National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 50 

1978 

BIOASSAY OF 

ACETOHEXAMIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 968-81-0 

NCI-CG-TR-50 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

ACETOHEXAMIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-850 

BIOASSAY OF
 

ACETOHEXAMIDE
 

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
 

acetohexamide conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer 

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

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

determine whether selected environmental chemicals have the 

capacity to produce cancer in animals. Negative results in which
 

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

control animals, do not necessarily mean that the test chemical
 

is not a carcinogen, inasmuch as the experiments are conducted
 

under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals
 

under the conditions of the test and indicate that exposure to
 

the chemical is a potential risk to man. The actual determination of the risk to man from animal carcinogens requires a wider
 

analysis.
 

CONTRIBUTORS: This bioassay of acetohexamide was conducted by
 

Southern Research Institute, Birmingham, Alabama, initially under
 

direct contract to the NCI and currently under a subcontract to
 

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

Testing Program.
 

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

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

Weisburger^'^. Ms. 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. S. D.
 

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

report represent their interpretation.
 

ill
 
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 the results
 

of the analyses were reviewed by Dr. C. W. Jameson^.
 

This report was prepared at Tracer Jitco^ under the direction of
 

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

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

Campbell, Deputy Director for Science; Drs. J. F. Robens and C.
 

H. Williams, toxicologists; Dr. R. L. Schueler, pathologist; Dr.
 

G. L. Miller, Ms. L. A. Waitz, and Mr. W. D. Reichardt,
 

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

assisted by Ms. Y. E. Presley.
 

The statistical analysis was reviewed by members of the
 

Mathematical Statistics and Applied Mathematics Section of NCI^:
 

Dr. John J. Gart, Mr. Jun-mo Nam, Dr. Hugh M. Pettigrew, and Dr.
 

Robert E. Tarone.
 

The following other scientists at NCI were responsible for
 

evaluating the bioassay experiment, interpreting the results, and
 

reporting the findings:
 

Dr. Kenneth C. Chu
 

Dr. Cipriano Cueto, Jr.
 

Dr. J. Fielding Douglas
 

Dr. Dawn G. Goodman
 

Dr. Richard A. Griesemer
 

Dr. Harry A. Milman
 

Dr. Thomas W. Orme
 

Dr. Robert A. Squire8
 

Dr. Jerrold M. Ward
 

^-Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

iv
 
^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

%ow with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

-"Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

"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.
 

^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.
 

SUMMARY
 

A bioassay of acetohexamide for possible carcinogenicity was
 

conducted by administering the test chemical in feed to Fischer
 

344 rats and B6C3F1 mice.
 

Groups of 35 rats of each sex were administered acetohexamide in
 

the diet at one of two doses, either 10,000 or 20,000 ppm, for
 

103 weeks and then observed for 2 to 4 additional weeks. Matched
 

controls consisted of 15 untreated rats of each sex. All
 

surviving rats were killed at 105 to 107 weeks.
 

Groups of 35 mice of each sex were administered acetohexamide at
 

one of two doses for 103 weeks and then observed for 4 or 5
 

additional weeks. Time-weighted average doses were 6,359 or
 

12,718 ppm. Matched controls consisted of 15 untreated mice of
 

each sex. All surviving mice were killed at 107 or 108 weeks.
 

Mean body weights of the dosed rats and mice of both sexes were
 

lower than those of the corresponding matched controls throughout
 

the study, and the depressions in weight were dose related.
 

Except for the female mice, sufficient numbers of animals 

survived long enough to be at risk for development of lateappearing tumors. 

In the rats, the only tumor occurring with greater incidence in
 

dosed than in matched-control animals was leukemia (males:
 

matched controls 0/15, low-dose 10/35, high-dose 4/35; females:
 

matched controls 0/14, low-dose 7/35, high-dose 4/34). Only the
 

incidence in the low-dose males was statistically significant
 

(P = 0.018). All of these animals had undifferentiated
 

(mononuclear cell) leukemia, which commonly occurs spontaneously
 

in Fischer 344 rats, except for two with lymphocytic leukemia.
 

The incidence of combined leukemia and lymphoma in historical-


control male rats at this laboratory in the bioassay program to
 

date is 24/235 (10.2%), which is higher than that in the matched
 

vii
 
controls. Thus, the incidence in the low-dose males cannot be
 

clearly associated with administration of the test chemical.
 

In the mice, the only neoplasms that occurred at a higher
 

incidence in dosed groups than in matched controls were lymphomas
 

in the males, but the incidences were not statistically significant (matched controls 1/15, low-dose 9/35, high-dose 3/34).
 

These types of lesions are found commonly in untreated B6C3F1
 

mice. The incidence of lymphomas in the historical-control male
 

mice is 28/536 (5.2%).
 

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

acetohexamide was not carcinogenic for either Fischer 344 rats or
 

B6C3F1 mice.
 

viii
 
TABLE OF CONTENTS
 

Page
 

I. Introduction	 1
 

II. Materials and Methods	 3
 

A. Chemical	 3
 

B. Dietary Preparation	 3
 

C. Animals	 4
 

D. Animal Maintenance	 5
 

E. Subchronic Studies	 7
 

F. Designs of Chronic Studies	 8
 

G. Clinical and Pathologic Examinations	 11
 

H. Data Recording and Statistical Analyses	 12
 

III. Results -Rats	 19
 

A. Body Weights and Clinical Signs (Rats)	 19
 

B. Survival (Rats)	 19
 

C. Pathology (Rats)	 22
 

D. Statistical Analyses of Results (Rats)	 23
 

IV. Results -Mice	 25
 

A. Body Weights and Clinical Signs (Mice)	 25
 

B. Survival (Mice)	 25
 

C. Pathology (Mice)	 28
 

D. Statistical Analyses of Results (Mice)	 30
 

V. Discussion	 33
 

VI.	 Bibliography 35
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Table Al Summary of the Incidence of Neoplasms in
 

Rats Fed Acetohexamide in the Diet 37
 

Male Rats Fed Acetohexamide in the Diet 39
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Fed Acetohexamide in the Diet 43
 

IX
 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Fed Acetohexamide in the Diet 47 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Acetohexamide in the Diet 49 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Acetohexamide in the Diet 52 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Acetohexamide in the Diet 55 

Table Cl Summary of the Incidence of Nonneoplastic Lesions 

in Male Rats Fed Acetohexamide in the Diet 57 

Table C2 Summary of the Incidence of Nonneoplastic Lesions 

in Female Rats Fed Acetohexamide in the Diet 60 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Acetohexamide in the Diet 63 

Table Dl Summary of the Incidence of Nonneoplastic Lesions 

in Male Mice Fed Acetohexamide in the Diet 65 

Table D2 Summary of the Incidence of Nonneoplastic Lesions 

in Female Mice Fed Acetohexamide in the Diet 68 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Acetohexamide in the Diet 71 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Acetohexamide in the Diet 73 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Acetohexamide in the Diet 77 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Acetohexamide in the Diet 83 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Acetohexamide in the Diet 85 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Acetohexamide in the Diet 88 
Page 

TABLES 

Table 1 Design of Acetohexamide Chronic Feeding 

Studies in Rats 9 

Table 2 Design of Acetohexamide Chronic Feeding 

Studies in Mice 10 

FIGURES 

Figure 1 Growth Curves for Rats Fed Acetohexamide 

in the Diet 20 

Figure 2 Survival Curves for Rats Fed Acetohexamide 

in the Diet 21 

Figure 3 Growth Curves for Mice Fed Acetohexamide 

in the Diet 26 

Figure 4 Survival Curves for Mice Fed Acetohexamide 

in the Diet 27 

xi
 

I. INTRODUCTION
 

Acetohexamide (CAS 968-81-0; NCI C03247) is an oral hypoglycemic
 

agent of the arylsulfonyl-urea group with a potency between that
 

of tolbutamide and chlorpropamide (Jackson, 1969).
 

These compounds function by stimulating the secretion of insulin
 

by the pancreas, and therefore, are effective only in patients
 

with at least minimal pancreatic function, as is the case with
 

maturity-onset diabetics (Larner and Haynes, 1975). Although
 

some oral hypoglycemics have been in use over the past 20 years
 

on a chronic basis in the treatment of maturity-onset diabetes,
 

acetohexamide has been used clinically for only about 10 years.
 

The chief advantage of these compounds over insulin is that they
 

may be taken orally where dietary modifications have failed to
 

control elevated blood glucose levels. However, since controlled
 

studies have shown that they are no more effective than dietary
 

modifications in controlling the symptoms of maturity-onset
 

diabetes on a long-term basis in the majority of patients and
 

that they may be associated with an increase in cardiovascular
 

mortality, their use in the future is expected to decline (Shen
 

and Bressler, 1977).
 

