
 

carcinomas or adenomas of 21/440 (4.7%), compared with 1/20 (5%)
 

in the female controls in this study, 16/46 (35%) in the low-dose
 

group, and 7/50 (14%) in the high-dose group. Thus, the occurrence of lung tumors in the low-dose female mice cannot clearly
 

be related to administration of the test chemical.
 

In previous studies by others, the effects of BHT in tumor
 

initiation, promotion, and protection have been investigated, and
 

the results indicate that the temporal sequence between BHT
 

administration and exposure to a known carcinogen may be
 

important. Administration of BHT in feed at doses of 2,000,
 

5,000, 8,000, or 10,000 ppm for 2 years to male and female rats
 

of unspecified strain induced no pathologic lesions; however,
 

weight gain in the animals administered 10,000 ppm was subnormal
 

indicating that a maximum tolerated dose may have been exceeded
 

(Deichmann et al., 1955). Administration of BHT in a single oral
 

dose of 200 mg in olive oil to female Sprague-Dawley rats prior
 

to oral administration of 12 mg of dimethylbenz(a)anthracene
 

(DMBA) in olive oil resulted in a decrease in the incidence of
 

38
 
mammary tumors when comparisons were made with incidences of the
 

tumors induced by DMBA alone (Wattenberg, 1972). Also, administration of BHT at 6,600 ppm for 24 weeks to male and for 32 weeks
 

to female CD SPF rats that were simultaneously administered
 

2-acetylaminofluorene (AAF) at 223 ppm or N-hydroxy AAF at 239
 

ppm decreased the incidences of hepatomas in the males
 

administered AAF or N-hydroxy AAF and the incidences of mammary
 

carcinomas in the females administered N-hydroxy AAF when these
 

organs were examined 12 to 13 weeks later and comparisons were
 

made with incidences of the tumors induced by AAF or N-hydroxy
 

AAF alone (Ulland et al., 1973). Administration of BHT alone in
 

feed under the same conditions induced no tumors of the liver or
 

mammary gland. In contrast, administration of BHT in feed at
 

5,000 ppm for 407 days to male Sprague-Dawley rats following
 

previous administration of AAF in feed at 200 ppm for 18 days
 

caused an increase in the incidences of liver tumors, compared
 

with the incidences of the tumors induced by AAF alone (Peraino
 

et al., 1977).
 

In a study using mice, administration of BHT alone in feed at
 

7,500 ppm to male BALB/c mice for 16 months increased the
 

incidences of tumors of the lung and of the stomach, compared
 

with incidences of the respective tumors in untreated controls,
 

but decreased the incidence of reticulum-cell sarcomas (Clapp et
 

39
 
al., 1974). Also, in another study using mice, administration of
 

BHT alone in feed to CFl mice at 1,000 ppm for the first 1 or 2
 

months, then at 1,000, 2,500, or 5,000 ppm for 22 to 23 months,
 

led to dose-related increases in the incidences of lung tumors;
 

in addition, the incidence of tumors of the ovary was reported to
 

be increased in the female CFl mice administered the BHT (Brooks
 

et al., 1977). When, however, BHT was administered in
 

tricaprylin by intraperitoneal injection at doses of 250 mg/kg
 

three times daily for 8 weeks to male and female A/He mice and
 

the animals held for an additional 16 weeks, it had no
 

significant effect on the incidence of lung tumors (Stoner et
 

al., 1973).
 

Administration of BHT in feed at 5,000 ppm for 2 weeks to female
 

A/HeJ mice simultaneously administered benzo(a)pyrene (BP) at
 

1,000 ppm decreased the incidence of the tumors induced by BP
 

alone (Wattenberg, 1972). Similarly, administration of BHT in
 

feed at 7,500 ppm for 7 weeks to male and female BALB/c mice
 

simultaneously administered diethylnitrosamine (DEN) in the
 

drinking water at 350 mg/kg body weight decreased the incidence
 

of carcinomas of the stomach in the females, but not in the
 

males, when comparisons were made with the incidences induced by
 

the DEN alone (Clapp et al., 1976).
 

40
 
However, when BHT was administered as a promoter, i.e., by intraperitoneal injection in corn oil to male Swiss-Webster mice at
 

doses of 250 mg/kg weekly for 13 weeks following intraperitoneal
 

injection of single doses of urethane at 1 mg/g, the numbers of
 

tumors per lung was increased when comparisons were made with the
 

numbers of tumors per lung induced by urethane alone. The
 

opposite effect was observed when 0.9% NaCl was injected instead
 

of the urethane, administration of the BHT then resulting in the
 

complete absence of lung tumors, compared with the occurrence of
 

lung tumors in the untreated controls (Witschi et al., 1977).
 

Thus, in previous studies, BHT administered alone did not
 

increase the incidence of tumors in rats, but the incidences of
 

tumors in mice were increased. In the present study, again using
 

BHT alone, lung tumors were observed at an increased but
 

equivocal incidence in female mice. In other previous studies,
 

BHT protected against carcinogenesis in rats and mice when it was
 

administered prior to or simultanously with exposure to a
 

carcinogen. In contrast, however, when BHT was administered to
 

rats and mice as a promoter, e.g., following a carcinogen, the
 

incidence of tumors was increased.
 

It is concluded that under the conditions of this bioassay,
 

increased incidences of focal alveolar histiocytosis in dosed
 

41
 
female rats and various nonneoplastic lesions of the liver in
 

dosed male mice may have been related to the administration of
 

BHT. BHT was not, however, carcinogenic for F344 rats or B6C3F1
 

mice of either sex.
 

42
 
VI. BIBLIOGRAPHY
 

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

& Sons, Inc., New York, 1971, pp. 362-365.
 

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

Panel on Carcinogenicity of the Cancer Research Commission of
 

UICC, Vol. 2_, International Union Against Cancer, Geneva, 1969.
 

Brooks, T. M., Hunt, P. F., Thorpe, E., and Walker, A. T. T.,
 

unpublished results, cited in Federal Register 42(104):27603­

27606, 1977. ~~~
 

Chapman, D. G. and Kertesy, Z. I., Food additives. In: Kirkothmer Encyclopedia of Chemical Technology, Vol. 10, Interscience
 

Publishers, New York, 1966, p. 14.
 

Clapp, N. K., Klima, W. C., and Satterfield, L. C., Sex-dependent
 

protection against diethylnitrosamine-induced squamous cell
 

carcinomas of forestomach by concomitant administration of food
 

additive, butylated hydroxytoluene. AACR Abstracts 17:168, 1976.
 

Clapp, N. K., Tyndall, R. L., Gumming, R. B., and Otten, J. A.,
 

Effects of butylated hydroxytoluene alone or with diethylnitrosamine in mice. Fd. Cosmet. Toxicol. j_2:367-371, 1974.
 

Code of Federal Regulations, 40 CFR 180.1001:363, 1976.
 

Code of Federal Regulations, 2l_ CFR .100.1J20:332-333, 1977.
 

Code of Federal Regulations, 21_ CFR 582/_l:484, 1977a.
 

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

Soc. B 34:187-220, 1972.
 

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

1970, pp. 48-52.
 

Deichmann, W. B., Clemmer, J. J., Rakoczy, R., and Bianchine, J.,
 

Toxicity of ditertiarybutylmethylphenol. AMA Archives Ind. Hlth.
 

1^:93-101, 1955.
 

Drews, Joel E., Allied Mills, Inc., Chicago, 111., personal
 

communication, 1978.
 

43
 
Dugan, L. R., Jr., Ancioxidancs. In: Kirk-othmer Encyclopedia
 

of_ Chemical Technology, Vol. 2^ Incerscience Publishers, New
 

York, 1963, pp. 588-604.
 

Federal Register, Food and Drug Administration, Butylated
 

hydroxytoluene. Use restrictions, U. S. Government Printing
 

Office, Washington, D. C., 42 (104):27603-27607, 1977.
 

Furia, T. E., ed., Regulatory status of direct food additives.
 

In: Handbook o£ Food Additives, CRC Press, Cleveland, Ohio,
 

1972, pp. 783-96~6"i
 

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

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Stat. Inst. 3Jh 148-169, 1971.
 

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

incomplete observations. J_. Am. Statist. Assoc. 5_3:457-481, 1958.
 

Lauffer, P. G. I., Lipsticks. In: Cosmetics—Science and
 

Technology, Vol. l^, Wiley-Interscience, New York, 1972, pp. 365­

376.
 

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

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

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

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

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

Noller, C. R., Phenols, aminophenols, and quinones. In:
 

Chemistry of Organic Compounds, W. B. Saunders Co., Philadelphia,
 

1966, pp. 560-561.
 

Peraino, C., Fry, R. J. M., Staffeldt, E., and Christopher, J.
 

P., enhancing effects of phenobarbitone and butylated
 

hydroxutoluene on 2-acetylaminoflourene-induced hepatic
 

tumorigenesis in the rats. Fd. Cosmet. Toxicol. JL5:93-96, 1977.
 

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

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

induced by different numbers of administrations of benzo(a)
 

pyrene and ferric oxide. Cancer Res. 32^:1073-1081, 1972.
 

Select Committee on GRAS Substances, Life Sciences Research
 

Office, Evaluation of_ the Health Aspects of Butylated
 

Hydroxytoluene a£ a_ Food Ingredient, Federation of American
 

Societies for Experimental Biology, Bethesda, Md., 1973.
 

44
 
Stecher, P. G., ed., The Merck Index, Merck & Co., Inc. Rahway,
 

N.J., 1968, p. 179.
 

Stoner, G. D., Shimkin, M. B., Kniazeff, A. J., Weisburger, J.
 

H., Weisburger, E. K., and Gori, G. B., Test for carcinogenicity
 

of food additives and chemotherapeutic agents by the pulmonary
 

tumor response in strain A mice. Cancer Res. ^3_: 3069-3085, 1973.
 

Stuckey, B. N., Antioxidants as food stabilizers. In: Handbook
 

£f_ Food Additives, Furia, T. E., ed., CRC Press, Cleveland, Ohio,
 

197 2~7Tp. 185-223.
 

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

Biometrika 62(3);679-682, 1975.
 

Ulland, B. M., Weisburger, J. H., Yamatnoto, R. S., and
 

Weisburger, E. K., Antioxidants and carcinogenesis: butylated
 

hydroxytoluene, but not diphenyl-p-phenylenediamine, inhibits
 

cancer induction by N-2-fluorenylacetamide and by N-hydroxy-N-2­

fluorenylacetamide in rats. Fd. Cosinet. Toxicol. 11;199-207,
 

1973.
 

