1977 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 31 

BIOASSAY OF 

TOLBUTAMIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS NO. 64-77-7 

NCI-CG-TR-31 

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

Public Health Service 

National Institutes of Health 
BIOASSAY OF
 

TOLBUTAMIDE
 

FOR POSSIBLE CARCINOGENI.CITY
 

Carcinogen Bioassay and Program Resources Branch
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

DREW Publication No. (NIH) 77-831
 

BIOASSAY OF
 

TOLBUTAMIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

CONTRIBUTORS: This report presents the results of the bioassay
 

of tolbutamide for possible carcinogenicity, conducted for the
 

Carcinogen Bioassay and Program Resources Branch, Carcinogenesis
 

Program, Division of Cancer Cause and Prevention, National Cancer
 

Institute (NCI), Bethesda, Maryland. The bioassay was conducted
 

by Southern Research Institute, Birmingham, Alabama, initially
 

under direct contract to NCI and currently under a subcontract to
 

Tracor Jitco, Inc., prime contractor for the NCI Carcinogenesis
 

bioassay program.
 

The experimental design and doses were determined by Drs. D. P.
 

GriswoldS J. D. Prejean^, E. K. Weisburger^, and J. H.
 

Weisburger^>3_ ^5^ j. Belzer^- and Mr. I. Brown were responsible
 

for the care and feeding of the laboratory animals. Data
 

management and retrieval were performed by Ms. C. A. Dominick-*-.
 

Histopathologic examinations were performed by Drs. R. B.
 

Thompson^- and J. C. Peckham^, and the diagnoses included in this
 

report represent their interpretation. Pathologists from NCI and
 

Tracor Jitco have reviewed selected slides and concur with the
 

overall pathologic evaluation of the study.
 

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

Mason Research Institute^. Statistical analyses were performed
 

by Dr. J. R. Joiner-*, using methods selected for the bioassay
 

program by Dr. J. J. Gart°. Chemicals used in this bioassay were
 

analyzed under the direction of Dr. E. Murrill', and analytical
 

results were reviewed by Dr. C. W. Jameson-3.
 

This report was prepared at Tracor Jitco under the direction of
 

NCI. Those responsible for the report at Tracor Jitco were
 

ill
 
Dr. Marshall Steinberg5

, Director of the Bioassay Program;
 

Drs. J. F. Robens5 and C. H. Williams5, toxicologists; Ms. L. A.
 

Waltz , bioscience writer; and Dr. E. W. Gunberg5, technical
 

editor, assisted by Ms. Y. E. Presley5.
 

The following scientists at the National Cancer Institute were
 

responsible for evaluating the bioassay experiment, interpreting
 

the results, and reporting the findings:
 

Dr. Kenneth C. Chu
 

Dr. Cipriano Cueto, Jr.
 

Dr. J. Fielding Douglas
 

Dr. Dawn G. Goodman
 

Dr. Richard A. Griesemer
 

Dr. Thomas W. Orme
 

Dr. Robert A. Squire8
 

Dr. Jerrold M. Ward
 

^•Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

^Garcinogenesis Program, Division of Cancer Cause and Prevention,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

%iow with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York. • • . , ' ' ''••''' '
 

^EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland. . • •
 

^Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

"Mathematical Statistics and Applied Mathematics Section,
 

Field Studies and Statistics Branch, Division of Cancer
 

Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

'Midwest Research Institute, 425 Volker Boulevard, Kansas City,
 

Missouri.
 

^Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

iv
 
SUMMARY
 

A bioassay of tolbutamide for possible carcinogenicity was
 

conducted by administering the test material in the diet to
 

Fischer 344 rats and B6C3F1 mice.
 

Groups of 35 rats of each sex were administered tolbutamide at
 

one of two doses, either 12,000 or 24,000 ppm,5 days a week for
 

78 weeks, then observed for an additional 28 weeks. Matched-


control groups consisted of 15 untreated rats of each sex. All
 

surviving rats were killed at 106 or 107 weeks.
 

Groups of 35 mice of each sex were administered tolbutamide at
 

one of two doses, either 25,000 or 50,000 ppm, 5 days a week for
 

78 weeks, then observed for an additional 24-26 weeks. Matched-


control groups consisted of 15 untreated mice of each sex. All
 

surviving mice were killed at 102-104 weeks.
 

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

those of the corresponding matched controls during the entire
 

study; however, survival was not significantly affected by
 

treatment 'in either species. In1 both sexes of both species,
 

survival was considered to be adequate for meaningful statistical
 

analyses of the incidence of tumors.
 

In both the rats and the mice, a variety of neoplasms were found
 

in both tolbutamide-treated and control groups. None of the
 

neoplasms were present at a statistically significant increased
 

incidence in treated groups of either species as compared with
 

control groups and were not considered to be compound related.
 

It is concluded that under the conditions of this bioassay,
 

tolbutamide was not carcinogenic for either Fischer 344 rats or
 

B6C3F1 mice.
 

TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals „ 4
 

D. Animal Maintenance 4
 

E. Subchronic Studies 7
 

F. Designs of Chronic Studies 8
 

G. Clinical and Pathologic Examinations 8
 

H. Data Recording and Statistical Analyses 11
 

III. Results -Rats 17
 

A. Body Weights and Clinical Signs (Rats) 17
 

B. Survival (Rats) 17
 

C. Pathology (Rats) 20
 

D. Statistical Analyses of Results (Rats) 21
 

IV. Results - Mice 23
 

A. Body Weights and Clinical Signs (Mice) 23
 

B. Survival (Mice) 23
 

C. Pathology (Mice) 26
 

D. Statistical Analyses of Results (Mice) 27
 

V. Discussion 29
 

VI. Bibliography 31
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed Tolbutamide in the Diet 33
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed Tolbutamide in the Diet 35
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Fed Tolbutamide in the Diet 38
 

vii
 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Fed Tolbutamide in the Diet 41 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Tolbutamide in the Diet 43 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Tolbutamide in the Diet 46 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Tolbutamide in the Diet 49 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Tolbutamide in the Diet... 51 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Tolbutamide in the Diet. 54 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Tolbutamide in the Diet 57 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Tolbutamide in the Diet... 59 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Tolbutamide in the Diet. 62 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Tolbutamide in the Diet 65 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Tolbutamide in the Diet 67 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Tolbutamide in the Diet 70 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Tolbutamide in the Diet 75 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Tolbutamide in the Diet 77 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Tolbutamide in the Diet 80 

viii
 
Page 

TABLES 

Table 1 Design of Tolbutamide Chronic Feeding 

Studies in Rats 9 

Table 2 Design of Tolbutamide Chronic Feeding 

Studies in Mice 10 

FIGURES 

Figure 1 Growth Curves for Rats Fed Tolbutamide 

in the Diet 18 

Figure 2 Survival Curves for Rats Fed Tolbutamide 

in the Diet 19 

Figure 3 Growth Curves for Mice Fed Tolbutamide 

in the Diet 24 

Figure 4 Survival Curves for Mice Fed Tolbutamide 

in the Diet 'V 25 

ix
 

I. INTRODUCTION
 

Tolbutamide (CAS 64-77-7; NCI C01763) was the first oral
 

hypoglycemic agent used in the management of diabetes. It is one
 

of the arylsulfonylurea hypoglycemics, a group which includes
 

tolazamide, chlorpropamide, and acetohexamide. All of these
 

compounds function by stimulating the secretion of insulin by the
 

pancreas and, therefore, are used only in patients with at least
 

minimal pancreatic function, as in maturity-onset diabetics
 

(Lamer and Haynes, 1975). Controlled studies have shown that
 

the oral hypoglycemics may be no more effective than dietary
 

modifications in controlling the symptoms of maturity-onset
 

diabetes on a long-term basis and may be associated with an
 

increase in cardiovascular mortality (Shen and Bressler, 1977).
 

Tolbutamide was selected for testing in the carcinogenesis
 

program in an attempt to evaluate the carcinogenicity of certain
 

drugs that are used extensively and for prolonged periods in
 

humans.
 

1 

• II. MATERIALS AND METHODS 

A. Chemical
 

Tolbutamide (l-butyl-3-(p-methylbenzenesulfonyl)urea) was
 

obtained from the Upjohn Company, North Haven, Connecticut. The
 

purity of Lot No. 656BC of tolbutamide used in the chronic study
 

was determined to be 99.6 +_ 0.6% by analysis at Midwest Research
 

Institute. The melting point of this material was 127-129°C
 

(literature: 126-127°C). Elemental analyses (C, H, N, S) were
 

correct for C^2^18^2(-)3S> tne molecular formula of tolbutamide.
 

The identity of the chemical was confirmed by nuclear magnetic
 

resonance, infrared, and ultraviolet spectra, which were in
 

agreement with the structure and matched the spectra given in the
 

literature.
 