Acetohexamide was selected for testing in the carcinogenesis
 

program as part of an attempt to evaluate the carcinogenicity of
 

drugs that may be used for prolonged periods in humans.
 

II. MATERIAL AND METHODS
 

A. Chemical
 

Acetohexamide (l-[(p-acetylphenyl)-sulphonyl]-3-cyclohexylurea)
 

was obtained in two batches (Lot Nos. 6RV07 and 7AD46) from Eli
 

Lilly and Company, Indianapolis, Indiana. The purity and
 

identity of Lot No. 7AD46, which was used in the chronic study,
 

was confirmed in analyses at Midwest Research Institute. The
 

melting point was 184.5-186°C and was consistent with that of
 

187-189°C reported in the literature (Aumueller et al., 1962).
 

Elemental analyses (C, H, N, S) were correct for C]_5H2()N204S' the
 

molecular formula of acetohexamide. Nuclear magnetic resonance,
 

infrared, and ultraviolet spectra were in agreement with those in
 

the literature (Salim and Hilty, 1967; Baltazar and Braga, 1966;
 

Kuhnert-Brandstatter et al., 1970) and with the structure.
 

The batch used for the chronic study was stored in the original
 

container at 5°C.
 

B. Dietary Preparation
 

Test diets were prepared every 2 weeks by mixing a known amount
 

of sifted acetohexamide 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. The
 

prepared diets were stored at room temperature in sealed plastic
 

containers.
 

The stability of acetohexamide in the feed mixture was determined
 

at Midwest Research Institute by measuring the concentration of
 

acetohexamide in formulated diets stored at room temperature
 

(25°C) for a 2-week period. The results of this analysis
 

indicated that acetohexamide mixed with animal meal is stable for
 

2 weeks at room temperature.
 

C. Animals
 

For the subchronic study, male Fischer 344 rats and female B6C3F1
 

mice were obtained from Charles River Breeding Laboratories,
 

Inc., Wilmington, Massachusetts.
 

For the chronic study, Fischer 344 rats and B6C3F1 mice of both
 

sexes were obtained from Charles River Breeding Laboratories
 

through contracts with the Division of Cancer Treatment, National
 

Cancer Institute. These rats and mice were received at 30 days
 

of age and were quarantined for 3-4 weeks. Animals with no
 

visible signs of disease were earmarked and then assigned to
 

control and dosed groups.
 
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%. Room air was changed 15 times
 

per hour and passed through both intake and exhaust fiberglass
 

roughing filters. In addition to natural light, illumination was
 

provided by fluorescent light for 9 hours per day. Food and
 

water were supplied daily and were 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 rat cages were provided with Iso-Dri® hardwood chip bedding
 

(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 cage tops were covered with disposable filter bonnets
 

beginning at week 56. Bedding was replaced once per week; cages,
 

water bottles, and feeders were sanitized at 82°C once per week;
 

and racks were cleaned once per week.
 

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

were housed with respective dosed animals. Animals administered
 

acetohexamide were maintained in the same rooms as animals of the
 

same species administered the following chemicals:
 
RATS
 

Feed Studies
 

anthranilic acid (CAS 118-92-3)
 

l-butyl-3-(p-tolylsulfonyl)urea (toIbutamide) (CAS 64-77-7)
 

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
 

(phenazopyridine 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)
 

pyrazinecarboxand.de (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
 

anthranilic acid (CAS 118-92-3)
 

1-buty1-3-(p-tolylsulfonyl)urea (tolbutamide) (CAS 64-77-7)
 

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
 

(phenazopyridine 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)
 
Gavage Studies
 

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

(phenesterin) (CAS 3546-10-9)
 

estradlol 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)
 

acronycine (CAS 7008-42-6)
 

5-azacytidine (CAS 320-67-2)
 

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

(CAS 789-61-7)
 

1,4-butanediol dimethanesulfonate (busulfan) (CAS 55-98-1)
 

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

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

hydrochloride [IPD] (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)tetrahydro-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 hydrochloride) (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)
 

E. Subchronic Studies
 

Subchronic studies were conducted to estimate the maximum
 

tolerated doses of acetohexamide, 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. In the subchronic studies, acetohexamide was
 

administered in the diet to male Fischer 344 rats at doses of
 

1,200, 3,000, 6,000, 15,000, or 30,000 ppm and to female B6C3F1
 
mice at doses of 2,500, 5,000, 10,000, 20,000, or 40,000 ppm.
 

Five animals of each species were used at each dose, and 5 rats
 

and 10 mice were maintained as untreated controls. Dosed animals
 

were fed diets containing acetohexamide 7 days per week for 45
 

days and then observed for an additional 45 days.
 

There were no deaths in the rats at any dose administered. At
 

the end of the 45-day administration period, mean body weight
 

gain of the dosed animals, compared with that of the controls,
 

was 98% at 1,200 ppm, 97% at 3,000 ppm, 87% at 6,000 ppm, 87% at
 

15,000 ppm, and 15% at 30,000 ppm. Mean body weight gains at 90
 

days were all within 15% of controls. The low and high doses for
 

the chronic studies using rats were set at 10,000 and 20,000 ppm.
 

In mice, all animals administered 40,000 ppm died by week 3. No
 

deaths occurred in any other dosed group. During the administration period, mean body weight gains of animals administered
 

2,500, 5,000, and 10,000 ppm were depressed compared with those
 

of controls, and at 20,000 ppm animals failed to gain weight.
 

Final body weights of the surviving dosed animals were, however,
 

comparable to those of controls. The low and high doses for the
 

chronic studies using mice were set at 10,000 and 20,000 ppm.
 

F. Designs of Chronic Studies
 

The designs of the chronic studies are shown in tables 1 and 2.
 
Table 1. Design of Acetohexamide Chronic Feeding Studies in Rats
 

Sex and Initial Acetohexamide Time on Studies 

Test No. of in Dietb Dosed Observed 

Group Animals3 (ppm) (weeks ) (weeks ) 

Male 

Matched-Control 15 0 107 

Low-Dose 35 10,000 103 3-4 

High-Dose 35 20,000 103 2-3 

Female 

Matched Control 15 0 107 

Low-Dose 35 10,000 103 4 

High-Dose 35 20,000 103 3 

a

Rats were 54 days of age when placed on study.
 

"The dosed animals were fed test diets 5 days per week and
 

control diets 2 days per week.
 
Table 2. Design of Acetohexamide Chronic Feeding Studies in Mice
 

Sex and Initial Ac e t oh exami d e Time on Study Time-Weighted
 c
 Test No . of in Dietb

» Dosed Observed Average Dose"
 

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

Male
 

Matched-Control 15 0 107
 

Low-Dose 35 10,000 28
 

5,000 75 4-5 6,359
 

High-Dose 35 20,000 28
 

10,000 75 4 12,718
 

Female
 

Matched-Control 15 0 107
 

Low-Dose 35 10,000 28
 

5,000 75 5 6,359
 

High-Dose 35 20,000 28
 

10,000 75 4 12,718
 

a

Mice were 55 days of age when placed on study.
 

dosed animals were fed test diets 5 days per week and control
 

diets 2 days per week.
 

°Doses were lowered at week 29, because weight gains were extremely
 

low, particularly in the females.
 

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

£(no. of weeks at each dose)
 

10
 
G. Clinical and Pathologic Examinations
 

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

animals that were moribund were killed and necropsied. Rats and
 

mice were weighed individually every 2 weeks for the first year
 

and once per month for the remainder of the study. Palpation for
 

masses was carried out at each weighing.
 

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,
 

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

brain, and sensory organs. Peripheral blood smears were prepared
 

from each animal whenever possible. 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.
 

11
 
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
 

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
 

12
 
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,
 

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
 

13
 
significantly higher proportion of tumors than did the control
 

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

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

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

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
 

14
 
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 < 0.05, two-tailed test)
 

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with its control was calculated
 

15
 
from the exact interval on the odds ratio (Gart, 1971). The
 

relative risk is defined as p^/Pc where pt is the true binomial
 

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

dosed 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 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 < 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
 

16
 
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.
 

17
 

III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of the low- and high-dose rats of both sexes
 

were lower than those of the corresponding matched controls
 

throughout the study, and the depressions in weight were dose
 

related (figure 1). No other signs of toxicity related to
 

administration of the test chemical were recorded in the rats.
 