United States International Trade Commission, Synthetic Organic
 

Chemicals - United States Production and Sales, 1976, USITC
 

Publication 833, U. S. Government Printing Office, Washington,
 

D.C., 1977, p. 299.
 

Wattenberg, L. W., Inhibition of carcinogenic and toxic effects
 

of polycyclic hydrocarbons by phenolic antioxidants and
 

ethoxyquin. J. Nat. Cancer Inst. 48:1425-1430, 1972.
 

Witschi, H., Williamson, D., and Lock, S., Enhancement of urethan
 

tumor igenesis in mouse lung by butylated hydroxy toluene. J_. Nat.
 

Cancer Inst. 52(2):301-305, 1977. ~
 

45
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED BHT IN THE DIET
 

47
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED BHT IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INIT1AIL Y I N STUD Y 2 0 50 50

 

ANIKAL S MISSIN G 1

 

ANIMAL S NSCtGESIE D 2 0 49 50 

ANIMAL S EXAMINE D HISTOPATHCIOGIC ALL Y 2 0 49 49

 

INTEGUMENTAR Y SYSTE M 

*SKII» (20) (49) <5C ) 

SQUAMOU S CELL CARCINCMA 2 (47.) 

E^SAL-CEI L CARCINOM A 1 (2%) 

*SUBcUT TISSU E (20) (49) (5C) 

FiBRCM A 2 (4/5) 

AfiELOBLASTI C CDONTOM A 1 (2% ) 

FESPIitATORY SYSTEM 

«LUN(J (20) (49) (49) 

SQUAMOU S CELL CARCINCMA , METAST A 1 (2%)

ALVEOLAR/BRONCHIOLA R ADENOM A 2 (4«) 

ALVEOLAR/EECNCHIGLA R CARCINOM A 1 (5% ) 1 (2%) 1 (2%) 

KEMATuPOIETI C SYSTE M 

#ERAI N (20) (49) (49)

 

MALIGNAN T EETICULOSIS 1 (2%) 

*MULIIPL E ORGAN S (20) (49) ( 5 C ) MALIGNAN T IYKPHOMA , NOS 1 (5«) MaLiG.LYMPfiCMA , UNDIFFER-TYPE 4 (20% ) 9 (18X) 1C (2055) 

ISPLiE N (20) (48) (47) 

HiMANGIOSASCCM A 1 (5X) 

MALIG-LYMPKOMA , U NDIFFEE-T Y FE 1 (2%) . 

*MANDIB'JLA F I. NCD E (20) (49) (46 )

 

SQUAMOU S CELL CARCINCMA , BETASTA 1 (2%) 

*SALIVAR Y GL/N D (20) (49) (4S) 

MAL1G. LYMPHOMA , HISTIOCYTIC TYPE -J.J2J1 

* NUiliiER OF ANIMAL S iilTH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMiiER OF ANIMAL S NECROPSIE D 

49 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

CIRCULATORY SYSTEM
 

NONE
 

EIGES1IVE .SYSTE M 

• IIVj-R	 (20) (48) (48)
 

EULE DUCT CARCINOMA 1 (2%)
 

NEOPLASTIC NOCULE 1 (2%) 1 (2%)
 

HhPATOCELIULAE CARCINOMA 1 (2.?) 1 (2%)
 

*SMA.LL INTESTINE (18) (48) (46)
 

LIPOMA 1 (2%)
 

UEINARY SYSTEC.
 

#KIDNEY (20) (49) (48)
 

NEPHROBLASTCMA 1 (2%)
 

((URINARY ELJIEEP (20) (47) (46)
 

TixANSITICNAL-CELL CARCINOMA 1
 

ENDOCKINS SYSTEM
 

#PITUITAP.Y (19) (47) (47) 

CrtRCINCMA.NO S 1 (5X) 

AOJ.NOBA, NCS 6 (32X) 9 (19%) 9 (19X) 

• ADRENA L (19) (49) (48) 

CORTICAL CfRCINOMA 2 (14%) 

PaSOCHBCBCCYTCHA 2 (1U) 8 (16%) 10 (21X) 

tADH^NAL/CAPSQLE (19) (49) (48)
 

PaRAGANGLlCSA, NOS 1 (2X)
 

STHYRCID (20) (49) (48) 

FOLLICULAE-CELI. ADENCM A 2 (««) 

FuLLICULAB-CEtL CARCINOBA 1 (5X) 2 (4%) 1 (2%) 

C-CELL ADENCM A 1 (5X) 5 (10X) 1 (2%) 

C-CELL CARCINOM A 1 (2X) 1 (2%) 

(18) (45) (43)
 

* NUMdE3 OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* HUKaEP OF ANIMALS NECROPSIED
 

50
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

#PAN«_RFATIC ISLETS

I^LET-CELI ADENOM A

ISLET-CELL CARCINOMA

 (19) (48)

 2 (4?)

 2 (4X)

 (US) 

1 (2%) 

1 (2%) 

REPRODUCTIVE SYSTEM 

*PREPUTIAL GLAND

CARCINOMA, NOS

ITES1IS

IMSR3TITIJSL-CELL TUMO R

 (20)

 (20)

 15 (75X)

 (49)

 3 (635) 

 (49)

 42 (86%)

 (50) 

 (49 ) 

 32 (65*) 

SERVOJ S SYSTE M 

«ERAIN/MENINGE S

MENINGIOM A

• ERAJ.N

Si,IOHA, NOS

 (20)

 1

 (20)

 (5%) 

 (49)

 (49)

 1 (2X) 

 (49) 

 (49) 

SPECIAL SENSE ORGANS 

*ZYMJAL'S GLA&D

CARCINOMA, NOS

SQUAMOUS CILL CARCINOMA

 (20) (49)

 1 (2%) 

 (50) 

1 (2%) 

HliSCULOSKEIETAL SYSTEM 

NONE 

ECDY CAVITIES 

*MESiNTERY

LIPOMA

*TUNiCA VAGINALIS

MiSOTHELICKA, NOS

 (20)

 (20)

 1 (5X) 

 (49)

 1 (2%) 

 (49)

 (50) 

 (50) 

ALL OTHER SYS1IBS 

*MOL1IPLE ORGANS

__ ZIBROS.A1CCJA _______________

 (20)

 J 

 (49) (50) 

t NUMBER OF ANIMALS 3ITH TISSUE EXAHINED MICROSCOPICALLY 

* NUMdER OF ANIMALS NECROPSIED 

51 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL
 

ANIMAi. D1SECSITICN SUMMARY 

ANIiULS INITIALLY IN STUDY 

NATURAL EEATH3 

MOPI3UNL SACRIFICE 

SCHEDJLEr SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

A«IMAL MISSING 

23 

13 

a INCLUDES AIJTCLYZED ANIMALS 

TUMOR SUMMAR Y
 

TOTAL \SIMALS WITH PRIMARY TUMORS* 19
 

TOTAL PRIMARY TUMORS 36
 

TOT^L ANIMALS WITH BENIGN TUMORS 18
 

TuTAL BENIGN TUMORS 25
 

TOTrtL ANIKALS VITH MALIGNANT TUMORS 9
 

TOTAL MALIGNAN T TUMORS 10
 

TOT^L ANIMALS KITH SECONDARY TUMORS*
 

TOTAL SECCKDARY TUMORS
 

ANIMALS WITH TUMORS UNCEBTAIN­ N OR flAIIGNANT
 

TuTAL UNCERTAIN TUMOPS
 

TOTnL ANIMAL S UITH TUMORS UNCERTAIN-


PPIuARY OR METASTATIC
 

TOTAL UNCEFTAIN TUMORS
 

* PRIMA3Y TUKCES: ALL TUMORS EXCEPT SECONDARY 7UMORS
 

LOW DOSE HIGH DOSE
 

5) 50
 

1 1
 

•3
 

39 36
 

1
 

46 44
 

1)) 6)
 

45 41
 

72 57
 

19 2C
 

26 22
 

* SECONDARY TUKORS: METASTATIC TUMORS OR TUMOES INVASIVE INTC AN ADJACENT ORGAN
 

52
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED BHT IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUCY 2J 5) 50 

ANIMALS MISSING 2 

ANIMALS NECROPSIED 18 50 50 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY 18 49 50 

INIEGJM-ENTAR Y SYSTEM
 

*SKId (18) (50) (50)
 

CARCINOMA,NOS 1 (2%)
 

*SUB«-UT TISSUE (18) (50) (5C)
 

FIBROMA 1 (2%)
 

CSTEOSARCCKA 1 (2%)
 

RESPIEATORY SYSTEM
 

tlUNCi (18) (48) (49)
 

AJ.VFOLAR/ERCNCHIOLA R ADENOK A 1 (6«) 2 (4S) 

ALVEOLAP/EFONCHIOLAR CARCINOMA 1 (2%) 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

#ERA.N (18) (49) (50)
 

MALIGNANT KETICULOSIS 1 (2%)
 

*MULTI?LE ORGAN S (18) (50) (5C)
 

MALIGNANT IYMPHCMA, NOS 1 (655) 2 (H%) 1 (2%)
 

MaLIG. LYMPHOMA, UNDIFFEE-T YPE 1 (65t) 8 (16X) 4 (3«)
 

*THYi'iUS (17) (43) (45)
 

THYMOMA 1 (2%)
 

CIKCULATORY SYSTEM
 

NONE
 

I NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY
 

* NUMdE R OF AKIMAL S NECROPSIED
 

53
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

UBINAS Y SYSTE M 

NCNi 

INCOCalN E SYS1FM 

#PITjITAR Y (18) (48) (49) 

AjINOMA , NOS 8 (44*) 9 (19%) 5 (10*) 

#ADRiNA L (17) (47) (49) 

PHEOCHROMOCYTOM A 2 (4S) 1 (2*) 

*1HY30ID (18) (48) (49) 

FOLLICULAR-CEL L ADENCM A 2 (4*) 

FOLLICULAR-CEL L CARCINOM A 1 (2*) 

C-CELL ADENOM A 2 (11X) 4 (8%) 4 (8*) 

•PANCREATIC ISLSTS (17) (46) (47)

 

ISLET-CELL AEENOM A 1 (2X)

 

REPRODUCTIV E SYSTE M 

*MAMMAR Y GLA M (18) (50) (50) 

ADENOCARCINOMA , NOS 2 (4*) 

FIBROADENOM A 5 (28*) 7 (14*) 5 (10*) 

*CLITORAL GLAN D (18) (50) (50)

 CARCINOMA , NOS 1 (2X)

 

*UTEi<US (17) (49) (IS) CARCINOMA , NOS 1 (2») 

ENDOHETRIA L S180MAL POLYP 2 (12*) 8 (16*) 6 (12*) 

#OVAfi Y (17) (49) (49)

 

THECOM A 1 (6X)

 

SEBVOUS SYSTEM 

SPECIAL SENSE ORGAN S 

*ZYMuAL'S GlAfcD (18) (50) (50) .. CiiRCINCHAy^OS __J J1Z1 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D HICROSCOPICALLY 

* NUMBER OF AKIMAL S NECROPSIED 

54 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MUSCULOSKELETA L SYSTSM 

NONr, 

£ODY CAVITIE S 

NCN*. 

