National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 140 

1978 

BIOASSAY OF 

PIVALOLACTONE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 1955-45-9 

NCI-CG-TR-140 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

PIVALOLACTONE
 

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. (NIK) 78-1395
 

REPORT ON THE BIOASSAY OF PIVALOLACTONE
 

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 pivalolactone conducted for the Carcinogenesis Testing Program, Division
 

of Cancer Cause arid Prevention, National Cancer Institute (NCI),
 

National Institutes of Health, Bethesda, Maryland· This is one of a
 

series of experiments designed to determine whether selected chemicals
 

have the capacity to produce cancer in animals· Negative results, in
 

which the test animals do not have a significantly greater incidence
 

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

chemical is not a carcinogen because the experiments are conducted
 

under a limited set of circumstances· Positive results demonstrate
 

that the test chemical is carcinogenic for animals under the conditions of the test and indicate a potential risk to man· The actual
 

determination of the risk to man from animal carcinogens requires a
 

wider analysis·
 

CONTRIBUTORS: This bioassay of pivalolactone was conducted by Litton
 

Bionetics, Inc., Bethesda, Maryland, initially under direct contract
 

to the NCI and currently under a subcontract to Tracor Jitco, Inc.,
 

prime contractor for the NCI Carcinogenesis Testing Program.
 

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

Weisburger (1,3)· The principal investigators for the contract were
 

Dr. S. M. Garner (4,5) and Dr. B. M. Ulland (4,5). Mr. S. Johnson
 

(4) was the coprincipal investigator for the contract. Animal treatment and observation were supervised by Mr. R. Cypher (4), Mr· D· S.
 

Howard (4) and Mr. H. D. Thornett (4); Mr. H. Paulin (4) analyzed
 

dosed feed mixtures. Ms. J. Blalock (4) was responsible for data
 

collection and assembly. Chemical analysis was performed by Midwest
 

Research Institute (6) and the analytical results were reviewed by
 

Dr. N. Zimmerman (7).
 

Histopathologic examinations were performed by Dr. A. DePaoli
 

(4) at Litton Bionetics, Inc·, the pathology narratives were written
 

by Dr. A. DePaoli (4), and the diagnoses included in this report
 

represent the interpretation of this pathologist· Histopathology
 

findings and reports were reviewed by Dr· R. L. Schueler (8).
 

Ill
 
Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (9); the
 

statistical analysis was performed by Mr. W. W. Belew (7,10) and
 

Mr· R. M· Helfand (7), using methods selected for the Carcinogenesis
 

Testing Program by Dr. J. J. Gart (11).
 

This report was prepared at METREK, a Division of The MITRE Corporation (7) under the direction of the NCI. Those responsible for
 

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

(7), task leader Ms. P. Walker (7), senior biologist Mr. M. Morse
 

(7), biochemist Mr. S. C. Drill (7), and technical editor Ms. P. A.
 

Miller (7). The final report was reviewed by members of the participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

Cueto, Jr. (1), Dr. J. F. Douglas (1), Dr. D. G. Goodman (1,12), Dr.
 

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

T. W. Orme (1), Dr. R. A. Squire (1,13), Dr. S. F. Stinson (1), Dr.
 

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

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 Now with the U.S. Environmental Protection Agency, 401 Μ Street
 

S.W., Washington, D.C.
 

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

4.	 Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Maryland.
 

5.	 Now with Hazleton Laboratories America, Inc., 9200 Leesburg
 

Turnpike, Vienna, Virginia.
 

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

Missouri.
 

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

Boulevard, McLean, Virginia.
 

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

Maryland.
 

iv
 
9.	 EG&G Mason Research Institute, 1530 East Jefferson Street,
 Rockville, Maryland·
 

10· Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

11· Now with The Solar Energy Research Institute, Cole Boulevard,
 

Golden, Colorado·
 

12· Now with Clement Associates, Inc·, 1010 Wisconsin Avenue, N.W.,
 Washington, D.C.
 

13· Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland·
 

SUMMARY
 

The bioassay of pivalolactone for possible carcinogenicity was
 

conducted using Fischer 344 rats and B6C3F1 mice. Pivalolactone in
 

water was administered by gavage, at either of two dosages, to groups
 

of 50 male and 50 female animals of each species. The high and low
 

dosages of pivalolactone utilized were, respectively, 300 and 150
 

mg/kg/day for rats and 150 and 75 mg/kg/day for mice. After a 103­

week period of compound administration for rats and a 102-week period of compound administration for mice, rats were observed for 2
 

additional weeks and mice for 1 additional week. Twenty animals of
 

each sex and species were placed on test as vehicle controls.
 

There was no significant positive association between dosage
 

and mortality for either rats or mice, and in both species, adequate
 

numbers of animals survived sufficiently long to be at risk from late-


developing tumors. Compound-related mean body weight depression was
 

not observed in either sex of either species. In addition, no adverse
 

clinical signs were observed among dosed mice. This evidence, plus
 

the relatively fast decomposition of pivalolactone in water, suggests
 

the possibility that the animals, and in particular the mice, may
 

have been able to tolerate a higher dose.
 

Statistically significant incidences of squamous-cell papillomas
 

and squamous-cell carcinomas of the forestomach were observed in rats
 

but not in mice. No other rare or unusual tumors were observed in
 

either species.
 

Under the conditions of this bioassay, pivalolactone was found
 

to be carcinogenic to both male and female Fischer 344 rats, producing
 

squamous-cell carcinomas and squamous-cell papillomas of the fore-


stomach. This study provided no evidence for the carcinogenicity of
 

pivalolactone in B6C3F1 mice of either sex.
 

vii
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 4
 

A. Chemicals 4
 

B· Dosage Preparation 5
 

C· Animals 5
 

D· Animal Maintenance 6
 

E· Gastric Intubation 7
 

F· Selection of Initial Dose Levels 8
 

G· Experimental Design 9
 

H· Clinical βηά Histopathologic Examinations 12
 

I. Data Recording and Statistical Analyses 14
 

III. CHRONIC TESTING RESULTS: RATS 19
 

A. Body Weights and Clinical Observations 19
 

B. Survival 19
 

C. Pathology 22
 

D. Statistical Analyses of Results 24
 

IV. CHRONIC TESTING RESULTS: MICE 34
 

A. Body Weights and Clinical Observations 34
 

B. Survival 34
 

C. Pathology 37
 

D. Statistical Analyses of Results 37
 

V. DISCUSSION 42
 

VI. BIBLIOGRAPHY 44
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH PIVALOLACTONE A-l
 

APPENDIX Β SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH PIVALOLACTONE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH PIVALOLACTONE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH PIVALOLACTONE D-l
 

IX
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 CHEMICAL STRUCTURE OF PIVALOLACTONE 2 

2 GROWTH CURVES FOR PIVALOLACTONE CHRONIC 

STUDY RATS 20 

SURVIVAL COMPARISONS OF PIVALOLACTONE 

CHRONIC STUDY RATS 21 

GROWTH CURVES FOR PIVALOLACTONE CHRONIC 

STUDY MICE 35 

SURVIVAL COMPARISONS OF PIVALOLACTONE 

CHRONIC STUDY MICE 36 

LIST OF TABLES 

Table Number Page 

DESIGN SUMMARY FOR FISCHER 344 RATS—PIVALOLACTONE GAVAGE EXPERIMENT 10 

DESIGN SUMMARY FOR B6C3F1 MICE—-PIVALOLACTONE GAVAGE EXPERIMENT 11 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

PIVALOLACTONE 25 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH PIVALOLACTONE 29 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

PIVALOLACTONE 38 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH PIVALOLACTONE 40 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH PIVALOLACTONE A-3 
LIST OF TABLES (Concluded) 

Table Number Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH PIVALOLACTONE A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH PIVALOLACTONE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH PIVALOLACTONE B-7 

CI SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH PIVALOLACTONE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH PIVALOLACTONE C-7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH PIVALOLACTONE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH PIVALOLACTONE D-7 

XI
 

I. INTRODUCTION
 

Pivalolactone (Figure 1) (NCI No. CO4126), an intermediate in
 

polymer preparation, was selected for bioassay by the National Cancer Institute because of the structural similarity of this compound
 

to β-propiolactone, a well-documented direct acting carcinogen (International Agency for Research on Cancer, 1974)·
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 3,3-dimethyl-2-oxethanone. It is
 

also called 3,3-dimethyl-2-oxetanone; 3,3-dimethy1-β-propiolactone;
 

dimethyl propiolactone; and pivalic acid lactone.
 

Pivalolactone has been used to prepare copolymers used in sutures, prosthetic devices, etc. (Schmitt and Epstein, 1976); to block
 

and graft copolymers with acrylics (Shor and Van Dyk, 1975), isoprene
 

and butadiene (Foss et al., 1976; Foss, 1975), and ethylenemethacrylic acid-vinyl acetate polymer (Sundet et al·, 1976); and to prepare
 

polyoxymethylenes (Radici et al·, 1975a; Radici et al.3 1975b).
 

Pivalolactone has also been used as a plasticizer for cyanoacrylate
 

adhesive (Brinkmann and Inoehl, 1975).
 

Specific production data for pivalolactone are not: available;
 

however, this compound does not appear to be produced or sold in
 

commercial quantities (in excess of 1000 pounds or $1000 in value
 

annually) in the United States (U.S. International Trade Commission,
 

1977).
 

The CAS registry number is 1955-45-9.
 
FIGURE 1

 

CHEMICAL STRUCTURE OF PIVALOLACTONE 
The potential for exposure to pivalolactone is greatest for
 

workers engaged in the preparation of this compound or pivalolactone
 

copolymers·
 
II. MATERIALS AND METHODS
 

A· Chemicals
 

Pivalolactone was purchased from Shell Laboratories, Amsterdam,
 

Holland· The manufacturer indicated that 15 ppm of the complex of
 

BF~ and tribenzylamine were added as a stabilizer to prevent polymerization· Chemical analysis was performed by Midwest Research Institute, Kansas City, Missouri· Thin-layer chromatographic plates
 

utilizing two solvent systems (petroleum ether:dioxane and methanol)
 

and visualized by hydroxylamine-ferric chloride each yielded one
 

spot· Infrared and nuclear magnetic resonance analyses were consistent with that expected on the basis of the structure· Ultraviolet/
 

visible spectrophotometric data indicated no absorbance between 210
 

and 800 nm, as would be expected· High-pressure liquid chromatography indicated the presence of two impurities·
 

A second batch of the compound was purchased from the same supplier· Thin-layer chromatography utilizing the same solvent systems
 

as the first batch showed only one spot· High-pressure liquid chromatography showed the presence of two impurities· Infrared and nuclear
 

resonance analyses were consistent with that expected on the basis of
 

the structure· Ultraviolet/visible analysis again showed no peaks,
 

as would be expected· These results suggested that the second batch
 

was similar in purity to the first·
 
A lactone colorimetric analysis of the stability of pivalolactone
 

in water was performed by Midwest Research Institute. The results
 

indicated that a 0.5 mg/ml solution of pivalolactone in water was
 

decomposed (97.1 percent) after 24 hours.
 

Throughout this report, the term pivalolactone is used to refer
 

to this material.
 

B. Dosage Preparation
 

Fresh solutions of pivalolactone in distilled water (Borden Polar
 

Water Company, Beltsville, Maryland) were prepared on each day that
 

intubation was performed. Excess portions of the mixtures were disposed of rather than stored. The concentration of pivalolactone in
 

distilled water ranged from 1.5 to 3 percent in rats, and from 0.68
 

to 1.36 percent in mice.
 

C. Animals
 

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

through contracts of the Division of Cancer Treatment, National
 

Cancer Institute. All rats were supplied by A. R. Schmidt, Madison,
 

Wisconsin. All mice were supplied by Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts.
 

Rats and mice were approximately 4 weeks old when received.
 

Upon receipt, animals were examined for visible signs of disease or
 
parasites· Obviously ill or runted animals were culled. The remaining animals were quarantined for 2 weeks prior to initiation of test.
 

Animals which did not manifest clinical signs of disease were placed
 

on test at this time. Animals were assigned to groups and distributed
 

among cages so that the average body weight per cage was approximately
 

equal for a given species and sex.
 

D. Animal Maintenance
 

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


controlled rooms. The temperature range was 20° to 26°C and the
 

relative humidity was maintained between 45 and 55 percent. Incoming
 

air was filtered through HEPA filters (Flanders Filters, McLean, Virginia) at a rate of 12 to 15 complete changes of room air per hour.
 

Fluorescent lighting was provided 8 hours per day (9:00 a.m. to 5:00
 

p.m.).
 