Rales and progressive weight loss were noted in individual
 

animals of both the dosed and control groups.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats fed acetohexamide in the diet
 

at the doses of this bioassay, together with those of the matched
 

controls, are shown in figure 2.
 

In each sex, the results of the Tarone test for positive dose-


related trend in mortality are not significant. At least 80% of
 

the male rats and over 70% of the female rats lived to the end of
 

the bioassay, providing sufficient numbers of male and female
 

rats at risk for development of late-appearing tumors.
 

19
 
0° 

360­

o 

—

I2 

g

 3 

 250­

D 

Q 

O

m 200­

UJ 150­

(I 

100­

50­

MALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

20 30 40 50 60 70 

TIME ON STUDY (WEEKS) 

80 90 100 110 

350­

O 

O

ffi 

 200­

O

o

 ° ° 

A

 00 

 A A 

0

AAA 

n

 O O O O ° 

AAA A A 

Dn 

o 

"J 150­

FEMALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

20 30 40 50 60 70 

TIME ON STUDY (WEEKS) 

90 100 

Figure 1. Growth Curves for Rats Fed Acetohexamide in the Diet 

20 
PROBABILITY OF SURVIVAL PROBABILITY OF 

p °>J e 3 1 * cn 

1§ cs !3 I t i; ^s 3 3 <3 S% § ?' c5 C3 g 3 C3 

p ­

n• i p 

to' > o a > o D 

3 s- M

i?

LOW DOSE

 

HIGH DOSE

 

1 

=EMALE RA 

oMALE RAl

 MATCHED COr

 LOW DOSE HIGH DOSE 

1 .1 

H _ 

~* c/> 0 & SL o 

£ g- g2

CO

 s" o 2 & 

"* m i

33 0 0

» ;* 

S.

 c 

^ o c

0 < § > - "* g- s s s

m

1 1 L.. 

m

X f> P" W °" pC - B) ~ 

3 ! j 

g- §- g- 

~T" 

U • rp

o "

O 

-^

?^ 

8- 1­

cll I 

MATCHED CON 

a s­ ^ 8­
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 occurred in both matched-control groups
 

and dosed groups. Except for leukemias, the neoplasms listed in
 

Appendix A appeared with approximately equal frequency in dosed
 

and control rats or appeared in insignificant numbers. Most of
 

these lesions are not uncommon in the Fischer 344 rat independent
 

of any treatment.
 

The incidence of hematopoietic neoplasms was higher in the dosed
 

than in the matched-control groups, and highest in the low-dose
 

rats. The incidences of the undifferentiated and lymphocytic
 

leukemias were as follows:
 

MALES FEMALES 

Matched Low High Matched Low High 

Control Dose Dose Control Dose Dose 

Number of Rats 

Necropsied (15) (35) (35) (14) (35) (34) 

Multiple Organs, 

Hematopoietic 

- undifferentiated 

leukemia 0 10 0 7 3 

- lymphocytic 

leukemia 0 0 0 0 1 

The undifferentiated or mononuclear leukemia (Davey and Moloney,
 

22
 
1970; Moloney et al., 1970) occurs spontaneously in the Fischer
 

344 rat with variable frequency.
 

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

proliferative, and inflammatory changes were encountered in
 

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

nonneoplastic lesions are commonly seen in aged Fischer 344 rats.
 

Although the incidence of hematopoietic neoplasms was higher in
 

Fischer 344 rats fed acetohexamide for 24 months at 20,000 and
 

10,000 ppm than in the control rats, this is considered by the
 

pathologists to be a spontaneous occurrence not associated with
 

administration of acetohexamide.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals in one group and with an incidence of at least
 

5% in one or more than one group.
 

In female rats, the results of the Cochran-Armitage test for
 

positive dose-related trend and of the Fisher exact test for
 

direct comparison of incidences between the matched-control group
 

and each of the dosed groups indicated no significant increases
 

in the tumors of the dosed groups. In male rats, the Cochran23
 
Armitage test result on the incidence of leukemia is not
 

significant, but the Fisher exact test shows that the incidence
 

in the low-dose group is significantly higher (P = 0.018) than
 

that in the matched controls. The incidence in the high-dose
 

group is not significant. The survivals of the groups are
 

comparable. The incidence of combined leukemia and lymphoma in
 

untreated-control male rats at this laboratory in this bioassay
 

program to date is 24/235 (10.2%), which is higher than that
 

(0/15) in the controls of this study. In females, the incidence
 

of these tumors in the laboratory historical controls is 19/235
 

(8.1%).
 

The combined incidence of islet-cell adenoma or carcinoma is
 

significantly higher in the control group of male rats than in
 

either dosed group. Significant results (P = 0.003) in the
 

negative direction are also observed in the incidence of
 

fibroadenoma of the mammary gland in female rats.
 

24
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of the low- and high-dose mice of both sexes
 

were lower than those of the corresponding matched controls
 

throughout the entire study, and the depressions in weight were
 

dose related (figure 3). Fluctuation in the growth curve may be
 

due to mortality; as the size of a group diminishes, the mean
 

body weight may be subject to variation. Progressive weight loss
 

and rales were noted in several individual animals, mostly in the
 

dosed groups.
 

To control respiratory disease, mice were given oxytetracycline
 

in the drinking water during week 36 (0.6 mg/ml) and during week
 

37 (0.3 mg/ml). To suppress the spread of the disease, propylene
 

glycol was vaporized in the mouse room during weeks 35 through
 

46.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice fed acetohexamide in the diet
 

at the doses of this bioassay, together with those of the matched
 

controls, are shown in figure 4.
 

In each sex, the results of the Tarone test for positive dose25
 
D D D D OD D D D D DDDD D D D DDDD

DD DD D D 

Q 

D D gOOO oo nO OL ° ° ° A A o o O oo°° o 

_O O O AA OAAA A AAAAAAA A 

D O O O AA

2 

w Q O O AA 

s AAA 

Q 

O 20­ CQ 

MALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

a a D a

a a ODOD D D a 

O 

O O I 

a DDDD

DC oo ooo;;o 0000000 0 A 

.,000 0 ^°AA°AAAAA A AAA A I8_o°oj A AA A 

A a AQO o 20 K )AO A m 

u 

5 

FEMALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Fed Acetohexamide in the Diet 

26 
PROBABILITY O

PROBABILITY OF SURVIVAL 

P P P 

V) 

I. 

n 

i 

X 

B)

3 

Si 

6> 
related trend in mortality over the period are not significant.
 

In male mice, 31/35 of the high-dose group, 34/35 of the low-dose
 

group, and 14/15 of the matched-control group lived beyond week
 

52 on study. In females, 24/35 of the high-dose group, 33/35 of
 

the low-dose group, and 11/15 of the matched-control group lived
 

at least as long as week 52 on study. Thus, sufficient numbers
 

of mice of each sex survived to week 52 to be at risk for development of tumors appearing within this period. Sufficient numbers
 

of the males survived to the end of the test to be at risk for
 

development of late-appearing tumors; however, the earlier deaths
 

of the females may have limited the development of late-appearing
 

tumors in this sex.
 

C. Pathology (Mice)
 

Histopatholpgic 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.
 

Excluding lymphoreticular neoplasms, the lesions listed in
 

Appendix B appeared with approximately equal frequency in dosed
 

and control mice or appeared in insignificant numbers. Most of
 

these lesions are not uncommon in the B6C3F1 mouse independent of
 

any treatment.
 

The incidences of lymphoreticular neoplasms were 9/35(26%) in the
 

28
 
low-dose males, 3/34(9%) in the high-dose males, and 1/15(7%) in
 

the matched-control males. In female mice, the incidences were
 

approximately equal for all three groups. The distribution and
 

type of the lesions were as follows:
 

MALES FEMALES 

Matched Low High Matched Low High 

Control Dose Dose Control Dose Dose 

Number of Mice 

Necropsied (15) (35) (34) (10) (35) (30) 

Brain
 

- malignant lymphoma,
 

histiocytic type 1 0
 

Multiple Organs, Lymphoreticular
 

- malignant lymphoma, 

lymphoblastic (undifferentiated) type 0 4 0 0 1 2 

- malignant lymphoma, 

lymphocytic type 0 1 2 0 0 0 

- malignant lymphoma, 

histiocytic type 1 2 1 1 2 0 

Mesenteric Lymph Node
 

- malignant lymphoma,
 

histiocytic type
 

Number of Mice with Tumors
 

of the Hematopoietic
 

System 1 1
 

The lymphoreticular neoplasms are a common finding in B6C3F1 mice
 

independent of any treatment, and vary in frequency from one
 

study to the next.
 