ALL OTHER SYSTIMS
 

NCNi
 

ANIMAL DISPOSITION SUMMARY
 

ANIilALS INITIALLY IN STUCY 20 5} 50
 

NATURAL DEATHS U 11 ?
 

MORIBUND SACEIFICE 1 2 2
 

SCHEDULES SACRIFICE
 

ACCIDENTALLY KILLED
 

TxiRaiNAL SACRIFICE 13 37 39
 

ANIMAL MISSING 2
 

* NUMJEF OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMcE? OF AKIMAL S NECROPSIED
 

55
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 

TOTAL PRIMAR Y TUMORS 

12 

21 

36 

53 

26 

31 

TOTAL ANIMAO S WITH BENIGN TUHOES 

TOTAL BENIGN TUMORS 

11 

19 

27 

36 

18 

22 

TOTAL ANIMALS WITH MALIGNANT TUMORS 

TOTAL MALIGNANT TUMORS 

2 

2 

16 

17 

S 

9 

TOTAL ANIMAL S WITH SECONDARY TUMOES* 

TOTAL SECONDARY TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

56
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED BHT IN THE DIET
 

57
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED BHT IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALL Y I N STUD Y 2 ) 5) 

ANIMAL S 5JECEOESIE D 2 0 50 50 

ANIMAL S EXAMINI C HISTOPATHOLOG1CALL Y 2 ) 50 49 

INTEGUMENTARY SYSTE.1
 

NCN.1
 

RESPIRATOR Y SYSTE M 

tl'JNJ (20) (50) (49 ) 

HEPATOCELLULA R CARCINOMA , METAST 3 (1558) 3 (6%) 

ALVEOLAE/EECNCHIOLA S ADENOM A 2 (105!) 9 (18*) 1C (20X ) 

Ai,VEOLAR/EFCNCHIOLA R CAECINOH A 5 (25*) 12 (2«X) 7 (145?) 

HEMATOPOIETI C SYSTE M 

*MULIIFLi CBGflN S (2)) (50) (50) 

BALIJSAN T LYMPHOMA , NO S 2 (1058) 5 (10X) 3 (635) 

KrtLI'J. LYMPHOMA , HISTIOCYTIC TYPE U (8S) 1 (255) 

M*iLIvJNAN T LYMPHOMA , KIXE D TYPE 2 (105!) 

HSPLnE N (19) (50) (48) 

AHGIO S ARCCP A 1 (5?.) 1 (2%) 

MALIG . LYMPHOMA , HISTIOCYTIC TYPE 1 (2%) 1 (2«) 

«nANJI2ULA S 1. NOE E (20) (1*9) (IS )

 

MALIGNAN T ZYMPhOBA , NO S 1 (2X ) 

*EPCJCHI^ L LYMP H NOD E (20) (U9 ) (US )

 

HEPATOCELLULA R CARCINOMA , METAST 1 (2X )

 

#flESi,NTtHIC 1. NOD E (20) (49) (49 ) 

"ULIJNAN T LYMPHOMA , NO S 1 (5X) 

MhLIG . LY f PHOMA , HISTIOCYTIC TYPE 2 (4*) 2 (4X) 

ISHA- L IN1ESIIS E (19) (48) (49 ) 

MaLIG.LYKPhCBA ^ HISTIOCYTIC TYPE 1 J2X1 

# NUH^EF OF AMMALS WITH TISSUE EXAMINED BICEOSCOPICALLY
 

* NUMBER OF AKIMAL S NECROPSIED
 

59
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

((THYMUS

MALI3.LYKPKOMA, LYMPHOCYTIC TYPE

MALIG. LYMPHOMA, HISTIOCYTIC TYPE

 (10)

 1 (US) 

 (39)

 1 (3X) 

 (*»£) 

CIRCULATORY SYSTEM 

NONi 

CIGESi'IVE SYSTIH 

ILIVJR

HEPATOCELLULAR

HEPATOCELLULA R

AttGIOSARCCK A

 ADENOM A

 CARCINOM A

 (20)

 2 (1JX )

 9 (45*)

 1 (5«)

 (48)

 11 (23S)

 12 (25%)

 (49) 

7 (14X ) 

6 (12*) 

1 (2% ) 

UHINAK Y SYSTE P 

4IKIDWE Y

HEPATOCELLULA R CARCINOMA , METAST

 (20)

 1 (5%) 

 (50) (49) 

INDOCtUNS SYS1IM 

IADRr.NAL

CORTICA L J.EENCM A

PhEOCHBOMCCYTCMA

 (20)

1 (5%) (49)

 1 (2*) 

 (49) 

#THYhOID

FOLLICULAR-CEL L ADENCM A

FOLLICULAR-CEL L CARCINOM A

 (18) (48)

2 (4X) 1 (2% ) 

 |49) 

2 

REPRODUCTIVE SYSTEM 

*SEMINAL VESICLE

SAhCOMA, NCS

 (20) (50)

 1 (2%) 

 (50) 

KERVOU3 3YSTEK 

#ERA1N (20) (50) (49) 

* NUMJEIt

* NUMJE H

 OF

 OF

 AKISAL S

 AMMAL S

 WITH TISSUF

 KECRCPSIE D 

 EXAMINE D MICROSCOPICALLY 

60
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL S£NS E ORGAN S 

+EYE/LACPIMA L GLAN D

AOLSCaA , NCS

*EAR

FIBROM A

 (20)

 (20)

 (50)

 (50)

 (5C) 

t (8*) 

 (5C) 

2 (H%) 

MUSCULOSKELETA L SYSTEH 

NCN E 

EOBY t-AVITIES 

*M£D1ASTINU M

SHRCCMA , NCS, METASTATIC (20) (50)i (2%) 

 (50) 

ALL OTHER SYSTEMS 

*MULIIPLE OFGASS

SARCOMA, NCS

 (20)

1 (S%)

 (50) (50) 

1 (2%) 

SNIKAi, EISPCSITICN SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL IEATH3 

MURIBUND SACEIFICE 

SCHEDULEE SACEIFICE 

ACCIDEN1AIIY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING 

20 

12 

50 50 

f NUMoEB OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY 

* NUfluER OF AKIMALS NECROPSIED 

61
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

TUMOR SUMMAR Y
 

TOTaL ANIMALS WITH PRIMARY TUMORS* 

TuTAL PRIMAR Y TUMORS 

17 

28 

39 

65 

j 2 

48 

TOTAL ANIMALS WITH BENIGN TUMOFS 

TOTAL EEKIGN TUMORS 

4 

5 

20 

23 

19 

25 

TOTAL ANIMALS WITH MALIGNANT TUMORS 

TOTAL MAIIGNANT TUMORS 

16 

23 

32 

42 

1S 

23 

TOTAL ANIMALS SilTH SECONDARY TUMORS* 

TOTAL SECC>DARY TUMOES 

3 

4 

4 

5 

TOTAL ANIMALS WITH TUMCRS UNCERTAIN­ EEN^GN OR rSUGNANT 

TUTAL UNCERTAIN TUMORS 

TOT«L ANIMAIE VJITH TUMORS UNCERTAIN- FRIiiARY OP KETASTATIC 

TOTAL UNCERTAIN TUMOfcS 

* PEI^AHY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMOHS
 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACEKT ORGAN
 

62
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED

ANIMALS INITIALLY IN STUDY
 

JNIMALS MISSING
 

ANIMALS NECPCESIFD
 

ANIMALS EXAMINED HISTOPATHOLOGIC ALLY
 

INTEGuMENTAfiY SYSTEM
 

NCNi
 

BESPIdATORY SYSTEK
 

#LUNG
 

Ai,VEOLAR/EBCNCHIOLAR ADENOMA
 

ALVFOLAR/BRCNCHIOLAR CAKCINOMA
 

HEMATOPOIETIC SYSIEM
 

"•MULTIPLE ORGANS
 

MALIGNANT IYMPHCMA, NOS
 

H«LI3. LYMPHOMA, HISTIOCYTIC TYPE
 

MALIGNANT IYMPHOMA, MIXED TYPE
 

#SPLi.EN
 

ANGI05 ARCCFA
 

MALIGNANT LYMPHOMA, NOS
 

*HESiNTESIC L. NODE
 

AiiJlOSAECCfA, METASTATIC
 

MALIGNANT LYMPHOMA, NOS
 

#SMALL INTESTINE
 

MALIG.LYCPtCMA, HISTIOCYTIC TYPE
 

((THYMUS
 

MALI3NANT LYMPHOMA, NOS
 

CIRCULATORY SYSTEM
 

NONE
 

 BHT IN THE DIET 

MATCHED
 

CONTROL


20


20


20


(20)


1	 (5X)


(20)


2 (1035)


2 (10X)


1 (5%)
 

(20)


2 (10$)
 

2 (10*)
 

(20)


1 (5%)
 

(20)


(17)


 LOW DOSE HIGH DOSE
 

 50	 50
 

3
 

 46 50
 

 U6 50
 

 (46) (50)
 

12 (26%) 3 (6*)
 

4 (9S) H (8X)
 

 (46)	 (50)
 

2 (455) 6 (12X)
 

5 (11S)
 

 (45)	 (50)
 

1 (2X)
 

 (44) (49)
 

1	 (2X)
 

 (45)	 (48)
 

1 (2X)
 

 (37)	 (33)
 

1 (3X)
 

# NURi/EE OF ANIMAL S UITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUNoE S O F ANIMAL S NECROPSIE D 

63 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

EIGESi'IVE SYSTE M 

*LIVtR (20) (46)

HEPATOCELLULAR ADENOMA

HJ.PATOCELIULA F CARCINOM A i (535)

 3

 1

 (78)

 (2%)

SARCOMA, NCS 1 (2%) 

A«GIOSARCCt" A 1 (5X) 1 (2%)

UEINAti Y SYST5 C 

NONi. 

