National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 98 

1979 

BIOASSAY OF 

dl-MENTHOL 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 89-78-1* 

*The Chemical Abstracts Service (CAS) Registry Number used to track this bioassay 

is 15356-70-4 which is determined to best define the material used in the conduct of 

this bioassay. 

NCI-CG-TR-98 

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

Public Health Service 

National Institutes of Health 
BIOASSAY OF
 

dl-MENTHOL
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 79-1348
 
ii
 
BIOASSAY OF
 

dl-MENTHOL
 

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
 

dl-menthol conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer
 

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

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

determine whether selected chemicals have the capacity to produce
 

cancer in animals. Negative results, in which the test animals
 

do not have a greater incidence of cancer than control animals,
 

do not necessarily mean that the test chemical is not a
 

carcinogen, inasmuch as the experiments are conducted under a
 

limited set of circumstances. Positive results demonstrate that
 

the test chemical is carcinogenic for animals under the
 

conditions of the test and indicate that exposure to the chemical
 

is a potential risk to man. The actual determination of the risk
 

to man from animal carcinogens requires a wider analysis.
 

CONTRIBUTORS; This bioassay of dl-menthol was conducted by
 

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

under direct contract to NCI and currently under a subcontract to
 

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

Testing Program.
 

The principal investigators for the dl-menthol study were Drs. M.
 

B. Powers^ and R. W. Voelker . Drs. Powers, C. Cueto, Jr. , and
 

0. G. Fitzhugh^ were responsible for the selection of the doses
 

administered during the chronic study. Dr. Powers prepared a
 

report of the methodology. Ms. K. J. Petrovics was responsible
 

for data management and Mr. G. Najarian^ for animal care.
 

Histopathologic examinations were performed by Drs. D. A. Banas1
 

and R. H. Habermann* and reviewed by Dr. Voelker, and the
 

diagnoses included in this report represent their interpretation.
 

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

Mason Research Institute^. The statistical analyses were
 

performed by Dr. J. R. Joiner^ and Ms. P. L. Yong3, using methods
 

selected for the bioassay program by Dr. J. J. Gart5. Chemicals
 

used in this bioassay were analyzed at Midwest Research Institute
 

under the direction of Dr. E. Murrill6} and feed mixtures
 

containing the test chemical were analyzed at Hazleton
 

Laboratories by Dr. C. L. Guytonl and Mr. E. Missaghil. The
 

results of these analyses were reviewed by Dr. S. S. Olin^.
 

This report was prepared at Tracor Jitco3 in collaboration with
 

Hazleton Laboratories and NCI. Those responsible for the report
 

at Tracor Jitco were Dr. L. A. Campbell, Director of the Bioassay
 

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

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

Miller, Ms. L. A. Waltz, and Mr. W. D. Reichardt, bioscience
 

writers; and Dr. E. W. Gunberg, technical editor, assisted by Ms.
 

Y. E. Presley and Ms. P. J. Graboske.
 

The following other scientists at NCI^ were responsible for
 

evaluating the bioassay experiment, interpreting the results, and
 

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

Jr., Dr. J. Fielding Douglas, Dr. Dawn G. Goodman?, Dr. Richard
 

A. Griesemer, Dr. Harry A. Milman, Dr. Thomas W. Orme, Dr. Robert
 

A. Squire^, Dr. Sherman Stinson, Dr. Jerrold M. Ward, and Dr.
 

Carrie E. Whitmire.
 

lHazleton Laboratories America, Inc. , 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

2carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

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

Maryland.
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

IV
 
^Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute,
 

National Institutes of Health, Bethesda, Maryland.
 

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

Missouri.
 

with Clement Associates, Inc., 1010 Wisconsin Avenue, N.W. ,
 

Suite 660, Washington, D. C.
 

%ow with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 
vi
 
SUMMARY
 

A bioassay of dl-menthol for possible carcinogenicity was conducted
 

by administering the test chemical in feed to Fischer 344 rats and
 

B6C3F1 mice.
 

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

administered dl-menthol at one of the following doses, either 3,750
 

or 7,500 ppm for the rats and either 2,000 or 4,000 ppm for the mice,
 

for 103 weeks, then observed for 1 or 2 additional weeks. Matched
 

controls consisted of 50 untreated rats of each sex and 50 untreated
 

mice of each sex. All surviving rats were killed at 105 weeks and
 

all surviving mice at 104 weeks.
 

Mean body weights of dosed rats and mice were only slightly lower
 

than those of corresponding controls. No other clinical signs
 

related to administration of the dl-menthol were noted in the dosed
 

groups of animals. A dose-related trend in mortality was observed
 

only in the female mice. Survival at the end of the bioassay was at
 

least 62% in all dosed and control groups of animals of each species,
 

and sufficient numbers of animals were at risk for the development of
 

late-appearing tumors.
 

In male rats, no tumors occurred at incidences which were considered
 

to be related to the administration of dl-menthol.
 

In female rats, no tumors occurred at higher incidences in the dosed
 

groups than in the control groups. Fibroadenomas of the mammary
 

gland occurred at lower incidences in the low-dose (10/49) and
 

high-dose (7/49) groups than in the control group (20/50), and
 

alveolar/bronchiolar adenomas or carcinomas of the lung occurred only
 

in the controls (3/50).
 

In mice of either sex, no tumors occurred in dosed groups at
 

incidences that were significantly different from those for
 

corresponding control groups.
 

It is concluded that under the conditions of this bioassay, 

dl-menthol was not carcinogenic for either Fischer 344 rats or B6C3F1 

mice. 

vii
 
viii
 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 4
 

C. Animals 5
 

D. Animal Maintenance 5
 

E. Subchronic Studies 8
 

F. Designs of Chronic Studies 9
 

G. Clinical and Pathologic Examinations 12
 

H. Data Recording and Statistical Analyses 13
 

III. Results -Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 19
 

C. Pathology (Rats) 21
 

D. Satistical Analyses of Results (Rats) 23
 

IV. Results - Mice 25
 

A. Body Weights and Clinical Signs (Mice) 25
 

B. Survival (Mice) 25
 

C. Pathology (Mice) 28
 

D. Statistical Analyses of Results (Mice) 29
 

V. Discussion 31
 

VI. Bibliography 33
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Administered dl-Menthol in the Diet 37
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Administered dl-Menthol in the Diet 39
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Administered dl-Menthol in the Diet.... 43
 

ix
 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Administered dl-Menthol in the Diet 49 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Administered dl-Menthol in the Diet 51 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Administered dl-Menthol in the Diet.... 55 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered dl-Menthol in the Diet 59 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered dl-Menthol 

in the Diet 61 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered dl-Menthol 

in the Diet 66 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered dl-Menthol in the Diet 71 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered dl-Menthol 

in the Diet 73 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered dl-Menthol 

in the Diet 77 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Administered dl-Menthol in the Diet 81 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Administered dl-Menthol in the Diet... 83 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Administered dl-Menthol in the Diet.. 90 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered dl-Menthol in the Diet 95 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Administered dl-Menthol in the Diet.... 97 
Table F2
 

Appendix G
 

Table 1
 

Table 2
 

Figure 1
 

Figure 2
 

Figure 3
 

Figure 4
 

Analyses of the Incidence of Primary Tumors 

in Female Mice Administered dl-Menthol in the Diet. 102 

Analysis of Formulated Diets for 

dl-Menthol Concentration 107 

TABLES 

Design of dl-Menthol Chronic Feeding Studies 

in Rats 10 

Design of dl-Menthol Chronic Feeding Studies 

in Mice 11 

FIGURES 

Growth Curves for Rats Administered dl-Menthol 

in the Diet 20 

Survival Curves for Rats Administered dl-Menthol 

in the Diet 22 

Growth Curves for Mice Administered dl-Menthol 

in the Diet , 26 

Survival Curves for Mice Administered dl-Menthol 

in the Diet 27 

xi
 
xii
 
I. INTRODUCTION
 

Menthol (CAS 89-78-1; NCI C50000)
 

is a naturally occurring mono-


cyclic terpene found in the oils
 

of the mint tree Mentha arvensis.
 

This species is native to China,
 

Japan, Brazil, and South Africa,
 

and imports of natural menthol
 

from these countries in 1976
 

dl - Menthol
 

amounted to 146 million pounds
 

(USITC, 1977). An additional 285
 

thousand pounds of menthol were
 

produced synthetically from either citronellal, thymol, or
 

3-menthene in the United States in 1974, the last year when
 

production data were made public (USITC, 1975). Peppermint oil
 

yields primarily the L isomer, as does synthesis from citronellal.
 

The racemic mixture, DL- (or dl-)menthol, is the end result of
 

synthesis from thymol and 3-menthene (Booth, 1965). USP standards
 

exist for both the L isomer and racemic menthol (USP, 1974).
 

Menthol is well known for its cooling effects and its mint flavor
 

and odor, which are the basis for the majority of its uses. The
 

single largest use for menthol is probably in cigarettes (Stanford
 

Research Institute, 1973). A survey of pharmaceutical products
 
indicates that menthol is formulated in over-the-counter rubs and
 

liniments (2-10% concentration), antipruritic lotions, nasal sprays,
 

expectorants, mouthwashes and sprays, cough drops, and foot powders
 

(Kastrup, 1976; Billups, 1977). Menthol is used on inflamed or
 

irritated skin and on oral mucosal membranes, since it is reported
 

to have mild anesthetic, antiseptic, and counterirritant properties
 

(Kastrup, 1976; Rosenthal, 1972), although it is possible that the
 

only real effect it produces is a cooling sensation (AMA, 1977).
 

Mentholated cosmetics include preshave and aftershave creams,
 

depilatories, cleansing creams, bath oils, perfumes, soaps,
 

toothpastes, and baby powder (Rosenthal, 1972; Bell, 1972; Opdyke,
 

1976).
 

Menthol is generally recognized as safe for use in foods as a
 

flavoring agent (FDA, 1976). It is used as a component of
 

peppermint and lime flavors (Swaine, 1975) in products such as
 

chewing gum (0.11%), baked goods (0.01%), candy (0.4%), ice cream
 

(0.007%), and nonalcoholic beverages (0.004%) (Fenaroli, 1971).
 