The batch of the chemical used for the chronic study was stored
 

in a cold room at 5°C.
 

B. Dietary Preparation
 

Test diets containing tolbutamide were prepared every 2 weeks by
 

mixing a known amount of sifted tolbutamide with a small amount
 

of Wayne® Lab Blox animal meal (Allied Mills, Inc., Chicago,
 

111.) in a portable mixer, then adding this mixture to the
 

required amount of animal meal and mixing in a twin-shell blender
 

for 10 minutes.
 

3 
No analyses of concentration or determinations of stability of
 

the chemical in feed were performed. The prepared diets were
 

stored at room temperature in sealed plastic containers.
 

C. Animals
 

For the subchronic study, Swiss mice and Sprague-Dawley rats were
 

obtained from Charles River Breeding Laboratories, Inc.,
 

Wilmington, Massachusetts.
 

For the chronic study, Fischer 344 rats were obtained from Harlan
 

Industries, Cumberland, Indiana, and B6C3F1 mice were obtained
 

from A. R. Schmidt, Madison, Wisconsin. These rats and mice were
 

30 days of age on arrival at the laboratory and were quarantined
 

for an acclimation period (rats for 23 days, mice for 8 days).
 

Animals with no clinical signs of disease were then assigned to
 

control and treated groups and earmarked for individual
 

identification.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. The temperature range was 20-24°C, and the relative
 

humidity was maintained at 40-60%. There were 15 changes of room
 

air per hour. The air was passed through both intake and exhaust
 

fiberglass roughing filters. In addition to natural light, illu4

 
mination was provided by fluorescent light for 9 hours per day.
 

Food and water were supplied daily and available ad libitum.
 

Rats and mice were housed five per cage in solid-bottom stainless
 

steel cages (Hahn Roofing and Sheet Metal Co., Birmingham, Ala.).
 

The bottoms of the rat cages were lined with Iso-Dri® hardwood
 

chips (Carworth, Edison, N.J.), and cage tops were covered with
 

disposable filter bonnets; mouse cages were provided with
 

Sterolit® clay bedding (Englehard Mineral and Chemical Co., New
 

York, N.Y.) and covered with filter bonnets for the latter part
 

of the chronic study. Bedding was replaced once per week; cages,
 

water bottles, feeders, and racks were sanitized once per week.
 

The rats and mice were housed in separate rooms. Control animals
 

were housed with respective treated animals. Animals treated
 

with tolbutamide were maintained in the same rooms as animals of
 

the same species being treated with the following chemicals:
 

RATS
 

Feed Studies
 

4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide
 

(acetohexamide) (CAS 968-81-0)
 

anthranilic acid (CAS 118-92-3)
 

4~chloro-N-((propylamino)carbonyl)benzenesulfonamide
 

(chlorpropamide) (CAS 94-20-2)
 

5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine
 

(pyrimethainine) (CAS 58-14-0)
 

2,6-diamino-'3-(phenylazo)pyridine hydrochloride (CAS 136-40-3)
 

L-tryptophan (CAS 73-22-3)
 

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)
 
N-(p-toluenesulfonyl)-N'-hexamethyleniminourea .
 

(tolazamide) (CAS 1156-19-0) .
 

1-phenethylbiguanide hydrochloride (phenformin) (CAS 114-86-3)
 

pyrazinecarboxamide (pyrazinamide) (CAS 98-96-4)
 

4,4'-sulfonyldianiline (dapsone) (CAS 80-08-0)
 

4,4'-thiodianiline (CAS 139-65-1)
 

ethionamide (CAS 536-33-4)
 

MICE
 

Feed Studies
 

4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide
 

(acetohexamide) (CAS 968-81-0)
 

anthranilic acid (CAS 118-92-3)
 

4-chloro-N-((propylamino)carbonyl)benzenesulfonamide
 

(chlorpropamide) (CAS 94-20-2)
 

5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine
 

(pyrimethamine) (CAS 58-14-0)
 

2,6-diamino-3-(phenylazo)pyridine hydrochloride (CAS 136-40-3)
 

L-tryptophan (CAS 73-22-3)
 

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)
 

N-(p-toluenesulfony1)-N'-hexamethyleniminourea
 

(tolazamide) (CAS 1156-19-0)
 

1-phenethylbiguanide hydrochloride (phenformin) (CAS 114-86-3)
 

pyrazinecarboxamide (pyrazinamide) (CAS 98-96-4)
 

4,4'-sulfonyldianiline (dapsone) (CAS 80-08-0)
 

4,4'-thiodianiline (CAS 139-65-1)
 

ethionamide (CAS 536-33-4)
 

Gavage Studies
 

cholesterol (p-(bis(2-chloroethyl)amino)phenyl)acetate
 

(phenesterin) (CAS 3546-10-9)
 

estradiol bis((p-(bis(2-chloroethyl)amino)phenyl)acetate)
 

(estradiol mustard) (CAS 22966-79-6)
 

Intraperitoneal Injection Studies
 

4'-(9-acridinylamino)methansulfon-m-aniside monohydrochloride
 

(MAAM) (NSC 141549)
 

acronine (CAS 7008-42-6)
 

5-azacytidine (CAS 320-67-2)
 

beta-2'-deoxy-6-thioguanosine monohydrate (beta-TGDR)
 

(CAS 789-61-7)
 

1,4-butauediol dimethanesult'onate (busulfan) (CAS 55-98-1)
 

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

6 
3,3'-iminobis-l-propanol dimethanesulfonate (ester)
 

hydrochloride (CAS 3458-22-8)
 

(+)-4,4'-(l-methyl-l,2-ethanediyl)bis-2,6-piperazinedione
 

(ICRF-159) (CAS 21416-87-5)
 

N,3-bis(2-chloroethyl) tetrahyclro-2H-l,3,2-oxazaphosphorin-2­

amine-2-oxide (isophosphamide) (CAS 3778-73-2)
 

N-(2-chloroethyl)-N-(l-methyl-2-phenoxyethyl)benzylamine
 

hydrochloride (phenoxybenzamine) (CAS 63-92-3)
 

N-(l-methylethyl)-4-((2-methylhydrazino)methyl)benzamide
 

monohydrochloride (procarbazine) (CAS 366-70-1)
 

tris(l-aziridinyl)phosphine sulfide (thio-TEPA) (CAS 52-24-4)
 

2,4,6-tris(dimethylamino)-s-triazine (CAS 645-05-6)
 

adriamycin (CAS 23214-92-8)
 

E. Subchronic Studies
 

Subchronic studies were conducted to estimate the maximum
 

tolerated doses of tolbutamide, on the basis of which low and
 

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

"high doses") were determined for administration in the chronic
 

studies. The drug was administered in feed at doses of 300, 800,
 

1,500, 3,000, or 6,000 ppm to male Sprague-Dawley rats; 12,000 or
 

24,000 ppm to female Sprague-Dawley rats; 500, 1,200, 2,500,
 

5,000, or 10,000 ppm to male Swiss mice; and 20,000 or 40,000 ppm
 

to female Swiss mice. Treated animals received the test diets 7
 

days per week for 45 days and were observed for an additional 45
 

days. Five animals were treated at each dose, and 20 animals of
 

each species were maintained as untreated controls.
 

No effects on body weight gain or survival and no gross
 

abnormalities at autopsy were seen in the rats or mice at any
 

7 
dose tested. For rats, the low and high doses for the chronic
 

studies were set at 12,000 and 24,000 ppm, respectively; for
 

mice, they were set at 25,000 and 50,000 ppm.
 

F. Designs of Chronic Studies
 

The designs of the chronic studies are shown in tables 1 and 2.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity, and
 

palpated for masses at each weighing. Rats and mice were weighed
 

individually every 2 weeks for 80 weeks and once per month for
 

the remainder of the study. Animals that died prior to day 100
 

were not necropsied, since it was assumed that they died of
 

toxicity due to the test chemical. Animals that were moribund at
 

the time of clinical examination were killed and necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

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

from killed animals and from animals found dead. The following
 

tissues were examined microscopically: skin, muscle, lungs and
 

bronchi, trachea, bone and bone marrow, spleen, lymph nodes,
 

thymus, heart, salivary gland, liver, gallbladder and bile duct
 

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

intestine, kidney, urinary bladder, pituitary, adrenal, thyroid,
 

8 
Table 1. Design of Tolbutamide Chronic Feeding Studies in Rats
 

Sex and Initial Tolbutamide Time on Study
 

Treatment No. of in Diet Treated Untreated
 

Group Animals3 (ppm)b (weeks) (weeks )
 

MALE
 

Matched-Control 15 0 106
 

Low-Dose 35 12,000 78 28
 

High-Dose 35 24,000 78 28
 

FEMALE
 

Matched-Control 15 0 107
 

Low-Dose 35 12,000 78 28
 

High-Dose 35 24,000 78 28
 

a

Rats were 53 days of age when placed on study.
 

b

The treated animals were fed the test diet 5 days per week and
 

the control diet 2 days per week.
 