All rats were housed four per cage by sex and all mice five per
 

cage by sex. Throughout the study, dosed and control animals of both
 

species were housed in polycarbonate cages (Lab Products, Inc., Garfield, New Jersey) suspended from aluminum racks. Racks were fitted
 

with a continuous stainless steel mesh lid over which a sheet of filter paper was firmly secured. Filter paper was changed at 2-week intervals, when the racks were sanitized. Clean cages and bedding were
 

(R)
 

provided twice weekly* Ab-sorb~dri hardwood chip bedding (Wilner
 

Wood Products Company, Norway, Maine) was used in polycarbonate cages
 

for the entire study*
 
Acidulated water (pH 2.5) was supplied to animals in water bottles filled by an automated metering device, which was checked daily
 

for diluting accuracy. Water bottles were changed twice weekly and
 

sipper tubes were washed at weekly intervals. All animals were
 

®
 supplied with Wayne Lab-Blox meal (Allied Mills, Inc., Chicago,
 

Illinois) in hanging stainless steel hoppers, which were refilled
 

three times per week and sanitized weekly. Food and water were
 

available ad libitum for both species.
 

All dosed and control rats were housed in a room with other
 

rats receiving diets containing EDTA trisodium salt (150-38-9);
 

rats receiving I.P. injections of methiodal sodium (126-31-8); and
 

other rats intubated with lithocholic acid (434-13-9).
 

All dosed and control mice were housed in a room with other
 

mice receiving diets containing 2,4-dimethoxyaniline hydrochloride
 

(54150-69-5); 4f-(chloroacetyl)-acetanilide (140-49-8); nithiazide
 

(139-94-6); p-phenylenediamine dihydrochloride (624-18-0); 4-nitroo-phenylenediamine (99-56-9); l-phenyl-3-methyl-5-pyrazolone (89­

25-8); and other mice intubated with trimethylphosphate (512-56-1);
 

3-(chloromethyl)pyridine hydrochloride (3099-31-8); and 2-(chloromethyl) pyridine hydrochloride (6959-47-3).
 

. Gastric Intubation
 

Intubation was performed for three days per week on a mg/kg body
 

weight basis, utilizing the most recently observed group mean body
 

CAS registry numbers are given in parentheses.
 
weight as a guide for determining the dose· All animals were weighed
 

and dosages adjusted once monthly, based on group mean body weight.
 

Animals of each sex within a dosed group received the same dosage·
 

Thus, although the ratio of dose to weight remained constant, the
 

total dosage administered fluctuated with an increase or decrease in
 

group mean body weight·
 

F. Selection of Initial Dose Levels
 

In order to establish the maximum tolerated dosages of pivalolactone for administration to dosed animals in the chronic study,
 

subchronic toxicity tests were conducted with both rats and mice.
 

Animals of each species were distributed among six groups, each
 

consisting of five males and five females· Pivalolactone mixed
 

with distilled water was introduced by gavage to five of the six rat
 

groups at dosages of 316, 464, 681, 1000, and 1470 mg/kg/day and to
 

five of the six mouse groups at dosages of 68, 100, 147, 215, and
 

316 mg/kg/day. The sixth group of each species served as a control,
 

receiving only distilled water by gavage. Intubation was performed
 

three times per week for 7 weeks, followed by a 1-week observation
 

period to detect any delayed toxicity. Individual body weights were
 

recorded weekly. At the end of the observation period, all survivors
 

were sacrificed and necropsied.
 

At a dosage of 1470 mg/kg/day, four male and four female rats
 

died; one additional male rat died at a dosage of 1000 mg/kg/day.
 

At the end of the subchronic test, the mean body weight gain of male
 

8
 
rats receiving 316 mg/kg/day was 23 percent greater than the mean
 

body weight gain of their controls, while female rats receiving the
 

same dosage displayed a mean body weight gain which was 4 percent
 

greater than that of their controls. The high dose selected for
 

administration to rats in the chronic bioassay was 300 mg/kg/day·
 

At a dosage of 316 mg/kg/day, two male and three female mice
 

died· At the end of the subchronic test, the mean body weight gain
 

of male mice receiving 215 mg/kg/day was 7 percent greater than the
 

mean body weight gain of their controls, while females receiving the
 

same dosage displayed a mean body weight gain which was 18 percent
 

less than that of their controls· The mean body weight gain of male
 

mice receiving 147 mg/kg/day was 5 percent greater than that of their
 

controls, while females receiving the same dosage displayed a mean
 

body weight gain which was 11 percent less than that of their controls. The high dose selected for administration to mice in the
 

chronic bioassay was 150 mg/kg/day.
 

G· Experimental Design
 

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

and untreated observation periods) are summarized in Tables 1 and 2.
 

All rats were approximately 6 weeks old at the time the test was
 

initiated and were placed on test simultaneously. The dosages administered were 300 and 150 mg/kg/day. Throughout this report, those
 

rats receiving the former dosage are referred to as the high dose
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

PIVALOLACTONE GAVAGE EXPERIMENT
 

INITIAL OBSERVATION PERIOD 

GROUP PIVALOLACTONE TREATED UNTREATED 

SIZE DOSAGEa (WEEKS) (WEEKS) 

MALE
 

CONTROL 20 0 - 105b
 

LOW DOSE 50 150 103
 

0 2
 

HIGH DOSE 50 300 103
 

0 2
 

FEMALE
 

CONTROL 20 0 105b
 

LOW DOSE 50 150 103
 

0 2
 

HIGH DOSE 50 300 103
 

0 2
 

Dosages, given in mg/kg body weight, were administered by gavage
 

three days per week.
 

Gavaged with distilled water 3 times per week for 103 weeks.
 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

PIVALOLACTONE GAVAGE EXPERIMENT
 

INITIAL OBSERVATION PERIOD 

GROUP PIVALOLACTONE TREATED UNTREATED 

SIZE DOSAGEa (WEEKS) (WEEKS) 

MALE
 

CONTROL 20 0 - 103b
 

LOW DOSE 50 75 102
 

0 1
 

HIGH DOSE 50 150 102
 

0 1
 

FEMALE
 

CONTROL 20 0 - 103b
 

LOW DOSE 50 75 102
 

0 1
 

HIGH DOSE 50 150 102
 

0 1
 

Dosages, given in mg/kg body weight, were administered by gavage
 

three days per week.
 

Gavaged with distilled water 3 times per week for 102 weeks.
 

11
 
groups and those receiving the latter dosage are referred to as the
 

low dose groups. All dosed rats were administered pivalolactone at
 

the dosages indicated for 103 weeks, followed by a 2-week observation
 

period.
 

All mice were approximately 6 weeks old at the time the test was
 

initiated and were placed on test simultaneously. The dosages administered were 150 and 75 mg/kg/day. Throughout this report, those
 

mice receiving the former dosage are referred to as the high dose
 

groups and those receiving the latter dosage are referred to as the
 

low dose groups. All dosed mice were administered pivalolactone at
 

the dosages indicated for 102 weeks, followed by a 1-week observation
 

period.
 

Vehicle control animals were intubated with 10 ml/kg distilled
 

water three times per week. All survivors were sacrificed and necropsied at the end of the observation period.
 

H. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the experiment. From the first day, all animals were inspected twice daily
 

for mortality. Food consumption data were collected at monthly
 

intervals from 20 percent of the animals in each group. Rats were
 

weighed at monthly intervals throughout the bioassay. Body weights
 

of mice were recorded once a week for the first 4 weeks, and once
 

monthly for the remainder of the bioassay.
 

12
 
All moribund animals or animals that developed large» palpable
 

masses that jeopardized their health were sacrificed· A necropsy
 

was performed on each animal regardless of whether it died, was sacrificed when moribund, or was sacrificed at the end of the bioassay.
 

The animals were euthanized by carbon dioxide asphyxiation, and were
 

immediately necropsied. The histopathologic examination consisted of
 

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

gross lesions taken from sacrificed animals and, whenever possible,
 

from animals found dead·
 

Tissues were preserved in a 10 percent neutral buffered formalin
 

solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to microscopic examination.
 

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

nodes, thymus, heart, salivary gland, liver, gallbladder (mice), pancreas, esophagus, stomach, small intestine, large intestine, kidney,
 

urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis,
 

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

A few tissues were not examined for some animals, particularly
 

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

preclude histopathologic interpretation. Thus, the number of animals
 

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

13
 
animals that were recorded in each group at the time that the test
 

was initiated·
 

I. Data Recording and Statistical Analyses
 

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

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

as recommended by the International Union Against Cancer (Berenblum,
 

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

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

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections·
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored· Statistical analyses for a possible dose-related effect
 

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

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

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

14
 
for all tests except the departure from linearity test, which is only
 

reported when its two-tailed P-value is less than 0·05·
 

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

(Miller, 1966, pp. 6-10) requires that the P-value for any comparison
 

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

15
 
used, it is discussed in the narrative section· It is not, however,
 

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

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

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

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

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

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

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

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

16
 
during which animals died naturally or were sacrificed was entered as
 

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

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

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

t c t
 

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

and ρ 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
 

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

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

17
 
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, Ρ < 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.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

There was no evidence of dose-related mean body weight depression in either male or female rats (Figure 2).
 

No abnormal clinical signs were recorded.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

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

For male rats the Tarone test for association between dosage and mortality and the Cox tests comparing both high dose to control and low
 

dose to control did not show any significant associations between
 

dosage and mortality. For females the Tarone test indicated a significant (P = 0.004) dose-related trend for accelerated mortality.
 

However, a significant (P = 0.026) departure from linear trend was
 

also present due to the relatively high survival of the low dose
 

group. The Cox tests comparing high dose to control and low dose to
 

control were not significant.
 

Adequate numbers of male rats were at risk from late-developing
 

tumors, as 62 percent (31/50) of the high dose, 76 percent (38/50) of
 

the low dose, and 75 percent (15/20) of the control group survived on
 

test until the termination of the study.
 

For female rats, 56 percent (28/50) of the high dose, 86 percent
 

(43/50) of the low dose, and 80 percent (16/20) of the control group
 

19
 
FIGURE 2

 

GROWTH CURVES FOR PIVALOLACTONE CHRONIC STUDY RATS 

20 
FIGURE 3

 

SURVIVAL COMPARISONS OF PIVALOLACTONE CHRONIC STUDY RATS 

21 
survived on test until termination of the study, thus providing adequate numbers at risk from late-developing tumors.
 

C· Pathology
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A (Tables Al and A2); findings on nonneoplastic lesions are
 

summarized in Appendix C (Tables CI and C2).
 

There was an increased incidence of gastric neoplasms in the
 

dosed groups when compared with the controls. Other neoplasms
 

occurred with a greater frequency in dosed rats than controls.
 

These lesions, however, often occur spontaneously in Fischer 344
 

rats.
 

In addition to the neoplastic lesions, a large number of degenerative, proliferative and inflammatory changes were encountered in
 

dosed and control animals (Appendix C) . Most of these nonneoplastic lesions are commonly seen in aged laboratory rats. However, a
 

greater number of proliferative changes occurred in the stomach of
 

dosed rats than in their controls.
 

The incidences of the neoplastic and proliferative lesions in
 

the stomach were as fellows:
 

22
 
MALES 1FEMALES
 

Low High Low High
 

Control Dose Dose Contro] L Dose Dose
 

Number of Animals with
 

Stomachs Examined
 

Histopathologically (19) (49) (48) (20) (50) (50)
 

Squamous-Cell
 

Care inoma 0 1 7 0 0 2
 

Squamous-Cell
 

Papilloma 0 5 14 0 2 9
 

Epithelial
 

Hyperplasia 5 39 14 1 32 26
 

Inflammation 3 11 3 3 9 4
 

Only the larger proliferative gastric lesions were noted during
 

the gross examination. These were usually reported as single papuliferous growths, although occassional multiple growths were noted
 

in the same animal· In most animals gross changes were not evident.
 

Microscopically, the proliferative lesions usually were focal,
 

located in the limiting ridge of the forestomach. Most squamous-cell
 

carcinomas were papillary. The lining epithelium was well differentiated, markedly thickened and variably keratinized. Chords of epithelial cells extended from the surface into the submucosa and the
 

muscularis mucosa. Invading cells were usually hyperchromatic and
 

somewhat atypical with variable mitotic activity. The squamous-cell..
 

papillomas were usually smaller, did not demonstrate local invasion,
 

cellular atypia or any degree of mitotic activity. Squamous-cell
 

hyperplasia varied greatly from minimal thickening of the squamous
 

23
 
epithelium to marked focal thickening of the lining epithelium with
 

early papillary proliferation. The hyperplastic changes, while
 

usually focal and involving the limiting ridge, occasionally were
 

multifocal and affected other portions of the forestomach. The hyper-


plastic changes were least severe (minimal to mild) in the affected
 

control animals, becoming more pronounced in the dosed groups· A
 

mild focal gastritis was associated with these proliferative changes.
 

The inflammation was characterized by infiltration of the mucosa in
 

the region of proliferation with a few granulocytes and lymphocytes.
 

The focal gastritis was most evident in the early proliferative lesions.
 