Menthol is also used as a denaturant in alcohol not intended for
 

human consumption (Penty and Lescisin, 1965).
 

dl-Menthol was selected for study by the Carcinogenesis Testing
 

Program because of its extensive use both as a medicinal and
 

flavoring agent.
 
II. MATERIALS AND METHODS
 

A. Chemical
 

USP-grade dl-menthol was obtained from Glidden Organics
 

International, Jacksonville, Florida (Lot No. 4-HTP-6), and from
 

Norda, Inc., New York, New York (Lot No. Nl 1-26-74-2054). Sub-


chronic studies were conducted using Lot No. 4-HTP-6, and chronic
 

studies were conducted with this lot (3 weeks only in rats) and
 

the rest of the study with Lot No. Nl1-26-74-2054.
 

The identity of the chemical in both cases was established on the
 

basis of elemental analyses (C, H) and infrared, ultraviolet, and
 

nuclear magnetic resonance spectra. Gas-liquid chromatography of
 

Lot No. 4-HTP-6 showed two impurities estimated at 0.3% each,
 

which were very similar in volatility to the major component, and
 

one less volatile impurity of about 1.3%. One minor impurity
 

(0.2%) was detected in Lot No. Nl1-26-74-2054 by the same
 

technique. No impurities were detectable in either lot by
 

thin-layer chromatography. Karl Fischer analysis showed 0.22 +
 

0.02% water in Lot No. 4-HTP-6, and less than 0.1% water in Lot
 

No. Nl1-26-74-2054. Infrared and nuclear magnetic resonance
 

spectra were consistent with spectra in the literature (Sadtler,
 

1965).
 
The bulk chemicals were stored at 1°C in their original
 

containers.
 

B. Dietary Preparation
 

The appropriate weight of the dl-menthol required for each
 

dietary concentration was dissolved in corn oil by stirring over
 

a low heat. This solution was then added to the Wayne® Lab Blox
 

animal meal (Allied Mills, Inc., Chicago, 111.) and thoroughly
 

mixed in a Patterson-Kelly twin-shell blender. The final concentration of corn oil in the test diets for the dosed groups of
 

animals and in the basal diets for the untreated groups of
 

controls was 2% by weight. The test diets and basal diets were
 

prepared each week and used within 1 week of preparation. These
 

diets were stored at room temperature.
 

As a quality control measure, samples of freshly mixed diets were
 

analyzed periodically during the study. The results of these
 

analyses are reported in Appendix G. At each dietary
 

concentration, the mean of the analytical concentrations was
 

within 10% of the theoretical concentration, and the coefficient
 

of variation was 13-16%. The relatively large coefficient of
 

variation was due to low recoveries obtained from samples that
 

were analyzed several weeks after mixing and had lost a
 

significant amount of the dl-menthol by vaporization. For
 
samples analyzed within 3 weeks of mixing (73% of the samples),
 

the average coefficient of variation was 9.28%, which is more
 

acceptable. Since the test diets were less than 1 week old when
 

fed to the animals, accurate dietary concentrations of dl-menthol
 

were maintained.
 

Temperature—dependent stability analyses conducted at Midwest
 

Research Institute also confirmed the stability of dl-menthol in
 

feed for 2 weeks at temperatures up to 45°C.
 

C. Animals
 

The Fischer 344 rats and the B6C3F1 hybrid mice of each sex were
 

supplied by the Frederick Cancer Research Center, Frederick,
 

Maryland, through contracts with the Division of Cancer
 

Treatment, National Cancer Institute. On arrival at the
 

laboratory, the rats were quarantined for approximately 4 weeks
 

and the mice for approximately 2 weeks; they were determined to
 

be free from observable disease or parasites and assigned to the
 

various dosed or control groups on the basis of initial
 

individual body weights so that a homogeneous distribution of
 

mean weights and weight ranges was obtained between groups.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. The temperature was generally maintained at 20-24°c and
 
the relative humidity at 45-55%. Incoming air was filtered
 

through 2-inch-thick disposable fiberglass filters at a rate that
 

allowed 12 changes of room air per hour. Fluorescent lighting
 

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

The rats and mice were each housed in polycarbonate cages covered
 

with stainless steel cage lids and non-woven fiber filter bonnets
 

(Filtek, Appleton, Wis.). The rats were initially housed five
 

per cage; however, at week 48, the males were divided into groups
 

of two or three per cage. The mice were housed five per cage
 

throughout the study. The rats and the mice were housed in
 

separate rooms.
 

All cages were furnished with heat-treated hardwood chip bedding
 

(Sani-Chips®, Shurfire Products Corporation, Beltsville,
 

Maryland); the bedding was changed twice per week. Diets and
 

county supplied water were made available ad libitum. Food
 

hoppers and water bottles were refilled twice per week.
 

Cages, water bottles and sipper tubes were sanitized at 81°C
 

twice per week, feed hoppers once per week, and cage racks once
 

per month. An industrial dish washer was used for the water
 

bottles and sipper tubes; a cage and rack washer was used for the
 

food hoppers, cages, and racks. The detergent used contained a
 

phosphate base (Acclaim®, Economics Laboratory, St. Paul, Minn.).
 
When racks were changed, clean racks were randomly repositioned
 

in the rooms.
 

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

were housed in the same room as the respective dosed animals.
 

Rats administered dl-menthol in the diet were maintained in the
 

same room as rats being administered the following chemicals:
 

Rats
 

Feed Studies
 

(CAS 13463-67-7) titanium dioxide
 

(CAS 119-53-9) benzoin
 

(CAS 120-61-6) dimethylterephthalate
 

Gavage Studies
 

(CAS 127-69-5) sulfisoxazole
 

(CAS 7488-56-4) selenium disulfide
 

(CAS 108-60-1) bischloroisopropyl ether
 

Drinking Water Studies
 

(CAS 108-95-2) phenol
 

At week 48, the rats fed dl-menthol, together with those fed
 

titanium dioxide and those fed benzoin, were moved to a separate
 

room for the remainder of the bioassay.
 

Mice administered dl-menthol in the diet were maintained in the
 

same room as mice being administered the following chemicals:
 
Mice
 

Feed Studies
 

(CAS 13463-67-7) titanium dioxide
 

(CAS 119-53-9) benzoin
 

(CAS 120-61-6) diraethylterephthalate
 

Gavage Studies
 

(CAS 127-69-5) sulfisoxazole
 

(CAS 7488-56-4) selenium disulfide
 

(CAS 108-60-1) bischloroisophropyl ether
 

Drinking Water Studies
 

(CAS 108-95-2) phenol
 

The control groups of rats and mice used for the dl-menthol
 

studies were used also for the titanium dioxide studies. The
 

control groups were maintained in the same rooms with the dosed
 

groups.
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses of dl-menthol, on the basis of which two
 

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

doses") were determined for use in the chronic studies. On the
 

basis of the results of a 14-day range-finding study, doses of
 

930, 1,870, 3,750, 7,500, and 15,000 ppm were selected to be
 

administered in the diet in the Subchronic studies. At each
 

dose, 10 males and 10 females of each species received the test
 

diets 7 days per week for 13 weeks, and 10 males and 10 females
 

8
 
of each species were given basal diets for the same period of
 

time. At termination of the subchronic studies, all animals were
 

necropsied and histopathologic examination was made of tissues
 

from controls, the highest-dose groups, and selected tissues from
 

the second highest-dose groups.
 

There were no deaths among the rats, and the mean body weight
 

gains in the dosed groups were comparable to those in the control
 

groups at all doses. There was a slightly increased incidence of
 

interstitial nephritis in the male rats in the highest-dose
 

groups. In the mice, the six deaths occurring during the study
 

could not be related to compound administration; however, females
 

receiving 15,000 ppm gained 2 grams less than did the controls.
 

There was a slightly increased incidence of perivascular lymphoid
 

hyperplasia and interstitial nephritis among the female mice in
 

the two highest-dose groups. The low and high doses for the
 

chronic studies using rats were set at 3,750 and 7,500 ppm, and
 

the low and high doses for the chronic studies using mice were
 

set at 2,000 and 4,000 ppm.
 

F. Designs of Chronic Studies
 

The test groups, doses administered, and times on study of the
 

chronic feeding studies are shown in tables 1 and 2.
 
Table 1. Design of dl-Menthol Chronic Feeding Studies in Rats
 

Sex and 

Test 

Group 

Initial 

No. of 

Animals3 

dl-Menthol 

Dosesb 

(ppm) 

Time 

Dosed 

(weeks) 

on Study 

Observed 

(weeks) 

Male 

Matched-Control 50 0 105 

Low-Dose 50 3,750 103 2 

High-Dose 50 7,500 103 2 

Female 

Matched-Control 50 0 105 

Low-Dose 50 3,750 103 2 

High-Dose 50 7,500 103 2 

a

Rats were 9 weeks of age when placed on study.
 

t>dl-Menthol was administered in test diets containing 2% corn oil
 

ad libitum 7 days per week. The control groups received a basal
 

diet containing 2% corn oil.
 

10
 
Table 2. Design of dl-Menthol Chronic Feeding Studies in Mice
 

Sex and 

Test 

Group 

Initial 

No. of 

Animals3 

dl-Menthol 

Doses^ 

(ppm) 

Time 

Dosed 

(weeks) 

on Study 

Observed 

(weeks) 

Male 

Matched-Control 50 0 104 

Low -Dose 50 2,000 103 1 

High-Dose 50 4,000 103 1 

Female 

Matched-Control 50 0 104 

Low-Dose 50 2,000 103 1 

High-Dose 50 4,000 103 1 

a

Mice were 6 weeks of age when placed on study.
 

^dl-Menthol was administered in test diets containing 2% corn oil
 

ad libitum 7 days per week. The control groups received a basal
 

diet containing 2% corn oil.
 

11
 
G. Clinical and Pathological Examinations
 

All animals were observed twice daily for signs of toxicity.
 

Clinical signs and the presence of palpable masses were recorded
 

every week. Mean body weights and food consumption were recorded
 

every 2 weeks for the first 12 weeks and every month thereafter.
 