9 
Table 2. Design of Tolbutamide Chronic Feeding Studies in Mice
 

Sex and 

Treatment 

Group 

Initial 

No. of 

Animals3 

Tolbutamide 

in Diet 

(ppm)b 

Time 

Treated 

(weeks ) 

on Study 

Untreated 

(weeks ) 

MALE 

Matched-Control 15 0 103 

Low-Dose 35 25,000 78 25 

High-Dose 35 50,000 78 24 

FEMALE 

Matched-Control 15 0 103 

Low-Dose 35 25,000 78 25-26 

High-Dose 35 50,000 78 25 

a

Mice were 38 days of age when placed on study.
 

^The treated animals were fed the test diet 5 days per week and
 

the control diet 2 days per week.
 

10
 
parathyroid, mammary gland, prostate or uterus, testis or ovary,
 

brain, and sensory organs. Peripheral blood smears were prepared
 

from each animal. Occasionally, additional tissues were also
 

examined microscopically. The different tissues were preserved
 

in 10% buffered formalin, embedded in paraffin, sectioned, and
 

stained with hematoxylin and eosin. Special staining techniques
 

were utilized when indicated for more definitive diagnosis.
 

A few tissues from some animals were not examined, particularly
 

from those animals that died early. Also, some animals were
 

missing, cannibalized, or judged to be in such an advanced state
 

of autolysis as to preclude histopathologic evaluation. Thus,
 

the number of animals from which particular organs or tissues
 

were examined microscopically varies, and does not necessarily
 

represent the number of animals that were placed on study in each
 

group.
 

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
 

11
 
Against Cancer (Berenblum, 1969). Data tables were generated for
 

verification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically
 

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

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

causes were not statistically censored. Statistical analyses for
 

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

(1972) 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,
 

12
 
the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic examination was required to detect lesions prior to histologic sampling
 

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

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

consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

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

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

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

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

made. The Bonferroni inequality (Miller, 1966) 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
 

13 3
of the dose-response curve is different from zero at the one-


tailed 0.05 level of significance. Unless otherwise noted, the
 

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

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

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

such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which animals died naturally or were sacrificed
 

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

methods of comparing these curves were used for two groups;
 

14
 
Tarone's extension to testing for linear trend was used for three
 

groups. The statistical tests for the incidence of tumors which
 

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

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P < 0.05, two-tailed test)
 

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each treated group compared to its control was calculated
 

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

relative risk is defined as Pt/pc where p^- is the true binomial
 

probability of the incidence of a specific type of tumor in a
 

treated group of animals and pc 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 treated group and the proportion in a
 

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

excess of unity represent the condition of a larger proportion in
 

the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

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

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

15
 
control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (a P < 0.025 one-tailed test when the control
 

incidence is not zero, P < 0.050 when the control incidence is
 

zero) has occurred. When the lower limit is less than unity, but
 

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

the absence of a significant result while the upper limit
 

indicates that there is a theoretical possibility of the
 

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

detected under the conditions of this test.
 

16
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of rats of each sex at both the low and high
 

doses were lower than those of the corresponding matched controls
 

throughout the study (figure 1). The body weights of the high-


dose groups were depressed more than those of the low-dose
 

groups. Weight differentials became narrower after administration of tolbutamide was terminated, especially in the females.
 

No signs of treatment-related toxicity were reported in the rats.
 

In an effort to control signs of respiratory disease, all rats in
 

the colony were treated with oxytetracycline in the drinking
 

water. Animals in this study were treated with oxytetracycline
 

at 0.6 mg/ml in the drinking water during weeks 34-38, and at 0.3
 

mg/ml during weeks 38-40.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

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

the doses of this experiment, together with those of the matched
 

controls, are shown in figure 2.
 

In both sexes, the Tarone test results for positive dose-related
 

trend in mortality over the period are not significant. In male
 

17
 
Figure 1. Growth Curves For Rats Fed Tolbutamide in the Diet 

18 
Figure 2. Survival Curves For Rats Fed Tolbutamide in the Diet 

19 
rats, 80% of the high-dose group, 77% of the low-dose group, and
 

43% of the matched-control group lived to the end of the study.
 

In females, 65% of the high-dose group, 63% of the low-dose
 

group, and 87% of the matched-control group survived to the end
 

of the study. Sufficient numbers of rats of both sexes were
 

available for meaningful statistical analyses of the incidence's
 

of late-developing 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.
 

There was a random occurrence of a variety of neoplasms in both
 

the control and treated groups which were not considered to be
 

compound related. The neoplasms listed in Appendix A appeared
 

with approximately equal frequency in treated and control rats,
 

or appeared in insignificant numbers. These lesions, however,
 

are not uncommon in this strain of rat independent of any
 

treatment. Few malignant tumors were observed, and no tumor
 

metastases were recorded.
 

In addition to the neoplastic lesions, a number of degenerative,
 

proliferative, and inflammatory changes were encountered in
 

20
 
animals of the control and treated groups (Appendix C). These
 

nonneoplastic lesions are commonly seen in aged rats.
 

In the judgment of the pathologists, tolbutamide did not appear
 

to induce neoplasms in Fischer 344 rats under the conditions of
 

this bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those specific primary tumors that were
 

observed in at least 5% of one or more treated groups of either
 

sex.
 

The results of the Cochran-Armitage test for positive dose-


related trend and of the Fisher exact test for the direct
 

comparison of incidences between the matched-control group and
 

each of the treated groups in the positive direction are not
 

significant in either sex. The incidences of mammary tumors in
 

female rats exhibited a significant (P < 0.001) negative trend
 

and both Fisher exact test results showed a significantly (P <
 

0.001) higher incidence in the controls than in either treated
 

group. There were also significant results in the negative
 

direction in the occurrence of thyroid tumors in male rats. In
 

each of the 95% confidence intervals of relative risk, shown in
 

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

21
 
indicates the absence of positive significant results. It should
 

also be noted that some of the intervals have upper limits
 

greater than one, indicating the theoretical possibility of the
 

induction of tumors by tolbutamide, which could not be detected
 

under the conditions of this test.
 

When groupings of types of tumors are made (as, for example,
 

C-cell adenoma and carcinoma of the thyroid), the incidences of
 

the individual components of the grouping are not included in the
 

statistical analyses in the tables unless the proportions in any
 

of the treated groups of either sex are 5% or more; however, a
 

list of the incidences of each type of tumor is provided in
 

tables Al and A2 of Appendix A.
 

22
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights for treated mice of both sexes were markedly
 

lower than those of the corresponding matched controls during
 

most or all of the tolbutamide administration period, and the
 

degree of depression was the same in the low- and high-dose
 

groups (figure 1). For unknown reasons, the surviving control
 

male mice lost weight after week 70, and their weights gradually
 

approximated those of the treated mice.
 

Except for effects on body weights, there were no signs of
 

treatment-related toxicity in the mice. As a part of treatment
 

of all mice at the laboratory to control signs of respiratory
 

disease, mice in this study received oxytetracycline at 0.6 rag/ml
 

in the drinking water during week 53, and 0.3 mg/ml during week
 •-,
 

54. During weeks 52-63, the mouse room was treated with
 

propylene glycol mist for the same purpose.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

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

the doses of this experiment, together with those of the matched
 

controls, are shown in figure 4.
 

23
 
Figure 3. Growth Curves For Mice Fed Tolbutamide in the Diet 

24 
Figure 4. Survival Curves For Mice Fed Tolbutamide in the Diet 

25 
In neither sex were the results of the Tarone test significant
 

for positive dose-related trend in mortality over the period of
 

the bioassay. In male mice, 43% of the high-dose group, 24% of
 

the low-dose group, and 29% of the matched-control group lived to
 

the end of the study, and the respective median times on study
 

were 88 weeks, 88 weeks, and 82 weeks. In the males, 29/35 high-


dose, 28/35 low-dose, and all of the 15 control mice lived at
 

least as long as 52 weeks. No tumor was observed before this
 

time.
 

In females, 61% of the high-dose group, 71% of the low-dose
 

group, and 47% of the matched-control group lived to the end of
 

the study, providing an adequate numbers of mice for meaningful
 

statistical analyses of the incidence of late-developing 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.
 

There was a random occurrence of a variety of neoplasms in both
 

the control and treated groups which were not considered to be
 

compound related. The tumors observed are not uncommon in this
 

strain of mouse independent of any treatment.
 