ODOCalNE SYST1.1 

4FITJITAR Y (20) (45)

ADENOMA , NOS 4 (935)

dADHSNA L (20) (46)

COPTICA L AIENCH A 1 (5X)

PiiECCHECMCCYTOM A 1 (255) 

)IC (20) (46)

FOLLICULAR-CEL L ADENCM A

BEPROJUCTIV E SYSTE M 

*KA(UAK Y GLAN L (20) (46) 

AijENOCASCINCMA , NO S 

KUTEtiU S (20) (45) 

PAPILLAR Y CYSTADENOCARCINCMA,NO S 

END01ETRIA L STROMA L POLYP 1 (5%) 1 (2*) 

AhGIOM A 

*OVAiiY/0/IDUCT (20) (45) 

PAPILLAR Y ADENOM A 1 (235) 

iCVArtY (19) (45) 

PAPILLAfiY ADENOM A 1 (235) 

P»PILLAP Y CYS1ADENOMA , NOS 1 (2%) 

NERVOU S SYSTEC 

_NQJLi 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NOMoEP. OF AMMAL S NECROPSIED 

64 

 (49)
 

2 (U%)


3 (6X) 

1 (235) 

 (47) 

1 (2%) 

 (48) 

1 (2%) 

 (49 ) 

1 (2SS) 

(50) 

2 (435) 

(49) 

1 (2%) 

1 (2%) 

(45) 

1 (2%) 

(47) 

1 (2») 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENS E CRSAN S 

*EYE/LACRIMAL GLAN D

ADENOMA , NCS

 (20) (46) .

 2 (4S) 

 (50) 

HOSCU1.0SKEIETAI SYSTEM 

NON E 

EODY CAVITIES 

NONfi 

ALL OTHER SYSTEMS 

*MULTIPLB OKGANS

SARCOMA, NCS , (20)3 (15*)

 (46)

1 (28) 

 (50) 

JNIMAi DISPOSITION SUMMARY 

ANIdALS INITIALLY IN STUDY

NATURAL DEATHS

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIMAL MISSING

 20

 3 

 17

 50

6 

 41

 3 

 50 

5 

 45 

t NUMJER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NOMuER OF ANIMALS NECROPSIED 

65 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

1UBOE SUMMAR Y
 

TOT^L ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIKAHY TUMORS


TOTAL ANIMALS KITH BENIGN TUMORS


TOTAL EEMGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL 1ALIGNANT TUMORS


TOTaL ANIMALS WITH SECONDARY TUMORSt


TOTAL SECCKDftRY TUMORS


TOTAL ANIMALS WITH TUMCRS UNCEBTAINEEN13N OR KAIIGNAN T
 

TuTAL UNCERTAIN TUMORS
 

TOT.iL ANIMAL S WITH TUMORS UNCERTAIN-
 PRIiiARY OB KETASTATIC
 

TuTAL JNCEETAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 14 32 23 

 17 42 31 

2 22 10 

2 26 11 

 13 16 17 

 15 16 2) 

1 

1 

* FRIdAPY TU1CRS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

t SLColOAFY TUhOPE: METASTATIC TUMORS OR TUMOF.S INVASIVE INTC AN ADJACIK1 ORGAN
 

66
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS ADMINISTERED BHT IN THE DIET
 

67
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED BHT IN THE DIET

 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIMALS MISSING 1
 

ANIMALS NICKOFSIEE 20 49 50
 

ANIMALS EXAMINED HISTOPATHOLOGICAL!Y 20 49 49
 

INTEGUM2NTAFY SYSTEM
 

NCNE
 

RESPIRATORY SYSTEK
 

SLUN u (20) (49) (49) 

HEMORRHAG E 1 (2%) 

BitCNCHCPliECMGNI A SUPPURATIV E 1 (5X) 

BitOSCHOPNEUHONIA , ACUTE 1 (5% ) 

HYPERPLASIA , ALVEOLA R EPITHELIU M 3 (6%) 3 (6%) 

ttLUNo/ALVEOII (20) (49) (49) 

U1STIOCYTCSIS 1 (5%) 4 (8%) 7 (14*) 

HEMATOPOIETI C SYSTEM 

#BONi HAS BOU (20) (48) (48) 

NYELOFIflRCSIS 1 (2%) 

#SPLiEN (20) (48) (47) 

HiiMOSIDEECSI S 1 (2%) 

HiMATOEOIESIS 9 (19%) 1 (2)8) 

#MANDIEULAB I. NODE (20) (49) (46) 

LYBPKANGIECTASIS 2 (10%) 5 (1)%) -= (6%) 

HYPERPLASIA, LYMPHOID 1 (2X) 1 (2X) 

*HES£.NTi:HIC I. NODE (20) (49) (4€) 

LYMPHANGIECTASIS 1 (2%) 1 (2%) 

CIHCOLATOHY SYSTE M 

#HEAR T (20) (49) (49)
 

PfiRIARTERITI S 

t NUBbER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

69
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

— ———* _-^—_— — — •————— ^ -*-­ — 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

#HEAiT/ATRIUH (20) (U9) (49)
 

THROMBOSIS, NCS 2 (10*) 1 (2%) 1 (2X)
 

#MYOCARDIUM (20) (49) (49)
 

INFLAMMATION, CHRONIC 1 (255)
 

INFLAMMATION, CHRONIC FOCAL 1 <5X)
 

FIBROSIS 1 (5X) 10 (20*) 8 (16X)
 

*COROHARY ARTERY (20) (49) (50)
 

ARTERIOSC1IBCSIS, NOS 1 (5%)
 

MiiDIAL CALCIFICATION 1 (2%)
 

'PULMONARY AE1ERY (20) (49) (5C)
 

MiDIAL C AICIFICATION 6 (12X)
 

*MES£.NTERIC ARTERY (20) (49) I5C)
 

ARTERIOSCLEROSIS, NOS 1 (2X)
 

DIGES1IV2 SYSTIM
 

*LIVi:R (20) (48) (48)
 

NECROSIS, NOS 1 (2%)
 

NECROSIS, FOCAL 2 (4%) 1 (2%)
 

HiTAMOSEHCSIS FATTY 2 (10X) 1 (2%)
 

CYTOPLASMIC VACUOLIZATION 13 (27X) 9 (19S)
 

HEPATOCYTOMEGALY 3 <15X) 11 (23%) 2 (4*)
 

HYPERPLASIA, FOCAL 1 (5%) 3 (6«)
 

tLIViR/CENTRIIOBULAR (20) (48) (48)
 

D£GfNERATICN, NOS 1 (535) 1 (2*)
 

NECROSIS, NOS 2 (4X)
 

NiCPOSIS, EIFFUSE 1 (2X)
 

#LIV£.P/FSRirCFTAL (20) (48) (48)
 

FIBROSIS 1 (2X)
 

#BILi: DUCT (20) (48) (46)
 

HYPERPLASIA, NOS 16 (80S) 8 (17S) S (UX)
 

((PANCREAS (19) (48) (46)
 

CYSTIC EDCTS 1 (2X)
 P-LRIARTERI-IIS 4 (8£) 2 (4«)
 

#?ANv.riEATIC ACINUS (19) (48) (46)
 

A'IROPHY, NCS 3 J6Si_ 1
 - i-J*!!
 

* NUMJER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMuER OF AKIMAL S NECROPSIED
 

70
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

AiBGPHY , FCCA L 2 (11«) 6 (13%) 

#STCrfAC:i (20) (49) 

OLCS'ft, FCCA L 

* SMALL INTESTIN E (18) (US)

 

HYPERPLASIA , LYMPHOI D 3 (6*)

 

#LAE'«Z INTESTIN E [19) (48)

 

NZBATODIASI S 2 (11*) 1 (255)

 

UEINAS Y SYSTE M 

t KIDNE Y (20) (49) 

PlELQNEPKBITIS , ACUTE 

IiNFLAMMATICN , CHRONIC 19 (95%) 48 (98X) 

*KIDNEY/COBTI X (20) (49)

 

CYST, NOS 

*PEOXIMA L CCKVOLUTE D (20) (49)

 

PiGKENTATICN , NOS 1 (2%)

 

IURIJJAR Y ELJEEEP . (20) (47)

 

IwFLAMMATICN , ACUTE HEMCREHAGI C 

ENDOCRIN E SYSTE M 

*PITUITAE Y (19) (47)

 

CYST, NOS 1 (5X) 1 (2%)

 

HiMOPEHAG E 1 (2X) 

INFARCT , KCS 1 (2X)

 

A«GIECTASI £ 1 (5X)

 

#ADEjiNA L CCR1IX (19) (49) 

L^POIDOSIS 2 (11X) 2 (4S)

 

HYPERPLASIA , NOS 

HYPERPLASIA , FCCAL 2 (11X) 3 (6S)

 

IADR£NA L HEEULL A {19) (49) 

HYPERPLASIA , NOS 

HYP2EPLASIA , FOCAL 1 (5X) 1 (2X) 

ANGIECTASI S 1 J2S1_ . 

* NUMuER OF AMHALS HITH TISSUE EXAMINED HICEOSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

€ (13X ) 

(46 ) 

2 (UX ) 

(48) 

(47) 

(48) 

1 (2X) 

46 (965!) 