Animals that were moribund and those that survived to the termination of the study were killed by exsanguination under sodium
 

pentobarbital anesthesia (Diabutal , Diamond Laboratories, Inc.,
 

Des Moines, Iowa).
 

The pathologic evaluation consisted of gross and microscopic
 

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

from killed animals and from animals found dead. The tissues
 

were preserved in 10% buffered formalin, embedded in paraffin,
 

sectioned, and stained with hematoxylin and eosin. The following
 

tissues were examined microscopically: brain (frontal cortex and
 

basal ganglia, parietal cortex and thalamus, and cerebellum and
 

pons), pituitary, spinal cord (if neurologic signs were present),
 

eyes (if grossly abnormal), esophagus, trachea, salivary gland,
 

mandibular lymph node, thyroid, parathyroid, heart, thymus, lungs
 

and mainstem bronchi, liver, gallbladder (mice), pancreas,
 

spleen, kidney, adrenal, stomach, small intestine, colon, urinary
 

bladder, prostate or uterus, testes or ovaries, sternebrae,
 

12
 
femur, or vertebrae including marrow, mammary gland, tissue
 

masses, and any macroscopic lesions.
 

A few tissues from some animals were not examined, particularly
 

from those animals that died early. Also, some animals may have
 

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

state of autolysis as to preclude histopathologic evaluation.
 

Thus, the number of animals from which particular organs or
 

tissues were examined microscopically varies, and does not
 

necessarily represent the number of animals that were placed on
 

study in each group.
 

H. Data Recording and Statistical Analyses
 

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

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

descriptive information on the chemicals, animals, experimental
 

design, clinical observations, survival, body weight, and
 

individual pathologic results, as recommended by the
 

International Union Against Cancer (Berenblum, 1969). Data
 

tables were generated for verification of data transcription and
 

for statistical review.
 

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental
 

13
 
results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically
 

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

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

causes were not statistically censored. Statistical analyses for
 

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

(1972) for testing two groups for equality and Tarone's (1975)
 

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

One-tailed P values have been reported for all tests except the
 

departure from linearity test, which is only reported when its
 

two-tailed P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

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
 

14
 
appeared at multiple sites (e.g., lymphomas), the denominators
 

consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

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

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

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

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

compared simultaneously with those for a control group, a correction to ensure an overall significance level of 0.05 may be made.
 

The Bonferroni inequality (Miller, 1966) requires that the P
 

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

cases where this correction was used, it is discussed in the narrative section. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

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

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

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

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


tailed 0.05 level of significance. Unless otherwise noted, the
 

direction of the significant trend is a positive dose relation15
 
ship. This method also provides a two-tailed test of departure
 

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

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

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

such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

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

methods of comparing these curves were used for two groups;
 

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

groups. The statistical tests for the incidence of tumors which
 

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

16
 
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 pt/pc where pt is the true binomial
 

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

dosed group of animals and pc is the true probability of the
 

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

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

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

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

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

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

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

17
 
significant result (P < 0.025 one-tailed test when the control
 

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

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

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

the absence of a significant result while the upper limit
 

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. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of the dosed male and female rats were slightly
 

lower than those of the corresponding controls throughout the
 

bioassay (figure 1). No other clinical signs related to administration of the dl-menthol were noted. Clinical signs commonly
 

observed among rats of this strain were noted at comparable rates
 

in the control and dosed groups, particularly during the second
 

year of the bioassay, and increased in incidence as the animals
 

aged. These signs included eye changes (redness, paleness,
 

cloudiness, lacrimination, a red discharge or bloody crust, and
 

an enlarged or protruding eye), a hunched and/or thin appearance,
 

urine stains on the abdominal fur, and occasionally, nasal
 

discharge, sores on the body or the extremities, soft feces, and
 

enlarged testes.
 

The incidence of palpable nodules and tissue masses in the dosed
 

males was generally comparable to that in the control males, but
 

was lower in the dosed females than in the control females.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered dl-menthol in the
 

19
 
D 

8 

§ A 

A A

D 

O 

A 

§ s o 8 @ g D 

" O

A A

 O 

A A 

8 8 

Dp

8

 A 

g 

I

(9 

 300-

Q 

O 

m 

150-

D

O

MALE RATS 

 MATCHED CONTROL 

 LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

I 300- C3 

LU 

> 250-

Q 

O 

m 

2 200-

UJ 

FEMALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered dl-Menthol in the Diet 

20
 
diet at the doses of this bioassay, together with those of the
 

matched controls, are shown in figure 2. The results of the
 

Tarone test for dose-related trend in mortality and the results
 

of the Cox test comparing the survival of the control group with
 

each dosed group are not significant in either sex.
 

In male rats, 34/50 (68%) of the high-dose group, 33/50 (66%) of
 

the low-dose group, and 31/50 (62%) of the controls were alive at
 

week 105. In females, 38/50 (76%) of the high-dose group, 35/50
 

(70%) of the low-dose group, and 36/50 (72%) of the controls were
 

alive at week 105. Sufficient numbers of rats of each sex were
 

at risk for the development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

are summarized in Appendix C, tables Cl and C2.
 

Each of the tumor types observed has been encountered previously
 

as a spontaneous lesion, and occurred with no appreciable differences in frequency between control and dosed rats with a few
 

exceptions. In female rats, chromophobe adenomas of the
 

pituitary gland and fibroadenomas of the mammary gland were
 

observed with greater frequency in female control rats.
 

Chromophobe adenomas occurred in 28/48 controls, 25/47 low-dose,
 

21
 
i.oo a 

-D

O

A

MALE RATS 

 MATCHED COIMTROl 

 LOW DOSE 

 HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKSI 

100 110 

CC 0.60-

co 

§ 0.40­

OD 

O

tr 

0­ 0.30­

FEMALE RATS 

-D MATCHED CONTROL 

O LOW DOSE 

_A HIGH DOSE 

1 10 20 40 50 60 70 80 90 100 

TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered dl-Menthoi in the Diet 

22
 
and 19/43 high-dose female rats. Mammary gland fibroadenomas
 

were diagnosed in 20/50 female controls, 10/49 low-dose, and 7/49
 

high-dose rats. Mammary adenocarcinomas were seen in 1/50
 

controls, 3/49 low-dose, and 0/49 high-dose rats.
 

Chronic inflammation of the kidney was observed with greater
 

frequency in the dosed males than in the control males (29/49
 

controls, 41/50 low-dose, 41/50 high-dose); however, this finding
 

is of questionable importance, since such lesions are often found
 

in aged male Fischer 344 rats.
 

All other inflammatory, degenerative, and hyperplastic lesions
 

that occurred were similar in incidence and kind to those
 

naturally occurring lesions found in aged Fischer 344 rats.
 

Based on the histopathologic examination, dl-menthol was neither
 

toxic nor carcinogenic to Fischer 344 rats under the conditions
 

of this bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Table El and E2 in Appendix E contain the statistical analyses of
 

the incidences of those primary tumors that occurred in at least
 

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

one or more than one group.
 

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

23
 
positive dose-related trend are not significant, and none of the
 

results of the Fisher exact tests are significant in the positive
 

direction. Significant results in the negative direction are
 

observed, however, in the Cochran-Armitage test on the incidence
 

of tumors of the lung in female rats and in all statistical tests
 

on the incidences of fibroadenomas of the mammary gland in the
 

females, due to lower incidences of those tumors in the dosed
 

groups than in the control groups.
 

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

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

this indicates the absence of significant positive results. It
 

should also be noted that each of the intervals has an upper
 

limit greater than one, except the incidence of fibroadenomas of
 

the mammary gland in high-dose females, indicating the
 

theoretical possibility of the induction of tumors by dl-menthol,
 

which could not be detected under the conditions of this test.
 

24
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of the dosed male and female mice were slightly
 

lower than those of the corresponding controls throughout the
 

bioassay (figure 3). The appearance and behavior of the dosed
 

and control groups of animals were generally similar, and clinical signs usually associated with aging were noted at comparable
 

rates in the control and dosed groups. These signs included
 

alopecia (generalized or localized) , sores on the back and other
 

parts of the body, particularly in the males, anal and/or penile
 

irritation, a hunched and/or thin appearance, and occasional
 

abdominal distention.
 

The incidences of palpable nodules and tissue masses in the dosed
 

male or female groups were generally comparable to those of
 

corresponding control groups.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered dl-menthol in the
 

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

matched controls, are shown in figure 4. In male mice, the
 

result of the Tarone test for dose-related trend in mortality,
 

25
 
g o D a n a a D D a 

D Q 0 Q a a A

a Q a n a a a o a O A O A O Q Q O 

C3 30- A Q QAO D 

DODQ 

o 

UJ 25­

5 f 

a 

O 20­ 00 

uj

S 

MALE MICE 

Q MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

n a a 

a a a D o 

a n aoooQO A
 

a D D O AA A A
 C3 30- DOnQAQO A O A
 I- n n n A
 Q O a O UJ 25 

DAQ Q _AOO O 2 g °- z 

UJ 

S 15­

FEMALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

TIME ON STUDY (WEEKS) 

! Figure 3. Growth Curves for Mice Adminstered dl-Menthol in the Diet 

26
 
•A 

~~?-\ CH kl JL... A 6

•Th 

C>i ^ »•"• y 7i 6

&---! 

**5­ > -^ D 

u. 

H 

CD 

O 

CC 

MALE M CE 

MATCHED CONTROL 

O LOW DOSE 

HIGH DOSE 

n nn10 20 40 50 60 70 90 100 110 

TIME ON STUDY (WEEKS) 

l.OOl w 

A 

A6- &-TL 

* A Ura A,-, 6 A-, ^" o)--T 

1 

I 

0.80­

'A-., A 

< 

> 

> 

n 

U) 

u_ 

° 050­ > 

H 

_l 

2 040­

m 

O 

cc 

"EMALE MICE 

HATCHED C 

O -OW DOSE 

0.10­ A ^1IGH DOSE 

0.00­

10 20 40 50 60 70 80 90 100 110
 

TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves for Mice Administered dl-Menthol in the Diet 

27
 
and the results of the Cox test comparing the survival of the
 

control group with each dosed group, are not significant. In
 

females, the result of the Tarone test is significant (P =
 

0.008). The result of the Cox test comparing the survival of the
 

control group with the high-dose group is significant (P =
 

0.020), but the comparison between the control and low-dose
 

groups is not significant.
 