26
 
In addition to the neoplastic lesions, a number of degenerative,
 

proliferative, and inflammatory changes were encountered in
 

animals of the control and treated groups (Appendix D). These
 

nonneoplastic lesions are commonly seen in aged mice; however,
 

the suppurative lesions involving the lungs were associated with
 

increased deaths or decreased life spans in the control and
 

treated groups of male mice. The incidence of suppurative
 

bronchopneumonia in the male mice was as follows: controls 8/14
 

(57%), low-dose 11/34 (32%), high-dose 18/32 (56%).
 

In the judgmemt of the pathologists, tolbutamide did not appear
 

to induce neoplasms in B6C3F1 mice under the conditions of this
 

bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those specific primary tumors that were
 

observed in at least 5% of one or more treated groups of either
 

sex.
 

The results of the Cochran-Armitage test for positive dose-


related trend and of the Fisher exact test for the direct
 

comparison of incidences between the matched-control group and
 

each of the treated groups in the positive direction are not
 

significant. The incidences of lymphomas and liver tumors in
 

27
 
male mice had negative trend statistics of P < 0.05. In each of
 

the 95% confidence intervals of relative risk, shown in the
 

tables, the value of one is included; this indicates the absence
 

of positive significant results. It should also be noted that
 

each of the intervals has an upper limit greater than one,
 

indicating the theoretical possibility of the induction of tumors
 

by tolbutamide, which could not be detected under the conditions
 

of this test.
 

When chromophobe adenoma and carcinoma of the pituitary in female
 

mice are combined into a grouping for analysis, the incidence of
 

the combined lesions in treated animals as compared with controls
 

is not statistically significant. A listing of each type of
 

^> '
 

tumor is provided in tables Bl and B2 of Appendix B.
 

28
 
V. DISCUSSION
 

Tolbutamide administration resulted in -decreased mean body
 

weights of rats and mice of both sexes. The degree of weight
 

depression was approximately equal regardless of the dose. Tests
 

for dose-related trend in mortality were not significant in
 

either sex of either species. Among rats, survival of both males
 

and females was adequate for meaningful statistical analyses of
 

the incidence of tumors. Among mice, survival at 106 weeks was
 

43% of the high-dose, 24% of the low-dose, and 29% of the
 

matched-control males, and 61% of the high-dose, 71% of the
 

low-dose, and 47% of the matched-control females. Adequate
 

numbers of the female mice survived for meaningful statistical
 

analyses of the incidence of tumors.
 

A variety of neoplasms were found in both the control and treated
 

rats and mice. The incidences of these neoplasms were not
 

statistically significant in rats or mice of either sex, and they
 

were not considered to be compound related.
 

Tolbutamide is a sulfonylurea compound which has been used as an
 

oral hypoglycemic agent for about 20 years. Long-term feeding
 

studies in rats were reported by Bander (1959). Rats treated by
 

oral gavage for 9 months at 250, 500, 1,000, and 2,000 mg/kg had
 

slightly fatty livers and hyperplasia of thyroids only at the
 

29
 
highest dose. No lesions attributable to treatment were observed
 

in the tissues of dogs administered 100 mg/kg of tolbutamide by
 

gavage for 9 months.
 

It is concluded that under the conditions of this bioassay,
 

tolbutamide was not carcinogenic for Fischer 344 rats or B6C3F1
 

mice.
 

30
 
VI. BIBLIOGRAPHY
 

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

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

Bander, A., Pharmacological studies of the sulfonylureas. Ann.
 

N.Y. Acad. Sci. 82:508-512, 1959.
 

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

Panel on Carcinogenicity of the Cancer Research Commission
 

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

Geneva, 1969.
 

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

Soc. B 34(2):187-220. 1972.
 

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

1970, pp. 48-52.
 

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

significance tests, confidence limits and adjustments for
 

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

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

incomplete observations. J. Amer. Statist. Assoc. 

j>3:457-481, 1958. 

Larner, J. and Haynes, R. C., Jr., Insulin and oral hypoglycemic
 

drugs; glucagon. In: The Pharamacological Basis of
 

Therapeutics, Goodman, L. S. and Gilman, A., eds., Macmillan
 

Publishing Co., Inc., New York, 1975, pp. 1507-1523.
 

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.
 

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

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

induced by different numbers of administrations of benzo(a)
 

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

31
 
Shen, S. W., and Brassier, R. , Medical Intelligence; Clinical
 

pharmacology of oral antidiabetic agents, Parts one and two.
 

New Eng. J_._ .of Med. 296 (9); 493-496 and 296 (14): 787-793. 

1977. 

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

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

32
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED TOLBUTAMIDE IN THE DIET
 

33
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMSIN MALE RATS 

FED TOLBUTAMIDE IN THE DIET 

CONTROL LOB DOSE 

ANIMAL S INITIALL Y I N STUDY 15 35 

ANIMAL S HISSING 1 

ANIMAL S NECPOPSIED 13 3U 

ANIHAL S EXAMINE D HISTOPATHOLOGIC ALLY 13 31 

INTEGUBENTAS Y SYSTEM 

*SUBCOT TISSUE (13) (34) 

SABCOMA , NOS 1 (8X) 

FIBROM A 1 (3») 

RESPIRATOR Y SYSTEM 

• LUN G (13) (33) 

ALVEOLAE/BRONCHIOLAR ADENOM A 1 (3%) 

SARCOMA , NOS 

HEMATOPOIETIC SYSTEM 

*BULTIPLE ORGAN S (13) (3t) 

MALIG.LYMPHOMA . ONDIFFER-TYPE 1 (3X) 

BALIG. LYMPHOMA. LYMPHOCYTIC TYPE 1 (8X) 2 (6X) 

UNDIFFERENTIATE D LEUKEMI A 1 (8X) 2 (6X) 

CIRCULATORY SYSTEM 

RON E 

DIGESTIVE SYSTEM 

NONE 

URINAB Y SYJTEB 

POSfi 

t NUMBER OF ANIHALS NITH TISSUE EXAMINED HICROSCOPICALLY
 

* NUMBER OF ANIHALS NECROPSIED
 

HIGH DOSE 

35 

3« 

3U 

(3U) 

1 (3X) 

1 (3X) 

(34) 

1 (3X) 

1 (3X) 

13«) 

3 <9*) 

35
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL tOH DOSE 

ENDOCRINE SYSTEM 

'•PITUITARY (11) (31) 

CHKOKOPHGBE ADENOM A 2 (18X) 5 (16X) 

#ADRENAL (12) (33)

 

PHEOCHROMOCYTOBA 1 (8X)

 

tTHYPOID (13) (33) 

FOLLICULAE-CBLt CARCINOMA 1 (3X) 

C-CELL ADENOMA t (31%) 1 (3X) 

C-CELL CARCINOM A 

*PANCP.FATIC ISLETS (13) (33) 

IStET-CELL ADENOMA 1 <3X) 

REPRODUCTIV E SYSTEM 

ITESTIS (13) (34) 

INTERSTITIAL-CELL TOMOR 11 (85X) 21 (62%) 

NERVCUS SYSTEM 

NON E 

SPECIAL SENSE ORGANS 

NONE 

HOSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NON E 

ALL OTHER SYSTEMS 

*MULTIPLE ORGANS (13) (3H)

2Jl£2JA££2jBi _ J18&L

 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOKBFR OF ANIMALS NECROPSIED
 

36
 

HIGH DOSE 

(26) 

5 <19X) 

(33) 

1 (3X) 

(32) 

3 (9X) 

1 (3X) 

(32) 

2 16%) 

(33) 

22 (67%) 

(34) 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

ANIMA L DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMA L MISSING
 

a INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ftNIMALS WITH SECONDARY TUMORSI
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUHORS UNCERTAINBENIGN OH MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIHALS WITH TUHORS UNCEETAINPEIMARY OR HETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL LOW DOSE HIGH DOSE 

15 35 35 

2 3 3 

6 5 4 

6 27 28 

1 

12 2<* 27 

22 36 41 

12 24 25 

18 30 35 

14 6 6 

4 6 6 

* PRIMARY TUMORS: ALL TUBOBS EXCEPT SECONDARY TOHOES
 

t SECONDARY TUMORS: HETASTATIC TUMORS OH TUMORS INVASIVE INTO AN ADJACENT ORGAH
 

37 
TABLE A2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

FED TOLBUTAMIOE IN THE DIET 

CONTROL LOi DOSE 

ANIMAL S INITIALL Y I N STUDY 15 35 

ANIMAL S MISSING 

ANIMAL S NECROPSIE D 14 31 

ANIMALS EXAMINE D HISTOPATHOLOGICftLLY 14 31 

INTEGUMENTAR Y SYSTEM 

*SUBCUT TISSUE H«J (31) 

ADNEXA L CABCINON A 

RESPIRATOR Y SYSTEM 

NON E 

HEMATOPOIETIC SYSTEM 

'MULTIPLE OBGANS (14) (31)

MALIG.LYMPHOMA , UNDIFFER-TYPE 2 (6X) 

UN0IFPERENTIATE D LEOKEMI A 2 (1<»X) 3 (10*) 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIVE SYSTEM 

NON E 

08IHAR Y SYSTE M 

*URINAR Y BLADDER (14) (30) 

PAPILLOHA , NOS 1 (3*) 

TRANSITIONAL-CELL PAPILLOMA 1 (3%) 

ENDOCRINE SYSTEM 

•PITUITARY (12) (30) . _ CHBOHOPgO^E 4DENOH1, .._.__ . .5 («2«, ., 8_J27S1_ . 