(48) 

2 (4X) 

(48) 

(46) 

2 (4X) 

(47) 

2 (4X) 

1 (2X) 

(48) 

1 (2X) 

(48) 

1 (2X) 

1 (2X) 

J_J2J1_ 

71
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

HTHYKOI D (20) (49) J48 ) 

CYSTIC FOLLICLES 4 (8X) 1 (2%) 

FOLLICULA R CYST, NOS 2 (4«) 

HYPERPLASIA , C-CELL 4 (2055) 15 (3U) 15 (3H) 

SPANCR'-ATIC ISLETS (19) (48) (48 )

 

HYPERPLASIA , NOS 1 (2%)

 

REPRODUCTIV E SYSTE M 

*MA.1KAPY GLAN I (2 )) (49) (50) 

DlLATATICN/EUCT S 2 (4%) 1 (2%) 

*PR03TATP (20) (49) (46 ) 

INFLAMMATION , SUPPURATIVE 2 (1J3S) 5 (1)*) 11 (2335) 

INFLAMMATION , ACUTE 4 (8J5) £. (4 % ) 

INFLAMMATION , ACUTE SUPPURATIV E 1 (5«) 3 (635) 

InFLAMMATICN , ACUTE HEMORKHAGI C 1 (2X) 

IflFLAHEATICN , CHRONIC 1 (2/S) 

#TES'i.IS (20) (49) (4S) 

ATROPHY, NCS 1 (535) 1 (255) 

HYPERPLASIA , INTERSTITIAL CELL 2 (455) 4 (835) 

KERVCU S SYSTE M 

#ERAI N (20) (49) (49) 

MINERALIZATIO N 1 (2?) 

HEMORRHAG E 2 (10*) 4 (8«) 

SPECIAL SENSE ORGAN S 

*EYE (20) (49) (50) 

CATARACT 4 (8») 3 (6*) 

*EYE/CORNEA (20) (49) (EC) 

ULCER, NOS 1 (2«) 

MUSCULOSKELETAL SYSTEM 

110 N£ 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

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

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

EODY CAVITIES 

*MEStNTERY

HiMORRHAG E

PtRIARTEEI-II S

 (20) (<*9)

 1 (2%) 

 (50) 

1 (2%) 

A LL OTHER SYSTEM S 

NO Hi 

SPECIAL JORPHCIOG Y SUMMAR Y 

Nu LESION F3EORTED

ANIMA L HISSIHG/SC NEC80ESY

AUTO/NECPOJSY/NO HISTO

* NUMBE R OF AMMAL S inlTH TISSUE

* NUMuE P OF AMMAL S NECROPSIED 

 EXAHINE D MICfiOSCOPICALLY 

1 

1 

1 

73 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED BHT IN THE DIET 

iNIMALS INIIIAILY IN STUDY

JNIMAi. 5 MISSIN G

ANIMAL S NECROPSIED

ANIMAL S EXAMINE D HISTOPATHOLOGICAL!.*

INTEGUMENTAR Y SYSTEM 

NCNji 

RESPIRATOR Y SYSTE M 

*LUN(i


BdCNCHCPNIUIONIA, ACUTS


HYPERPLASIA, ALVEOLAP EPITHEIIUM


tlUNG/ALVECII


HJ.STIOCYTCSIS


HEMATOPOIETIC SYSTEM
 

#SPL.iEN
 

HjiKOSIDEPCSIS 

LYMPHOI D CEPLETIO N 

H-aATOEOIESI S 

((MANJIEULAR 1. NODE 

LYMPHANGIECTASI S 

HYPERPLASIA , LYMPHOI D 

#MES£.NTERIC L. NODE 

LfMPhANGIECTASI S 

CIRCULATORY SYSTEM 

#HEAtiT 

P..RIARTERITI S 

MATCHED 

CONTROL

 23

 2 

 18

 18

 (18)


 3 (17X)


 (18)


 2 (11X)


(17) 

1 (6X) 

2 (12?) 

(18) 

1 (6X) 

(18) 

(18) 

1 (6*) 

(18) 

 LOW DOSE HIGH DOSE 

 50 50 

 51 50 

 49 50 

 (48)	 (49)
 

1 (2%) 1 (2X)
 

2 (4*) 4 (8X)
 

 (48)	 (4S)
 

 12 (25X) 21 (43*)
 

(48) (49) 

2 (4X) 

1 (2«) 

5 (10X) 4 (8«) 

(48) (49) 

1 (2«) 1 (2X) 

(48) (49 ) 

1 (2%) 

(49) (50) 

1 (235) 

(49) (50) 

# NUauE F 0? ANIMAL S WIT H TISSUE EXAMINE D KICBOSCOPICALLY 

* NUMuS P OF AJilMAL S NECROPSIE D 

74 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

INFLAMMATION , CHRONI C FCCAL 

*PULuC1AR Y JHIER Y 

MEDIA L CAICIFICATIO N 

EIGESriVZ SYSTE M 

*LIV-R 

INFLAMMATION , NECROTIZIN G 

GRANULOMA , NOS 

CrIOLA:iGICFlBROSI S 

B^lAMdEPHCEI S FATTY 

LiPOIDOSIS 

CYTOPLASMI C VACUOLIZATIO N 

H^PATOCYTCKEGAL Y 

hlPEPPLASIA , FOCAL 

A;,GIZCTASIS 

#EIL^ DJCT 

HYPEPPLASIA , NO S 

#PAN(_REA S 

P^RIAfiTERITI S 

(tPANuEEATIC ACINU S 

AiPOPHY , FCCA I 

IGASTRIC MUCOS A 

SxHEa UIZATIC N 

#SMALL INTESTINE 

HYPERPLASIA , LYMPHOI D 

#SMAj.L INTEST. /SEROSA 

INFLAMMATICN , ACUTE FOCA L 

#LARli E INTESTIN E 

NtMATOBIASIS 

HYPERPLASIA , LYMPHOI D 

UPINAii Y SYSTE M 

*KIDiJEY 

H^MQRRHAGIC_CYST__ 

t HUHbE R OF ANIMAL S WITH TISSUE

* NUMBER OF AMMAL S NECROPSIE D 

MATCHED 

CONTROL LOW DOSE 

1 (2X) 

(18) 

1 (6?) 

(50) 

3 (6X) 

(17) (48) 

2 (4'*) 

1 (6X) 

1 (6X ) 

1 (6X) 

1 (656) 3 (6*) 

4 (8%) 

11 (65*) 16 (33%) 

1 (2X) 

(17) (48) 

2 (12X) 15 (31%) 

(17) (46) 

(17) (46) 

5 (11*) 

(17) (48) 

1 (6S) 

(17) (46) 

1 (2X) 

(17) (46) 

1 (2X) 

(17) (46) 

1 (2X) 

1 (2X) 

(17) (48) 

 EXAMINE D MICROSCOPICALLY 

HIGH DOSE 

(50) 

1 (235) 

(49) 

1 (2X) 

2 (4X ) 

5 (10X ) 

|4S) 

9 (18!J) 

(47) 

1 (2*) 

(47 ) 

2 (455) 

(49 ) 

(49) 

1 (2*) 

(49) 

(49) 

1 (2X) 

2 (4X) 

(49) 

75 
11

TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

" J- ' ' ~ ~" — ——- —— - r -i — — i .- T- ] 

GLOMERULCKIPHPITIS , ACOTE 

PYELONEPHRITIS , ACUTE 

INFLAMMATION , CHRONIC 

NjiPHROSIS, NOS 

GLOMERULOSCLEROSIS , NOS 

*PERIR£NAL TISSUE 

HEMORRHAG E 

tOBIMAB Y ELAEEER 

INFLAMMATICN ,

INFLAMMATION ,

HYPERPLASIA ,

ENEOCfilN E SYSTE M 

•PITUITARY 

CYST, NOS 

 ACUTE HEHORRHAGIC 

 ACUTE/CHRCNIC 

 EPITHELIAL 

HKM08RHAGI C CYST 

ANGIECTASI S 

#ADRENA L 

NiCROSIS, IOCAL 

SADRLNA L CORTE X 

LJ.POIDOSIS 

HYPERPLASIA , NOS 

HiPSflPLASIA , FOCAL 

#ADRiNA L KEEULL A 

HYPERPLASIA , EOCA L 

ANGIECTASI S 

ITHYhOID 

CYSTIC FCI1ICIES 

FOLLICULA R CYST, NOS 

HYPERPLASIA , C-CELL 

HYPERPLASIA , FOLLICULAR-CELL 

#PARATHYRCI E 

HYPERPLASIA , NO S 

REPRODUCTIVE SYSTEM
 

*BABdARY GLAN E
 

'~ ' L.M.I. Jl _ ~~"~"*~ ­ - ­ ­ ­ """ TM~ """•' 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

8

1

 (Ul%) 

 (6X) 

1 (255) 

23 (48X) 

1 (2%) 

1 (2% ) 

28 (57%) 

(17) (48) (49 ) 

1 (2%) 

(16) (47) (46 ) 

1 (2%) 

1 (2X) 

2 (4%) 

(18) 

2 (11%) 

4 (22%) 

(48) 

1

1

3

 (25) 

 (2*) 

 (6X) 

(49) 

4 (8X) 

4 (3%) 

(17) (47) J4S) 

1 (2%) 

H7) 

3 (18%) 

(47) 

2

2

 (H%) 

 (4X) 

(49) 

2 (4%) 

1 (2X) 

(17) 

1 (6X) 

1 (6%) 

(47) (IS ) 

(18) 

a (22%) 

(48) 

1 (2%) 

1 (15%) 

1 (27u) 

(49) 

3 (6%) 

1 (2*) 

12 (24%) 

(16) (41) 

1 (2%) 

(38) 

(18) (50) (50) 

* NUMBER OF AKIKALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF AKIMALS NECROPSIED
 

76
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC

#CEEVIX UTERI


EPIDERMAL INCLUSION CYST


FOLY?


fUTEHUS/ENDCMHTEIUM


INFLA.1MAIICN, ACUTE


HYPERPLASIA, CYSTIC


#OVAhY


CYSTIC FCIIICLES


KEPVOUS SYSTEK
 

«BBAJ.N


HEMORRHAGE


NiCROSIS, IOCAL


SPECIAL SENSE CRGANS
 

NONE
 

KUSCULOSKELETAL	 SYSTEH
 

NONt
 

ECCY CAVITIES
 

*MEStNTERY


FIBROSIS, FOCAL


OTHER SYS1EMS
 

SPECIAL MORPHCI03Y SUHMABY
 

 LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

 (17)	 (49) (US)
 

1	 (2%)
 

1 (2S)
 

 (17)	 (49) (4S)
 

1 (2%)
 

3 (6X)
 

 (17)	 (49) (4S)
 

2 (12%) 2 (H%) 3 (63!)
 