In male mice, there were 35/50 (70%) of the high-dose group,
 

32/50 (64%) of the low-dose group, and 32/50 (64%) of the
 

controls still alive at week 104. In female mice, there were
 

36/50 (72%) of the high-dose group, 40/50 (80%) of the low-dose
 

group, and 45/50 (90%) of the controls still alive at week 104.
 

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

development of late-appearing tumors.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

A low incidence of neoplasia was observed in both control and
 

dosed groups of mice. These neoplasms were of the usual number
 

and type observed in mice of this age and strain. A slightly
 

increased incidence of hepatocellular carcinomas was observed in
 

28
 
the high-dose males (8/47 controls, 8/49 low-dose, 14/48 high-


dose); however, the incidence was not increased over that
 

observed occasionally in historical-control groups of mice of
 

this age and strain. Alveolar/bronchiolar adenomas or carcinomas
 

of the lung occurred primarily in the dosed females (1/49
 

controls, 3/47 low-dose, 5/48 high-dose). The incidence of lung
 

neoplasms was not considered indicative of a carcinogenic effect,
 

as this neoplasm has been commonly seen at a similar low
 

incidence in historical-control groups.
 

Other degenerative, proliferative, and inflammatory lesions
 

observed were also of the usual incidence and kind observed in
 

aged B6C3F1 mice, and incidences in dosed groups were comparable
 

with those in control groups.
 

Based on the histopathologic examination, dl-menthol was neither
 

toxic nor carcinogenic to B6C3F1 mice under the conditions of
 

this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

29
 
The results of the Cochran-Armitage test for dose-related trend
 

and those of the Fisher exact test comparing the incidence of
 

tumors in the control group with that in each of the dosed groups
 

are not significant in either sex.
 

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

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

absence of significant positive results. It should also be noted
 

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

indicating the theoretical possibility of the induction of tumors
 

by dl-menthol, which could not be detected under the conditions
 

of this test.
 

30
 
V. DISCUSSION
 

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

than those of corresponding controls. No other clinical signs
 

related to administration of dl-menthol were noted in any dosed
 

groups of rats and mice. A dose-related trend in mortality was
 

observed only in the female mice. Survival at the end of the
 

bioassay was at least 62% in all dosed and control groups of
 

animals of each species, and sufficient numbers of animals were
 

at risk for the development of late-appearing tumors.
 

In male rats, no tumors occurred at incidences which were
 

considered to be associated with the administration of
 

dl—menthol.
 

In female rats, no tumors occurred at higher incidences in dosed
 

groups than in control groups. Fibroadenomas of the mammary
 

gland occurred at lower incidences in the low-dose (10/49) and
 

high-dose (7/49) groups than in the control group (20/50), and
 

alveolar/bronchiolar adenomas or carcinomas of the lung occurred
 

only in the controls (3/50).
 

In mice of either sex, no tumors occurred in dosed groups at
 

incidences that were significantly different from those for
 

corresponding control groups.
 

31
 
The acute oral LD5Q of menthol in Osborne-Mendel rats has been
 

reported as 3,180 mg/kg body weight (Jenner et al. , 1964) and as
 

2,900 mg/kg body weight (Herken, 1961). When administered in the
 

diet to male and female rats for 5.5 weeks, d- or dl-menthol at
 

100 or 200 mg/kg body weight caused no adverse effects on gain in
 

weight (Herken, 1961). No long-term studies have been reported
 

previous to the present bioassay.
 

It is concluded that under the conditions of this bioassay,
 

dl-menthol was not carcinogenic for either Fischer 344 rats or
 

B6C3F1 mice.
 

32
 
VI. BIBLIOGRAPHY
 

AMA Department of Drugs, Dermatologic agents. AMA Drug
 

Evaluations, PSG Publishing Co., Inc., Littleton, Mass.,
 

1977, p. 915.
 

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

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

Bell, S. A., Preshave and aftershave preparations. In:
 

Cosmetics, Science and Technology, Vol. _2_, Balsam, M. S. and
 

Sagarin, E. , eds. , Wiley-Interscience, New York, 1972, pp.
 

22-23 and 26-27.
 

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

Panel on Carcinogenicity of the Cancer Research Commission
 

of UICC, Vol. _2_, International Union Against Cancer, Geneva,
 

1969.
 

Billups, N. F. , American Drug Index, J. B. Lippincott Co.,
 

Philadelphia, 1977, p. 392.
 

Booth, A. B. , Terpenes and terpenoids. In: Kirk-Othmer
 

Encyclopedia of Chemical Technology, Vol. 19, Standen A. ,
 

ed., Interscience Publisher, Inc., New York, 1965, pp.
 

823-825.
 

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

Soc. B 34(2);187-220, 1972.
 

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

1970, pp. 48-52.
 

Fenaroli, G. , Mentha arvensis. In: Fenaroli's Handbook of
 

Flavor Ingredients, Furia, T. E. and Bellanca, N., eds., The
 

Chemical Rubber Co., Cleveland, Ohio, 1971, pp. 165-166 and
 

492.
 

Food and Drug Administration, Food and Drugs. _2J_ CFR 121.101,
 

April 1, 1976.
 

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

significance tests, confidence limits and adjustments for
 

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

33
 
Herken, H. , Report to Schering AG, 1961. Cited by Opdyke, D. L.
 

J., Monographs on fragrance raw materials. Fd. Cosmet.
 

Toxicol. 14(5).-473-474. 1976.
 

Jenner, P. M., Hagan, E. C., Taylor, J. M., Cook, E. L. , and
 

Fitzhugh, 0. G., Food flavourings and compounds of related
 

structure. I. Acute oral toxicity. Fd. Cosmet. Toxicol.
 

_2:327-343, 1964.
 

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

incomplete observations. J. Am. Statist. Assoc. _53:457-481,
 

1958.
 

Kastrup, E. K. , ed., Facts and Comparisons, Facts and
 

Comparisons, Inc., St. Louis, Mo., 1976, pp. 184a, 185, 524,
 

564, 572, 577, 618-619, and 633.
 

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

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

and Biomed. Res. 7:230-248. 1974.
 

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

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

Opdyke, D. L. J., Fragrance raw materials monographs. Fd.
 

Cosmet. Toxicol. 14(5);473-474. 1976.
 

Penty, C. A., Jr., and Lescisin, G. A., Ethanol. In:
 

Kirk-Othmer Encyclopedia of Chemical Technology Vol. _8,
 

Mark, H. F., McKetta, J. J. , and Othmer, D. F., eds., John
 

Wiley & Sons, Inc., New York, 1965, p. 450.
 

Rosenthal, M. W., The essential oils. In: Cosmetics: Science
 

and Technology, Vol. 1, Balsam, M. S. and Sagarin, E., eds.,
 

Wiley-Interscience, New York, 1972, pp. 540 and 546.
 

Sadtler Research Laboratories, Sadtler Standard Spectra, IR No.
 

586, Sadtler Research Laboratories, Philadelphia, 1965.
 

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

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

induced by different numbers of administrations of benzo (a)
 

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

Stanford Research Institute, dl-Menthol. Chemical Dossier,
 

Stanford Research Institute, Menlo Park, Calif., 1973.
 

34
 
Swaine, R. L., Natural and synthetic flavorings. In: Handbook
 

of Food Additives, second edition, Furia, T. E., ed., CRC
 

Press, Cleveland, Ohio, 1975, pp. 470-472 and 503.
 

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

Biometrika 62:679-682, 1975.
 

United States International Trade Commission, Imports of
 

Benzenoid Chemicals and Products, 1976. USITC Publication
 

828, Washington, D.C., 1977.
 

United States International Trade Commission, Synthetic Organic
 

Chemicals. United States Production and Sales, 1973, ITC
 

Publication 728, Washington, B.C., 1975.
 

United States Pharmacopeial Convention, Menthol. In: The United
 

States Pharmacopeia XIX, United States Pharmacopeial
 

Convention, Inc., Rockville, Md., 1974, p. 302.
 

35
 
36
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED dl-MENTHOL IN THE DIET
 

37
 
38

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED dl-MENTHOL IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

*SKIN
 

SQUAMOtJS CELL CARCINOMA
 

BASAL-CELL CARCINOMA
 

ANGIOMA
 

*SUBCUT TISSUE
 

SQUAMOUS CELL CARCINOMA
 

FIBROMA
 

FIBROSARCOMA
 

LIPOSARCOMA
 

HEMANGIOSARCOMA
 

HEMANGIOPERICYTOMA, B4LIGNANT
 

OSTEOSARCOMA
 

RESPIRATORY SYSTEM
 

ILUNG
 

ALVEOLAH/BRONCHIOLAB CARCINOMA
 

HEMANGIOPERICYTOMA, METASTATIC
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

HONOCYTIC LEUKEMIA
 

ISPLEEN
 

HEMANGIOSARCOMA
 

tCERVICAL LYMPH NODE
 

1WATCHED
 

(:ONTROL LOW DOSE HIGH DOSE
 

50	 5 0 ,5 0
 

49 50	 50
 

49 50	 50
 

(49)	 (50) (50)
 

1 (2%) 1 (2X)
 

1 (2»)
 

(50) (50)
 

1 (2X) 1 (2X)
 

1 (2*) 2 (4X)
 

1 (2X) 1 (2*)
 

1 (2»)
 

1 (2*) 1 (2X)
 

1 (2*) 1 (2X)
 

1 (2*)
 

(49) (50) (50)
 

1 (2X) 1 (2X)
 

1 (2*) 1 (2X)
 

(09) (50) (50)
 

1 (2X)
 

14 (29%) 14 (28X) 11 (22X)
 

(49)	 (50) (50)
 

1 (2X) 1 (2X)
 

(49) (49) (50)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

39
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

tLIVER (49) (50) 