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

29
 
proliferative, and inflammatory changes were encountered in
 

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

nonneoplastic lesions are commonly seen in aged B6C3F1 mice;
 

however, the severe suppurative bronchopneumonias were associated
 

with increased deaths or decreased life spans. The incidence of
 

bronchopneumonias was high in all the control and dosed female
 

groups, and in the low-dose males.
 

Although the incidence of lymphoreticular neoplasms was higher in
 

B6C3F1 male mice fed acetohexamide for 24 months at the original
 

and reduced low doses (10,000 and 5,000 ppm) than in the control
 

mice, they were considered by the pathologists to be spontaneous
 

neoplasms not associated with administration of acetohexamide.
 

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 in one group and with an incidence of at least
 

5% in one or more than one group.
 

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

positive dose-related trend and of the Fisher exact test for
 

direct comparison of incidences between the matched controls and
 

each of the dosed groups are not significant. In each of the 95%
 

confidence intervals of relative risk, shown in the tables, the
 

30
 
value of one is included; this indicates the absence of
 

significant positive 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 acetohexamide, which could not be detected under the conditions of
 

this test. In male mice, significant trends (P = 0.049) in the
 

negative direction are observed in the incidences of liver
 

tumors, where the incidences in the controls exceed those in the
 

dosed animals. These significant negative results may be due to
 

the somewhat higher mortality of the dosed animals compared with
 

the controls in the last 20 weeks on study.
 

31
 

V. DISCUSSION
 

In this bioassay, administration of acetohexamide in feed caused
 

a dose-related depression in mean body weights of rats and mice
 

of both sexes. No other clinical signs related to administration of the test chemical were observed; however, some animals in
 

all groups of mice developed severe suppurative bronchopneumonia.
 

Except for the female mice, adequate numbers of animals survived
 

long enough to be at risk for development of late-appearing
 

tumors.
 

In the rats, the only tumor occurring with greater incidence in
 

dosed than in matched-control animals was leukemia (males:
 

matched controls 0/15, low-dose 10/35, high-dose 4/35; females:
 

matched controls 0/14, low-dose 7/35, high-dose 4/34). Only the
 

incidence in the low-dose males was statistically significant
 

(P = 0.018). All of these animals had undifferentiated
 

(mononuclear cell) leukemia, which commonly occurs spontaneously
 

in Fischer 344 rats, except for two with lymphocytic leukemia.
 

The incidence of combined leukemia and lymphoma in historical-


control male rats at this laboratory in the bioassay program to
 

date is 24/235, which is higher than that in the matched
 

controls. Thus, the incidence in the low-dose males cannot be
 

clearly associated with administration of the test chemical.
 

33
 
In the mice, the only lesions that appeared at a higher frequency
 

in the dosed than in the matched-control animals were lymphomas
 

in the males. These incidences were not statistically significant (controls 1/15, low-dose males 9/35, high-dose males 3/34)
 

and are found commonly in untreated B6C3F1 mice. In historical-


control male mice, the incidence of lymphoma is 28/536.
 

It is concluded that under the conditions of this bioassay, acetohexamide was not carcinogenic for either Fischer 344 rats or
 

B6C3F1 mice.
 

34
 
VI. BIBLIOGRAPHY
 

Aumueller, G. K., Weyer, R., Baender, A., and Herr, H. for
 

Fabwerke Hoechst A-G, Ger. 1,135, 891 (Cl. 120), September
 

6, 1962.
 

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

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

Baltazar, J., and Ferriera Braga, M. M., Physical-chemical
 

characteristics and methods of measurement of acetohexamide.
 

Rev. Port. Farm. 16(3):167-174, 1966.
 

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

Panel on Carcinogenicity of the Cancer Research Commission
 

of UICC, Vol. J2, International Union Against Cancer, Geneva,
 

1969.
 

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.
 

Davey, F. R. and Moloney, W. C., Postmortem observations on
 

Fischer rats with leukemia and other disorders. Lab.
 

Invest. 23(3):327-334, 1970.
 

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

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Stat. Inst. 39(2);148-169. 1971.
 

Jackson, W. P. U., Sulphonylureas in the management of human
 

diabetes. Oral Hypoglycaemic Agents; Pharmacology and
 

Therapeutics. Academic Press, London, 1969, pp. 150-163.
 

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

incomplete observations. J. Amer. Statist. Assoc.
 

531:457-481, 1958.
 

Kuhnert-Brandstatter, M., Koffer, A., Vlachopoulos, A., and
 

Lobenwein, A., Microscopic characterization and identification of drugs by UV spectrophotometry. Sci. Pharm. 

38:154-163, 1970. 

35
 
Larner, J. and Haynes, R. C., Jr., Insulin and oral hypoglycemic
 

drugs, glucagon. In: The Pharmacological Basis of
 

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

Publishing Co., Inc., New York, 1975, pp. 1507-1533.
 

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.
 

Moloney, W. C., Boschetti, A. E., and King, V. P., Spontaneous
 

leukemia in Fischer rats. Cancer Res. 30:41-43, 1970.
 

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.
 

Salim, E. F. and Hilty, W. W. Qualitative and quantitative tests
 

for acetohexamide. J. Pharm. Sci. 56(3):385-386. 1967.
 

Shen, S. W. and Bressler, R., Medical Intelligence: Clinical
 

pharmacology of oral antidiabetic agents (Part II). N.
 

Engl. J. Med. 296(14):787-793. 1977.
 

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

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

36
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED ACETOHEXAMIDE IN THE DIET
 

37
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

FED ACETOHEXAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIMALS EXAMINED HISTOPATHOLOGICALLY

 15

 15

 15

 35

 35

 35

 35 

 35 

 35 

INTEGUHENTABY SYSTEM 

*SUBCUT TISSUE

SARCOMA, NOS

FIBBOMA

 (15)

 1 (1%) 

 (35)

 1 (3*) 

 (35) 

RESPIRATORY SYSTEM 

#LUN G

ALVEOLAR/BRONCHIOLA R ADENOM A

C-CELL CARCINOMA , METASTATIC

 (15)

 1 (75!)

 (34)

 1 (3%)

 (35) 

1 (3%) 

1 (3%) 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGANS

UNDIFFERENTIATJED LEUKEMIA

LYMPHOCYTIC LEUKEMIA

 (15) (35)

 10 (29%)

 (35) 

3 (9X) 

1 (3%) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

ILIVER

HEPATOCELLULAR ADENOMA

 (15) (35) (35) 

1 (3S) 

•STOMACH

SQUAMOUS CELL PAPILLOMA

 (14) (35)

 1 {351) 

 (35) 

tCOLON

A£ENOMATOUS_POLYPA_NQS

 (14)

 JL1221. 

 (35) (35) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED

* NUHBER OF ANIMALS NECROPSIED 

 MICROSCOPICALLY 

39
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE 

UBINARY SYSTEM
 

IKIDNEY (15) (35)
 

MIXED TUMOR. MALIGNANT 1 (3X)
 

ENDOCRINE SYSTEM
 

•PITUITARY	 (11) (33)
 

CHROMOPHOBE ADENOMA 1 (3S)
 

CHROMOPHOBE CARCINOMA 2 (6%)
 

#ADRENAL (15) (35)
 

PHEOCHROMOCYTOMA. MALIGNANT 1 (7X) 1 (3X)
 

tTHYROID (14) (35)
 

FOLLICULAR-CELL ADENOMA 1 (7X)
 

C-CELL ADENOMA 1 (7X) 2 (6X)
 

C-CELL CARCINOMA 1 (7X) 2 (6X)
 

*PANCR£ATIC ISLETS (14) (35)
 

ISLET-CELL ADfiNOMA 2 (14X)
 

ISLET-CELL CARCINOMA 1 (7X)
 

REPRODUCTIVE SYSTEM 

*MAMMARY GLAND 

FIBROADENOMA 

(15) 

1 (7X) 

(35) 

*PREPUTIAL GLAND 

CARCINOMA, NOS 

ADENOMA, NOS 

(15) (35) 

tTESHS 

INTERSTITIAL-CELL TUMOR 

SARCCMA, NOS 

(15) 

15 (100X) 

(35) 

34 (97X) 

NERVOU S SYSTEM 

• PERIPHERAL NERVE (15) (35) 

MOBOFIB10SARCOMA. 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

40 

HIGH DOSE 

(35)
 

(33)
 

4 (12X)
 

1 (3%)
 

(35)
 

(35)
 

4 (11X)
 