While mild hyperplastic changes were noted in the control groups,
 

the increased incidence and severity of these hyperplastic lesions in
 

the dosed groups, coupled with the occurrence of papillomas and carcinomas in the dosed animals, suggests that pivalolactone was responsible for both neoplasms and increased hyperplasia.
 

The results of this pathologic examination indicate that
 

pivalolactone was carcinogenic, inducing squamous-cell carcinomas,
 

papillomas and focal hyperplasia in the forestomach of Fischer 344
 

rats.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

24
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH PIVALOLACTONE5
 

TOPOGRAPHY:MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

 Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma*3
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Neoplastic Nodule*3
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW HIGH 

CONTROL DOSE DOSE 

1/20(0.05) 4/50(0.08) 2/50(0.04) 

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

1.600 0.800 

0.175 0.045 —__ 77.169 46.273 

105 105 105 

3/20(0.15) 5/50(0.10) 4/50(0.08) 

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

——— 0.667 0.533 

— 0.147 0.102 

4.014 3.410 

84 95 69 

0/20(0.00) 3/50(0.06) 2/50(0.04) 

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

——_ Infinite Infinite 

0.250 0.123 

___ Infinite Infinite 

105 105 
TOPOGRAPHY: MORPHOLOGY
 

Stomach: Squamous-Cell Carcinoma
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Stomach: Squamous-Cell Papilloma or
 

Squamous-Cell Carcinoma*5
 

) 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

CONTROL
 

0/19(0.00)
 

 = 0.011
 

_——
 

0/19(0.00)
 

 < 0.001
 

2/18(0.11)
 

N.S.
 

84
 

LOW
 

DOSE
 

1/49(0.02)
 

N.S.
 

Infinite
 

0.021
 

Infinite
 

98
 

6/49(0.12)
 

N.S.
 

Infinite
 

0.649
 

Infinite
 

85
 

2/47(0.04)
 

N.S.
 

0.383
 

0.030
 

5.033
 

103
 

HIGH
 

DOSE
 

7/48(0.15)
 

N.S.
 

Infinite
 

0.804
 

Infinite
 

98
 

21/48(0.44)
 

 < 0.001
 

Infinite
 

2.823
 

Infinite
 

76
 

3/44(0.07)
 

N.S.
 

0.614
 

0.079
 

6.973
 

105
 
___ 

TABLE 3 (CONTINUED)
 

TOPOGRAPHY:MORPHOLOGY . CONTROL 

Adrenal: Pheochromocytoma or Pheochromocytoma, Malignant" 3/20(0.15) 

 Values0 N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 105 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma** 1/20(0,05)
 

 Values0 N.S.
 

Relative Risk (Control)
 

Lower Limit .
 

Upper Limit
 

Weeks to First Observed Tumor 105
 

Testis: Interstitial-Cell Tumor 12/20(0.60)
 

 Values0 N.S.
 

Relative Risk (Control)
 

Lower Limit —
 

Upper Limit —
 

Weeks to First Observed Tumor 92
 

LOW
 

DOSE
 

4/48(0.08)
 

N.S.
 

0.556
 

0.106
 

3.546
 

105
 

6/48(0.13)
 

N.S.
 

2.500
 

0.339
 

112.370
 

105
 

42/50(0.84)
 

 - 0.035
 

1.400
 

0.980
 

2.115
 

69
 

HIGH
 

DOSE
 

2/49(0.04)
 

N.S.
 

0.272
 

0.025
 

2.233
 

105
 

2/46(0.04)
 

N.S.
 

0.870
 

0.049
 

50.196
 

105
 

40/49(0.82)
 

N.S.
 

1.361
 

0.946
 

2.096
 

64
 
00 

TABLE 3 (CONCLUDED)
 

Treated groups received doses of 150 or 300 mg/kg by gavage.
 

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

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

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

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

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

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

ho
 
TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH PIVALOLACTONEa
 

TOPOGRAPHY:MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Hematopoietic System:

Malignant Lymphoma*3 

 Values0 

 Leukemia or 

4/20(0.20) 

 = 0.010(Ν) 

1/50(0.02) 

 = 0.021(N) 

1/50(0.02) 

 = 0.021(10 

Departure from Linear Trend = 0.032 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.100 

0.002 

0.944 

0.100 

0.002 

0.944 

Weeks to First Observed Tumor 70 105 83 

Stomach:

 Values0 

 Squamous-Cell Carcinoma 0/20(0.00) 

N.S. 

0/50(0.00) * 2/50(0.04) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Infinite 

0.123 

Infinite 

Weeks to First Observed Tumor 97 

Stomach: Squamous-Cell Papilloma 

or Squamous-Cell Carcinoma^ 

 Values0 

0/20(0.00) 

 = 0.002 

2/50(0.04) 

N.S. 

11/50(0.22) 

 = 0.017 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Infinite 

0.123 · 

Infinite 

Infinite 

1.384 

Infinite 

Weeks to First Observed Tumor _____ 105 94 
_____ 

____ 

TOPOGRAPHY:MORPHOLOGY
 

Pituitary: Chromophobe Adenoma
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma
 

u> VaLues
0
 

ο
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4


Ρ


 (CONTINUED)
 

CONTROL
 

8/19(0.42)
 

= 0.026(Ν)
 

70
 

1/19(0.05)
 

N.S.
 

— —
 

.
 

105
 

0/18(0.00)
 

N.S.
 

LOW
 

DOSE
 

22/47(0.47)
 

N.S.
 

1.112
 

0.608
 

2.423
 

100
 

3/49(0.06)
 

N.S.
 

1.163
 

0.103
 

59.809
 

105
 

2/47(0.04)
 

N.S.
 

Infinite
 

0.118
 

Infinite
 

105
 

HIGH
 

DOSE
 

10/46(0.22)
 

N.S.
 

0.516
 

0.231
 

1.302
 

18
 

0/48(0.00)
 

N.S.
 

0.000
 

0.000
 

7.392
 

3/47(0.06)
 

N.S.
 

Infinite
 

0.241
 

Infinite
 

96
 
TABLE 4 (CONCLUDED)
 

LOW HIGH
 

TOPOGRAPHY:MORPHOLOGY CONTROL DOSE DOSE
 

Mammary Gland: Fibroadenoma 0/20(0.00) 1/50(0.02) 3/50(0.06)
 

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

Relative Risk (Control) — — Infinite Infinite
 

Lower Limit 0.022 0.250
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 105 105
 

Uterus: Endometrial Stromal Polyp 8/20(0.40) 6/50(0.12) 10/49(0.20)
 

 Values0 N.S. = 0.012(N) N.S.
 

Departure from Linear Trend = 0.019
 

Relative Risk (Control) ___ 0.300 0.510
 

Lower Limit — 0.104 0.224
 

Upper Limit 0.871 1.298
 

Weeks to First Observed Tumor 69 105 28
 

Treated groups received doses of 150 or 300 mg/kg by gavage.
 

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

c
 The probability level for the C6chran=Armitage test is given beneath the incidence of tumors in
 

the control group when Ρ < 0,05; otherwise, not significant (N.S.) is indicated. The probability
 

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

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

(N) indicates a lower incidence in the treated group(s) than in the control group.
 

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

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

group when Ρ < *0.05.
 
observed in at least one of the control or pivalolactone-dosed groups
 

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

group.
 

The Cochran-Armitage test indicated a significant positive
 

association between dose and the combined incidence of squamous-cell
 

papillomas or squamous-cell carcinomas of the forestomach for both
 

males (P < 0.001) and females (P = 0.002). The Fisher exact tests
 

comparing high dose to control were also significant in both males
 

(P < 0.001) and females (P = 0.017). Based on these results the
 

administration of pivalolactone was associated with the increased
 

incidence of squamous-cell neoplasms of the forestomach in both male
 

and female rats.
 

For male rats the Fisher exact test comparing the incidence of
 

interstitial-cell tumors of the testis in the low dose group to that
 

in the control group had a probability level of Ρ = 0.035, a marginal
 

result which was not significant under the Bonferroni criterion.
 

For female rats the possibility of a negative association between
 

chemical administration and tumor incidence was observed for the combined incidence of leukemia or malignant lymphoma.
 

A significant negative trend was also indicated by the Cochran-


Armitage test for pituitary chromophobe adenomas in female rats. The
 

Fisher exact tests, however, were not significant. A significant
 

negative Fisher exact test for the incidence of endometrial stromal
 

32
 
polyps of the uterus was also found for low dose female rats, but no
 

other tests were significant·
 

In summary, the statistical findings were that the administration of pivalolactone was associated with the increased incidence of
 

squamous-cell neoplasms of the forestomach in both male and female
 

rats.
 

33
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

No consistent dose-related mean body weight depression was
 

apparent in either male or female mice (Figure 4) .
 

No abnormal clinical signs were recorded·
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

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

For both male and female mice the Tarone test did not indicate a positive association between dosage and mortality· For male mice, a
 

surprisingly significant (P = 0.0152) negative trend was indicated
 

by the Tarone test, with the survival of the high dose and low dose
 

males higher than that of the control group.
 

Although one low dose male mouse was missing in week 14, adequate
 

numbers of male mice were at risk from late-developing tumors, as 90
 

percent (45/50) of the high dose, 84 percent (42/50) of the low dose,
 

and 65 percent (13/20) of the control group survived on test until
 

the termination of the study.
 

Although two low dose female mice were missing in week 16, 72
 

percent (36/50) of the high dose, 72 percent (36/50) of the low dose,
 

and 80 percent (16/20) of the control group survived on test until
 

termination of the study, providing adequate numbers at risk from
 

late-developing tumors.
 

34
 
TIME ON TEST (WEEKS)

 

FIGURE 4 

GROWTH CURVES FOR PIVALOLACTONE CHRONIC STUDY MICE 

35 
TIME ON TEST (WEEKS)

 

FIGURE 5 

SURVIVAL COMPARISONS OF PIVALOLACTONE CHRONIC STUDY MICE 

36 
C· Pathology
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix Β (Tables Bl and B2); findings on nonneoplastic lesions are
 

summarized in Appendix D (Tables Dl and D2)·
 

A variety of tumors occurred spontaneously, both in the control
 

and dosed groups· Most frequently affected were the liver, lung and
 

hematopoietic system. These lesions, however, are not uncommon in
 

aged mice of this strain, as can be seen from their occurrence in
 

controls· The significant proliferative gastric lesions present in
 

rats were not noted in mice.
 

In addition to the neoplastic lesions, a number of proliferative, inflammatory and degenerative changes were also encountered
 

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


neoplastic lesions are seen commonly in aged B6C3F1 mice, and were
 

not related to administration of the chemical·
 

The results of this pathologic examination indicate that
 

pivalolactone had no observable carcinogenic effect in mice.
 

D· Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

were observed in at least one of the control or pivalolactone-dosed
 

groups and where such tumors were observed in at least 5 percent of
 

the group·
 

37
 
___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH PIVALOLACTONE*
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Adenoma
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma^
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Adenoma or
 

Hepatocellular Carcinoma^
 

 Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

2/20(0.10)
 

N.S.
 

—
 

—
 

103
 

10/20(0.50)
 

N.S.
 

61
 

4/20(0.20)
 

N.S.
 

—
 

103
 

LOW
 

DOSE
 

6/49(0.12)
 

N.S.
 

1.224
 

0.248
 

11.802
 

87
 

10/49(0.20)
 

 = 0.017(N)
 

0.408
 

0.195
 

0.936
 

72
 

10/49(0.20)
 

N.S.
 

1.020
 

0.346
 

4.068
 

72
 

HIGH
 

DOSE
 

10/49(0.20)
 

N.S.
 

2.041
 

0.498
 

18.154
 

103
 

12/50(0.24)
 

 - 0.035(N)
 

0.480
 

0.244
 

1.059
 

87
 

4/50(0.08)
 

N.S.
 

0.400
 

0.085
 

1.984
 

103
 
TABLE 5 (CONCLUDED)
 

Treated groups received doses of 75 or 150 mg/kg by gavage.
 

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

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

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

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

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

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

39 
TABLE 6 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN FEMALE MICE TREATED WITH PIVALOLACTONEA 

LOW HIGH 

TOPOGRAPHY -.MORPHOLOGY CONTROL DOSE DOSE 

Lung: Alveolar/Bronchiolar Adenoma or 

Alveolar/Bronchiolar Carcinoma^ 0/20(0.00) 4/48(0.08) 2/50(0.04) 

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

Relative Risk (Control) —__ Infinite Infinite 

Lower Limit 0.402 0.123 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor 96 103 

Hematopoietic System: Leukemia or 

Malignant Lymphoma*5 4/20(0.20) 13/48(0.27) 19/50(0.38) 

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

Relative Risk (Control) — _ 1.354 1.900 

Lower Limit — 0.495 0.752 

Upper Limit — 5.170 6.909 

Weeks to First Observed Tumor 82 83 75 

Treated groups received doses of 75 or 150 mg/kg by gavage.
 