KUPFFER-CELL SARCOMA 1 (2X) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

tSALIVAR Y GLAN D (47) (48) 

HEMANGIOSARCOHA , INVASIV E 1 (2*) 

HEMANGIOPERICYTOMA , NETASTATIC 

tLIVER (49) (50) 

NEOPLASTIC NODULE 1 (2*) 

HEPATOCELLOLAR CARCINOM A 3 (6*) 

• PANCREA S	 (49) (50) 

ADENOCARCINOMA , NOS, METASTATIC 1 (2X) 

#SMALL INTESTINE (49) (50) 

ADENOCAHCINOMA , NOS 1 (2X) 

URINAR Y SYSTE M 

#KIDNE Y (49) (50) 

TRANSITIONAL-CELL CARCINOM A 1 (2X) 

TUBULAR-CEL L ADENOM A 1 (2*| 

ENDOCRIN E SYSTEM 

fPITUITAR Y (48) (49) 

CHROMOPHOBE ADENOM A 5 (10X) 7 (14*) 

tADRENAL (49) (50) 

PHEOCHROMOCYTOMA 7 (14%) 7 (14*) 

tTHYROID (49) (48) 

FOLLICULAR-CELL ADENOM A 

FOLLICULAR-CEL,L_CAgCINQHA 1 J2S) 1.12SL 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPIC/ILLY
 * NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

(50) 

(50) 

1 (2X) 

(50) 

2 (4)1) 

(50) 

(50) 

(50) 

1 (2X) 

(46) 

2 (4X) 

(50) 

7 (14X) 

(48) 

1 (2X) 

2_iiSJ._, 

40
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

C-CELL ADENOMA
 

C-CELL CARCINOMA
 

fPANCREATIC ISLETS
 

ISLET-CELL ADENOMA
 

ISLET-CELL CARCINOMA
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND
 

ADENOCARCINOMA, NOS
 

FIBROADENOMA
 

*PREPUTIAL GLAND
 

CARCINOMA, NOS
 

tTESTIS
 

INTERSTITIAL-CELL TUMOR
 

INTERSTITIAL-CELL TUMOR, HALIGNA
 

*EPIDIDYMIS
 

INTERSTITIAL-CELL TUMOR, INVASIV
 

NERVOUS SYSTEM
 

#ERAIN
 

ASTROCYTOMA
 

SPECIAL SENSE ORGANS
 

*ZYMBAL'S GLAND
 

CARCINOMA, NOS
 

MUSCULOSKELETAL SYSTEM
 

*BONE
 

OSTEOSARCOMA
 

*SKELETAL MUSCLE
 

MATCHED 

CONTROL LOW 

1 

4 (8%) 

(49) (50) 

1 (2%) 2 

(49) (50) 

1 

1 (2%) 4 

(49) (50) 

2 (4%) 2 

(49) (50) 

44 (90%) 47 

1 (2%) 

(49) (50) 

1 (2%) 

(49) (50) 

1 

(49) (50) 

1 

(49) (50) 

1 (2*) 

(49) (50) 

DOSE HIGH 

(2%) 

(4X)

1 

(50) 

1 

1 

(2*) 

(8X)

(4*)

(94X)

(50) 

2 

(50) 

4 

(50) 

 50 

(50) 

(2X)

(50) 

1 

(2X) 

(50) 

(50) 

(50) 

DOSE
 

(2X)
 

(2%)
 

(2%)
 

(1%)
 

(3%)
 

(100%)
 

(2*)
 

* BOMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

41
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

BOD Y CAVITIE S 

*ABDOMINA L CAVIT Y
 

MESOTHELIOMA , NOS
 

ALL OTHEF SYSTEMS
 

*MULTIPLE ORGANS
 

MESOTHELIOMA, NOS
 

MESOTHELIOMA, MALIGNANT
 

ANIMA L DISPOSITION SUMMAR Y
 

ANIMALS INITIALLY IN STUDY
 

NATUEAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMA L MISSING
 

5)	 INCLUDES AUTOLYZED ANIMALS
 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS * 

TOTAL PRIMAR Y TUMORS 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS HITH SECONDARY TUMORS*


TOTAL SECONDARY TDMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS HITH TtJHOSS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL 

(49)
 

(49)
 

2 (4%)
 

50 

18 

1 

31 

47
 

90
 

46
 

59
 

 24


 28


 3


 3
 

3


 3
 

LOW DOSE HIGH DOSE 

(50)	 (50)
 

1 <2X) 1 (2X)
 

(50)	 (50)
 

2
 

2 (4X)
 

50 50

 

13 15 

4 1 

33 

48 50
 

106 97
 

48 50
 

70 66
 

 28 26
 

 33 30
 

2 1
 

2 3
 

3 1
 

3 1
 

* PRIMARY TUMOfiS: ALL TUMORS EXCEPT SECONDARY TUHORS
 

# SECONDARY TUMORS: HETASTATIC TUMORS OB TOMOBS INVASIVE INTO AN ADJACENT OBSAN
 

42
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED dl-MENTHOL IN THE DIET 

1 

1 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

50

50

50

 50

 49

 49

 50 

 49 

 49 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (49) (49)
 

SQUAMOUS CELL CARCINOMA 1 (2*)
 

FIBROUS HISTIOCYTOMA, METASTATIC 2 (<*X)
 

*SUBCUT TISSUE (50) (49) (49)
 

SQUAMOUS CELL CARCINOMA 1 (2%)
 

FIBROMA 1 (2%)
 

FIBROUS HISTIOCYTOMA, MALIGNANT 1 (2*)
 

LIPOMA 1 (21)
 

RESPIRATORY SYSTEM
 

#LUNG (50) (49) (48)
 

ALVEOLAH/BRONCHIOLAR ADENOMA 2 (4*)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2*)
 

FIBROUS HISTIOCYTOMA, METASTATIC 2 (4*)
 

HEHATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS (50) (49) (49)
 

MYELOMONOCYTIC LEUKEMIA 1 (2*)
 

MONOCYTIC LEUKEMIA 10 (20*) 5 (10*) 6 (12*)
 

SSPLEEN (50) (49| (49)
 

HEMANGIOSARCOMA 1 (2*)
 

*CERVICAL LYMPH NODE (50) (47) (47)
 

FIBROUS HISTIOCYTOMA, METASTATIC 1 (2X)
 

tBEONCHIAL LYMPH NODE (50) (47) (47)
 

FIBIOUS_HISTIQCITOMAJL_HETASTATIC 1.12SL
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

43
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

#MESENTERIC L. NODE

FIBROUS HISTIOCYTOMA, HETASTATIC

 (50) (47)

 1 (2%) 

 (1*7) 

CIRCULATORY SYSTEM 

*HEART

FIBROUS HISTIOCYTOMA, METASTATIC

 (50) (49)

 1 (2X) 

 (48) 

DIGESTIVE SYSTEM 

#LIVER

NEOPLASTIC NODULE

FIBROUS HISTIOCYTOHA, BETASTATIC

 (50)

 1 (2%)

 (49)

 1 (2XJ

 1 (2%) 

 (49) 

1 (2X) 

#PANCREAS

FIBROUS HISTIOCYTOBA, HETASTATIC

 (50) (47)

 1 (2X) 

 (49) 

URINARY SYSTEM 

tKIDNEY

FIBROUS HISTIOCYTOBA, METASTATIC

 (50) (19)

 1 (2X) 

 (49) 

tRIGHT KIDNEY

MIXED TUBOR, MALIGNANT

 (50) (49)

 1 (2*) 

 (49) 

#LEFT KIDNEY

MIXED TUMOR, METASTATIC

 (50) (49)

 1 (2%) 

 (49) 

ENDOCRINE SYSTEM 

tPITUITARY 

CHROMOPHOBE ADENOMA 

(48) 

28 (58X) 

(47) 

25 (53») 

(43) 

19 (44X) 

fADRENAL 

PHEOCHROMOCYTOBA MIXED TUHOR, BETASTATIC 

(50) (49) 

1

1

 (2X) 

 (2*) 

(49) 

3 (6S) 

• THYROID

FOLLICOLAR-CELL ADENOMA

JQHICflLAB-CELt CABCINOHft

 (48)

 2 (4«)

 _

 (47) 

2 (UX) 

 1.12SL 

(46) 

2 (4S) 

t NUBBER OF ANIMALS WITH TISSUE EXABIHED BICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

44
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

C-CELL CARCINOM A 1 (2%) 2 (<•*) 

#PANCREATIC ISLETS 

ISLET-CELL ADENOM A 

(50) (47) 

REPRODUCTIV E SYSTEM 

*MAMMAR Y GLAN D (50) (U9) 

ADENOMA , NO S 1 (2%) 

ADENOCARCINOMA , NOS 1 (2%) 3 (6X) 

CYSTADENOMA, NOS 1 (2%) 

FIBROADENOM A 20 (i*0») 10 (20*) 

*PEEPUTIA L GLAN D (50) (U9) 

CARCINOMA , NOS 2 (US) 2 (U*) 

#UTERO S (50) («9) 

ADENOCARCINOMA , NOS 1 (2%) 

ENDOMETRIA L STROMA L POLYP 6 (12%) 6 (12*) 

ftUTERUS/ENDOMETRIU M (50) (U9) 

SARCOMA , NOS 1 (2%) 

ENDOMETRIA L STROMAL POLYP 1 (2X) 

IOVAR Y (<*9) (49) 

FIBROM A 1 (2%) 

SEMINOMA/DYSGERMINOH A 1 (2X) 

NERVOU S SYSTE M 

#BRAI N (48) (49) 

GLIOMA , NOS 1 (2*) 

ASTROCYTOM A 1 (2*) 

SPECIAL SENSE ORGAN S 

*HARDERIA N GLAN D (50) (49) 

SQOAMOU S CELL CARCINOMA , METAST A 1 (2*) 

*ZYHBAL' S GLAN D (50) (49) 

CARCINOMA.JJOS J.J2SI 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECHOPSIED
 

HIGH DOSE 

2 (4%) 

(49) 

1 (2*) 

(49) 

7 (14%) 

(49) 