2 (6X)
 

(35)
 

1 (3%)
 

(35)
 

(35)
 

1 (3X)
 

1 (3X)
 

(35)
 

29	 (83X)
 

1 (3X)
 

(35) 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL S£NSE ORGAN S 

*HARDERIAN GLAND (15) (35) (35) 

ADENCHA , NOS 1 (3») 1 (3S) 

HOSCULOSKEIETAL SYSTEM 

NON E 

BODY CAVITIES
 

OTUNICA VAGINALIS (15) (35) (35)
 

MESOTHELIOMA, NOS 1 (3«)
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 15 35 35
 

NATURAL DEATHS 2 2
 

MORIBUND SACRIFICE 1 5 6
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 12 28 29
 

ANIMAL MISSING
 

I_INCLODES_AUTOLYZED_ANiaALS
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

41
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

TUHOB SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TOHORS* 15 35 33
 

TOTAL PRIMARY TUMORS 26 57 53
 

TOTAL ANIHALS HITH BENIGN TUHORS 15 3U 31
 

TOTAL BENIGN TUMORS 23 40 41
 

TOTAL ANIMALS WITH MALIGNANT TUMOES 3 15 11
 

TOTAL MALIGNANT TUMORS 3 17 11
 

TOTAL ANIMALS HITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OF MALIGNANT 1
 

TOTAL UNCERTAIN TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPBIMARY OB METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUflORS
 

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

42
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

FED ACETOHEXAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIHALS INITIALLY IN STUDY 15 35 35
 

ANIMALS NECBOPSIED 14 35 34
 

ANIHALS EXAMINED HISTOPATHOLOGICALLY 14 35 34
 

INTEGUMENTARY SYSTEM
 

NONE
 

BESEIBATORY SYSTEM
 

tLUNG (14) (35) (34)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 2 (6S) 1 (3S)
 

C-CILL CARCINOMA. METASTATIC 1 (3%)
 

HEMATOPOIETIC SYSTEM
 

*BULTIPLE ORGANS (14) (35) (34)
 

UNEIFFEBENTIATED LEUKEMIA 7 (20%) 3 (9X)
 

LYHPHOCYTIC LEUKEMIA 1 (3%)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tSTOHACH (14) (34) (34)
 

SQUAMOUS CELL CARCINOMA 1 (3*)
 

URINABY SYSTEM
 

tURINARY BLADDER (14) (35) (34)
 

TRAliITIONAL=CELL_CAR£IfiOJ!i_ I_il21
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

43
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

ENDOCRINE SYSTEM
 

#PITUITARY (12) (27) (30)
 

CHROMOPHOBE ADENOHA 1 (8») 7 (2655) 5 (1755)
 

CHROMOPHOBE CARCINOMA 1 (855) 3 (1155) 2 (751)
 

•THYROID	 (13) (32) (34) 

C-CELL ADENOM A 1 (355) 1 (3X) 

C-CELL CARCINOM A 1 (855) 1 (355) 

REPRODUCTIVE SYSTEM
 

*flAKMARY GLAND (14) (35) (34)
 

PAPILLARY ADENOCARCINOMA 1 (3%)
 

FIBRCADENOMA 4 (2955) 4 (1156)
 

IUTERUS (14) (33) (34)
 

ADENOCARCINOMA, NOS 1 (3%)
 

SARCCMA, NOS 1 (3«)
 

ENDOMETRIAL STBCMAL POLYP 1 (7X) 5 (1555) 4 (1255)
 

ENDOMETRIAL STROMAL SAHCOMA 2 (1455) 1 (355)
 

fOVARY (14) (33) (34)
 

GRANULOSA-CELL CARCINOMA 1 (355)
 

NERVOUS SYSTEM
 

tBRAIN (13) (35) (34)
 

ASTROCYTOMA 1 (335)
 

*TRIGEMINAL NERVE (14) (35) (34)
 

CHRCMOPHOBE CARCINOMA, METASTATI 1 (355)
 

SPECIAL SENSE ORGANS
 

*EAR CANAL (14) (35) (34)
 

SQUAMOUS CELL CARCINOMA 1 (355)
 

MUSCULOSKEIETAL SYSTEM
 

NONE
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES
 

*MESENTERY


SARCOHA, NOS, METASTATIC


ALL OTHER SYSTEMS
 

*HOITIPLE ORGANS


C-CELL CARCINOMA, BETASTATIC


ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATURAL DEATHa
 MORIBUND SACRIFICE


SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL EENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAI MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS HITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

 (14) (35) (34) 

1 (3») 

 (14) (35) (34) 

1 (7X) 

 15 35 35 

3 1 1 

1 8 5 

 11 26 29 

9 23 18 

11 33 22 

5 15 10 

6 19 11 

4 13 11 

5 14 11 

1 1 2 

1 1 2 

* PRIMARY TUMORS: ALL TUHORS EXCEPT SECONDARY TUMORS
 « SECONDARY TUMORS: METASTATIC TUMORS OR TOMORS INVASIVE INTO AN ADJACENT ORGAN
 

45
 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED ACETOHEXAMIDE IN THE DIET
 

47
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED ACETOHEXAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIHALS INITIALLY IN STUDY 15 35 35
 

ANIMALS KECBOPSIED 15 35 34
 

ANIMALS EXAMINED HIS1 ^PATHOLOGICALLY 15 35 34
 

INTEGUMENTARY SYS1EM
 

NONE
 

RESPIBATOEY SYSTEM
 

tLUNG (15) (35) (33)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (1%) 1 (3%) 2 (6X)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (1%) 2 (6%)
 

HEMATOPOIETIC SYSTEM
 

tERAIN (15) (35) (34)
 

MALIG. LYMPHOMA, HISTIOCYTIC TYPE 1 (3X)
 

*MULTIPLE ORGANS (15) (35) (34)
 

MALIG. LYMPHOMA, UNDIFFER-TYPE 4 (11%)
 

MALIG. LYMPHOMA, LYMPHOCYTIC TYPE 1 (3X) 2 (6X)
 

MALIG. LYMPHOMA, HISTIOCYTIC TYPE 1 (7%) 2 (6X) 1 (3X)
 

#MESENTERIC L. NODiS (14) (32) (29)
 

MALIG. LYMPHOMA, HISTIOCYTIC TYPE 1 (3X)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER (15) (33) (34) 

_ _ HEPATOCJLLULAR_A£ENOMA ______________ _ 

f NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIHALS NECROPSIED
 

49 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

HEPATOCELLULAR CARCINOMA 2 (13«) 1 (3%) 

UHINAKY SYSTEM 

NONE 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

NONE 

NERVOUS SYSTEM 

NONE 

SPECIAL SENS-E ORGANS 

*HARDERIAN GLAND

FAPIILARY ADENOMA

PAPIILARY CYSTADENOMA. NOS

 (15) (35)

 1 (3S) 

1 (3») 

 (34) 

MUSCOIOSKEIETA1 SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

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

50
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

ANIKAL DISEOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 15 35 35
 

NATURAL DEATHS) 2 10 8
 

MORIBUND SACRIFICE 4 8 9
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 17 18
 

ANIMA L MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 6 14 5
 

TOTAL PRIMARY TUMORS 6 16 6
 

TOTAL ANIMALS WITH BENIGN TUMORS 2 U 3
 

TOTAL EENIGN TUMORS 2 4 3
 

TOTAL ANIMALS WITH MALIGNANT TUMORS a 11 3
 

TOTAL MALIGNANT TUMORS H 12 3
 

TOTAL ANIMALS WITH SECONDARY TUMORS#
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINEENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PEIMAEY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

51
 
TABLE B2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

FED ACETOHEXAMIDE IN THE DIET 

CONTROL LOW DOSE


ANIMALS INITIALLY IN STUDY 15 35
 ANIMALS KECHOPSIED 10 35


ANIMALS EXAMINED HISTOPATHOLOGICALLY 10 35


INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

tLUNG (10) (34)


ALVEOLAR/BRONCHIOLAR ADENOMA 1 (3%)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (10) (35)


MALIG.LYHPHOMA, ONBIFFER-TYPE 1 (3S)


MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (10X) 2 (6X)
 

CIHCUIATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER (10) (35)


HEPATOCELLULAR ADENOMA


HEPATOCELLULAR CARCINOMA 1 (10%) 1 (3%)
 

tEUODENUH (8) (34)


SARCOMA, NOS 1 (3*)
 

URINARY SYSTEM .
 