* NUMBER OF ANIHALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF 4NIHALS NECHOPSIED
 

HIGH DOSE 

35 

1 

31 

31 

(31)

1 (3X) 

(31) 

1 (3X) 

2 (6X) 

(27) 

1 (M) 

(23) 

U_iMiL 

38
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)

 

CONTROL LOU DOSE HIGH DOSE 

•THYROID (14) (3D (30) 

C-CELL ADENOM A 2 (14*) 

C-CELL CARCINOM A 1 (7X) 

REPRODUCTIV E SYSTEM 

*NAHMAR Y GLAN D (14) (31) (31) 

ADENOCARCINOMA . NOS 1 (3%) 

FIBROADENOM A 8 (57X) 2 (6X) 

*PPEPUTIA L GLAN D (14) (31) (31) 

ADNEXA L CARCINOMA 1 (7%) 

*VAGIN A (14) (31) (31) 

SARCOMA , NOS 1 (3*) 

tUTERUS (14) (30) (30) 

SARCOMA, ' NOS 

ENDOMETRIAL STROHAL POLYP 4 (29%) 2 (7S) 

1

2

 (3X) 

 (7%) 

*UTER US/EN DOM ETRIU H (14) (30) (30) 

ADENOCARCINOHA , NOS 1 (7X) 

PAPILLAR Y ADENOCARCINOM A 1 (3X) 

NERVOU S SYSTEM 

NON E 

SPECIAL SENSE ORGANS 

NON E 

HUSCULOSKELETAL SYSTEM 

NON E 

BODY CAVITIE S 

NONE 

A LL CTHEB SYSTEMS 

NONE 

t NUMBE R 0? ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIHUL S NECROPSIED 

39 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)

 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOF. SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUHORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIHALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUHORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUBOHS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUHOBS UHCERTAINPRIMARY OR HETASTATIC
 

TOTAL UNCERTAIN TUHORS
 

CONTROL
 

15
 

2
 

13
 

13
 

24
 

12
 

19
 

(1
 

5
 

LOW DOSE HIGH DOSE
 

35 35
 

6 7
 

7 5
 

22 22
 

1
 

•
 

15 16
 

22 19
 

10 13
 

1U 14
 

7 5
 

8 5
 

* PBIHARY TUMORS: ALL fUHORS EXCEPT SECONDARY TUHORS
 

t SECONDARY TUHORS: BETASTATIC TUBOHS OR TOBORS INVASIVE INTO AN ADJACENT ORGAN
 

40 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED TOLBUTAMIDE IN THE DIET
 

41
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED TOLBUTAMIDE IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS HISSING
 

ANIHALS NECBOPSIED
 

ANIHALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEB
 

*SUBCUT TISSUE
 

SARCOHA, SOS
 

FIBEOSARCOHA
 

RESPIRATOBY SYSTEH
 

tLONG
 

ALVEOLAR/BHOHCHIOLAH ADENOMA
 

ALVEOLAR/BRONCHIOLAR CARCINOMA
 

HBHATOPOIETIC SYSTEM
 

*HULTIPLE ORGANS
 

HALIG.LYHPHOHft. HISTIOCYTIC TYPE
 

MALIGNANT LYMPHOMA. MIXED TYPE
 

MALIGNANT RETICOLOSIS
 

tHEDIASTINAL L.NODE
 

ALVEOLAR/BRONCHIOLAR CA, HBTASTA
 

CIRCOLAT08Y SYSTEH
 

NOHE
 

DIGESTIVE SYSTEM
 

tLIVER
 

HEPATOCELLOLAR ADENOMA
 

HEPATOCELLOLAR CARCINOMA
 

HEMANGIOBA
 

ORINARY SYSTEB
 

. «o»B
 

CONTROL
 

15
 

1
 

14
 

1U
 

(1<»)
 

1 (7%)
 

(14)
 

1 (7%)
 

(14)
 

1 (7%)
 

1 (7«)
 

(14)
 

(14)


2 (14*)
 

2 (14X)
 

LOU DOSS
 

35
 

1
 

34
 

34
 

(34)
 

1 <3X)
 

(34)
 

1 (3*)
 

1 (3X)
 

(34)
 

5 (15%)
 

1 (3X)
 

1 <3X)
 

(25)
 

1 (4X)
 

(34)
 

2 (6X)
 

2 (6X)
 

HIGH DO SB
 

35
 

32
 

32
 

(32)
 

(32)
 

(32)
 

(21)
 

(32)
 

1 (3X)
 

1 (3X)
 

t NUMBER OF AHIHALS MITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIHALS NBCROPSIED 

43 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)

 

CONTROL LOB DOSE HIG H DOSE 

ENDOCRIN E SYSTE M 

iPITUITARY (12) (30) (18)

 

CHROMOPHOBE ADENOMA 1 (3*)

 

•THYROI D (14) (29) (2U)

 

FOLLICULAR-CELL ADENOM A 1 <7X ) 1 (3*)

 

REPRODUCTIVE SYSTEM 

NON E 

NERVOU S SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*HAPDRPIAN GLAN D (14) (3i») (32) 

ADENOMA , NOS ' 2 (14%) 

MUSCULOSKELETAL SYSTEM 

NON E 

BODY CAVITIES 

NONE 

A LL OTHER SYSTEMS 

__,E9.iJE. ,^^M n,^ , f r „_, -a_^ 1-. JM— 

» NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

44 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUHORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUHORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUflORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS DNCEETAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL
 

15
 

H
 

6
 

<»
 

1
 

8
 

11
 

it
 

5
 

5
 

6
 

LOW DOSE HIGH DOSE
 

35 35
 

10 9
 

16 10
 

2
 

8 14
 

1
 

13 2
 

16 2
 

5 2
 

5 2
 

9
 

11
 

1
 

1
 

* PRIMARY TUMOBS: ALL TUHORS EXCEPT SECONDARY TUMORS
 * SECONDABY TUMORS: BETASTATIC TUMORS OR TOHORS INVASIVE IHTO AN ADJACENT OBGAM
 

•45
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

FED TOLBUTAMIDE IN THE DIET 

CONTROL LOU DOSE
 

ANIMAL S INITIALLY IN STUDY 15 35
 

ANIMAL S K'ECROPSIED 12 3i»
 ANIMAL S EXAMINE D HI STOP ATHOLOGIC ALLY 12 314
 

INTEGUMENTAR Y SYSTEM
 

*SUBCUT TISSUE (12) (3U)
 

KSMANGIOSARCOM A 1 (8%)
 

RESPIRATORY SYSTEM
 

#LUNG (11) (34)
 

ALVEOLAB/ERCNCHIOLAR ADENOMA 2 (6%)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (12) (34) 

MALIGNANT LYMPHOMA, DOS 1 (3X) 

MALIG. LYMEHOMA, UNDIFF ER-TYP E 

KALIG. LYMPHOMA, HISTIOCYTIC TYPE 

1

2

 (3%) 

 (6%) 

&PEYEF.S PATCH 

MALTG. LYMPHOMA, HISTIOCYTIC TYPE 

(11) (33) 

1 (3*) 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

•LIVER	 (12) (34)
 

HEPATOCELLULAR ADENOMA 2 (6%)
 

HEPATOCELLULAR CARCINOMA 1 (8%)
 

URINAR Y SYSTEM
 

NOJ1E _,_... _ ,,... .
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALL?
 