1 (2*) 

(48) 

8 (17%) 

(48) 

2 (4*) 

(48) 

(49) 

(49) 

(49) 

45
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

HUSCULOSKELETAL SYSTEM
 

*FEHOB


FIBROUS HISTIOCYTOMA, MALIGNANT


BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

ANIHAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATUBAL DEATHS


MORIBUND SACRIFICE


SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIMAL MISSING
 

1_I1CLUDES_AUTQLYZED_ANIMALS
 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

 (50)	 (49) (49)
 

1 (2*)
 

 50 50 50
 

 11 14 8
 

3 1 4
 

 36 35	 38
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

46
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUHORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUHORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR HETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

4 1 36 32 

83 67 53 

38 32 29 

62 46 40 

19 15 10 

20 20 12 

1 3 

1 13 

1 1 1 

1 1 1 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUM3RS
 

t SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT OR3AN
 

47
 
48

 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED dl-MENTHOL IN THE DIET
 

49
 
50

 
TABLE 81.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED dl-MENTHOL IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 50 50
 ANIMALS NECROPSIED 47
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 47 49
 

INTEGUMENTAR Y SYSTE M 

*SKIN 

FIBROSARCOMA, METASTATIC 

(47) (49) 

*SUBCUT TISSUE 

FIBROMA 

FIBROSARCOMA 

HEMANGIOSARCOMA 

(47) 

4

8

1

 (9%) 

 (17%) 

 (2%) 

(49) 

1

6

2

RESPIRATORY SYSTEM
 

tLUNG (46) (49)
 

HEPATOCELLULAR CARCINOMA, METAST
 

ALVEOLAR/BRONCHIOLAR ADENOMA 5 (11%) 6


ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2%) 2


FIBROSARCOMA, METASTATIC
 

HEMANGIOSARCOMA, METASTATIC 1


HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (47) 

MALIG.LYHPHOMA , LYMPHOCYTIC TYPE 4 (9%) 1

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

GRANULOCYTIC LEUKEMI A 2

tSPLEEN (47) (49) 

HEMANGIOSARCOMA
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

#MESENTERIC L. NODE (47) (49)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1


-HEI­

* NUMBER OP ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECHOPSIED
 

 (2%)
 

 (12%)
 

 (4%)
 

 (12%)
 

 (»*)
 

 (2%)
 

 (2%)
 

 (2%)
 

 {"»%)
 

 (2%)
 

50
 

48
 

(48) 

1 (2%) 

(48) 

6

4

1

 (13%) 

 (8%) 

 (2%) 

(48)
 

1 (2%)
 

6 (13%)
 

1 (2%)
 

(48)
 

2 (4%)
 

2 («%)
 

1 (2%)
 

1 (2%)
 

(48)
 

2 («%)
 

51
 
TABLE B1 . MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ISHALL INTESTINE (47) (49) (47)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2J)
 

tPEYERS PATCH (47) (49) (47)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2X)
 

MALIG.LYHPHOMA, HISTIOCYTIC TYPE 1 (2%)
 

CIRCULATORY SYSTEM
 

• HEART (46) (49) (48)
 

HEMANGIOSARCONA 1 (2%)
 

DIGESTIVE SYSTEM
 

#LIVER (47) (49) (48)
 

HEPATOCELLULAR CARCINOMA 8 (17X) 8 (16J) 14 (29»)
 

HEMANGIOSARCOMA 1 (2X)
 — •_V>-~~>.~..«I.~*B«a.O« _ __ •.«•••.•••.*—•_ •— ••—.— —*«—••,——•*—•———— —«. •_«.•___•> .^~_»*_____«._«__*.
 

URINARY SYSTEM
 

NONE
 

ENDOCEINE SYSTEM
 

tTHYROID (43) (46) (44)
 

FOLLICULAR-CELL ADENOMA 1 (2»)
 

FOLLICULAR-CELL CARCINOMA 1 (2»)
 

REPRODUCTIVE SYSTEM
 

tTESHS (47) (49) (U8)
 

INTERSTITIAL-CELL TOMO R 1 (21)
 

NEFVOUS SYSTEM 

NON E 

SPECIAL SENSE ORGANS 

*EYE/LACRIMAL GLAN D (47) (49) (48) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

52
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

•HARDERIAN GLAND (47) (49) (48) 

ADENOMA , NOS 1 (2i) 3 (6X) 

MUSCULOSKELETAL SYSTEM
 

*ABDONINAL MUSCLE (47) (49) (48)
 

FIBROSAHCOMA, BETASTATIC 1 (2X)
 — •» — — «»——•_ — _ — •_ — — «— *»•—•••»•• — —_*_•___..___..__•»•_«._.._.___•»__..______«__•—.__.._____._«_.
 

BODY CAVITIES
 

NONE
 

ALL OTHEE SYSTEMS
 

*MULTIPLE ORGANS (47) (49) (48)
 

MESOTHELIOMA, NOS 1 (2»)
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

NATURAL DEATHS 17 18 15
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED 1
 

TERMINAL SACRIFICE 32 32 35
 

ANIMAL MISSING
 

!_!NCLUDES_AUTOLYZED_AN!HALS
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

53
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)

 

TUMOR SUMMARY
 

TOTAL ANIBALS WITH PRIMARY TUBORS*
 

TOTAL PRIMARY TUBORS
 

TOTAL AtJIHALS WITH BENIGN TUBORS
 

TOTAL BENIGN TUBORS
 

TOTAL ANIBALS WITH BALIGNANT TUBORS
 

TOTAL BALIGNANT TUBORS
 

TOTAL ANIBALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIBALS WITH TUMORS UNCERTAIN­ BENIGN OR BALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIBALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUHORS
 

MATCHED
 

CONTROL
 

29
 

36
 

10
 

10
 

22
 

25
 

1
 

1
 

LOW DOSE HIGH DOSE 

28 33 

35 i|8 

9 15 

9 16 

23 26 

25 32 

2 2 

2 U 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUHORS
 

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

54
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED dl-MENTHOL IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS 

ANIMALS 

ANIMALS 

ANIMALS 

INITIALLY IN 

MISSING 

NECROPSIED 

STUDY 

EXAMINED HISTOPATHOLOGICALLY 

50 

1 

49 

49 

50 

47 

47 

50 

48 

48 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

ILUN G (49) (47) 

ALVEOLAR/BRONCHIOLAR ADENOMA 3 (6X) 5 (10%) 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2«) 

FOLLICOLAR-CELL CARCINOMA, METAS 1 (2%) 

LEIOMYOSARCOHA, METASTATIC 1 (2«) 

HEMATOPOIETIC SYSTEM
 

*HOLTIPLE ORGANS (49) (47) (48)
 

MALIG.LYMPHOMA, LYHPHOCYTIC TYPE 6 (12%) 5 (11%) 7 (15%)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 12 (24%) 8 (17%) 3 (6%)
 

MALIGNANT LYHPHOMA, MIXED TYPE 2 (4%)
 

• SPLEEN (49) (47) (48)
 

HEMANGIOSARCOHA 1 (2%)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%)
 

fCERVICAL LYMPH NODE (48) (47) (48)
 

HEHANGIOSARCOHA 1 (2%)
 

tMESENTERIC L. NODE (48) (47) (48)
 

HALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%)
 

*THYMUS (23) (26) (29)
 

MALIG.LYMPHOMA. LYMPHOCYTIC TYPE 1 (3%)
 

CIRCULATORY SYSTES
 

NONE
 

* NUMBER OP ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

55
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIVE SYSTEM 

tLIVER (49) (<*7) (48)
 

HEPATOCELLULAH CARCINOMA 1 (2%) 3 (6X) 3 (6%)
 

#STOHACH (48) (47) (48)
 

LEIOMYOSARCOMA 1 (2X)
 

ILARGE INTESTINE (48) (46) (48)
 

LEIOMYOSARCOMA, HETASTATIC 1 (2X)
 

URINARY SYSTEM
 

• KIDNEY (49) (47) (48)
 

LEIOMYOSARCOMA, METASTATIC 1 (2X)
 

#URINARY BLADDER (47) (US) (47)
 

LEIOHYOSARCOMA, BETASTATIC 1 (2X)
 

ENDOCRINE SYSTEH
 

IPITUITARY (33) (35) (39)
 

CHROMOPHOBE ADENOMA 3 (9%) 1 (3%)
 

#THYROID (43) (46) (46) 

FOLLICDLAR-CELL ADENOM A 3 (7%) 3 (7X) 

FOLLICULAR-CELL CARCINOM A 1 (2%) 

REPRODUCTIV E SYSTEM 

*«AMMARY GLAND (49) (47) (48) 

ADENOCABCINOMA, NOS 1 (2%) 3 2 (4S) 

IUTEHUS (48) (46) (48)
 

LEIOMYOSARCOMA, METASTATIC 1 (2*)
 

ENDOMETRIAL STROMAL POLYP 1 (2J)
 

HEMANGIOSARCOMA 1 (2%)
 

IOVARY (47) (47) (48)
 

PAPILLARY CYSTADENOMA, NOS
 

TERATOMA, NOS 1 (2%)
 

NERVOUS SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

56
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

*ABDOHINAL CAVITY

HEMANGIOSARCOMA

 (49)

 1 (2%) 

 («7) (48) 

*HESENTERy

LEIOMYOSARCOHA, METASTATIC

 (49)

 1 (2X) 

 (47) (48) 

ALI CTHEF SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY

NATURAL DEATHS

MORIBUND SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIMAL MISSING

 50

 4

 45

 1 

 50

 10

 40

 50 

 12 

2 

 36 

8 INCLUDES AUTOLYZED ANIMALS 

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

57
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS*

TOTAL PRIMARY TUMORS

 30

 34

 20 

 27 

2U 

26 

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAL BENIGN TOMORS

 6

 7

 7 

7 

5 

6 

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

 26

 27

 17 

 20 

19 

19 

TOTAL ANIMALS WITH SECONDARY TUMORS* TOTAL SECONDARY TUMORS 1 

6 

1 

1 

TOTAL ANIMALS WITH TUMORS

BENIGN OR MALIGNANT 

TOTAL UNCERTAIN TUMORS 

 UNCERTAIN1 

1 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

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

58
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED dl-MENTHOL IN THE DIET
 