NOHI ,
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF AHIMALS NECROPSIED
 

52
 

 HIGH DOSE
 

 35
 

 30
 

 30
 

 (29)
 

 (30)
 

2 (7X)
 

 (30)
 

1 (3%)
 

 (28)
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ENDOCRINE SYSTEM 

NONE 

BEEECDUCTIVE SYSTEM 

tUTERUS

ENDOMETRIAL STROMAL POLYP

 (10) (35)

 1 (3%) 

 (30) 

NEEVCUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKEIETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 15 35

NATURAL DEATHS 10 5

MORIBUND SACRIFICE 2 17

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

1EBMINAL SACRIFICE 3 13

ANIMAL MISSING 

a mcjLUDES AUTQLY2ED ANIMALS 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

 35 

 13 

 15 

7 

53
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

TUBOR SUMMAR Y 

TOTAL ANIMALS WITH PRIMAR 2 Y TUMORS*

TOTAL PRIMARY TUMORS 2 

7 

7 

3 

3 

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAL BENIGN TUMORS

 2

 2

 1 

1 

TOTAL ANIMALS WITH MALIGNAN 2 T TUMORS

TOTAL MALIGNANT TUMORS 2 

5 

5 

2 

2 

TOTAL ANIMALS WITH SECONDARY TUMORS* 

TOTAL SECONDARY TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMAEY OR METASTATIC 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

54
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED ACETOHEXAMIDE IN THE DIET
 

55
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

FED ACETOHEXAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 15 35 35
 

ANIMALS NECROP5IED 15 35 35
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 15 35 35
 

_•
 

INTEGUMENTARY SYSTEM
 

*SKIN (15) (35) (35)
 

HPIDERMAL INCLUSION CYST 1 (755) 1 (3%)
 

*SUECUT TISSUE (15) (35) (35)
 

INFLAMMATION, SUPPURATIVE 1 (3%)
 

INFLAMMATION, GRANULOMATOUS 2 (6%)
 

RESPIRATORY SYSTEM
 

*NASAL CAVITY (15) (35) (35)
 

INFLAMMATION, SUFPURATIVE 1 (3%)
 

tTRACHEA (15) (35) (35)
 

INFLAMMATION, SUPPURATIVE 1 (755) 4 (1155) 1 (355)
 

INFLAMMATION, CHRONIC 1 (3%)
 

ILUNG (15) (34) (35)
 

PNEUMONIA, CHRONIC MURINE 1 (355)
 

HEMATOPOIETIC SYSTEM
 

IE-ONE MARROW (15) (33) (34)
 

ATROPHY, NOS 1 (315)
 

#THYMUS (14) (35) (35)
 

HEMORRHAGE 1 (755)
 

CIRCULATORY SYSTEM
 

NONE. ;
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

57
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

DIGESTIVE SYSTEM
 

*PHABYNX (15) (35) (35)
 

INFLAMMATION, SUPPURATIVE 1 (38)
 

URINARY SYSTEM
 

#KIDNEY (15) (35) (35)
 

INFLAMMATION, CHRONIC 1« (93X) 29 (83%) 20 (57%)
 

ENDOCRINE SYSTEM
 

fTKYROIB (14) (35) (35)
 

HYP1RPLASIA, FOLLICULAR-CELL 1 (3%)
 

REPRODUCTIVE SYSTEM
 

tPROSTATE (15) (32) (35)
 

INFLAMMATION, CHRONIC SUPPURATIV 1 (7%)
 

#TESIIS (15) (35) (35)
 

HEMORRHAGE 1 (3%)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/CORNEA (15) (35) (35)
 

INFLAMMATION, CHEONIC 1 <3%)
 

MUSCOLOSKEIETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDCMINAL CAVITY (15) (35) (35)
 

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

58
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL HCEPHOLOGY SUMMARY
 

NONE
 

t NUMEER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMEER OF ANIMALS NECROPSIED
 

59
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

FED ACETOHEXAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 15 35 35
 

ANIMALS NECROPSIED 14 35 34
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 14 35 34
 

INTEGUMENTARY SYSTEM
 

NCNE
 

RESPIRATORY SYSTEM
 

#TRACHEA (13) (35) (34)
 

INFLAMMATION, SUPPURATIVE 1 (B%) 2 (6%) 2 (6%)
 

HEMATOPOIETIC SYSTEM
 

NCNE
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER (13) (35) (34)
 

HYPERPLASIA, FOCAL 1 (3%)
 

HYPEEPLASIA, RETICULUM CELL 1 (3X)
 

URINARY SYSTEM
 

#KIDNEY (14) (35) (34)
 

INFLAMMATION, CHRONIC 7 (50*) 4 (11X) 1 (3%)
 

ENDOCRINE SYSTEM
 

_ NO_NE
 

# NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

60
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

REPRODUCTIVE SYSTEM
 

#UTEEUS (14) (33) (31)
 

HYCROMETRA 2 (6%) 2 (6»)
 

HEMORRHAGE 1 (3%)
 

BECIDUAL ALTERATION, NOS 1 (3X)
 

fUTERUS/ENDOMETRIUM (14) (33) (34)
 

INFLAMMATION, SUPPURATIVE 3 (21S)
 

INFLAMMATION, CHKONIC SUPPURATIV 2 (14X) 7 (21X) 1 (3%)
 

HYPEEPLASIA, NOS 1 (1%)
 

HYPEEPLASIA, FOCAL 1 (3X)
 

tOVARY/OVIDUCT (14) (33) (34)
 

INFLAMMATION ,AMMATION, CHRONIC SUPPURATIV 1
 (38)
 

#OVAR Y (14) (33) (34)
 

INFLAMMATION, CHRONIC SUPPURATIV 2 (6») 1 (3%)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/CORNEA (14) (35) (34)
 

INFLAMMATION, CHRONIC SUPPURATIV 1 (3%)
 

*MIDELE EAR (14) (35) (34)
 

INFLAHMATION, CHRONIC SUPPURATIV 1 (3%)
 

MOSCULOSKEIETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTBER SYSTEMS
 

•MULTIPLE ORGAN S (14) (35) (34)
 

t NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF AHIHALS NECROPSIED
 

61
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL MOEPHOLOGY SUHBARY 

HO LESION BEPOBTED 2 6

AUTCLYSIS/NO NECEOPSY 1

* NUMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMEER OF ANIMALS NECROPSIED 

 14 

1 

62
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED ACETOHEXAMIDE IN THE DIET
 

63
 

TABLE D1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

FED ACETOHEXAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIBALS INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIHALS EXAMINED HISTOPATHOLOGICALLY

 15

 15

 15

 35

 35

 35

 35 

 34 

 34 

INTEGUMENTARY SYSTEM 

*SKIN

INFLAMMATION, PYOGBANULOMATOUS

 (15) (35)

 1 (3%) 

 (34) 

*SUBCUT TISSUE

INFLAMMATION, ACUTE SUPPURATIVE

 (15) (35) (34) 

1 (3X) 

RESPIHATOBY SYSTEM 

iLUNG/ERCNCHUS

HYPfRPLASIA, LYMPHOID

 (15) (35)

 1 (3%) 

 (33) 

#LUNG

ERONCHOPNEUflONIA SUPPURATIVE

INFLAMMATION, ACUTE SUPPURATIVE

ERCNCHCPNEUMONIA ACUTE SUPPURATI

 (15)

 3 (20%)

 1 (73S)

 (35)

 7 (20%)

 1 (3X) 

 (33) 

3 (9S) 

1 (3%) 

HEMATOPCIETIC SYSTEM 

#BCNE HAEROW

HYPOPLASIA, HEMATOPOIETIC

 (15) (32)

 1 {3%) 

 (34) 

tMESENTERIC L. NODE

HYPERPLASIA, LYMPHOID

 (14) (32)

 1 (3S) 

 (29) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

fLIVER

CYTOPLASMIg VACUQLIZATigN

 (15) (33) (34) 

i_I3»l. 