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

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

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

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

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

The 95% confidence interval on the relative risk of the treated group to the control group.
 
None of the statistical tests for any site in mice of either
 

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

levels administered in this experiment there was no evidence that
 

pivalolactone was a carcinogen in B6C3F1 mice.
 

In male mice the Cochran-Armitage test indicated a significant
 

negative association between dose and the incidence of hepatocellular
 

carcinomas. The Fisher exact tests, however, were not significant.
 

Also, the Fisher exact test indicated a negative association between
 

the incidence of leukemia or malignant lymphoma in low dose male mice
 

and that in the control group. No other tests, however, were significant under the Bonferroni criterion.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

Tables 5 and 6, the value one is included; this indicates the absence
 

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in mice by
 

pivalolactone that could not be established under the conditions of
 

this test.
 

41
 
V. DISCUSSION
 

The survival among dosed rats was not significantly less than
 

the survival among their controls, and there were no positive associations between dosage and mortality for mice of either sex. Adequate numbers of rats and mice of both sexes survived long enough
 

to be at risk from late-developing tumors· Depression of mean body
 

weight was not observed in dosed groups of either species. In addition, no adverse clinical signs were observed among dosed mice. This
 

evidence, plus the relatively fast decomposition of pivalolactone in
 

water, suggests the possibility that the animals, and in particular
 

the mice, may have been able to tolerate a higher dose·
 

The administration of pivalolactone to male and female rats was
 

associated with increased incidences of hyperplasia, squamous-cell
 

papillomas, and squamous-cell carcinomas of the forestomach. The
 

Cochran-Armitage test indicated significant positive associations
 

between pivalolactone dosage and the combined incidence of squamouscell papillomas and squamous-cell carcinomas of the forestomach in
 

male and female rats. The Fisher exact tests indicated that the
 

number of high dose rats with squamous-cell papilloma or squamouscell carcinoma of the forestomach was significant for both males and
 

females. These tumors were combined for statistical analysis since
 

they are assumed to have a common pathogenesis. They do not commonly
 

occur in control Fischer 344 rats and are, therefore, considered
 

indicative of a carcinogenic effect of pivalolactone.
 

42
 
No tumors occurred in mice at significantly higher incidences
 

in dosed groups than in control groups· No rare or unusual tumors
 

were observed during the histopathologic examination of mice.
 

No indications were found in the literature for the carcinogenicity in rodents of BF3 or tribenzylamine, stabilizers added to
 

the chemical by the manufacturer.
 

These results indicate that under the conditions of this bioassay, pivalolactone was carcinogenic in male and female Fischer 344
 

rats, causing a significant increase in the combined incidence of
 

squamous-cell carcinomas and squamous-cell papillomas at the site of
 

chemical administration, the forestomach. This study provided no
 

evidence for the carcinogenicity of pivalolactone in B6C3F1 mice of
 

either sex.
 

43
 
VI. BIBLIOGRAPHY
 

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Brinkmann, B· and W. Imoehl, ftCyanoacrylate Adhesive·11

 Ger. Offen.
 

2,349,799 (Schering A.-G.), April 24, 1975; Chemical Abstracts
 

8_3, 115898e.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS)
 

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

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

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

Co., Ltd., London, 1970.
 

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

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

Foss, R.P., "Copolymers of Pivalolactone and Isoprene or Butadiene."
 

U.S. Patent 3,907,933 (E.I. duPont de Nemours and Co.), September
 

23, 1975; Chemical Abstracts 84, 18921v.
 

Foss, R.P., H.W. Jacobson, Η·Ν. Cripps, and W.H. Sharkey, "Block and
 

Graft Copolymers of Pivalolactone. II. ABA and ABA-g-A Copolymers with Dienes." Macromolecules 9(2):373-374, 1976.
 

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

Tests, Confidence Limits, and Adjustments for Stratification."
 

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

International Agency for Research on Cancer, IARC Monographs on the
 

Evaluation of Carcinogenic Risk of Chemicals to Man: Some
 

Aromatic Amines, Hydrazine and Related Substances, N-Nitroso
 

Compounds, and Miscellaneous Alkylating Agents, Volume 4. IARC,
 

Lyon, France, 1974.
 

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

Observations." Journal of the American Statistical Association
 

53:457-481, 1958~ "~~ ~~~~~
 

44
 
Linhart, M.S., J.A. Cooper, R.L. Martin, N.P· Page, and J.A. Peters,
 

"Carcinogenesis Bioassay Data System.11

 Computers and Biomedical
 

Research 7:230-248, 1974.
 

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

Co., New York, 1966.
 

Radici, P., D. Colombo, and P. Colombo, flPoly(oxymethylenes)." Ger.
 

Qffen. 2,461,559 (Societa Italiana Resine S.p.A.)3, July 10,
 

1975a; Chemical Abstracts 83, 179948d.
 

Radici, P., R. Croce, and P. Colombo, "Preparation and Stabilization
 

of Polyoxymethylene.11

 Ger. Offen. 2,460,465 (Societa Italiana
 

Resine S.p.A.), July 10, 1975b; Chemical Abstracts 83, 179947c.
 

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

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

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

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

Schmitt, E.E. and M. Epstein, "Reducing Capillarity of Polyglycolic
 

Acid Sutures." U.S. Patent 3,982,543 (American Cyanamid Co.),
 

October 19, 1976; Chemical Abstracts 85, 198151w.
 

Shor, A.C. and J.W. VanDyk, "Coating Substrates with Q"-Substitutedβ-Propiolactone Graft Copolymers." U.S. Patent 3,925,574 (E.I.
 

duPont de Nemours and Co.), December 9, 1975; Chemical Abstracts
 

84·, 75932y.
 

Sundet, S.A., R.C. Thamm, J.M. Meyer, W.H. Buck, S.W. Caywood, P.M.
 

Subramanian, and B.C. Anderson, "Block and Graft Copolymers of
 

Pivalolactone. I. A New Class of Elastoplastics and Thermoplastic Elastomers." Macromolecules 9(2):371-373, 1976.
 

Tarone, R.E., "Tests for Trend in Life-Table Analysis."1 Biometrika
 

62^:679-682, 1975.
 

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

United States Production and Sales, 1976. USITC Publication
 

833, U.S. Government Printing Office, Washington, D.C., 1977.
 

45
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH PIVALOLACTONE
 

TABLE Al
 

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

CONTROL (VEH) 

11-150 5 

LOW 

11 ­

DOSS 

1503 

hIGH DOSE 

11-150 1 

?MhAi.S 

ANIMALS 

ANIMALS 

INITIALLY IN SI'JDY 

NECFCESIED 

EXAMINED ¥ ISTOPAI HOLOG1CALLY** 

2 0 

2 0 

2 0 

5 0 

50 

5 0 

5 0 

5 0 

INTtGJMENTAPY SYSIZM 

•SKIN 

SubACEOUS ADENOCARCINOMA 

(20) (50) (50) 

1 (2%) 

*SUFLUT IISSU E 

StEACEOUS ADENOMA 

SAFCCKA , NCS 

Fx3P0i1A 

LIPOSASCCPA 

(20) 

1 (5%) 

(50) 

1 

H 

1 

(2%) 

(8%) 

(2%) 

(50 ) 

1

1

2

 (2??) 

 (21 ) 

 (43 ) 

BESPIRATO^Y SYSTEM 

ffLUNu 

NEOPLASM, NO3, METASTATIC 

CAFCINCMA, NOS, ".ST^bTATIC 

AX.VFOLAR/EPCNCHIOLAP ACtNOfA 

SiiSACjiOUS ADENOCARCINOMA, ^ETAST 

Ai^FNOSQUAfCUS CA3CISCMA 

SARCOMA, NCS, METASTATIC 

OoTEOSAFCC^^V/ METASTATIC 

(20) (50) 

1 

1 

2 

1 

1 

1 

(2%) 

(2%) 

(4%) 

(2%) 

(295) 

(2*) 

(49 ) 

1 (2%) 

BEMATuFOlETIC SYSTfcM 

#DPA^.N 

MALIGNANT FETICULOSI S 

(20) (49) (49) 

1 (2%) 

*MULIIFLE CFCANS 

MALIw.LYMPhCMA, JNDItFEF-TYFΕ 

MHLIG.LYMPHOMA, LYMPHOCYTIC TY°2 

LhUKEMIA,NCS 

UhDlFFEFEMIATED LFUKEMIA 

(20) 

1

1

1

 (5%) 

 (5%) 

 (5%) 

(50) 

1 

3 

1 

(2%) 

(6%) 

{2%) 

(5C) 

1

3

 (255) 

 (6%) 

CIRCULATORY SYSTEM 

# NUIOJCR OF ANIMALS WITH TISSUE EXAMINED

• NUWu^R OF ANIMALS NECROFSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

A-3
 
TABLE Al (CONTINUED) 

CON TFOL (VEi: ) LOW DOSi f-1Gb DOSE 

1 1 -1505 11-1503 11­ '511 

EIGESilV E SYSII M 

tSALIVA^Y GLAND (20) (41>) (44) 

S ArCCMA, NCS 1 (5%) 

(20) (50) (50) 

NEOPLASTIC NOPOLΕ 3 (6%) (4%) 

«FANcF^AS (20) (48) (46) 

AcINAP-CELI ADENC1A 1 (2%) 

#S£CHACH (19) (•49) (48) 

SQUAMOUS CILL PAPILLOMA 5 (10%) 14 (29%) 

SwUA'OQOS C£LL CARCINOMA 1 (27o) 7 (15%) 

#LAFuE INIISIIN E (20) (49) OC) ALLMOCA IN ADENOMATOUS TOLYF 1 12%) 

UPINA^Y SYSTEM 

NONi. 

ENCOCuINF SYSliM 

(1b) (47) (44) 

CnPOMOE-POEI ADcND^IA 2 (11%) 2 (Ui) 3 (7%) ACIDOPHIL ADuNCMA 1 (6X) 

(20) (48) (49) 

CAPCINCMA,NOS 1 (2%) 

PriEOCHROMCCYIC^iV 2 (107) U (tsw

) 2 (47) 

FnEOCHFCMCCYTCfU, 1 (5%) 

(z0) (••8) (46) 

C-CELL 

C-CiLL CAECIKCMP 

1 (5X) i) 

1 (ίο-) (21) 

1 

1 (2·.) (2 "ζ) 

#PA D AlriYROIE (13) 

ADENOMA, NCS 1 (8%) 

«PANCPEATIC ISLETS (20) (48) (46) 

1__ . L2%L___ 

# NUMuEp OF ANIMALS WITH IISSUc , LXA'IINhD MICROSCOPICALLY 

• NUMBER O^ AMMAIS N5CPCFSIED 

A-4
 
TABLE Al (CONTINUED) 

CONTROL (VilH) LOW DOSE
 11-1505 11-1503
 

EEPEO£UCTIVE SYSIfM 

#!ESi.IS (20) (50) 

INTERSTITIAL-CELL T'JMOR 12 (60%) 42 (84%) 

NERVOUS SYSTEM 

#EFAIN (20) (49)

 

LPENDYMCPA (5%)

 

SPECIAL SENSE CP3ANS 

NON£ 

MUSCULOSKELETAL SYSTEM 

•MUSv-LE OF LEG (20) (50)

C^TECSAFCCKA 1 (2%) 

EODY CAVITIES 

•ABDui'iINAL VISCIiFA (20) (50) SAPCOMA, NCS 1 (2*) 

(20)	 (50) 

1 (2%) 

•TUNICA VAGINALIS (20) (50) MUSOTHELICPA , NOS

 

ALL OTHER SYSTIMS
 

L^ ORGANS	 (20) (50)
 

# NUM3EF OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER UF ANIMALS NTCROFSIED
 

hIGK DOSE 

11-150 1 

(49) 

40 (82%) 

(49) 

 (50)
 

(50) 

<5C) 

(5C) 

1 (2%) 

(50)
 

A-5
 
TABLE Al (CONCLUDED)
 

CONTPOL(VEH) LOW DOSE HIGH DOSE
 

11-1505 11-1503 11-1501
 

ANIMAL DISE0SI1ICN SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

NAl'JRAL DEATH} 15
 

MORIBUND SACRIFICE 4
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 15 38 2 1
 

ANIMAL MISSING
 

2 INCUDES AUTCLYZED ANIMALS
 

SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 19 46 U6
 TuTAL PFIMARY TUMORS 26 fa3 84
 