 (18)	 (49) (50)
 

1 (2*) H (9%)
 

1 (2X)
 

 (18)	 (50) (50)
 

1 (655)
 

*	 NUMBER OF ANISALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUM3ER OF ANIMALS NECROPSIED
 

77
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

AuIMA L MI££:UG/NC NECROPS Y 

AJTC/NECRCPSY/N O HISTO 

t NUMBE R OF AMHAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMliE P OF ANIMAL S NECROPSIED 

78 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE ADMINISTERED BHT IN THE DIET
 

79
 

TABLE D1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED BHT IN THE DIET 

MATCHED 

CONTROL LOW

ANIMAL S ISITIJIL Y I N STUD Y 21 5 ) 

ANIMAi- S HECECP£IE B 20 50 

ANIMAL S ..XAMINi D HISTOPATHOLOGIC ALL Y 2 ) 50 

INTESJHENTAP Y SYSTE M 

*SKIi» (20) (50) 

EPID^mA L INCLUSIO N CYST 1 

INFLAMMATION , NO S 

*SU!3wl)T TISSU I (20) (50) 

HiiKOHailASIC . CYST 

PESPIhATOB Y SYSTE M 

I7KA..KEA (19) (49) 

H^MOaPHAG I 4 

*TRAUHEA L GLAN D (19) (49 ) 

DILATATION , NOS 1 [5%) 

*LUN u (20) (50) 

H^SOPRHAG E 1 

liFLAM.IATICS , NO S 4 (203!) 3 

PivOTiilNOSIS, ALVEOLA E 2 (105!) 6 

HiP"S?LAFIfl , LYMPHOI D 1 

hEKATOPOIiLTI C SYSTE M 

*BLOJD (2)) (50) 

L-.OKOCY1CS3S, NOS 1 

EtTIC JLOCYTOSI S 1 

#SPL-E N (19) (50) 

COHG3STICN , NC S 

HYPE;!?IASIA , BETICOLCB CELI 1 (5% ) a 

H^MATOFOIESI S 5 (26JE) 12 

f NU,«DES OF ANIMALS WITH TISSUE EXAMINED HICBOSCOPICALLY
 

* NUMjEF. OF AKI1ALS NECROPSItD
 

IDOSE 

(2%) 

(8)5) 

(2% )

(6%) 

(12X) 

(2%) 

(2X ) 

(2X) 

(8X) 

J24X1 _ 

HIGH DOSE 

5T 

50 

49 

(50) 

1 (2X ) 

(5C)

1 (2*) 

(49) 

(45) 

(US) 

3 (635) 

5 (105t) 

3 (636) 

(50) 

(46 ) 

1 (2%) 

1 (159EL 

81
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

*LYMi?H NOD E (2)) (1*9)

 

HYPERPLASIA , LYMPHOI D 

• MANDIBULA R I. NODE (20) (49)

 

MINERALIZATIO N 

HiMOSIDEFCSIS 1 (5X)

 

HYPEPPLASIA , LYMPHOI D 1 (51) 2 (4«)

 

*MES£NTERIC L. NOE E (20) (49)

 

CONGESTION, NOS 1 (2X)

 

LIPOIDCSIS 

HYPERPLASIA , BETICULUa CELL 1 (5S) 2 (US) 

HYPERPLASIA , LYMPHOI D 

HjiMATOFCIESIS 1 (5X)

 

#THYMU S (10) (39)

 

HYPERPLASIA , LYMPHOI D 1 (3XJ 

CIBCULATURY SYSTEM 

IHEAnT (20) (50) 

MxNERALlZATIC N 1 (5X)

 

IMYOLAKDIU M (20) (50) 

INFLiflHATICN , NOS 

CIGESilVt. SYS1IM 

*LIV^ F (20) (48) 

HEMORRHAG E 

luFLAMMATICN , NO S 2 (4^ )

 

IwFLAMMATICN , FOCAL 11 (55X) 21 (44S) 

GnANJLCMA , NO S 1 (2< )

 

P1.LIOSIS HIPATI S 34 (71*) 

NiCPOSIS, FOCA L 2 (10S) 1 (21)

 

Ni.CPOSIS, CYTODEGENSRATIV E 33 (6<»X )

 

CiTOPLASMI C VACUOLIZATIO N 3 (15X) 20 (42X)

 

BASOPHILI C CYT O CHANG E 2 (45) EOSINOPHILIC CYTO CHANG E

 

HiPATOCYlCREGAL Y 9 (19X )

 HI.MATOPCIESI S 1 (2*)

 

*GALi.BL^DTE? (20) (50) 

CwST^ JOS 1_J2%J_ . 

t NUMuER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NU".iT!P OF AM14LS NECHCESIED
 

HIGH DOSE 

(4S ) 

1 (2%) 

(49) 

1 (2X) 

3 (6X) 

(49) 

2 (455) 

1 (2S) 

1 (2«) 

4 (8%) 

(46 ) 

1 (2% ) 

(49 ) 

(49) 

1 (2%) 

(49) 

1 (2% ) 

27 (55*) 

43 (88*) 

2 (45? ) 

43 (88%) 

2 2 (45X) 

1 (2%) 

20 (41*) 

(5C ) 

82
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

InFLAKKATICB ,

HYPERPLASIA ,

SBILi EUCT 

IHFLA1MATICN ,

HYPEkPLASIA ,

*PANv.3EAS 

INFLAKKATICN ,

INFLAMMATION ,

NLCPOSIS, FAT 

A1ROPHY, NOS 

 NO S 

 PAPILLAR Y 

 NOS 

 NOS 

 NOS 

 FOCAL 

#PANl.HEATIC ACINU S 

D^GENERAIICN ,

*ESOPhAG'JS 

HEMORRHAG E 

ISTOiiACK 

CYST, NOS 

INFLAMMATION ,

INFLAMMATION ,

*SMAi.L INTESTIN E 

HYPERPLASIA ,

*LARG £ INTESTIN E 

HYPERPLASIA ,

URINAR Y SYSTEfi 

(IKIDNE Y 

 NOS 

 NOS 

 FOCAL 

 LYMPHOI D 

 LYHPHOIE 

HYDRONEPHECSIS 

PYELONEPHFITIS , NOS 

INFLANBATICN , INTERSTITIAL 

INFARCT, NCS 

INFARCT, HALE D 

CALCINOSIS, NCS 

HYPERPLASIA , TUBULA R CELL 

#KIDNEY/TUBUL E 

DiLATATICli, NOS 

tUBINAP Y ELJEEER 

CASTX NOS__ . _ 

MATCHED 

CONTROL 

(20) 

(17) 

1 (6%) 

2 (12*) 

(17) 

1 (6%) 

(19) 

(18) 

(19) 

(18) 

1 [6%} 

(20) 

1 (5S) 

2 (10*) 

2 (10*) 

1« (70S) 

(20) 

(18) 

_ — — 

LOW DOSE 

1 (2% ) 

(48) 

(«7) 

1 (2%) 

1 (2«) 

(47) 

(«6) 

1 (2%) 

(149) 

(48) 

(48) 

(50) 

1 (2X) 

3 (6«) 

2 (<i%) 

36 (72%) 

(50) 

3 (6X) 

(50) 

7 J14?I 

HIGH DOSE 

1 (2X ) 

(49 ) 

2 (4X ) 

1 (2*) 

(46 ) 

1 (W 

3 <7X ) 

1 (2%) 

1 (2X) 

(46 ) 

(47 ) 

(46) 

1 (2X ) 

1 (2% ) 

1 (2X ) 

(47 ) 

1 (2*) 

(46 ) 

2 (4% ) 

* 

(49) 

1 (2S) 

40 (82X) 

(IS) 

2 (4S) 

(49) 

4_(8X1 

* NUHdER OF ANIMALS HITH TISSUE EXAHINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

83
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

IKFLAKKATICN , FYOGRANULCHATOU S 1 (2%) 

£SDOCcIN2 SYST1.1 

*PITuITAFY (1U) (46) 

CYST , NOS 

((ADRENA L CCRTI X (20) (49 ) 

FIBROSI S 

HYPERPLASIA , NODULAR 4 (8*) 

hiPSF^LASIA , NO S 16 (80%) 43 (88%) 

#ADRr,NA L diDUIL A (20) [49) 

CYST, NOS 

LiuEN-JRATICN , NOS 

IThYttCID (18) (48) 

HYPERPLASIA , FCCAL 

HYPERPLASIA , C-CELL 1 (2«) 

tEANi-fcEATI C IELJ1S (17) (47) 

HYPERPLASIA , NO S 4 (24*) 1 (23) 

BEFRCUL'CTIV E SYSTE H 

*PR£?UTIA L GIAN C (20) (50) 

CiST, NOS 4 (G%) 

INFLAMMATION , NOS 

#?RODTAIE: (18) (48) 

CAST, NO S 1 (6%) 8 (17%) 

Ii.FLAi1BATICN, SUPPURATIV E 1 (2X) 

*SZ«INAL VESICL E (20) (50) 

CAST, NCS 1 (5X) 

#lESiIS (20) (50) 

GivANULCMA , SPERMATIC 1 (231) 

ATROPHY , NC S 

HYPERPLASIA , INTERSTITIAL CELL 

#TESi'IS/TUBUlI (20) (50) 

DiGENERATICJLt.NOS, _ __ 

* NUMDE P OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMiiER OF AMMAL S NECROPSIE D 

HIGH DOSE 

(45) 

1

(49 ) 

1

2

46

(49 ) 

1

1

(«S ) 

2

(46 ) 

(50) 

3

1

(41 ) 

7

<5C ) 

(49) 

1

1

(49) 

(2%) 

(2%) 

(H%) 

 (98*) 

 (2X) 

 (2X) 

 (4% ) 

 (6*) 

(2%) 

 (17*) 

(2%) 

 (2*) 

_ _ J_1?J1 

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

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

*EPILIDY1IS (20) (5J ) (50) 

INFLAMMATION , PYOGRANULCMATOU S 1 (2») 

NERVOU S SYSTE M 

#ERAiN/«ENINGE S (20) (50) (49) 

INFLAMMATION , FOCAL 1 (2S) 

#ERAJ. N (20) (50) (49) 

MINERALIZATIO N 5 (25*) 19 (388) 15 (31S) 

HiCROCEPHALUS , INTERNAL 4 (8!S) 3 (6%) 

HiMORRHAG I 1 (2%) 

SPECIAL SENS E ORGAN S 

NONi, 

flUSCULOSKELETAI SYSTEM 

NON t 

EOCY CAVITIE S 

*ABDU1IN'AL CAVIT Y (20) (50) (50) 

LJ.P03RANUICMA 1 (5%) 