* NUKBEE OF ANIHALS NECROPSIED
 

HIGH COSE
 

35
 

34
 

34
 

(34)
 

(34)
 

(34)
 

(33)
 

(34)
 

1 (3%)
 

_«_«-m --— — nn-TT- TTI- _—i
 

46 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

HIGH DOSE 

(26) 

(34) 

(32) 

(34) 

(34) 

1 (3*) 

(34) 

1 (3*) 

ENDOCRIN E SYSTE M 

#PITOITAR Y 

CHROMOPHOBE ADENOMA 

CHROMOPHOBE CARCINOM A 

#ADRENA L 

PHEOCHROBOCYTOMA, MALIGNANT 

ITHYROID 

FOLLICULAR-CELL ADENOMA

 

8EPRODUCTIVE SYSTEM 

#UTERUS 

SAECONA , NOS

 

tOVAR Y 

PAPILLAR Y ADENOMA 

NERVCO S SYSTEM 

NON E 

SPECIAL SENSE OBGAN S 

*HARDERIA N GLAN D 

ADENOMA , NO S 

MtJSCULOSKELETAl SYSTEH 

NON E 

BODY CAVITIES 

NON E 

A LL OTHER SYSTEMS 

MON g ni _ 

CONTROL LOW DOSE 

(11) (26)

 

1 (9X) 2

 (8*) 

(11) (34) 

1 OX) 

(12) (25)

 

1 (8X) 

(11) (3U) 

1 (9%) 1 (3X) 

(11) (34) 

(12) (34) 

t	 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

47
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

a) INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

CONTROL
 

15
 

5
 

3
 

7
 

3
 

5
 

2
 

2
 

1
 

3
 

LOH DOSE HIGH DOSE 

35 35 

7 U 

3 9 

1 2 

2U 20 

11 3 

13 3 

U 3 

u 3 

9 

9 

TOTf.L ANIMALS WITH SECONDARY TUMORS1 i
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBEN" I GN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS tflTH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMOR S
 

# SECONDARY TUMORS: HETASTATIC TUMORS OR TOHOBS INVASIVE INTO AN ADJACENT ORGA N
 

48 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED TOLBUTAMIDE IN THE DIET
 

49
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE 

RATS FED TOLBUTAMIDE IN THE DIET 

CONTROL LOB DOSE 

ANIMAL S INITIALL Y I N STUDY 15 35 

ANIMAL S MISSIN G 1 

ANIHAL S NECROPSIED 13 34 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY 13 34 

INTEGUMENTAR Y SYSTEM 

*SKIN (13) (34) 

EPIDERMA L INCLUSION CYST 1 (8%) 2 (6X) 

RESPIRATOR Y SYSTEM 

tTBACHE A (13) (34) 

LYMPHOCYTIC INFILTRATE 1 (3%) 

INFLAMMATION , SUPPURATIV E 2 (15X) 

tLUNG/BRONCHU S (13) (33) 

HYPERPLASIA , LYMPHOI D 6 (18X) 

#LUN'G/BRONCHIOLE (13) (33) 

HYFERPLASIA, LYMPHOID 

tLUN G (13) (33) 

EFONCHOPNEUMONI A SUPPURATIV E 1 (3X) 

PNEUMONIA , CHfiONIC HURIN E 6 (18X) 

FIBROSIS, FOCAL 1 (3X) 

HEMATOPOIETIC SYSTEM 

• BONE HARRO H (12) (33) 

ATROPHY, NOS 

MYELOFIBROSIS 1 (3X) 

•SPLEEN (13) (33) 

HYPERPLASIA , RETICULUM CELL 

HEMATOPOIESIS 

CIRCULATORY SYSTEM 

• MYOCARDIU M (13) (3i») 

_-TMINELAMHATIO»U_INTJRSTI.TI>L « 431XJ. 5_J15J1 

t NUMBER OF ANIHALS WITH TISSUE EXAMINE D BICROSCOPICALLY
 

* NUMBER OF ANIBALS NECBOPSIED
 

HIGH COSE 

35 

3«» 

34 

(34) 

H (12X) 

(34) 

1 (3«) 

(34) 

(34) 

1 (3X) 

(34) 

(31) 

1 (3%) 

(34) 

1 (3X) 1 (3X) 

(34) 

8 I24X) 

51
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE
 

DIG tSTI V E SYSTEM
 

*EILS DUCT (13) (3U)
 

HYPERPLASIA, FOCAL 1 (8X)
 

SPANCPEAS (13) (33)
 

INFLAMMATION, INTERSTITIAL 1 (3X)
 

INFLAMMATION, CHRONIC 1 (8%) 3 (9X)
 

INFLAMMATION, CHRONIC FOCAL 2 (6*)
 

PEBIARTERITIS 1 (3*)
 

•STOMACH	 (13) (33)
 

MINERALIZATION 1 (8%)
 

URINAR Y SYSTEM
 

"KIDNEY (12) (33)
 

INFLAMMATION , CHRONIC 2 (17X) 5 (15X)
 

INFLAMMATION , CHRONIC DIFFUSE 7 (58X) 4 (12X)
 

IKICrEY/TUPULE (12) (33)
 

MINERALIZATION 1 (8«)
 

ENDOCRIN E SYSTEM
 

*T HYROI D (13) (33)
 

KYPEPPLASIA, C-CELL 1 (8%) 1 <3X)
 

REPRODUCTIVE SYSTEM
 

frPFOSTATE (11) (32)
 

INFLAMMATION, SUPPUHATIVE
 

_
 

NERVOU S SYSTEM 

tCEREBRUM 

HEMORRHAG E 

(12) (33) 

1 (3X) 

•SPINAL CORD 

J3AIACIA - (13) (34) 

t NUMBE R OF ANIMAL S WITH TISSUE EXAHINED MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECROPSIED
 

HIGH DOSE
 

(34)
 

(32)
 

2 (6X)
 

1 (3X)
 

1 (3%)
 

(33)
 

(33)
 

3 (9X)
 

9 (27X)
 

(33)
 

(32)
 

(33)
 

1 (3X)
 

(34)
 

(34)
 

1 (3SL
 

52
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

SPECIA L SENS E ORGAN S 

*EYE/RETINA 

ATROPHY , NOS 

BUSCULOSKELETA L SYSTEM 

NON E 

BODY CAVITIES 

*MESENTEB Y 

NECROSIS , FAT 

A LL CTHER SYSTEMS 

NON E 

SPECIA L MORPHOLOG Y SUMMAR Y 

NO LESION REPORTE D 

ANIMA L MISSING/NO NECROPSY 

NO NECROPS Y PERFORMED 

AUTOLYSIS/N O NECROPSY 

CONTROL L OH DOSE HIGH DOSE 

(13) (34) (34)

 

1 (3X) 

(13) (34) (34)

 

1 (8X) 

1 2 2 

1 

1 1 

1 

»	 NUMBER OF ANIMALS BITH TISSUE EXAHIHED MICROSCOPICALLY
 

*	 NUMBER OF ANIHALS NECROPSIED
 

53
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE 

RATS FED TOLBUTAMIDE IN THE DIET 

CONTROL LOB DOSE
 

ANIMALS INITIALLY IN STUDY 15 35
 

ANIMALS HISSING
 

ANIMALS NECROPSIED U 31
 

ANIMALS EXAMINED HISTOPATHOLOGIC ALLY 14 31
 

INTEGUMENTARY SYSTEM
 

NONE
 

EESPIPATORY SYSTEM
 

*TRACHEA (i<0 (31)
 

LYMPHOCYTIC INFILTRATE 1 (3%)
 

INFLAMMATION, SUPPURATIVE 1 (7X) 2 <6X)
 

*LUNG/BRONCHUS (14) (31)
 

HYPEP.PLASIA, LYMPHOID
 

#LUNG (14) (31)
 

PNEUMONIA, CHRONIC MURINE
 

FIBROSIS
 

HEHATOPOIETIC SYSTEM
 

tBONE HARROW (13) (29)
 

MYELOFIBROSIS
 

tSPLEEN (14) (31)
 

HYPERPLASIA, RETICULUH CELL 1 (3X)
 

HEMATOPOIESIS
 

CIECULATORY SYSTEM
 

fHYOCARDIOM <!<•) (30)
 

INFLAMMATION, INTERSTITIAL 2 (7X)
 

DIGESTIVE SYSTEM
 

tLIVEH (14) (31)
 

HYPERPLASIA, NODOLftB J-JJS1—
 i


• NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMAL S NECROPSIED
 

54
 

HIGH DOSE
 

35
 

1
 

31
 

31
 

(31)
 

(31)
 

1 (3X)
 

(31)


3 (10X)
 

1 (3X)
 

(29)
 

1 <3X)
 

(30)
 

2 (7X)
 

(30)
 

2 (7*)
 

(30)
 

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

URINARY SYSTEM
 

*KTDNEY
 

INFLAMMATION, CHRONIC
 

INFLAMMATION, CHRONIC DIFFUSE
 

ENDOCRINE SYSTEM
 

tT HYROID
 

CYSTIC FOLLICLES
 

HYPERPLASIA, C-CELL
 

REPRODUCTIVE SYSTEM
 

*PREPUTIAL GLAND
 

HYPERPLASIA, EPITHELIAL
 

tUTERUS/ENDOMETRIUM
 

INFLAMMATION, SUPPURATIVE
 

HYPERPLASIA, CYSTIC
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/PETINA
 

ATROPHY, NOS
 

*LENS CAPSULE
 

MINERALIZATION
 

MUSCULCSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

CONTROL LOW DOSE HIGH COSE 

(14) (31) (31) 