TOTAL ANIMALS WITH BENIGN [UMO^S 17 43
 TOTAL BENIGN TUMORS 20 b5 65
 

TOTrtL ANIMALS WITH MALIGNANT TUMORS 6 13 15
 

TOTAL MALIGNANT TUMORS 6 16
 

IOT«L ANIMALS WITH StCONDAPY TUMORS* 1
 

TOTAL SECONDARY IUMOFS 4 1
 

TOTAL ANIMALS WITH TUMOPS UNCERTAINEENIGN OR MALIGNANT 4 λ
 

TOTAL UNCEFTAIN IUMO°S 4 3
 

TOTH L ANIKALS WITH TUKO CS UNCEFTAIN-
 PFlnARY OP F11 ASTA1 1C
 TOTAL UhCEFTAiN TUMOFS
 

* PRIMARY TUMCFS: ALL TUMO°S EXCEPT SECONDAFY TUMOFS
 

# SECONDARY TUfOFS: METASTATIC TUI1OPS O*> TUMORS INVASIVE INTO AN ADJACENT OPGAN
 

A-6
 
TABLE A2
 

SUMMARY OF THE INCIDENCF OF NEOPLASMS IN FEMALE RATS TREATED WITH PIVALOI ACTONE
 

CONTROL (VEH) LOW DOSE HIGH DO1E
 

11-1506 11 1504 11-1502
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIMALS NECFCISIID 20 50 50
 

ANIMALS EiAMIMD HISTOPATHCLOGICALLY** 20 50 50
 

INTEGUilENTAFY SYSTEM 

• SKIN 

SQUAMOUS CELL CARCINOMA 

ALENOCA/SCUAMOUS METAPLASIA 

(20) (50) 

1 (2%) 

(50) 

1 (2%) 

•SUBoTJI TISSUE 

FIBROMA 

FIB^OADLNCfA 

(20) (50) 

1 (2%) 

(5C) 

1 (2%) 

RESPIRATORY SYSTEM
 

#LUNo (20) (50) (50) 

ALtNOCAFCINCMA, NOSf METASTATIC 1 (2%) 

A^VEOLAR/EFCNCHIOLAR CARCINOMA 1 (2%) 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE	 ORGANS (20) (50) (50)
 

MALIGNANT IYFFHOMA, NOS 3 (15%) 1 (2%)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%)
 

LfiUKEaiA,NCS	 1 (5%) 

CIRCULATORY SYSTEM 

NONL 

LIGESilVE SYSTEM 

#STCMACH (20) (50) (50) 

S^ ϋ AM Ο US _C El. L_ PAPILLOMA 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMtiER OF ANIMALS NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7
 
TABLE A2 (CONTINUED)
 

HIGH DOSE
 

11-1502
 

1 (2%)
 

(47)
 

1 (2%)
 

(46)
 

10 (22%)
 

1 (2%)
 

(46)
 

(47)
 

1 (2%)
 

2 (4%)
 

3 (6%)
 

48)
 

2 (4%)
 (


(50)
 

1 (2%)
 

3 (SI)
 

(49)
 

10 (20%)
 

(49)
 

SQUAMOUS CILL CARCINOMA
 

ADENOCAPCIKMA, NOS
 

SAPCOMA, NCS
 

URINARY SYSTEM
 

#USINARY BLAEEEP
 

LEIOMYOMA
 

ENDOCtUNE SYSTEM
 

«PITUITARY
 

SQUAMOUS CELL CARCINOMA, METASTA
 

ChFOMOPHOEE ADLNOMA
 CHHOMOFHOEE CARCINOMA
 

#ADPZNAL
 

PHEOCH^OaCCYTCMA
 

#THYKOID
 

AJENOMA , NCS
 

FOLLICULAE-CELL ADENCMA
 

C-CELL ADEi^CMA
 

#PANLREATIC ISLETS
 

ISLET-CLLL ADENOMA
 

REPRODUCTIVE SYSTEM
 

•ΜΑΚΛΑΡ. GLANI
 

ADENOMA, NCS
 

CYSTADENCKA, NOS
 

INTRADUCTAL PAPILLOMA
 

FIB»OADENCKA
 

tUTEriUS
 

Ai^ENOCAPCINCMA NOS
 

LEIOMYOSAFCOMA #


ENDOMETRIAL STPOMAL POLYP
 

ENDOMETRIAL STROMAL SARCOMA
 

*UTERUS/£NDCKETRIUM
 

CONTROL(VEH)
 

11-1506
 

(19)
 

(19)
 

8 (42%)
 

(19)
 

1 (5%)
 

(18)
 

(20)
 

(20)
 

1 (5%)
 

(20)
 

1 (5%)
 

8 (40%)
 

(20)
 

LCW DOSE
 11-1504
 

1 (2%)
 

(44)
 

(47)
 

1 (2%)
 22 (47%)
 

(49)
 

3 (6%)
 

(47)
 

2 (4«ζ)
 

(46)
 

(SO)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

(50)
 

1 (2%)
 

6 (12%)
 

1 (2%)
 

(SO)
 

# NUM3ER OF AKIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMJ3E° OF ANIMALS NECRCPSIED
 

A-8
 
ACENOCARCIKf A, NOS 

NERVOUS SYSTEM 

NONE 

SFECIAL SiNS E CfcGANS 

ΝΟΝΔ 

MUSCULOSKELETAL SYSTEM 

NONi. 

EODY CAVITIES 

•MES^NTEPY 

SAPCCMA, NCS, METASTATIC
 

AIL OTHER SYS1IMS
 

NONE
 

ANIMAi, DISEOSI1ICN SUMMAPY
 

AMiALS INITIALLY IN STUDY
 

NATURAL DhATHa)
 

MORIBUND SACRIFICE
 

SCHEDULEE SACRIFICE
 

ACCIDENTALLY KILLLD
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

TABLE A2 (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

11-150 6 11-150 4 11-150 2 

1 (2%) 

(20)	 (50) (50)
 

1 (2%)
 

20 50 50
 

19
 

3
 

16	 28
 

# NUMdER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF AMMALS NECPQFSIE D
 

A-9
 
1LK0R SUMMARY
 

TO^H L ANIMALS rfITH FPIMARTc TUMOPS*


TuTAL PRIFiRY TUMORS


TOTH L ANIMALS rfITL BENIGN TUMOPS


TOTAL EENICN TUMOPS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TuTAL MAIICNANT TUhORS


TOTAL ANIMALS WITH SECONDARY TUMORS*


TOTAL SECONDARY TUMOPS


TOTAL ANIMALS WITH TJMORS UNCE^TAIN-


ELNiCN OR MALIGNANT
 

TOIAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMOPS
 

TABLE A2 (CONCLUDED)
 

CONTROL (VEH)
 

11-1506
 

 14


 23


 1 _>


 18


 4


 5


* PRIUAPY TUICFS : ALI. TUMORS LXCE^T SECJNDAPY TUMOPS 

# SECGNDAaY TUFOPS: METASTATIC TUMORS OP TUdOPS INVASIVE


Α-IO
 

LOW DOSE hIGH DOSE
 1 1-1504 11-1502
 

 35 34
 

 48 5 1
 

 30 32
 

 40 4C 

> 

8 5 

8 6 

2 1 

2 I 

 INTO AN ADJACEM ORGAN
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH PIVALOLACTONE
 

TABLE B1
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICF TREATED WITH PI VALOLACI ONE
 

CONTROL (VSri)
 22-2505
 

ANIMALS INITIAILY IN STUDY 20
 

ANIMALS MISS1NC
 

ANIMALS NECFO?SI£D 20
 

ANIMALS EXAMINED FISTOPATHCLOG1CALL Υ** 20
 

INTEGUMENTARY SYSTEM
 

NCNr,
 

FESPIRATO^Y SYSTEF
 

#L'JNJ
 

HEPATOCELLULAR CAFCINOMA, MEIAST
 

Ai-VEOLAF/EECNCHIOLAR ADENOMA
 

HEMATOPOIETIC 5YSTEM
 

«MULTIPLE 0FCAN3
 

MALIG.LYMPPOMA, UNDIFFEF-TYPE
 

MALIG.LYMFHOMA, LYMPHOCYTIC TYPE
 

HiiLIG. LYMPHOMA, HISTIOCYTIC TYPF
 

L~UKEMIA,NCS
 

UNDIFFERENTIATED


#SPLtEN 

HEMANGIOSAFCCPA 

MALIG.LYMPHOMA,

#LYMPF NODE 

MALIG.LYMPKMA,

MALIG . LYMPHOMA,

«MESENTERIC L. NODE 

MHLIG.LYMFPCKA , MALIG.LY1FH3WA,

HALIG.LYrFPCMA,

#PEY^FS PATCH 

MALIG-LYPPPCMA,

# NUMtiER OF AMMALS

* NUMBER OF AMMALS

 LEUKEMIA
 

 LYMPHOCYTIC TYPE 

T

J NDIFFEF-TYPL 

 LYMPHOCYTIC TYPE 

 UNDIFFEP-TYPE 

 LYMPHOCYTIC TYPE 

 HISTIOCYTIC 1YPE 

 UNDIFFEE-TYPF 

(20)
 

2 (10%)
 

(20)
 

1 (5%)
 

2 (10%)
 

2 (10%)
 

1 (5%)
 

1 (5%)
 

(18) 

1 (6%) 

(19) 

(19 ) 

1 (5%) 

1 (5%) 

(18) 

 WITH TISSUE EXAMINED MICROSCOPICALLY 

 NECROPSInD ** EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

B-3 

LOW COSE
 22-2503
 

50
 

1
 

49
 

U9
 

(49)
 

1 (2%)
 6 (12%)
 

(49)
 

2 (4%)
 

3 {€%)
 

1 (2%)
 

1 (2%)
 

(1*7) 

(48) 

2 

1 (2%) 

(48) 

(46) 

HIGH DOSE
 22-2501
 

50
 

50
 

50
 

(49)
 

10 (20%)
 

(50)
 

2 (4%)
 

3 (6%)
 

2 (4%)
 

1 (2%)
 

<5C) 

1 (2X) 

(46 ) 

(46) 

2 (4%) 

1 (2%) 

(49) 

1 12%) 
TABLE Bl (CONTINUED) 

CONTHOL(VEK) LOW DOSS HIGH DOSE 

22-2505 22-2503 22-2501 

CIRCULATORY SYSTEM
 

NONi.
 

riGLSIIVE SYSliM
 

«LIVER (20)
 (49) (5 0) 

SQUAMOUS CELL CAPCINCMA, MEIASTA 1 (2%) HuPATOCEILULAR ADENOMA 1 (5%) 2 (4%) 3 (ό%) HEPATOCELLULAR CARCINOMA 3 8 (16%) 1 (2%) 

tSTOriACH (20) (48) (50ι
 

SQUAMOUS CELL CARCINOMA 1 (2%)
 

URINARY SYSTEM
 

NCN£
 

ENDOCuIME SYSTEM 

#ADE£NAL (16) (37) (48)
 CORTICAL AEIINCMA 1 (3%)
 

«THYhOID (13) (23) (36) 

FJLLICULAP-CELL ADZNCUA 1 (3%) 

REPRODUCTIVE SYSTEM
 

#TESIIS (17) (45) (<*S) 

IfcTERSTITI AL-CELL TIIMO9 1 (2%) SuMINOMA/EYSGtRMINOMA 1 {2%) 

HEKANGICMA 1 {2%) 

l^EPVOUS SYSTEM 

SPECIAL SENSE CSGANS 

# NUMbER OF AMMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMuE° OF AMMALS NECROPSIED 

B-4 
KUSCULOSKJELETAI SYSTEM
 

NONi,
 

EOCY CAVITIES
 

NONi.
 