* NUMBER OF ANIMALS WITH TISSUE EXAHINED HICROSCOPICALLY 

* NUMBER OF ANIHALS NECROPSIED 

65
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

HYPEBPLASIA, NODULAR . 1 (3%)
 

HYPIBPLASIA, IYMPHOID 1 (3X)
 

*EUCDENUM (11) (30) (32)
 

HYPSRPLASIA, LYMPHOID 1 (3X)
 

URINAR Y SYSTEM
 

#KIDNEY (15) (33) (34)
 

INFLAMMATION, CHRONIC FOCAL 1 (7%)
 

ENDOCRINE SYSTZM
 

ITfiYROID (15) (35) (33)
 

HYPERPLASIA, FOLLICULAR-CELL 1 (38)
 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/CORNEA (15) (35) (34)
 

ULCER, NOS 1 (3%)
 

*MIEtLE EAR (15) (35)
 

INFLAHHATION, SUPPURATIVE 1 (7%) 1 (3S)
 

HUSCOIOSKELETAL SYSTEM
 

NONB
 

BODY CAVITIES
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIHALS NECROPSIED
 

66
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ALL OTHER SYSTEMS 

*HULTIPLE ORGANS 

HYPEBPLASIA, LYMPHOID 

(15)

1 (7JJ) 

 (35) (34) 

SPECIAL MOFPHOLOGY SUMMARY 

NC LESION REPORTED 

NO NECROPSY PERFORMED 

U 12

# NUMEER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

 21 

1 

67
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

FED ACETOHEXAMIDE IN THE DIET 

HIGH DOSE
 

35
 

30
 

30
 

(30)
 

(29)
 

(29)
 

9 (31%)
 

1 (3%)
 

1 (3%)
 

5 (17%)
 

1 (3%)
 

(28)
 

ANIMAL S INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

*SKIN
 

ULCER, CHBONIC
 

RESPIRATORY SYSTEM
 

#LUNG/ERCNCHUS
 

HYPERPLASIA, LYMPHOID
 

tLUNG
 

ERONCHOPNEUHONIA SUPPORATIVE
 

ERONCHOPNEUMONIA NECROTIZING
 

ERCNCHOPNEUHONIA ACUTE SUPPORATI
 

ERCNCHOPNEUMONIA CHRONIC SUPPUHA
 

NECROSIS, FOCAL
 

HYPERPLASIA, ALVEOLAE EPITHELIUM
 

HEMATOPOIETIC SYSTEM
 

NONE
 

CIRCUIATOBY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

•DUODENUM
 

METAPLASIA, OSSEOUS
 

HYPERPLASIA, LYMPHOID
 

UBINARY SYSTEM
 

__NONI
 

CONTROL
 

15
 

10
 

10
 

(10)
 

1 (10%)
 

(10)
 

1 (10%)
 

(10)


4 (40«)
 

1 (10%)
 

(8)
 

LOW DOSE
 

35
 

35
 

35
 

(35)
 

(34)
 

(34)
 

6 (18%)
 

4 (12%)
 

1 (3X)
 

1 (3%)
 

(34)
 

1 (3%)
 

1 (3%)
 

* NUMBER OF ABIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

68
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

ENDOCRINE SYSTEM
 

NONE
 

REPBCCUCTIVE SYSTEM
 

tUTEROS (10) (35) (30)
 

ANGIECTASIS 1 (10X)
 

fUTERUS/ENDOMETRIOB (10) (35) (30)
 

HYPiRPLASIA, EPITHELIAL 1 (10X)
 

HYPEBELASIA, CYSTIC 4 (no*) 9 (26X)
 

#OVARY (9) (34) (28)
 

INFLAMMATION, SUPPURATIVE 2 (6%)
 

NERVOUS SYSTEM
 

#BRAIN (10) (34) (30)
 

INFLABMATION, ACUTE SUPPURATIVE 1 (3X)
 

ABSCESS, NOS 1 (3%)
 

*TRIGEMINAL GANGLION (10) (35) (30)
 

INFLAMMATION, NOS 1 (3X)
 

SPECIAL SENSE ORGANS
 

*HIDDLE EAR (10) (35) (30)
 

INFLAMMATION, SOPPUHATIVE 1 (10%)
 

INFLAMMATION, CHHONIC SOPPDRATIV 1 (3%)
 

mJSCULOSKElETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

f NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

69
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ALL OTHER SYSTEMS
 

*HULTIPLE ORGANS (10) (35) (30)
 

HYPEBPIASIA, LYMPHOID 1 (10*) 2 (6X) 2 (7%)
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 9 8
 

NO NECROPSY PERFORMED 2
 

AUTOLYSIS/NO NECROPSY 5 3
 

* NUKBER OF ANIMALS WITH TISSUE EXAMINEE MICROSCOPICALLY
 

* NUMEER OF ANIHALS NECROPSIED
 

70
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED ACETOHEXAMIDE IN THE DIET
 

71
 

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

Fed Acetohexamide in the Diet3
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Hematopoietic System: Leukemia*5 0/15 (0) 10/35 (29) 4/35 (11)
 

P Values0 >d N.S. P = 0.018 N.S.
 

Departure from Linear Trend6 P = 0.007
 

Relative Risk (Matched Control)f Infinite Infinite
 

Lower Limit 1.362 0.423
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor — 77 93
 

Pituitary: Chromophobe Carcinomab 0/11 (0) 2/33 (6) 1/33 (3)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f Infinite Infinite
 

Lower Limit 0.108 0.019
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor — 106 105
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

(continued) 

Topography: Morphology


Pituitary: Chroinophobe Adenoma
 

or Carcinoma5


P Valuesc»d


Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Thyroid: C-cell Carcinoma5


P Valuesc«d


Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Fed Acetohexaraide

Matched
 

 Control
 

 0/11 (0)
 

 N.S.
 

—
 

 1/14 (7)
 

 N.S.
 

 107
 

 in the Diet3 

Low
 

Dose
 

3/33 (9)
 

N.S.
 

Infinite
 

0.221
 

Infinite
 

106
 

2/35 (6)
 

N.S.
 

0.800
 

0.047
 

45.853
 

106
 

High
 

Dose
 

5/33 (15)
 

N.S.
 

Infinite
 

0.464
 

Infinite
 

105
 

2/35 (6)
 

N.S.
 

0.800
 

0.047
 

45.853
 

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

Fed Acetohexaraide in the Dieta 

(continued) 

Topography: Morphology


Thyroid: C-cell Adenoma
 

or Carcinomab


P Valuesc»d


Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Ul Pancreatic Islets: Islet-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


Matched Low High 

 Control Dose Dose 

 2/14 (14) 4/35 (11) 6/35 (17) 

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

0.800 1.200 

0.135 0.257 

8.252 11.299 

 107 106 105 

 3/14 (21) 0/35 (0) 1/35 (3) 

 P = 0.039(N) P = 0.020(N) N.S. 

P = 0.014 

0.000 0.133 

0.000 0.003 

0.648 1.528 

 103 106 — 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Acetohexamide in the Dieta
 

(continued)
 

Matched Low High
 

Topography; Morphology Control Dose Dose
 

Testis: Interstitial-cell Tumorb 15/15 (100) 34/35 (97) 29/35 (83)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Control) f 0.971 0.829
 

Lower Limit 0.000 0.000
 

Upper Limit 1.066 1.046
 

Weeks to First Observed Tumor 99 77 86
 

^J aDosed groups received 10,000 or 20,000 ppm in feed.
 

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

G

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 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 < 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 < 0.05 for any comparison.
 

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 

Fed Acetohexamide in the Diet3 

Matched Low High
 

Topography; Morphology Control Dose Dose
 

Lung: Alveolar/Bronchiolar
 

Adenomab 0/14 (0) 2/35 (6) 1/34 (3)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Matched Control) ^ Infinite Infinite
 

Lower Limit 0.125 0.023
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor — 86 106
 

Hematopoietic System: Leukemia*5 0/14 (0) 7/35 (20) 4/34 (12)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Matched Control) f Infinite Infinite
 

Lower Limit 0.834 0.410
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 79 73
 
00

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

(continued)
 

Topography^: Morphology


Urinary Bladder: Transitional-cell
 

Carcinomab


P Valuesc'd


Relative Risk (Matched Control) f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


 Pituitary: Chromophobe Carcinomab


P Valuesc»d


Relative Risk (Matched Control) ^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Fed Acetohexamide in the Diet3 

Matched Low High 

 Control Dose Dose 

 1/14 (7) 0/35 (0) 0/34 (0) 

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

0.000 0.000 

0.000 0.000 

7.42L 7.633 

__ __ 

 88 

 1/12 (8) 3/27 (11) 2/30 (7) 

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

1.333 0.800 

0.125 0.047 

67.223 45.610 

 72 85 70 
__ 

Table E2. Analyses

(continued) 

Topography: Morphology

Pituitary: Chromophobe Adenoma 

or Carcinomab


P Valuesc»d


Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Thyroid: C-cell Carcinomab


P Values0. d


Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


 of the Incidence of

Fed Acetohexamide in

Matched
 

 Control
 

 2/12 (17)
 

 N.S.
 

 72
 

 1/13 (8)
 

 N.S.
 