1 (7%) 

1 (3%) 1 (3*) 

(I'M (31)

1 (3%) 

(30) 

1 (3X) 

(14) (31)

1 (3X) 

(31) 

(14) (30) (30) 

1 (7X) 

1 (3X) 

1 (3%) 

(14) (31) (31) 

1 (7X) 

(14) (31)

1 (3*) 

(31) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

55
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

ALL OTHER SYSTEMS 

*MULTIPLE ORGANS (1I») (31) (31) 

FJBROSIS 1 (3%) 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 1 11 9
 

ANIMAL MISSING/NC NECROPSY 1
 

NO NECROPSY PERFORMED 1 1
 

AUTOLYSIS/NG NECROPSY 1 3 2
 

t NUMBE R O F ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUflBE K OF ANIMAL S NECBOPSIED 

56 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED TOLBUTAMIDE IN THE DIET
 

57
 

TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

FEDTOLBUTAMIOE IN THE DIET

 

ANIHALS INITIALLY IN STUDY
 

ANIHALS HISSING
 

ANIHALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUHENTARY SYSTEH
 

NONE
 

BESPIRATORY SYSTEH
 

tTRACHEA
 

INFLAHHATION, SOPPURATIVE
 

•LUNG/ BRONCHIOLE
 

HYPERPLASIA, PLASHA CELL
 

• LONG
 

ERONCHOPNEOHONIA SUPPURATIVE
 

BRONCHOPNEUHONIA CHRONIC SOPPURA
 

HEHATOPOIBTIC SYSTEH
 

NONE
 

CIRCULATORY SYSTEH
 

•HYOCARDIUH
 

INFLAHHATION, INTERSTITIAL
 

INFLAHHATION, CHROMIC DIFFUSE
 

DIGESTIVE SYSTEH
 

•LIVER
 

HEHORRHAGE
 

HEHORRHAGE, CHRONIC
 

NECppSIS, CQiGVLftXlYE ._,
 

CONTROL
 

15
 

1
 

14
 

H4
 

(14)
 

1 (7X)
 

(1U)
 

(14)
 

8 (57X)
 

(1U)
 

(14)
 

1 (7%)
 

1 (7X)
 

LOW DOSE HIGH EOSE
 

35	 35
 

1
 

34 32
 

34 32
 

(34)	 <31)
 

7 (21X) 3 (10X)
 

(34)	 (32)
 

3 (9X)
 

(34) (32)
 

10	 (29%) 18 (56X)
 

1 (3*)
 

(34) (32)
 

1 (3X)
 

1 (3X)
 

(34) (32)
 

3 I?BL
 

t	 NUHBER OF ANIHALS WITH TISSUE EXAHINED MICROSCOPICALLY
 

*	 NUHBER OF ANIHALS HECROPSIED
 

59
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

HYPERPLASIA, NODULAR 

#LTVEE/CENTRILOBCLAR 

NECROSIS, COAGULATIVE 

(14) 

1 (7%) 

(34) 

1 (3X) 

(32) 

URINAR Y SYSTEM 

#KIDNEY 

HYDRONEPHEOSIS 

PYELONEPHRITIS SUPPDRATIVE 

*KIDNEY/CORTEX 

NECROSIS, COAGULATIVE 

(14) 

(14) 

(34) 

1 (3*) 

(34) 

1 (3*) 

(31)

1 (3*) 

(31) 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

NONE 

HERVCUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*EYE/COPNEA 

ULCER, CHRONIC 

*EYE/CONJUNCTIVA 

ULCER, CHRONIC 

(14) 

(14) 

(34) 

1

(34) 

1

 <3X) 

 (3X) 

(32) 

(32) 

HUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

_ N.O_N_fi _ _ 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY 

* NUMBER OF ANIBALS NECBOPSIED 

, B , 

60 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ALL OTHER SYSTEMS 

NONE 

SPECIAL HORPHOLOGY SUMMARY 

NO LESION REPORTED 

ANIMAL MISSING/HO NECROPSY 

ACCIDENTAL DEATH 

NO NECrOPSY PERFORMED 

AUTOLYSIS/NC NECROPSY 

1 

9 

1 

11 

1 

1 

1 

61
 
TABLE 02.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

FED TOLBUTAMIDE IN THE DIET 

HIGH DOSE
 

35
 

34
 

34
 

(34)


6 (18*)
 

(34)
 

1 (3X)
 

(34)


11	 (32X)
 

1 (3%)
 

(34)
 

(34)


1 (3%)
 

(34)
 

I J6*l
 

ANIMAL S INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HI STOP ATHOLOGICALLY
 

IVT EGUMENTAFY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

#TEACHEA
 

INFLAMMATION, SUPPURATIVE
 

&LUNG/BRONCHU5
 

INFLAMMATION, SUPPURATIVE
 

*LUNG
 

ERONCHOPNEUMONIA SUPPURATIVE
 

ERONCHOPNEUMONIA CHRONIC SOPPORA
 

HEKATOPOIETIC SYSTEM
 

NONE
 

CIRCULATORY SYSTEM
 

#MYOCARDIU«
 

INFLAMMATION, INTERSTITIAL
 

DIGESTIVE	 SYSTEM
 

#LIVER
 

HYFERPLASIA, NODULAR
 

URINAR Y SYSTEM
 

*KIDNEY
 

HI.pRQN£P_ti£OSIS „ _
 

CONTROL LOB DOSE
 

15 35
 

12 31*
 

12 34
 

(12)	 (34)
 

3 (25%)
 

(11) (34)
 

(11) (34)
 

4 (36S) 1 (3%)
 

(11) (34)
 

1 (3*)
 

(12) (34)
 

(12)	 (34)
 

„_.....•..*___«_«——*».
 

t	 NUMBER OF ANIMALS WITH TISSUE EXAHIHED BICBOSCOPICJLLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

62 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

# UTERUS/EN DOM ETR I UM
 

INFLAMMATION, SUPPURATIVE
 

•OVARY
 

HEMORRHAGE, CHRONIC
 

INFLAMMATION, CHRONIC


NERVOUS SYSTEM
 

tBRAIN
 

FOIVASCULAR CUFFING
 

SPECIAL SENSE OBGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

ADIPOSE TISSUE
 

INFLAMMATION, CHRONIC


SPECIAL MORPHOLOGY SUHHARY
 

$Q L£SION_£JE£0£11£
 

 SOPPURATIV
 

 FOCAL
 

CONTROL LOH DOSE HIGH DOSE
 

(11) (34) 

1 (3%) 

(34) 

1 (3«) 

(11) 

1 (9X) 

(34) 

1 (3X) 

(34) 

(12) (34) (34) 

2 (6X) 

1 

3 20 16 

t NUMBE R OF ANIMAL S WITH TISSUE EXASI8BD MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

63 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOS DOSE HIGH COSE 

NC NECROPSY PEBFORHED 2 1 

AUTOLYSIS/HO NECROPSY 1 1 

64 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED TOLBUTAMIDE IN THE DIET
 

65
 

Table El.


Topography: Morphology
 

Hematopoietic System:
 

Lymphoma or Leukemia^*
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe
 

Adenoma^
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Tolbutamide in the Diet3 

Matched Low High 

Control Dose Dose 

2/13 (15) 5/34 (15) 3/34 (9) 

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

0.956 0.574 

0.188 0.077 

9.325 6.438 

95 95 90 

2/11 (18) 5/31 (16) 5/26 (19) 

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

0.887 1.058 

0.183 0.217 

8.485 9.996 

100 106 104 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

(continued)
 

Topography: Morphology
 

Thyroid: C-cell Adenomab
 

P Valuesc>d
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-cell Adenoma
 

or Carcinomab
 

P Valuesc'd
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolbutamide in the Diet3
 

Matched Low 

Control Dose 

4/13 (31) 1/33 (3) 

N.S. P = 0.018 (N) 

P = 0.019 

0.098 

0.002 

0.895 

103 106 

4/13 (31) 1/33 (3)
 

N.S. P = 0.018 (N)
 

P = 0.015
 

0.098
 

0.002
 

0.895
 

103 106
 

High
 

Dose
 

3/32 (9)
 

N.S.
 

0.305
 

0.055
 

1.593
 

•104
 

4/32 (13)
 

N.S.
 