ALL OIHEH SYSIIMS
 

NONii
 

iNIMAL DISPOSITION SUMHAPY
 

ANIMALS INITIALLY IN STUDY
 

NATUPAL DEPTHS
 

MuFIBUNE SACRIFICE
 

SLH£DrIL£C SACRIFICE
 

ACCIDENTALIY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL HISSING
 

2 INCLUDES AUTCLYZFD ANIMALS
 

TABLE Bl (CONTINUED) 

CONTROL(VEH) LOW DOSE EIGH DOSE 

22-2505 22-2503 22-2501 

20 50 50
 

7 5 3
 

2 2
 

13 1*2 45
 

1
 

# N"»!bER OF ANIMALS WITH TISSUE EXAMINED MICKOSCOPICALLY
 

• NUMuE? OF AMMALS NECROFSIED
 

B-5
 
TABLE Bl (CONCLUDED)
 

CONTROL(VEH) LOW DOSE HIGH DOSE
 22-2505 22-2503 22-2501
 

ΊϋΡΟ SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 12 24 25
 TOTAL PRIKARY TUMORS 16 30 29
 

TOTAL ANIMALS WITH BENIGN TUMORS 2 10 14
 

TuTAL EENICN TOHOPS 3 10 15
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 11 17 13
 

TOTAL MALICNANT THMOES 13 20 14
 

TOIH L ANIMALS WITH SECONDARY TUMORSt 2
 

TJTAL SECChDABY TUMOFS 2
 

TOTAL ANIMALS WITH IUMOPS UNCEPTAINEEN1GN OR MALIGNANT
 

TOTAL UNCEEIAIN TUMOP3
 

TOTAL ANIMALS WITH TUMORS UNCEPTAIN-


FFInAPY OF KETASTATIC
 

ToTAL HNCEFTAIN TUMORS
 

FFIiiASY 1UMCFS: ALL TUMOFS EXCIPT SECONDAPY TUMOFS
 

SECONDARY T0UJ5S: METASTATIC TUMOFS OP TUMOFS INVASIVE INTO AN ADJACENT ORGAN
 

B-6
 
TABLE B2

 

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

HIGH DOSE
 22-2502
 

50
 

50
 

50
 

(50)
 

1 (2%)
 

(50)
 

2 [4%)
 

1 (.1%)
 

(50)
 

3 (t> X)
 

4 (HX)
 

5 (10X)
 

2 (4%)
 

1 (2X)
 

(48)
 

1 (2X)
 

1 (2*)
 

(49)
 

1 <2X)
 

(49)
 

CONTROL(VEH)


22-2506
 

ftNIMALS INITIALLY IN STUDY 20
 

RNIKAuS NECIOfSItD 20
 

SMMAL S EXAKIMD HISTOPAIHCLOGICALLY ** 20
 

[NTEGUMENTAFY SYSTEM
 

•SKIN


Ai,NFXAL CAFCINOMA
 

PESPInATCHY SYSTEM
 

#LHNo


AI.VEOLAP/EECNCHIOLAR ADENOMA
 

A^VEOLAF/EFCNCHIOLAP CARCINOMA
 

AI>NEXAL CAECINOMA, METASTATIC
 

FUABDOMYOSAFCCMA, METASTATIC
 

HEMATOPOIETIC SYSTEM
 

*MULIIPLE OFGANS


MALIGNANT LYMPHOMA, NOS


MALIG.LYMPHOMA, UNDIFFEE-TYPE


MALIG.LYMPHOMA, LYMPHOCYTIC IYPE
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE


MALIGNANT IYMFHOMA, MIXED IYPE


LU0KEMIA,NC S
 

«SPLEEN


MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

MALIG.LYKFFCMA, HISTIOCYTIC TYPE
 

#LY!!PH NODE


MaLIGNANT LYMPHOMA, NOS
 

MALIG.LYMFFOMA , LYMPHOCYTIC TYPE
 

UIALIG.LYKFHCMA, HISTIOCYTIC TYPE
 

«MESENTERIC L. NODE
 MALIGNANT LYMPHOMA. NOS
 

 (20)
 

 (20)
 

 (20)
 

 1 (5%)
 

1 (5%)
 

 1 (5%)
 

1 (5%)
 

 (19)
 

 (19)
 

 (19)
 

LOW COSE
 22-2504
 

50
 

2
 

U8
 

48
 

(48)
 

(48)
 

3 (6«)
 

1 (2%)
 

1 (2*)
 

(48)
 

4 (8%)
 

2 (4%)
 

1 (2%)
 

1 (2%)
 

(43)
 

(47)
 

1 (2%)
 

1 (2%)
 

(47)
 

1 (2%)
 

# NUMBER OF ANIMALS WITH TISS'IE EXAMINED MICROSCOPICALLY 

* NUMoEh OF AMMALS NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-7
 
TABLE B2 (CONTINUED) 

CONTROL(VEh) LOd DOSE hIGP DO3E 

22-2506 22-2504 22-250? 

KALIG. LYMPf-CIA, LYMPHOCYTIC IYPE 1 {2%) 

MaLlG.LYMFFO*lAf

 HISTIOCYTIC TYPE 1 {2%) 

#LIVEP (19) [US) <5C)
 

MHLIG.LYMPHOMA, UNDIFFE5-TYFE 1 (2%)
 

CIFCU^ATOPY SYSTEI 

NO Ν r. 

DIGESTIV E SYSTi M 

#LIVr.P (19) (4 8) (50)
 

Hi.PATOCELIULAF CARCINOMA 2 (U%)
 

U^INArtY SYSTEM 

NGN,. 

ENDOCitlNE SYSTEM 

#ADREWAL (18) (40) (46)
 

AbENOCARCINCMA, NUS 1 (6%)
 

EEPPOJUCTIVE SYSTEM 

(19) (47) (49) 

LUIOMYCM A 1 (5%) 

LtIOMYCSAFCCIA 1 (2*) 

# OVA IX Υ (19) (46) (46)
 

CxSTADENC^P , NOS 1 (2%)
 

KEHVOJS SYSTEC 

NONr 

SPhCIuL bcNSE CRGANS 

*EYi2/LAC^IMAL GLAND (20) (48) (5C) 

Αυ£ΝΟΜΑ NCS 1 (2%) 

# NU^luEP OF AMMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NTJ*flL·* OF ANIMALS NECROPSIED 

B-8
 
TABLE B2 (CONTINUED) 

CONTROL (VEH) LOW DOSfi HIGH DOSE 

22-2506 22-2504 22-2502 

MUSCULOSKELETAL SYSTEM 

NONt 

EOEY CAVITIES 

NONL· 

ALL OiHER SYSTIMS 

•MULTIPLE OFGANS (20) (48) (50) RHABDOMYOSARCOMA 1 (2%) 

ANIMAL DISEOSI1IC N SUMMAFY 

ANINALS INITIALLY IN STUDY 2 0 5 0 5 0 

NATURAL DitATili 3 7 9 

MORIBUND SACRIFICE 1 5 5 

SCHEDULED SACRIPIC2 

ACCIDENTALLY KILLED 

•L&RMINAL SiCaiFIC E 16 3 6 36 

AiMMAL MISSING 2 

 INCLUDES AMTCLY2ED ANIMALS 

# NUMiiER OF AMMALS WITH TISSUE EXAMINED MICPOSCOPICALLY 

• NUMbEE OF AMMALS NECROFSIED 

B-9
 
TABLE B2 (CONCLUDED)
 

CONTEOL(VEH) LOW DOSE HIGH DOSE
 

22-2506 22-2504 22-2502
 

lUMOP, SUM ΑΡΥ
 

TOTHL ANIMALS WITH PFIMA^Y TUMORS* 6 20 22
 

TOTAL PBIKHRY IUMOPS 6 22 23
 

TOTAL ANIMALS WITH BENIGN TUMOPS 1 4 2
 

TOTAL EEMCN TUMORS 1 5 2
 

TOTH L ANIMALS WIIH MALIGNANT 1UMOF.S 5 16 21
 

TOTAL MALICNANT TUMORS 5 17 2i
 

TOTAL ANIMALS WIIH SECONDARY TUMORS» 1 1
 

TOTAL SECChDARY TUMOTS 1 1
 

TOTAL ANIMALS WITH TUMOFS UNCERTAINEENJ.GN OP MALIGNANT
 

TOTAL UNCEFIAIN TUMOBS
 

TOTH L ANIMALS WITH TUIOBS UNCFFTAINFRIHARY OP fITASTATIC
 

TOTAL UNCEETAIN TUMORS
 

* PRIMARY TUMOFS: ALL TUMORS EXCEPT SECONDARY T'THOPS
 

# SECONDARY TUfORS: METASTATIC TUMORS OP TUMOFS INVASIVE INTO AN ADJACENT OR3AN
 

Β-IO
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH PIVALOLACTONE
 

TABLE CI
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS
 

TREATED WITH PIVALOLACTONE 

ANIMALS
 

ANIHAL S
 

ANIMALS
 

INITIALLY IN SIUDY
 

NECPCFSIEC
 

EXAMINED HISTOPATHOLOGICALLY**
 

1NTEGUMENTAPY SYSTEM 

•SKIN
 

INFLAMMATION, CHPONIC
 

FESPIaATORY	 SYSTEM 

#LUNu/b»CNCHICLE 

INFLAMMATION, ACUTE FOCAL
 

#LUNG
 

MINEPALIZATION 

CONGESTION, NCS
 

EDLMA, NCS
 

HUMOPHHAGE 

FutEUMONIA, CHFCNIC

HEMATOPOIETIC SYSTEM
 

«30NE MAPPOW
 

MYELOFIBROSIS
 

#SPLLEN
 

FIBROSIS, FOCAL
 

ICLRVICAL LYPFH NODZ
 

OMPHANGIECTASIS
 

•MESENTERIC	 L. NODE
 

LiMPHANGIECTASIS
 

CIRCULATORY SYSTEM
 

tMYOcARDIUM
 

 MURINE 

CONTEOL(VEH)
 

11-1505
 

20
 

20
 

20
 

(20) 

(20)
 

(20)
 

14 (10%)
 

(20)
 

1 (5%)
 

(20)
 

(20)
 

(20)
 

(20) 

LOW DOSS HIGH DOSE 

11­ 1503 11­ 1501 

50 50 

50 50 

50 50 

(50) (50) 

1 [2%) 

(50) 

1 (2%) 

(50) <4S) 

1 (2%) 

1 (2%) 

1 (2%) 1 (2%) 

1 (2%) 

10 (20%) 3 (6?>) 

(48) (48) 

(50) (49) 

1 (2%) 

(49) (48) 

1 (2%) 

(49) (48) 

1 (2%) 

(50) (48) 

# NUMBER OF ANIMALS WITH IISSH E EXAMINED MICFOSCOPICALLΥ 

* NUMuEF OF ANIMALS NECHOPSIED 

* * EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3
 
TABLE CI (CONTINUED) 

CONTROL(VEH) LOW DOSE 11-1505 11-1503 

FIBROSIS 5 (25%) 13 (26%) 

, KOS 1 (5%) 

• VEIN (20) (50) 

TiiPOMBOSIS, NUS 

EIGESfIVE SYSTIM
 

#LIVJ>* (20) (50)
 

CJNGFSTICN, NCS 1 (2%)
 

HhPATITIS, TOXIC 1 (5%)
 

BuTAMOFFHOSIS FATTY 1 (5%) 3 (6%)
 

3ASOPHILIC CYTO CHANGF 3 (6%)
 

FOCAL CELLDLA3 CHANGE 3 (6%)
 

ANGIECTASIS	 1 (2%)
 

#LIVrF/CENIRIIOECLiP (20) (50)
 

NECROSIS, NOS 2 (4%)
 

«PANCREAS (20) (48)
 

INFLAMMATION, CHPONIC
 

FIBROSIS, FOCAL
 

tPANCPEAIIC ACINUS (20) (48)
 

ATROPHY, NCS 1 (2%)
 

#ESOt?HAGUS (20) (49)
 

INFLAMMATION, ACUTS NECROTIZING
 

PARAKERATOSIS 1 (2%)
 

• STCiiACH	 (19) (49)
 

IN FLAK MAT 1C Ν, NOS 3 (16%)
 

INFLAMMATION, FOCAL 10 (20fo)
 

INFLAMMATION, CHRONIC 1 (2%)
 

INFLAMMATION, CHRONIC FCCAL
 

hYFERPLASIA, EPITHELIAL 5 (26%) 39 (80%)
 

HiPLRPIASIA, PAPILLARY
 

#£MALL INTESTINE (20) (50)
 

CONGESTION, NCS
 

HEMORRHAGE
 

INFLAMMATION, ACUTE/CHRONIC
 

#LAPGE INTESTINE (20) (49)
 

NiMATODIASIS _ 2 . (4^)
 

# NuKoEF OF ANIMALS WI'IH TISSUE EXAMINED MICROSCOPICALLY 

* MTHaEP OF ANIMALS NECP.OFSIED 

C-4
 

HIGH
 11-1
 

13
 

1
 

(50)
 

1
 

(50)
 

1
 

1
 

<5C)
 

(46)
 

1
 

1
 

(*»*)
 

(47)
 

2
 

(48)
 

1
 

1
 

1
 

14
 

1
 

(49)
 

1
 

1
 

1
 

(5C)
 

DOSE
 501
 

(27%)
 

(258)
 

12%)
 

(2%)
 

(2%)
 

(2%)
 

(2%)
 

(4%)
 

(?*)
 

(2%)
 

(?%)
 

(29%)
 

(?*)
 

(2%)
 

(2%)
 

(2%)
 
PARASITISM
 

«COLON
 

PARASITISM
 

UBINAhY SYSTEf.
 