ALL OIHER SYSTEMS
 

*J1ULIIPLS OFGANS (20) (50) (50)
 

HYPERPLASIA, LYMPHOID 1 (5%) 2 («*)
 

MaSTOCYTCSIS 1 (2%)
 

SEECIrtL MOEFHCICGY SUMMAPY
 

AUTG/NBCFCESY/NO HISTO 1
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUKuES OF ANIMAL S NECROPSIED
 

85
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED

ANIMALS INITIALLY IN STUEY

ANIBAL S MISSIN G

ANIHAL S NECROPSIES

ANIMAL S EXAMINE D HISTOPATHCLOGICA1LY

INTEGUMENTAR Y SYS1EH 

*SUBCUT TISSUE

NfiCROSIS, IAT

RESPIRATORY SYSTEM 

#LUNG


INFLAMMATION, NOS


INFLAMMATION, FOCAL


LYMPHOCYTIC INFLAMMATORY INFILTR


INFLAMMATION, FOCAL GRANULCMATOU


PKOTEINOSIS, ALVEOLAP


HEMATOPOIETIC SYSTEM
 

KBONj i MARRO H

MYELOFIBROSIS


«S"Li.EN

H^MATOPOIJSI S

IMANUIBULAR I. NOEE


HYPERPLASIA, LYMPHOID


*MES~NTERIC L. NODE


INFLAMMATION, GRANULOMATOUS


HYPERPLASIA, RETICUL'JM CELL


HYPERPLASIA, LYMPHOID


Hi-KATOEOIESIS


tTHYMUS


HYPERPLASIA, LYMPHOID


CIRCULATORY SYSTEM
 

 BHT IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 20 5) 50 

3 

 2) 46 50 

 20 46 50 

 (20) (46) (50) 

1 (2*) 

 (20) (46) (5C) 

1 (5*) 4 (Q%) 

1 (2%) 1 (255) 

1 (2%) 

1 (2%) 1 (2%) 

1 (5X) 

 (20) (46) (50) 

 15 (75*) 34 (74S) 28 (563!) 

 (20) (45) (5C) 

6 (30S) 20 (44«) 13 (26% ) 

 (20) (44) (49) 

1 (2*) 

 (20) (44) (49) 

1 (5%) 1 (2X) 

2 (5S) 

1 (2%) 

1 (555) 

 (17) (37) (32) 

1 (6%) 

# NUMuER OF ANI3ALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHjER OF AMMAL S NECROPSIED
 

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

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIVE SYS1F.1 

ISAL^-VAEY GIAN D (19) (44) (50) 

HYPERPLASIA , LYMPHOI D 1 (1%) 

(20) (46) (IS) 

CYST, NOS 1 (2%) 

INFLAMMATION , NO S 1 (2*) 

INFLAMMATION , FOCAL 12 (6 OX) 27 (59.*) 36 (73*) 

NiCROSIS, FOCAL 1 (5*) 3 (75) 2 (4X) 

EUSINOEHIIIC CYTO CHANG E 1 (2X ) 

HEPATOCYTOMEGAL Y 1 (2S) 1 (2X) 

IjiUKEMOIE FEACTION 1 (25!) H^MATOPOIESI S 2 (1058) 2 (<**) 

*EILii EUCT (20) (46) (19)

INFLAMMATION , NOS 1 (25!) 

•PANCREAS (18) (45) (48)

 

DILATATICN/DUCTS 1 (2X)

 

INFLAMMATION , FOCAL 1 (6X) 1 (2X)

 

ATROPHY, NCS 1 (6X) 3 (7*)

 

ATROPHY , DIFFUSE 1 (6X)

 

IPEYiRS PATC E (20) (45) (48) 

INFLAMMATION , NOS 1 (2%) 

HYPERPLASIA, LYMPHOID 1 (2X) 

URINAflY SYSTEM 

JKIDNE Y (20) (46) 

HYDHONEPHRCSI S 1 (5*) 

INFLAMHAT.ICN, NOS 1 (2X) 

INFAECT, KCS 

HYPERPLASIA , TUBULA R CELL 2 (10X) 6 (13X) 8 (16X) 

HYPERPLASIA, LYMPHOID 4 (8X) 

• URINAR Y ELftECER (19) (45) (47) 

IfiFLAMMAIICN , NOS 1 (2X) 

ENDOCKINE SYS1JM
 

•PITUITARY (20)	 (47)
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUM3ER OF ANIMALS NECROPSIED
 

87
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

(lADRhNAL (20) (46) (46)
 

HYPERPLASIA, NODULAR 1 (2S)
 

LEUKSMCIC IEACTICN 1 (2%)
 

fADRiNAL COETIX (20) (46) (48)
 

HYPERPLASIA, NODULAR 2 (tX) 1 (2%)
 

HYPERPLASIA, NOS 19 (955E) 39 (85%) 44 (92X)
 

ITIIYKOID (20) (46) (4S)
 

HYPERPLASIA, FOLLICULAR-CELL 3 (7?) 3 (6%)
 

*PANv.REATIC ISLETS (16) (45) (46)
 

HYPERPLASIA, NOS 1 (2X)
 

REFBOUUCTIVE SYSTEM
 

«UTEhUS (20) (45) (49)
 

HiSOPRHAGI 1 (2«)
 

PYCMETEA 1 (2%)
 

tUTEo'JS/ENDCKETBIUM (20) (45) (49)
 

HYPERPLASIA, CYSTIC 6 (3JK) 24 (53%) 16 (335!)
 

*OVAhY (19) (45) (47)
 

CYST, NOS 1 (5%) 12 (27X) 4 (9X)
 

NERVOUS SYSTEM
 

*ERAIN (20) (46) (49)
 

MINERALIZATION 7 (35 X) 15 (33«) 6 (16X)
 

HYDROCEPHALUS, INTERNAL 2 (105!) 4 (9*)
 

SPECIAL SENSE CRGANS
 

NO Hi
 

BUSC'JLOSKELETAI SYSTEM
 

* NOBBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NOMuER OF ANIMALS -NECROPSIED
 

88
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC

EODY CAVITIES
 

*MES£NTE3Y


NtCROSIS, FAT


ALL CfHER SYSltMS
 

*MULTIPL£ OEGANS


HYPERPLASIA, LYMPHOID


IUMATOPOIESIS


SPECIAL aOEFBCIOGY SUMMARY
 

NO LtSICN EEPCRTEE


ANIMAL NISSING/NC NECROPSY


AuTO/NECECFSY/HISTO PERF


AUTOLYSIS/KG NECROPSY


 LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

 (20)	 (<*6) (50)
 

1 (5X)
 

 (20)	 (46) (50)
 

2 {<*%)
 

1 (2%)
 

1 1
 

3
 

1
 

1
 

I	 NUMBER OF AMflALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

89
 

APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS ADMINISTERED BHT IN THE DIET
 

91
 

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

Administered BHT in the Diet (a)
 

Topography; Morphology
 

Lung: Alveolar/Bronchiolar
 

Carcinoma or Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

vo
 

Hematopoietic System:
 

Lymphoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/20(5)
 

N.S.
 

105
 

5/20(25)
 

N.S.
 

88
 

Low
 

Dose
 

1/49(2)
 

N.S.
 

0.408
 

0.005
 

31.413
 

105
 

9/49(18)
 

N.S.
 

0.735
 

0.262
 

2.517
 

100
 

High
 

Dose
 

3/49(6)
 

N.S.
 

1.224
 

0.108
 

62.958
 

105
 

12/50(24)
 

N.S.
 

0.960
 

0.376
 

3.124
 

76
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered BHT in the Diet (a)
 

(continued)
 

Topography ; Morphology
 

Pituitary: Carcinoma, NOS,
 

or Adenoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

VD
 

Adrenal: Pheochromocytoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

7/19(37)
 

N.S.
 

90
 

2/19(11)
 

N.S.
 

91
 

Low High 

Dose Dose 

9/47(19) 9/47(19) 

N.S. N.S. 

0.520 0.520 

0.212 0.212 

1.440 1.440 

76 102 

8/49(16) 10/48(21) 

N.S. N.S. 

1.551 1.979 

0.355 0.486 

14.223 17.573 

105 94 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered BHT in the Diet (a)
 

(continued)
 

Topography ; Morphology
 

Thyroid: Follicular-cell Carcinoma
 

or Adenoma (b)
 

P Values (e,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

VO
 

cn
 Thyroid: C-cell Carcinoma or
 

Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Matched Low High
 

Control Dose Dose
 

1/20(5) 4/49(8) 1/48(2)
 

VT ft
 N.S. N.S. L!. O •
 

1.633 0.417
 

0.179 0.006
 

78.704 32.058
 

105 100 94
 

1/20(5) 6/49(12) 2/48(4)
 

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

2.449 0.833
 

0.332 0.047
 

110.166 48.155
 

Weeks to First Observed Tumor 105 103 94
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered BHT in the Diet (a)
 

(continued)
 

Topography: Morphology


Pancreatic Islets: Islet-cell
 

Carcinoma or Adenoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


VO
 

Preputial Gland:
 

Carcinoma, NOS (b)


P Values (c,d)


Departure from Linear Trend (e)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

 Control
 

 0/19(0)
 

 N.S.
 

—
 

 0/20(0)
 

 N.S.
 

P = 0.044
 

Low High
 

Dose Dose
 

4/48(8) 2/48(4)
 

N.S. N.S.
 

Infinite Infinite
 

0.383 0.122
 

Infinite Infinite
 

105 105
 

3/49(6) 0/50(0)
 

N.S.
 —
 

Infinite
 —
 0.255
 —
 Infinite
 —
 

90
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered BHT in the Diet (a)
 

(continued) 

TOt>OBr*phyt Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

festiil Ittterstitial-cell 

Tumor (b) 15/20(75) 42/49(86) 32/49(65) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.143 

0.883 

1.577 

0.871 

0.653 

1.333 

Weeks to First Observed Tumor 73 90 75 

(a) Dd^ec) groups received 3,000 or 6,000 ppm.
 

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

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

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

indicated. Beneath the incidence of tumors in a dosed group is the probability level for
 

the Fisher exact test for the comparison of that dosed group with the matched-control
 

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

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

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

for any comparison.
 

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

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

Administered BHT in the Diet (a)
 

Topography ; Morphology
 

Lung: Alveolar /Bronchiolar
 

Carcinoma or Adenoma (b)
 

P	 Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

oo
 

Hematopoietic System:
 

Lymphoma (b)
 

P	 Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/18(6)
 

N.S.
 