0.406
 

0.095
 

1.914
 

104 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed ToIbutamide in the Diet3
 

(continued)
 

Matched Low High
 

Topography; Morphology Control Dose Dose
 

Testis: Interstitial-cell
 

Tumor

b 11/13 (85) 21/34 (62) 22/33 (67)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.730 0.788
 

Lower Limit 0.579 0.628
 

Upper Limit 1.205 1.280
 

Weeks to First Observed Tumor 75 96 95
 

a

Treated groups received doses of 12,000 or 24,000 ppm in feed.
 

"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 < 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that treated group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each treated group and the
 

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

Topography: Morphology
 

Hematopoietic System:
 

Leukemiab
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System:
 

Lymphomab
 

P Valuesc'd
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolbutamide in the Dieta
 

Matched Low
 

Control Dose
 

2/14 (14)	 3/31 (10)
 

N.S.	 N.S.
 

0.677
 

0.091
 

7.510
 

107 69
 

0/14 (0)	 2/31 (6)
 

N.S.	 N.S.
 

Infinite
 

0.142
 

Infinite
 

100
 —
 

High
 

Dose
 

2/31 (6)
 

N.S.
 

0.452
 

0.037
 

5.825
 

87
 

1/31 (3)
 

N.S.
 

Infinite
 

0.026
 

Infinite
 

106
 
Table E2. Analyses of the Incidence of Primary .Tumors in Female Rats
 

(continued)
 

Topography: Morphology
 

Hematopoietic System:
 

Lymphoma or Leukemia"
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe
 

Adenoma*5
 

P Values0>d
 

Relative Risk (Matched Control) ^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolbutamide in the Dieta 

Matched Low High 

Control Dose Dose 

2/14 (14) 5/31 (16) 3/31 (10) 

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

1.129 0.677 

0.221 0.091 

11.053 7.510 . 

107 69 87 

5/12 (42) 8/30 (27) 11/23 (48) 

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

0.640 1.148 

0.253 0.511 

2.079 3.308 

107 96 87 
__ 

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

Fed Tolbutamide in the Dieta
 

(continued)
 

Topography; Morphology
 

Thyroid: C-cell Adenomab
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-cell Adenoma
 

or Carcinoma^
 

P Values0 »d
 

Daparture from Linear Trend6
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

2/14 (14)
 

P = 0.031 (N)
 

107
 

3/14 (21)
 

P = 0.006 (N)
 

P = 0.022
 

107
 

Low
 

Dose
 

0/31 (0)
 

N.S.
 

0.000
 

0.000
 

2.276
 

0/31 (0)
 

P = 0.026 (N)
 

0.000
 

0.000
 

1.011
 

High
 

Dose
 

0/30 (0)
 

N.S.
 

0.000
 

0.000
 

2.336
 

0/30 (0)
 

P = 0.027 (N)
 

0.000
 

0.000
 

1.042
 

—
 
__ 

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

(continued)
 

Topography; Morphology
 

Mammary Gland:
 

Fibroadenomab
 

P Valuesc»d
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Uterus: Endometrial
 

Stromal Polyp*5
 

P Valuesc>d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolbutamide in the Diet3
 

Matched
 

Control
 

8/14 (57)
 

P < 0.001 (N)
 

P = 0.006
 

99
 

4/14 (29)
 

N.S.
 

107
 

Low
 

Dose
 

2/31 (6)
 

P < 0.001 (N)
 

0.113
 

0.015
 

0.479
 

106
 

2/30 (7)
 

N.S.
 

0.233
 

0.024
 

1.452
 

106
 

High
 

Dose
 

0/31 (0)
 

P < 0.001 (N)
 

0.000
 

0.000
 

0.234
 

2/30 (7)
 

N.S.
 

0.233
 

0.024
 

1.452
 

106
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Tolbutamide in the Diet3
 

(continued)
 

a

Treated groups received doses of 12,000 or 24,000 ppm in feed.
 

^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 < 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that treated group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each treated group and the
 

control group.
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE FED TOLBUTAMIDE IN THE DIET
 

75
 

__ 

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

Fed Tolbutamide in the Dieta
 

Matched Low High
 

Topography : Morphology Control Dose Dose
 

Hematopoietic System:
 

Lymphomab 2/14 (14) 6/34 (18) 0/32 (0)
 

P Values0 »d P = 0.046 (N) N.S. N.S.
 

Relative Risk (Matched Control)f 1.235 0.000
 

Lower Limit 0.266 0.000
 

Upper Limit 11.532 2.200
 

Weeks to First Observed Tumor 103 76
 

Liver: Hepatocellular
 

Adenoma^ 2/14 (14) 2/34 (6) 1/32 (3)
 

P Valuesc'd N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.412 0.219
 

Lower Limit 0.034 0.004
 

Upper Limit 5.351 3.927
 

Weeks to First Observed Tumor 88 88 102
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice 

Fed Tolbutamide in the Diet3 

(continued) 

Matched
 

Topography: Morphology Control
 

Liver: Hepatocellular
 

Carcinoma" 2/14 (14)
 

P Valuesc'd P = 0.045 (N)
 

Relative Risk (Matched Control)^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 85
 

Liver: Hepatocellular
 

Adenoma or Carcinoma'3 3/14 (21)
 

P Values0>d P = 0.046 (N)
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 85
 

Low High 

Dose Dose 

2/34 (6) 0/32 (0) 

N.S. N.S. 

0.412 0.000 

0.034 0.000 

5.351 2.200 

__ 

76 

4/34 (12) 1/32 (3) 

N.S. N.S. 

0.549 0.146 

0.112 0.003 

3.399 1.668 

76 102 
__ 

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

Fed Tolbutamide in the Diet3
 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Pituitary: Chromophobe
 

Adenomab 0/12 (0) 1/30 (3) 0/18 (0)
 

P Valuesc>d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f Infinite
 

Lower Limit 0.023
 —
 Upper Limit Infinite
 —
 

Weeks to First Observed Tumor 83
 —
 —
 

a

Treated groups received doses of 25,000 or 50,000 ppm in feed.
 

^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 < 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that treated group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

"A negative trend (N) indicates a lower incidence in a treated group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

*The 95% confidence interval of the relative risk between each treated group and the
 

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

Topography: Morphology
 

Hematopoietic System:
 

Lymphomab
 

P Valuesc'd
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

00
 

° Weeks to First Observed Tumor
 

Liver: Hepatocellular
 

Adenomab
 

P Valuesc>d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolbutamide in the Diet3
 

Matched Low
 

Control Dose
 

0/12 (0)	 5/34 (15)
 

N.S.	 N.S.
 

P = 0.012
 

Infinite
 

0.487
 

Infinite
 

79
 —
 

0/12 (0)	 2/34 (6)
 

N.S.	 N.S.
 

Infinite
 

0.112
 

Infinite'
 

103
 —
 

High
 

Dose
 

0/34 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

1/34 (3)
 

N.S.
 

Infinite
 

0.020
 

Infinite
 

103 
__ __ 

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

Fed Tolbutamide in the Diet3

 

(continued) 

Matched Low High 

Topo#raphyj Morphology Control Dose Dose 

Liver: Hepatocellular
 

Carcinomab 1/12 (8) 0/34 (0) 0/34 (0)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.000 0.000
 

Lower Limit 0.000 0.000
 

Upper Limit 6.492 6.492
 

Weeks to First Observed Tumor 103
 

Liver: Hepatocellular
 

Adenoma or Carcinoma'3 1/12 (8) 2/34 (6) 1/34 (3)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.706 0.353
 

Lower Limit 0.042 0.005
 

Upper Limit 40.714 26.851
 

Weeks to First Observed Tumor 103 103 103
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice 

Fed Tolbutamide in the Dieta 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Pituitary: Chromophobe 

Carcinoma0 0/11 (0) 2/26 (8) 0/26 (0) 

P Valuesc » d N.S. N.S. N.S. 

Relative Risk (Matched Control)f Infinite •.« 

Lower Limit 0.138 _­

Upper Limit Infinite «-

__ __ 

Weeks to First Observed Tumor 83 

Pituitary: Chromophobe
 

Adenoma or Carcinoma" 1/11 (9) 2/26 (8) 0/26 (0)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.846 0.000
 

Lower Limit 0.052 0.000
 

Upper Limit 47.845 7.805
 

Weeks to First Observed Tumor 103 83
 —
 a

Treated groups received doses of 25,000 or 50,000 ppm in feed;
 

^Number of tumor-bearing animals/number of animals examined at site (percent).
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice 

Fed Tolbutamide in the Diet3 

(continued) 

c

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

Cochran-Armitage test when P < 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that treated group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison,
 

^The 95% confidence interval, of the relative risk between each treated group and the
 

control group.
 


CD 

DHEW Publication No. (NIH) 77-831 xj