«KIDNEY
 

MINERALIZATION
 

INiLAMMATICN, CHRONIC
 

«URINAFY ELAEIEP
 

MUCOCELE
 

INFLAMMATION, AC'JTE
 

HYPERPLASIA, EPITHELIAL
 

ENDOCaINE SYSTEM
 

«PITUITARY
 

CYST, NOS
 

ANGIECTASIS
 

#ADRr,NAL
 

MINERALIZAIICN
 

HYPERPLASIA, FOCAL
 

«ADRENAL CORIIX
 

MLTAMOBFHCSIS FATTY
 

ANGIECTASIS
 

#ADRi.NAL MEDULLA
 

HYPEFPLASIA, NOS
 

«THYROID
 

HYPERPLASIA, C-CELL 

PEPRODUCTIVE SYSTEM
 

GLANE
 

CYSTIC DUCTS
 

tPROoTAT*1
 

#	 NUMoEP OF ANIMALS WITH TISSUE

• NUMiiER OF AMMALS NECRCPSIED 

TABLE CI (CONTINUED) 

CONTROL (VEH) LOW DOSE 

11-1505 11-1503 

6 (30%) 12	 (24%) 

(20)	 (49) 

1 (2%) 

(20)	 (48) 

10 (50%)	 30 (63%) 

(17) (43) 

1	 (6%) 

1 (2%) 

1 (2%) 

(18)	 (47) 

1 (2%) 

(20)	 (48) 

1	 (2%) 

(20)	 (48) 

1 (2%) 

(20)	 (46) 

1 (2%) 

(20)	 (48) 

1 (5%) 

(20)	 (50) 

(16)	 (42) 

 EXAMINED MICROSCOPICALLY 

C-5
 

EJGH DOSE
 11-1501
 

 (165 

(50) 

2 (4%) 

(49) 

1 (2%) 

16 (33%) 

(4 2) 

(44) 

1 (?. %) 

1 (A%) 

(49) 

1 (**) 

(49) 

1 

1 (2%) 

(49) 

(<A6) 

1 (2%) 

(50)
 

1 (2%)
 

(43)
 
*S£MINAL VESICLE
 

INFLAMMATION, SUPPURATIVE
 

MINERALIZATION
 

AIROPHY, NCS
 

SYSTEM
 

NONE
 

SPECIAL SENSE CPGANS
 

NONL
 

KUSCULOSKSLETA I SYSTEM
 

NONL
 

EOEY CAVITIES
 

•MEDIASTINUM
 

INFLAMMAIICN, NOS
 

*KESuNTERY
 

N£,C?05IS# PAT
 

ALL OTHEF SYSTEMS
 

•MULTIPLE ORGANS
 

LnUKEMOID FEACTION
 

ADTPCS* TISSt11·; 

INFLAMMATION, NECROTIZING 

SEECIAL MOFEHCIOGY SU^IMAFY 

NO LESICN FEECFTED 

# NUMBER OF AKIMALS WITH TISSUE

* NUMuE0

 OF AisIMAtS NEC^OPSIL·^ 

TABLE CI (CONCLUDED)
 

CONTROL(VEH) LOW DOSE
 

11-1505 11-1503
 

(20) (50) 

(20) (50) 

1 (5S) 3 (15%) 3 (695) 

(20) (50) 

(20) (50) 

(20) (50) 

 EXAMINED hICEOSCOPICALLY 

C-6
 

HIGH DOSE
 

11-1501
 

(50) 

1 (2*) 

(49) 

1 (2%) 3 (6S) 

(50) 

2 (4%) 

( = 0) 

2 (6S) 

(50) 

1 (2*) 
TABLE C2
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS
 

TREATED WITH PIVALOLACTONE
 

CON1FOL (VEH) 

11-1506 

LOW 

11­

DOSS 

1504 

HIGH DObE 

11-1532 

ANIMALS 

ANIMALS 

ANIMALS 

INITIAILY IN STUDY 

NECBCESIED 

JiXAMIMD HISTOFATHOLOGICALLY 

20 

20 

** 20 

50 

50 

50 

50 

50 

50 

INTEGUf£N1ARY £YSTF*I 

NO ΝI 

FESPIuATORY SY5IEM 

#LUNG 

MANERALIZATIC N 

CuNGESIICN, NOS 

Ex/tMA, NCS 

HAHORBXIAGE 

BriCNCHCFNEMCNIA, A UTE 

PNEHMONIA, CHFONIC rlHRINE 

(20) 

5 (25%) 

(50) 

1 

1 

1 

16 

(2%) 

(2%) 

{2%) 

(32%) 

(50) 

3 

1 

8 

(5%) 

(2%) 

(16? 

PEMATOFOIZTIC SYSTEM 

OSTEOSCLHBCSIS 

(19) {Hβ) 1 (2%) (^5) 

tSPLEEN 

NLCHOSIS, ICCAL 

HiiMOSIDERCSIS 

(20) 

1 (5%) 

(47) 

1 (2%) 

(49) 

1 (2%) 

TORY SYSTEM 

«ΗΞΑΛ Τ 

P^RIARTEFIIIS 

(20) (50) 

1 (2%) 

(48) 

•MYOCARDIUM 

INFLAMMATION, FOCAL 

IiJFLAMMATICN, MULTIFOCAL 

(20) (50) 

1 12%) 

(48) 

1 (2%) 

# NUMBEP OF ANIMALS WITH TISSUE EXAMINED

• NUMBEP OF AMMALS NECRCFSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

C-7
 
TABLE C2 (CONTINUED) 

CONTROL(VEH) LOvf DOSS UGH DO JE 

11-1506 11-1504 11-150^ 

DIGESTIVE SYSTIM
 

#LIVER


INFLAMMATION, NECROTIZING


INFLAMMATION, ChRONIC NECPOTIZIN
 

riaPATITIS, TCXIC
 

NtCFOSIS, NOS


PiTAMORPHCSIS FATTY


BASOPHILIC CYTO CHANGE
 

FOCAL CELLLLAF. CHANGE
 

HiFERPLASIA, FOCAL
 

HYPERPLASIA, EIFFUSE
 

#LIViF/CEN1PIICEULAP


MfcTAMOPPHCEIS FATTY
 

*EIL U DUCT


HiPERPLASIA, NOS
 

#EANC*EATIC ACINUS


ΑΓΡΟΡΗΥ, NCS
 

ATROPHY, FCCAL
 

#STCMACH


INFLAMMATION, NOS
 

INFLAMMATION, FOCAL


INFLAMMATION, CHRONIC


HYPERPLASIA, EPITHELIAL


HYPERPLASIA, PAPILLARY
 

HiPEPKtPATCSIS
 

#SMALL INTESTINE


HYPEPPLASIA, FCCAL
 

#LARoE INTESTINE


PARASITISM


uFINAuY SYSTiM
 

#K1DNEY
 

MINuRALIZAlICN
 

INFLAMMATION, NOS
 

I^FLAMMATICN^CHPONIC
 

#	 NUM^EP OF AM1ALS WITH TISSUE

*	 NUMuE* OF AMMALS NECROPSIED 

 (20)
 (47) 1 (5%)
 

1	 (2%) 

 1 (5*) 

 1 (5%) 3 (6%) 

13 (28%) 1 (2%) 1 (2%) 1 (2%) 

 (20) (47)
 

 (20)	 (50) 

1 (2%) 

 (20)	 (4b) 

1 (2%) 

 (20)	 (50) 

1 (2%) 2 (10%) 7 (14%) 

 1 (5%) 1 (2%) 

 1 (5%) 32 (64%) 

 (20) (50)
 

 (20)	 (49)
 

 8 (40%)
 10 (20%)
 

(20) (49)
 

 EXAMINED MICROSCOPICALLY 

C-8
 

(50)
 

1 (2-5)
 

1 (2%)
 

3 (6a

;)
 

1 (2%)
 

(5C)
 

1
 

(50)
 

(46)
 

1 (2V)
 

(5C)
 

1 {2%)
 

26 (52%)
 

1 (2J.)
 

(5C)
 

1 (2%)
 

(49)
 

17 (V>%)
 

(50) 

2 (4*,) 

1 (2%) 

LilPil. 
TABLE C2 (CONTINUED) 

UGH DOSE 

11-1502 

(5C) 

1 (2%) 

CONTPOL (V2H) 

11-1506 

LOW DOSS 

11-1504 

l^PHFOPATHY, ICXIC 1 (2%) 

#KIBNEY/PELVIS 

MINERALIZATION 

(20) (49) 

ENDOCRINE
 

#PITU1TARY (19) (47)
 CYST, NOS 1 (5%) 2 (4%)
 

h^MOPRHAGIC CYST 1 (2%)
 

AdGIECTASIS 2 iW)
 

#ADRUNA L (19) (49)
 

FIBROSIS 1 U%)
 METAMORPHOSIS FATTY
 

AN3IECTASIS 1 (2%)
 

#ADP^NAL CORTEX (19) (49)
 

laTAMURFHOSIS FATTY 1 (2%)
 HYPERPLASIA, FOCAL 1 (2%)
 

•AD^nNAL	 MEDULLA (19) (49)
 

HYPERPLASIA, NOS
 

(18) (47) 

HiPERPLASIi, C-CILL 1 (6%) 

REPRODUCTIVE SYSTEM
 

• MAKIiARY bLANE (20) (50)
 

CiSTIC DUC1S 1 (2%)
 

tLIEKUS (20) (50)
 

CfbT, NOS
 

PiCMSIRA 3 (15%) 1 (2%)
 

#LTERUS/ENDCMETRIU^1 (20) (50)
 

CiSTr NOS 1 (2%)
 IWFLAMMATICN, NOS 3 (151

*) 2 (4%)
 

INFLAMMATION, SUPPURATIVE 1 (2%)
 INFLAMMATION, ACUTE 1 (2%)
 HYPERPLASIA, NOS 1 [5%) 3 (6%)
 

HYPERPLASIA. CYSTIC
 

# NUMBER OF AMMALS WITH TISSUE FXAMINED MICROSCOPICALLY
 

* NUMBER OF AMMALS NECROPSILD
 

(46)
 

1
 

(48)
 

2
 

(48)
 

(48)
 

1
 

(47)
 

1
 

(50)
 

1
 

(49)
 

1
 

1
 

(49)
 

1
 

1
 

(2%)
 

(4%)
 

(2%)
 

(2%)
 

(2%)
 

(2%)
 

(2%)
 

(2%)
 

(2%)
 

2 .14%) _
 

C-9
 
TABLE C2 (CONCLUDED) 

CONTROL (VEH) LOW DOSE EIGh DOSS 

11-1506 11-1504 11-1502 

#OVAitY/OVIDfJCT 

INFLAMMATION, NOS 

(20) (50)

1 (2%) 

J49) 

IWFLAMMATICN, SUPPURATIVE 1 K2%) 

#OVARY (20) (47) (5C) 

CIST, NOS 3 (β*) PAROVARIAN CYSI 1 (5%) 4 (9%) 2 (4%) 

NESVOUS SYSTEM 

#ERAIN 

HiDPOCEPHALfJS, INTERNAL 

(20) (49) (50) 

1 (2*) 

SPECIAL SENSE CFGANS 

NON£ 

PUSCU1.OSKELETAI SYSTEM 

NONE 

EOEY CAVITIES 

•A3DOMINAL CAVITY 

NuCROSIS, FAI 

(20) (50) 

1 (2%) (50) 

•HESiiNIERY (20) (50) (50) NhC^OSIS, ΪΑΤ 1 (5%) 1 (2%) 

ALL OTHER SYSIIflS 

•MULTIPLE ORGANS (20) (50) (50) LuTJKEMOID iZAClION 

SPECIAL MOFFKCIOGY SUKMAFY 

NO LESION FEFCRTED 

# NUMxiER OF AMMALS WITH TISSUE EXAMINED MICECSCOPICALLY 

• NUMdER OF AMMALS NECRCFSIED 

C-IO
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH PIVALOLACTONE
 

TABLE Dl
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE
 

TREATED WITH PIVALOLACTONE 

CONTROL (VErt)

22-2505

 LUF DOSL·

 22-2503

 HIGh DOSE 

22-250! 