105
 

2/18(11)
 

N.S.
 

92
 

Low	 High
 

Dose	 Dose
 

3/48(6)	 1/49(2)
 

N.S.	 N.S.
 

1.125	 0.367
 

0.100	 0.005
 

57.811	 28.279
 

105 105
 

10/50(20) 5/50(10)
 

N.S.	 N.S.
 

1.800	 0.900
 

0.445	 0.168
 

15.993	 8.989
 

87 73
 
__ 

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

Administered BHT in the Diet (a)
 

(continued)
 

Matched Low High
 

Topography ! Morphology Control Dose Dose
 

Pituitary: Adenoma, NOS (b) 8/18(44) 9/48(19) 5/49(10)
 

P Values (c,d) P - 0.003(N) P » 0.038(N) P - 0.004(N)
 

Relative Risk (f) 0.422 0.230
 

Lower Limit 0.184 0.074
 

Upper Limit 1.086 0.697
 

Weeks to First Observed Tumor 87 78 84
 

VO
 

VO Thyroid: Follicular-cell
 

Carcinoma or Adenoma (b) 0/18(0) 3/48(6) 0/49(0)
 

P Values (c,d) N.S. N.S.
 —
 

Departure from Linear Trend (e) P » 0.049
 

Relative Risk (f) Infinite
 

Lower Limit 0.236
 

Upper Limit Infinite
 —
 

—
 

Weeks to First Observed Tumor 105
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered BHT in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Thyroid: C-cell Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

o

o	 Mammary Gland: Fibroadenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

2/18(11)
 

N.S.
 

105
 

5/18(28)
 

N.S.
 

87
 

Low High 

Dose Dose 

4/48(8) 4/49(8) 

N.S. N.S. 

0.750 0.735 

0.122 0.119 

7.883 7.727 

105 105 

7/50(14) 5/50(10) 

N.S. N.S. 

0.504 0.360 

0.165 0.098 

1.814 1.416 

101 98 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered BHT in the Diet (a)
 

(continued)
 

Topography ; Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Uterus: Endometrial Stromal 

Polyp (b) 2/17(12) 8/49(16) 6/49(12) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.388 

0.322 

12.696 

1.041 

0.215 

10.000 

Weeks to First Observed Tumor 105 105 93 

(a) Dosed groups received 3,000 or 6,000 ppm.
 

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

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

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

indicated. Beneath the incidence of tumors in a dosed group is the probability level for
 

the Fisher exact test for the comparison of that dosed group with the matched-control
 

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

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

group.
 

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

for any comparison.
 

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

group.
 

APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE ADMINISTERED BHT IN THE DIET
 

103
 

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

Administered BHT in the Diet (a)
 

Topography; Morphology
 

Lung: Alveolar/Bronchiolar
 

Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar
 

Carcinoma or Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

5/20(25)
 

N.S.
 

75
 

7/20(35)
 

N.S.
 

75
 

Low High 

Dose Dose 

12/50(24) 7/49(14) 

N.S. N.S. 

0.960 0.571 

0.376 0.184 

3.124 2.068 

81 107 

21/50(42) 17/49(35) 

N.S. N.S. 

1.200 0.991 

0.609 0.482 

2.876 2.452 

81 107 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered BHT in the Diet (a)
 

(continued)
 

Topography; Morphology
 

Hematopoietic System:
 

Lymphoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular
 

Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

5/20(25)
 

N.S.
 

108
 

9/20(45)
 

P - 0.003(N)
 

91
 

Low
 

Dose
 

14/50(28)
 

N.S.
 

1.120
 

0.457
 

3.556
 

74
 

12/48(25)
 

N.S.
 

0.556
 

0.271
 

1.283
 

81
 

High
 

Dose
 

8/50(16)
 

N.S.
 

0.640
 

0.218
 

2.250
 

107
 

6/49(12)
 

P - 0.005(N)
 

0.272
 

0.098
 

0.749
 

107
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered BHT in the Diet (a)
 

(continued) 

Topography; Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Liver: Hepatocellular Carcinoma 

or Adenoma (b) 11/20(55) 23/48(48) 13/49(27) 

P Values (c,d) P = 0.009(N) N.S. P ­ 0.025(N) 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.871 

0.537 

1.624 

0.482 

0.262 

1.002 

Weeks to First Observed Tumor 91 81 107 

Thyroid: Follicular-cell
 

Carcinoma or Adenoma (b) 0/18(0) 3/48(6) 2/49(4)
 

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

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.236 0.113
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 108 107
 
table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered BHT in the Diet (a)
 

(continued) .
 

Matched Low High
 

Topography» Morphology Control Dose Dose
 

Eye/Lacrimal Gland:
 

Adenoma, NOS (b) 0/20(0) 0/50(0) 4/50(8)
 

P Values (c,d) P = 0.039 — N.S.
 

Relative Risk (f) — Infinite
 

Lower Limit — 0.386
 

Upper Limit — Infinite
 

Weeks to First Observed Tumor — — 107
 

(a) Dosed groups received 3,000 or 6,000 ppm.
 

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

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

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

indicated. Beneath the incidence of tumors in a dosed group is the probability level for
 

the Fisher exact test for the comparison of that dosed group with the matched-control
 

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

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

group.
 

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

0.05 for any comparison.
 

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

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

Administered BHT in the Diet (a)
 

Topography: Morphology


Lung: Alveolar/Bronchiolar
 

Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


o
 

vo
 

Lung: Alveolar/Bronchiolar
 

Carcinoma or Adenoma (b)


P Values (c,d)


Departure from Linear Trend (e)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

 Control Dose Dose 

 1/20(5) 4/46(9) 4/50(8) 

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

1.739 1.600 

0.191 0.175 

83.697 77.169 

 108 108 107 

 1/20(5) 16/46(35) 7/50(14) 

 N. S. P = 0.009 N.S. 

P = 0.002 

6.957 2.800 

1.231 0.403 

282.404 123.407 

108 101 107 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

(continued)
 

Topography; Morphology
 

Hematopoietic System:
 

Lymphoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular
 

Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Administered BHT in the Diet (a) 

Matched Low High 

Control Dose Dose 

7/20(35) 8/46(17) 8/50(16) 

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

0.497 0.457 

0.191 0.175 

1.419 1.312 

70 108 105 

1/20(5) 1/46(2) 3/49(6) 

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

0.435 1.224 

0.006 0.108 

33.420 62.958 

108 108 107 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered BHT in the Diet (a)
 

(continued) 

Matched Low High 

Topography; Morphology Control Dose Dose 

Liver: Hepatocellular Carcinoma
 r»v A/lAm/vmQ i K i
 w*. **u^_»«.vr*uu \** f 1/20(5) 4/46(9) 5/49(10)
 

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

Relative Risk (f) 1.739 2.041
 

Lower Limit 0.191 0.254
 

Upper Limit 83.697 94.440
 

Weeks to First Observed Tumor 108 108 107
 

Pituitary: Adenoma, NOS (b) 0/20(0) 4/45(9) 1/47(2)
 

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

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.429 0.023
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 108
 107 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered BHT in the Diet (a)
 

(continued)
 

Topography! Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Multiple Organs: Sarcoma, 

NOS (b) 3/20(15) 1/46(2) 0/50(0) 

P Values (c,d) P ­ 0.007(N) N.S. P = 0.021(N) 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.145 

0.003 

1.700 

0.000 

0.000 

0.659 

Weeks to First Observed Tumor 79 103 — 

(a) Dosed groups received 3,000 or 6,000 ppm.
 

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

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

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

indicated. Beneath the incidence of tumors in a dosed group is the probability level for
 

the Fisher exact test for the comparison of that dosed group with the matched-control
 

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

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

group.
 

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

for any comparison.
 

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

group.
 
Review of the Bloassay of Butylated Hydroxytoluene (BHT)* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

December 13, 1978
 

The Clearinghouse on Environmental Carcinogens was established
 

in May, 1976, in compliance with DREW 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 on the Institute's bioassay program to identify and 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, and State health officials.
 

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 Butylated Hydroxytoluene (BHT).
 

The reviewer for the report on the bioassay of BHT raised a question
 

regarding the possible significance of the increased incidence of lung
 

tumors observed in low-dose treated female mice. He wondered if the
 

lung tumors in the high-dose treated females might become statistically
 

significant when compared with historic controls. He pointed out other
 

studies, referenced in the report, indicating that BHT may induce lung
 

tumors. Given the data from this bioassay and other studies, the
 

reviewer expressed concern that the conclusionary statement in the
 

report (". . . BHT was not carcinogenic . . ."in rats and mice) was
 

worded too strongly. Finally, he noted that almost 9 million pounds
 

of BHT were produced in 1976 for use in foods. Because of the large
 

exposure to BHT, he emphasized the need to gain the best understanding
 

of the significance of the bioassay data.
 

A Program staff pathologist said that the mean Program-wide
 

incidence of lung tumors in male historic controls was about 11.7
 

percent and in females about 4.4 percent. He added that there is
 

considerable variation around the mean for lung tumors. In regard
 

to the significance of the response, the staff member said that
 

greater credence could have been given to the findings if the high-


dose treated female mice also had had a statistically significant
 

increase in lung tumors. Without it, however, the possibility of
 

113
 
a false positive in the low-dose treated females was increased.
 

It was pointed out that BHT appears to be a promoting agent in the
 

experimental induction of liver and lung tumors.
 

In view of the widespread human exposure to BHT in foods, evidence
 

of its hepatotoxicity, and a suggestion of its tumorigenic effect in
 

the lung, it was moved that the compound be considered for retest by
 

the NCI Chemical Selection Working Group. It was further moved that
 

the report on the bioassay of the compound be accepted as written.
 

The motion was seconded and approved without objection.
 

Clearinghouse Members Present:
 

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

Joseph Highland, Environmental Defense Fund
 

William Lijinsky, Frederick Cancer Research Center
 

Henry Pitot, University of Wisconsin Medical Center
 

Verne A. Ray, Pfizer Medical Research Laboratory
 

Verald K. Rowe, Dow Chemical USA
 

Michael Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

Kenneth Wilcox, Michigan State Health Department
 

*	 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.
 

114
 

US. GOVERNMENT PRINTING OFFICE 1979 281-217/3150 1-3 

NIH Publication No. 79-1706