59
 
60

 
TABLE C1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED dl-MENTHOL IN THE DIET 

•"'" JJ L L
 •" ' - - '-••» — II.-' •—••Lii-iiwin-.-TtTM- «-_.•»___.«»
 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 50 5D 50
 

ANIMALS NECROPSIED 49 50 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 49 50 50
 

INTEGUMENTARY SYSTEM
 

*SKIN (49) (50) (50)
 

EPIDERMAL INCLUSION CYST 1 (2X) 1 (2*) 1 (2X)
 

METAPLASIA. SQUAHOUS 1 (2X)
 

*SUBCUT TISSUE (49) (50) (50)
 

EPIDERMAL INCLUSION CYST 1 (2X) 1 (2X)
 

ABSCESS, NOS 1 (2X)
 

GRANULOHA, NOS 1 (2X)
 

NECROSIS, FAT 1 (2X)
 

RESPIRATORY SYSTEM
 

ILUNG (49) (50| (50)
 

PNEUHONIA, CHEONIC MORINE 5 (10*) 1 (2X) 7 (14X)
 

HEHATOPOIETIC SYSTEH
 

NONE
 

CIRCULATORY SYSTEH
 

fHEART (49) (50) (50)
 

THROMBOSIS, NOS 1 (2X)
 

THROMBUS, ORGANIZED 1 (2X)
 

FIBBOSIS 1 (2*)
 

DEGENERATION, NOS 1 (2*)
 

IAURICULAR APPENDAGE (49) (50) (50)
 

THROMBOSIS, NOS 1 (2»)
 

•MYOCARDIUM	 (49) (50) (50)
 

INFLAMMATION, NOS
 J_I2iL___.
 

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

61
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

INFLAMMATION, CHRONIC 1 (2X)
 

FIBROSIS 6 (12*)
 

DEGENERATION, NOS 1 (2*) 6 (12%)
 

DIGESTIVE SYSTEM
 

tLIVER (1*9) (50) (50)
 

PELIOSIS HEPATIS 1 (2X)
 

METAMORPHOSIS FATTY 1 (2X)
 

FOCAL CELLULAR CHANGE 1 <2X)
 

ANGIECTASIS 1 (2%)
 

ILIVER/CENTRILOBULAR (49) (50| (50)
 

NECROSIS, NOS 1 (2%) 1 (21)
 

*BILE DUCT (49) (50) (50)
 

HYPERPLASIA, NOS 1 (2J) 1 (2%)
 

•PANCREAS	 (49) (50) (50)
 

PERIARTERITIS 2 (»*) 3 (6S) 1 (2%)
 

ATROPHY, NOS 1 (2X)
 

ISTOMACH C*9) (48) (48)
 

ULCER, FOCAL 2 (<U) 1 (2X)
 

CALCIUM DEPOSIT 1 (2%)
 

HYPERKERATOSIS 2 (K»)
 

ACANTHOSIS 2 (4X)
 

#COION (49) (50| (50)
 

PARASITISM 3 (6X)
 

URINARY SYSTEM
 

tKIDNEY (49) (50) (50)
 

HINERALIZATION 4 (8%)
 

HYDRONEPHROSIS 1 (2»)
 

PYELONEPHRITIS, NOS 1 (2*)
 

INFLAMMATION, CHRONIC 29 (59%) 41 (82%) 41 (82X)
 

AMYLOIDOSIS 1 (2%)
 

PIGMENTATION, NOS 1 (2X) 1 (2X)
 

ATROPHY, NOS 1 (2X)
 

tKIDNEY/PELVIS (49) (50| (50)
 

MINERALIZATION _1_i2«l. 1 J2»l_.
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPIC!LLY
 * NUMBER OF ANIMALS NECROPSIED
 

62
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

ENDOCRINE SYSTEM
 

IADRENAL
 

ANGIECTASIS
 

fADRENAL CORTEX
 

DEGENERATION, NOS
 

HYPEHPLASIA, NOS
 

tTHYROID
 

CYSTIC FOLLICLES
 

HYPEEPLASIA, C-CELL
 

HYPEEPLASIA, FOLLICUL AR-CELL
 

tPARATHYROID
 

HYPERPLASIA, NOS
 

IPANCREATIC ISLETS
 

HYPEEPLASIA, NOS
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND
 

GALACTOCELE
 

LACTATION
 

#PROSTATE
 

INFLAMMATION, NOS
 

INFLAMMATION, SUPPORATIVE
 

ITESTIS
 

ATROPHY, NOS
 

*EPIDIDYMIS
 

GRANULOMA, SPERMATIC
 

NECROSIS, FAT
 

*SCHOTUM
 

EPIDERMAL INCLUSION CYST
 

NERVOUS SYSTEM
 

#BRAIN
 

#	 NUMBER OF ANIMALS WITH TISSUE


*	 NUMBER OF ANIMALS NECROPSIED
 

MATCHED
 

CONTROL LOW DOSE
 

(49) (50)
 

(50)
 

1 (2X)
 

(49) (48)
 

1	 (2*) 1 (2%)
 

(31) (39)
 

(49)	 (50)
 

1 (2%)
 

(49)	 (50)
 

1 (2%)
 

1 (2X)
 

(47) (48)
 

1 (2X) 1 (2X)
 

1 (2S)
 

(49)	 (50)
 

3 (6») 1 (2X)
 

(49)	 (50)
 

1 (2X)
 

(49)	 (50)
 

1 (2X)
 

(49)	 (50)
 

1-12.SL
 

 EXAMINED MICROSCOPICALLY
 

HIGH DOSE
 

(50)
 

1 (2*)
 

(50)
 

1 (2%)
 

(48)
 

1 (2*)
 

2 (4%)
 

1 (2*)
 

(35)
 

1 (3%)
 

(50)
 

(50)
 

(50)
 

3 (6X)
 

(50)
 

(50)
 

1 (2%)
 

(50)
 

(50)
 

63
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

SPECIAL SENSE ORGANS
 

*£YE (49) (50) (50)
 

SYNECHIA, ANTERIOR 1 (2%)
 

SYNECHIA, POSTERIOR 1 (2X) 1 (2%)
 

CATARACT 1 (2X) 2 (4X)
 

LENTICULAR OPACITIES 1 (2X)
 

*EYE/CORNEA (49) (50) (50)
 

PANNUS 1 (2X)
 

CATARACT 1 (2X)
 

VASCULARIZATION 1 (2X)
 

*EYE/CBYSTALLINE LENS (50) (50)
 

RUPTURE 1 (2X)
 

*HARDERIAN GLAND (49) (50) (50)
 

HYPERPLASIA, NOS 1 (2X)
 

HUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY (49) (50) (50)
 

NECROSIS, FAT 3 (6%) H (8X)
 

*PERICARDIUM (l»9) (50) (50)
 

INFLAHHATION, NOS 1 (2X)
 

*MESENTERY (49) (50) (50)
 

PERIARTERITIS 1 (2X) 2 (UX) 2 (4X)
 

NECROSIS, FAT 2 (4X) 3 (6X) 3 (6X)
 

ALL OTHER SYSTEMS
 

'DIAPHRAGM
 

HERNIA, NOS 2 4
 

SPECIAL BORPHOLOGY SUHHARY
 

AUTO^NECROPSY/HISTO PERF 1
 

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

64
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

AOTOLYSIS/NO NECROPS* 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NEC80PSIED
 

65
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED dl-MENTHOL IN THE DIET

 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

ANIMALS NECROPSIED 50 49 49
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 50 49 49
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (50) (49) (49)
 

NECROSIS, FAT 1 (2X)
 

RESPIRATORY SYSTEM
 

#LUNG (50) (49) (48)
 

CONGESTION, NOS 1 (21)
 

HEMORRHAGE 1 (2X)
 

PNEUMONIA, CHRONIC MURINE 3 (6*) 4 (8X) 1 (2*)
 

HEHATOPOIETIC SYSTEM
 

tBONE MARROW (50) (49) (49)
 

HYPERPLASIA, NOS 1 (2*)
 

#SPLEEN (50) (49) (49)
 

HEMORRHAGE 1 (2%)
 

PIGMENTATION, NOS
 

HEMATOPOIESIS 1 (2%) 1 (2%)
 

#CERVICAL LYMPH NODE (50) (47) (47)
 

INFLAMMATION, NOS 3 (6)1)
 

CIRCULATORY SYSTEM
 

#HEART (50) (49) (48)
 

PERIARTERITIS 1 (2X)
 

CALCIUM DEPOSIT 1 (2X)
 

#MYOCARDIUM (50) (49) (48)
 

INFLAMMATION,NOS_
 

* NOMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

66
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

FIBBOSIS
 

DEGENERATION, NOS
 

DIGESTIVE SYSTEH
 

•LIVER
 

INFLAHHATION, NOS
 

PELIOSIS HEPATIS
 

METAMORPHOSIS FATTY
 

FOCAL CELLULAR CHANGE
 

ANGIECTASIS
 

#STOMACH
 

ULCER, NOS
 

ULCER, FOCAL
 

#COLON
 

PARASITISM
 

URINARY SYSTEM
 

#KIDNEY
 

PYELONEPHRITIS, NOS
 

INFLAMMATION, CHRONIC
 

tKIDNEY/PELVIS
 

INFLAMMATION, NOS
 

#DRINARY BLADDER
 

CALCULUS, NOS
 

INFLAMMATION, NOS
 

INFLAMMATION, CHRONIC
 

HYPEPPLASIA, EPITHELIAL
 

ENDOCRINE SYSTEH
 

IPITDITARY
 

CYST, NOS
 

ANGIECTASIS
 

*ACRENAL
 

_ CJfSTj,. NOS
 

MATCHED
 

CONTROL
 

1 (2%)
 

1 (2X)
 

(50)
 

2 (4X)
 

3 (6X)
 

1 (2X)
 

(50)
 

1 (2%)
 

1 (2X)
 

(50)
 

(50)
 

1 (2X)
 