ANIMALS INITIALLY IN STUDY

ANIMALS MlbSINC

AMKALS NECROPSIED

iNIMA^S EXAMTNiD HISlOPAIHCLOGIcALLΥ**

 20

 20

 20

 50

 1 

49

 49

 5C 

50 

50 

INTEGUMENTARY SYSTEM 

•SUB^TTT TISSUE

FuiSIGN ECEY, NOS

AJSCESS , NCS

 (20) (49)

 1 (2%) 

1 (2%) 

(50) 

PESPIiiATOPY SYSTEM 

rjNo 

PNEUMONIA, ASPIRATION 

PNEUMONIA, CHRONIC 'MURINE 

PERIVASCULITIS 

(20) 

1 (5%) 

(49) 

7 (14%) 

(49) 

1

1 1

 (21 ) 

(22%) 

HEMATOPOIETIC SYSTEM 

«SPLEEN 

FIBROSIS, FCCAL 

HYPERPLASIA, RETICULUM

HYPERPLASIA, LYMPHOID

'UMATOPGItSTS 

CELL 

(18) )

1 (6%) 

)

 (2%)

 (47 (47) 

1 

11 

1 

(2%) 

[2%) 

{2%) 

(50) 

1 (2%) 

#LYMJ?H NODE

HYPERPLASIA, LYMPHOID 

(19)) ) 

2 (4%) 

(48 ) (46) 

«MEStNTEPIC L. MODE

HiPERPLASIA, RETICULUM

HYPERPLASIA, LYMPHOID

 CELL 

(19))

 1 (5%)

) 

1 (2%) 

(48 ) (46) 

1 {2%) 

CIRCULATOP Y SYSTEM 

tMlTuAL VALVE (19) (48) (49) 

# NUMJE R OB ANIMALS WITrf TISSU E EXAMINED

* NUMBER OF AMMALS N£C1OPSI£ D 

* * EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

MICROSCOPICALLY 

D-3
 
•TESTICULAR

 flETERY 

DrGENERATICN, HYALINE 

CIGESIIVE SYSTiH
 

#LIVUR
 

EiiEOLISM, NOS 

NuCROSIS, NOS 

NiC^OSIS, fOCAL 

HuPATOCYTCf^GALY 

MLTAMOPPHCSI S FATTY
 

«LIVUR/HEPATCCYIE S
 

HiPEPPLiSIA, NOS
 

«ESOPHAGUS
 

IAFLAMMAIICN, NOS 

#STCrfACH 

INFLAMMATION, NOS
 

«SMALL INTESTINE
 

NhMATODIASIS
 

S PA1CF
 

AobCISSr NCS
 

HYPERPLASIA, LYMPHOID
 

#LAPoE INTESTINE
 

NLMATODIASI S
 

UFINA£,1 SYSTEM
 

«KIDNEY
 

Gi-CMERULCNED

HRITIS,
 

SCLEROSIS
 

PLBIVASCOIIIIS
 

ENDOCRINE SYSIEM
 

«THYROID
 

# NUMi3ER OF ANIMALS WITH TISSUE

* NOMijEF OF ANIMALS NECROPSIED 

TABLE Dl (CONTINUED) 

CONTROL(V2H) LOW DOS*! 

22-2505 22-2503 

(20) (49) 

1 (2%) 

(^0) (49) 

1 (2%) 

1 (5%) 

2 (109?) 

1 (2%) 

(20) (49) 

1 (5%) 

(20) (49) 

(19) (48) 

1 (2") 

(20) (48) 

(18) (46) 

(18) <<•*) 

1 (2%) 

1 (6%) 3 (7%) 

(19) (49) 

7 (14*) 

(20)	 (48)
 

1 {2%)
 

1	 {2%)
 

(13) (23)
 

 EXAMINED MICROSCOPICALLY 

D-4 

HIGh DOSE 

/:Z-,501 

(50) 

(50) 

(50) 

(50) 

1 {2%) 

(48) 

(50) 

1 (2%) 

(49) 

1 (2%) 

(49) 

3 (6%) 

(49) 

14 (29%) 

(5C)
 

1	 (2%)
 

(36) 
FEPFOCnCTIVE SYSTEM
 

•SEMINAL	 VESICLE
 

CYST, NCS
 

flFSIIS
 

AIFOPHY, NCS
 

KERVOUS SYSTEM
 

«EFAiN
 

FSAMMOΜA ECDIES
 

SPECIAL SENSE C«GANS 

NCNi: 

MUSCULOSKELETAL SYSTEM 

•SKELETAL	 MUSCLE
 

PrtFASIIISP
 

EODY cAVITItS
 

• MESENTERY
 

PERIARTE
 

N£.C*OSIS, FAT
 

ALL OIHER
 

•MULTIPLE OFGANS
 

PLPIVASCULITIS
 

SPECIAL lORFhCLOGY SUMMAFY
 

NO LESION FEECEIED
 

TABLE Dl (CONTINUED)
 

CONTROL (VEH) LOW DOSE
 

22-2505 22-2503
 

(20)
 

(17)
 

1 (2*)
 

(19)	 (49)
 

2 (4%)
 

(2C) (49 ) 

1 (2%) 

(20) (49)
 

2
 

(20) (49)
 

12
 

HIGH DOSE
 

22-2501
 

(50)
 

1 (2%)
 

(4S)
 

(49)
 

1C (20%)
 

(50) 

(50)
 

1 (2%)
 

1 (2%)
 

(50)
 

1 (2%)
 

* N"^aER OF AMMALS WITH TISSUE EXAMINED MICEOSCOPICALLY
 

* NUMBER OF AMMALS NECROESILD
 

D-5
 
TABLE Dl (CONCLUDED) 

CONTROL (VEH) LOW DOSE

22-2505 22-2503

AUTO/NECBCFSY/HI3TO FERF 

OF AMMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

OF AMMALS NEC^OPSIED 

 HIGH DOSE 

 22-2501 

D-6
 
TABLE D2
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE
 

TREATED WITH PIVALOLACTONE
 

CONTROL (VEH) 22-2506 LOW EOSE 22-2504 hIGh DOSE 22-2502 

ANTMAi,b INITIALLY IN STUDY 

iNIMAi.S iUSSING 

ANIMALS N-CRCFSIED 

PNIMAi-S EXAMINED HISTOPAIHOLOGICALL **

20 

20 

20 

50 

2 

48 

48 

50 

50 

50 

INTEGUMENTARY SYSTEM 

•SUBcUT TISSUE

AriSCESS, NCS

 (20) (48)

 1 (2%) 

(50) 

FESPIrtATORY SYSTEM 

«LUNG 

PMUMONIA, ASPIRATION 

PNEUMONIA, CHRONIC MURINE 

(20) 

10 (50%) 

(48) 

1 (2%) 

5 (10%) 

(50) 

12 (24*) 

HEMATOPOIETIC SYSTEM 

«SPLEEN 

HYPERPLASIA, PETICULUM CELL 

HYPERPLASIA, LYMPHOID 

(19) 

1 (5%) 

C+3) (48) 

1 (2%) 

«L1MPF NODE 

HiPEPPLASI?, LYMPHOID 

(19) (47) 

1 (2%) 

(49) 

1 (2%) 

«MANDIBULAR L. NCDE 

HiPERDLASIA, LYMPHOID 

(19) (47) (49) 

1 {2%) 

«MESENTERIC L. NOEE 

HKPEPPLASIA, NOS 

HYPEFPLASIA, PETICULUM CELL 

(19) 

1 (5%) 

C*7) < 45 ) 

1 (2%) 

CIRCULATORY SYSTEM 

«HYOcARDIUB (20) (46) (49) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED

* NUMoEF OF AMMALS NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

MICROSCOPICALLY 

D-7
 
TABLE D2 (CONTINUED) 

CONTROL(VEri) LOW DOSS HIGfc D >SE 

22-2506 22-2504 Z2-25Q2 

LIGESTIVE SYSTEM 

(19) (48) (50) 

LYMPHOCYTIC INFLAMMATORY INFILTF 2 (47,) 

NECROSIS, FOCAL 1 (.·%) 

M-TA^OPFiiCSIS FATTY 2 (4%) 

ri^MATOEOIESIS 2 (k%) 

«PANCPEAS (19) (43) (49)
 

DIL^TATICN/DUCTS 1 (5%)
 

#STCMACH (19) (48) (50)
 

HYPERPLASIA, EPITHELIAL 1 (2%)
 

#PEYLPS PATCH (19) (47) (50) 

HYPERPLASIA, LYMPHOID <;%) 

#CULuN (19) (47) |5C)
 

NJEIATODIASIS 1 (2%)
 

SYSTEM 

#KIDNEY (20) (48)
 

HYDRONEPHROSIS 1
 1	 (<X ) LYMPHOCYTIC INFLA ΊΜ1ΤΟ * ENFILTR 5 (10%)
 

INFLAMMATION, SUPPURATIVE 1 (2%)
 

PERIARTERITIS 1 ΡΧ)
 INFARCT, NCS 1 (2%)
 

*URINARY ELiEIEP (18) (43) (45)
 

INFLAMMATION, NOS 1 (2%)
 

ENDOCrtlNE SYSTEM
 

•PITUITARY	 (11) (34) (36) 

ANGIECTASIS 1 (1%) 

#THYi*OID (7) (31) (29) 

CYSTIC FOIIICLES 1 (3%) 2 0%) HYPERPLASIA, FOLLICULAR-CELL 1 (3%) 

FEPROCUCTIVE SYSTEM
 

• MAMMARY GLAlvC	 (20) (48) (50)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NTJ^ER OF ANIMALS NECROPSItD 

D-8
 
TABLE D2 (CONTINUED) 

CONT&OL(VEH) LOW D0SJ5 HIGH DOSE 

22-2506 22-2504 22-2502 

#UTEKUS/ENDOMETRIUM (19) (47) (49) INFLAMMATION, SUPPURATIVE 1 (2%) 

HYPERPLASIA, NOS 1 (5%) 1 (2%) 3 (6%) 

HXPERPLASIA, CYSTIC 11 (58%) 16 (34%) 22 (45%) 

#OVAnY (19) (46) (Uβ) 

C*ST, NOS 4 (21%) 3 (7%) 9 (20%) 

KEFVOUS SYSTEK
 

*ERA1N (19) (48) (50)
 

PS AMMO MA ECDIES 2 (11%) 3 (6%)
 

SPECIAL SENSE C^GANS
 

•EYE/LACRIMAI GLAND	 (20) (48) (50)
 

HYPERPLASIA, EPITHELIAL 1 (5%)
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

EODY cAVITIES
 

•MESENTERY	 (20) (48) (50)
 

NECROSIS, PAT 1 (2%)
 

ALL OTHER SYSTEMS
 

•MULTIPLE ORGANS	 (20) (48) (50)
 

HYPERPLASIA, NOS 1 (5%)
 

HYPERPLASIA, LYMPHOID 1 (5%) 2 (4%)
 

SPECIAL MOfEHOlOGi SUMMARY
 

NO LESION FEICRTED 6 4
 

A^IMAL_MISSING/NO_NECROPSI 2
 

* NUMriER OP ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-9
 
TABLE D2 (CONCLUDED) 

CONTPOL(VEH) LOW DOSE

22-2506 22-2504

AUTO/NECFCESY/HISTO FEFF 

# NUM3EF OF AMMALS WITH TISSUE EXAMINED MICPOSCOPICALLY 

* NUMBER OF AMMALS NECKOPSIiD 

 HIGH DOSE 

 22-2502 

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

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

June 29, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed.
 The members of the Clearinghouse have been drawn from
 

academia, industry, organized labor, public interest groups,
 State health officials, and quasi-public health and research
 

organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and,
 

collectively, provide expertise in chemistry, biochemistry,
 

biostatistics, toxicology, pathology, and epidemiology.
 

Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment
 

Subgroup of the Clearinghouse is charged with the responsi­ bility of providing a peer review of reports prepatred on
 

NCI-sponsored bioassays of chemicals studied for carcinogenicity. It is in this context that the below critique is given
 

on the bioassay of Pivalolactone for carcinogenicity.
 

The reviewer noted that the substance is a structural
 

analog of the human carcinogen Beta-Propiolactone. He
 

agreed with the conclusion in the report that Pivailolactone
 

was carcinogenic in treated rats under the conditions of
 

test. He expressed surprise that it also was not carcinogenic in the treated mice. The reviewer conjectured that
 

the Pivalolactone may have hydrolyzed to an innocuous
 

substance by the time it was administered to the mice, thus
 

accounting for its lack of carcinogenic activity. He pointed
 

out that Beta-Propiolactone hydrolyzes to propionic acid in
 

the presence of water. The reviewer recommended that an
 

analysis be done to determine the half-life of Pivalolactone
 

in water. The results of such an analysis might be helpful
 

in assessing the dose of the test substance administered to
 

the mice. The reviewer noted the small control group size
 

and the route of exposure as experimental shortcomings.
 Despite these deficiencies, he said that Pivalolactone
 

should be considered to pose a carcinogenic risk to man. He
 

moved that the report on the bioassay of Pivalolactone be
 

accepted as written.
 

47
 
A Subgroup member suggested that the lack of a
 

carcinogenic response in the mice may have been due to a
 

vitamin deficiency* Another Subgroup member expressed
 

concern regarding the lack of knowledge about the precise
 

identity of the test substance administered. The reviewer
 

explained that his concern was not in its identity but
 

rather whether the Pivalolactone decomposed to an innocuous
 

substance. A staff member said that the program could
 

undertake some analysis of the stability of Pivalolactone
 

in water. It was agreed to accept the report on the bioassay
 

Pivalolactone as written. It was noted, however, that
 

additional information on the half-life of the compound in
 

water was desired.
 

Clearinghouse Members present:
 

Arnold L. Brown (Chairman), Mayo Clinic
 

Paul Nettesheim, National Institute of Environmental
 

Health Sciences
 

Verne Ray, Pfizer Medical Research Laboratory
 

Verald K. Rowe, Dow Chemical U.S.A.
 

Michael B. Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

48
 

<HJ.S GOVERNMENT PRINTING OFFICE 1978 260-899/3214 1-3 

DHEW Publication No. (NIH) 78-1395