 107
 

 Primary Tumors in Female Rats 

 the Diet3 

Low
 

Dose
 

10/27 (37)
 

N.S.
 

2.222
 

0.600
 

18.632
 

85
 

1/32 (3)
 

N.S.
 

0.406
 

0.006
 

30.928
 

86
 

High
 

Dose
 

7/30 (23)
 

N.S.
 

1.400
 

0.334
 

12.617
 

0/34 (0)
 

N.S.
 

0.000
 

0.000
 

7.090
 

70 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Acetohexamide in the Diet3
 

(continued)
 

Topography: Morphology
 

Thyroid: C-cell Adenoma
 

or Carcinoma^3
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

00
 

o
 Mammary Gland: Fibroadenoraa^
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched


Control


1/13 (8)


N.S.


107


4/14 (29)


P = 0.003(N)


72


 Low
 

 Dose
 

 2/32 (6)
 

 N.S.
 

0.813
 

0.048
 

46.435
 

 86
 

 4/35 (11)
 

 N.S.
 

0.400
 

0.091
 

1.912
 

 107
 

High
 

Dose
 

1/34 (3)
 

N.S.
 

0.382
 

0.005
 

29.166
 

106
 

0/34 (0)
 

P = 0.005(N)
 

0.000
 

0.000
 

0.430
 

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

Fed Acetohexamide in the Dieta
 

(continued)
 

Topography; Morphology


Uterus: Endometrial Stromal
 

Polypb


P Valuesc»d


Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


oo
 

Uterus: Sarcoma, NOS (not otherwise
 

specified), or Endometrial
 

Stromal Sarcomab


P Valuesc»d


Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 1/14 (7)
 

 N.S.
 

 107
 

 2/14 (14)
 

 N.S.
 

 107
 

Low High 

Dose Dose 

5/33 (15) 4/34 (12) 

N.S. N.S. 

2.121 1.647 

0.279 0.190 

96.931 78.533 

86 106 

1/33 (3) 1/34 (3) 

N.S. N.S. 

0.212 0.206 

0.004 0.004 

3.821 3.714 

107 99 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Acetohexamide in the Dieta
 

(continued)
 

a

Dosed groups received 10,000 or 20,000 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 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 < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

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

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 FED ACETOHEXAMIDE IN THE DIET
 

83
 

Table Fl.


Topography: Morphology


Lung: Alveolar/Bronchiolar
 

Carcinoinab


P Valuesc»d


Relative Risk (Matched Control)^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


oo
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma*3


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 Mice
 

Fed Acetohexamide in the Diet3 

Matched Low High 

 Control Dose Dose 

 1/15 (7) 2/35 (6) 0/33 (0) 

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

0.857 0.000 

0.050 0.000 

49.128 8.417 

__ 

 107 103 

 2/15 (13) 3/35 (9) 2/33 (6) 

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

0.643 0.455 

0.085 0.037 

7.208 5.871 

 107 98 107 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

(continued)
 

Topography; Morphology
 

Hematopoietic System: Malignant
 

Lymphoma"
 

P Values0 »d
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)^
 

Lower Limit
 

Upper Limit
 

oo
 Weeks to First Observed Tumor
 

Liver: Ilepatocellular Carcinoma'3
 

P Values0 »d
 

Relative Risk (Matched Control )f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Acetohexamide in the Diet3 

Matched Low High 

Control Dose Dose 

1/15 (7) 9/35 (26) 3/34 (9) 

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

P = 0.030 

3.857 1.324 

0.627 0.120 

162.855 67.356 

101 75 99 

2/15 (13) 1/33 (3) 0/34 (0) 

P = 0.04KN) N.S. N.S. 

0.227 0.000 

0.004 0.000 

4.101 1.463 

103 93 — — 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice 

Fed Acetohexamide in the Diet3 

(continued) 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Liver: Hepatocellular Adenoma
 

or Carcinoma*3 3/15 (20) 2/33 (6) 1/34 (3)
 

P Values0 »d P = 0.049(N) N.S. N.S.
 

Relative Risk (Matched Control)f 0.303 0.147
 

Lower Limit 0.029 0.003
 

Upper Limit 2.421 1.688
 

Weeks to First Observed Tumor 103 93 107
 

oo 

a

Dosed groups received time-weighted average doses of 6,359 or 12,718 ppm in feed.
 

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

G

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 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 < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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 < 0.05 for any comparison.
 

95% confidence interval of the relative risk between each dosed group and the control
 

group.
 
Table F2. Analyses of the Incidence of

Fed Acetohexamide in

 Primary Tumors

 the Diet3 

 in Female Mice 

Tomography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Hematopoietic System:

Lymphomab

 Malignant 

 1/10 (10) 3/35 (9) 2/30 (7) 

P Valuesc » d N.S. N.S. N.S. 

Relative Risk (Matched Control) f 

Lower Limit 

Upper Limit 

0.857 

0.083 

43.687 

0.667 

0.041 

38.024 

Weeks to First Observed Tumor 101 88 98 

oo 

00 Liver: Hepatocellular Carcinoma*3

 1/10 (10) 1/35 (3) 0/30 (0) 

P Values0 » d N.S. N.S. N.S. 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 107 

0.286 

0.004 

21.825 

108 

0.000 

0.000 

6.165 

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

Fed Acetohexamide in the Diet3
 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Liver: Hepatocellular Adenoma
 

or Carcinoma" 1/10 (10) 1/35 (3) 1/30 (3)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.286 0.333
 

Lower Limit 0.004 0.005
 

Upper Limit 21.825 25.331
 

Weeks to First Observed Tumor 107 108 101
 OO
 

VO
 a

Dosed groups received time-weighted average doses of 6,359 or 12,718 ppm in feed.
 

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

G

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 dosed group is the probability level for the Fisher exact test for
 

the comparison of that uosed 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 dosed group than in the control group.
 

e

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

95% confidence interval of the relative risk between each dosed group and the control
 

group.
 

Review of the Bioassay of Acetohexamide* for Carcinogenlcity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

March 6, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with 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 (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academia, industry, organized labor, public interest
 

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

research organizations. Members have been selected on the
 

basis of their experience in carcinogenesis or related fields
 

and, collectively, provide expertise in chemistry, biochemistry,
 

biostatistics, toxicology, pathology, and epidemiology.
 Representatives of various Governmental agencies participate
 

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

of the Clearinghouse is charged with the responsibility of
 

providing a peer review of reports prepared on NCI-sponsored
 

bioassays of chemicals studied for carcinogenicity. It is in
 

this context that the below critique is given on the bioassay
 

of Acetohexamide for carcinogenicity.
 

The primary reviewer briefly described the experimental
 

design and the conditions under which Acetohexamide was
 

studied. He noted an increase in the incidence of leukemia
 

in treated rats and lymphomas in treated mice. However,
 neither of the lesions was statistically significant when
 

compared to the historical control incidence. A Program
 

pathologist commented that lymphomas frequently present a
 

problem to interpret since their spontaneous incidence is
 

so variable. The tendency is to draw a conservative
 

conclusion when a response is observed in the low dose
 

group but not in the high dose one.
 

A discussion ensued on the mononuclear cell leukemia
 

reported in treated rats. Program staff noted the variability
 

of this lesion from one study to another, as well as between
 

laboratories. In the laboratory conducting this particular
 

study, it was noted that the leukemia incidence observed
 

in treated rats was within the spontaneous tumor range.
 

91
 
Further discussion occurred on the probability of a
 

false positive or negative, given the induced and spontaneous
 

tumor rates in the mice. A Subgroup member suggested that
 

the conclusion be reworded as follows: Under the conditions
 

of test, a statistically significant increase in the incidence
 

of lymphomas was found in the treated male mice; however,
 

it cannot be determined if the increased incidence was
 

compound related because of the spontaneous occurrence of
 

this tumor type. It was moved and seconded that the report
 

be accepted as modified by this statement. (No vote was
 

taken on the motion.)
 

After further discussion, a Subgroup member moved
 

that the results of the study were inadequate to reach
 

a conclusion regarding the carcinogenicity of Acetohexamide.
 

The motion was seconded and approved by all the members
 

except Dr. Highland and Dr. Weisburger, who abstained.
 

Members present were:
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Arnold Brown, Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

E. Cuyler Hammond, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Henry Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Michael Shimkin, University of California at San Diego
 

John Weisburger, American Health Foundation
 

Sidney Wolfe, Health Research Group
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

U. S. GOVERNMENT PRINTING OFFICE : 1978 — 260-899/3119 

92 

DHEW Publication No. (NIH) 78-850