19 (38X)
 

(50)
 

1 (2*)
 

(47)
 

1 (2X)
 

1 <2X)
 

(48)
 

2 (4%)
 

(50)
 

LOW DOSE
 

4 (8X)
 

4 (8X)
 

(49)
 

1 (2X)
 

(48)
 

(49|
 

1 (2X)
 

(49)
 

5 (10X)
 

(49)
 

(44)
 

1 (2X)
 

(47)
 

1 (2X)
 

(49)
 

HIGH DOSE
 

4 (8*)
 

4 (8X)
 

(49)
 

1 (21)
 

1 (2X)
 

2 (4X)
 

(48)
 

(49)
 

(49)
 

2 ("»*)
 

2 (4X)
 

(49)
 

(39)
 

1 (3X)
 

2 (5X)
 

(43)
 

2 (5X)
 

(49)
 

i_12iL_,
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

67
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE
 

ANGIECTASIS	 1 (2X) 1 (2J)
 

•ADRENAL	 MEDULLA (50) (49)
 

HYPERPLASIA, HOS
 

•THYROID	 (48) (47)
 

CYSTIC FOLLICLES
 

REPRODUCTIVE SYSTEM 

*MANNARY GLAND 

GALACTOCELE 

LACTATION 

(50) 

2

1

 (4X) 

 (2X) 

(49) 

9

8

 (18X) 

 (16X) 

*VAGINA 

PROLAPSE 

INFLAMMATION, NOS 

INFLAMMATION, CHRONIC 

(50) 

1 (2X) 

(49| 

ICTERUS 

HYDROMETRA 

CYST, NOS 

THROMBUS, ORGANIZED 

(50) 

7

2

1

 (14X) 

 (4X) 

 (2X) 

(49| 

1 (2X) 

tUTERUS/ENDOHETRIUH 

INFLAMMATION, NOS 

HYPERPLASIA, CYSTIC 

(50) (49) 

1 (2X) 

tOVARY/PAROVARIAN 

NECROSIS, FAT 

(50) (49) 

*OVARY 

CYST, NOS 

FOLLICDLAR CYST, NOS 

PAEOVARIAN CYST 

FIBROSIS 

CALCIUM DEPOSIT 

(19) 

1 (2X) 

(49) 

3

1

1

1

 (6X) 

 (2X) 

 (2X) 

 (2X) 

NERVOUS SYSTEM
 

* BRAIN (48) (49)
 

THROMBOSIS. NOS i_I2il
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

(49)
 

1 (2X)
 

(46)
 

1 (2X)
 

(49) 

8

5

 (16X) 

 (10X) 

(49) 

1 (2X) 

1 (2X) 

(48) 

5 (10X) 

(48)
 

2 (4X)
 

(48)
 

1 (2X)
 

(48)
 

2 (4X)
 

(49)
 

68
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

SPECIAL SENSE ORGANS
 

*EYE


CONGENITAL MALFORMATION, NOS
 

INFLAMMATION, NOS
 

SYNECHIA, ANTERIOR
 

CATARACT


*EYE/CORNEA


VASCULARIZATION
 

*HARDERIAN GLAND


HYPERPLASIA, NOS


MOSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

'ABDOMINAL CAVITY


NECROSIS, FAT


'ABDOMINAL HALL


NECROSIS, FAT
 

'PERITONEAL CAVITY


NECROSIS, FAT


'MESENTERY


NECROSIS, FAT
 

ALL OTHER SYSTEMS
 

DIAPHRAGM
 

HERNIA, NOS
 

SPECIAL MORPHOLOGY SUMMAR Y 

HO_IJSIQN_JEPORTEfi 

t NUMBEfi OF ANIMALS WITH TISSUE

* NUMBER OF ANIMALS NECROPSIED 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 (50) (49) (49) 

1 (2*) 

1 (2X) 

1 (2X) 

3 (6X) 

3 (6*) 1 (2%) 

 (50) (49) (49) 

1 (2X) 

 (50) (49) (49) 

1 (2%) 

 (50) (49) {49) 

3 (6X) 2 (4X) 1(2%) 

 (50) (49) (49) 

1 (2X) 

 (50) (49) (49) 

1 (2») 

 (50) (49) (49) 

1 (2X) 

3 3 

 EXAMINE D MICROSCOPICALL Y 

69 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

AOTOLYSIS/NO NECROPSY
 

f NUMBEfi OF ANIMALS WITH TISSUE EXAHINED MICBOSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

70
 
APPENDIX U
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE ADMINISTERED dl-MENTHOL IN THE DIET
 

71
 
72
 
TABLE D1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED dl-MENTHOL IN THE DIET 

MATCHED
 

CONTROL LOW DOSE
 

ANIMALS INITIALLY IN STUDT 50 50
 

ANIMALS NECBOPSIED 47 49
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY H7 49
 

INTEGUMENTARY SYSTEM
 

*SKIN (47) (49)
 

EPIDERMAL INCLUSION CYST 1 (2%)
 

INFLAMMATION, NOS 3 (6X) 3 (6%)
 

FIBHOSIS
 

ACARIASIS
 

HYPERKERATOSIS 2 (4X)
 

ACANTHOSIS 2 (4X) 1 (2X)
 

METAPLASIA. OSSEOUS
 

*SUBCUT TISSUE C*7) (49)
 

GRANDLOHA, NOS 1 (2*)
 

FIBROSIS 1 (2X)
 

NECROSIS, FAT 1 (2X)
 

RESPIRATORY SYSTEM
 

tLUNG/BRONCHIOLE (46) (49)
 

INFLAMMATION, CHRONIC 1 (2X)
 

*LUNG (46) (49)
 

PNEUMONIA, CHRONIC MURINE 1 (2X) 1 (2X)
 

HEMATOPOIETIC SYSTEM 

•SPLEEN 

AMYLOIDOSIS 

HEMATOPOIESIS 

(U7) 

3 (6%) 

(49) 

3 (6X) 

tLUHBAR LYMPH NODE 

INFLAMMATION, NOS 

(47) (49) 

tMESENTERIC L. NODE 

LYMPHANGIECTASIS 

(47) 

1 I2X) _ 

(49) 

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

HIGH DOSE
 

50
 

48
 

48
 

(48)
 

1 (2X)
 

1 (2%)
 

1 (2X)
 

1 (2X)
 

(48)
 

(48)
 

(48)
 

5 (10X)
 

(48)
 

1 (2X)
 

2 (4X)
 

(48)
 

1 (2X)
 

(48)
 

73
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

HEMORRHAGE
 

INFLAMMATION, NOS
 

ANGIECTASIS
 

CIRCULATORY SYSTEM
 

#AURICULAR APPENDAGE
 

THROMBOSIS, NOS
 

DIGESTIVE SYSTEM
 

#LIVER
 

THROMBUS, ORGANIZED
 

INFARCT, FOCAL
 

#LIVER/CENTRILOBULAR
 

NECROSIS, NOS
 

*GALLBLADDER
 

THROMBUS, ORGANIZED
 

•PANCREAS
 

DILATATION/DUCTS
 

CYST, NOS
 

INFLAMMATION, SUPPUKATIVE
 

tSTOMACH
 

ULCER, FOCAL
 

HYPERKERATOSIS
 

ACANTHOSIS
 

#LARGE INTESTINE
 

NEMATODIASIS
 

PARASITISM
 

NECROSIS, FAT
 

*ANUS
 

PROLAPSE
 

URINARY SYSTEM
 

IKIDNEY
 

HYDRONEPHROSIS
 

MATCHED
 

CONTROL LOW DOSE
 

1 (2%)
 

4 (9%)
 

11 (22%)
 

(1*6) (49)
 

1 (2%)
 

(47)	 (49)
 

1 (2%)
 

1 (2*)
 

(47)	 (49)
 

2 (4X)
 

(47)	 (49)
 

1 (2%)
 

(47)	 (48)
 

1	 (2%)
 

1 (2X)
 

(47)	 (49)
 

2 (4X) 1 (2*)
 

2 (4%) 1 (2%)
 

(45)	 (49)
 

1 (2«)
 

(47)	 (49)
 

1 (2%)
 

(47)	 (49)
 

1.12.11
 

HIGH DOSE
 

3 (6*)
 

4 (8X)
 

(48)
 

(48)
 

1 (2%)
 

(48)
 

(48)
 

(48)
 

1 (2X)
 

(48)
 

1 (2X)
 

(46)
 

1 (2*)
 

1 (2X)
 

(48)
 

(48)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

74
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE


PYELONEPHRITIS, NOS 3 (6X)
 

PYELONEPHRITIS SUPPURATIVE
 

INFLAMMATION, CHRONIC 2 (4X)
 

tUEINARY BLADDER (46) (47)
 

CALCULUS, NOS 1 (2%)
 

INFLAMMATION, NOS 1 (2%)
 

INFLAMMATION, CHRONIC 5 (11S)
 

HYPERPLASIA, EPITHELIAL 1 (2%)
 

ENDOCRINE SYSTEM
 

#THYROID (43) (46)
 

INFLAMMATION, NOS
 

REPRODUCTIVE SYSTEM
 

*PENIS (47) (49)
 

INFLAMMATION, CHRONIC 2 (4*)
 

*PREPUCE (47) (49)
 

INFLAMMATION, CHRONIC 2 (4X)
 

*PREPUTIAL GLAND (47) (49)
 

INFLAMMATION, NOS
 

HYPERPLASIA, NOS 1 (2X)
 

#PROSTATE (46) (47)
 

INFLAMMATION, SUPP08ATIVE 3 (6%)


*EPIDIDYMIS (47) (49)
 

NECROSIS, FAT 1 (2X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/COENEA (47) (49)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

 HIGH DOSE
 

1 (2%)
 

(48)
 

(44)
 

1 (2S)
 

(48)
 

(48)
 

(48)
 

(47)
 

1 (2X)
 

(48)
 

(48)
 

75
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

VASCULARIZATION 1 (2*)
 

MUSCULOSKELETAL SYSTEM
 

*KNEE JOINT (1*7) (49) (48) 

OSTEO ARTHRITIS 4 (8%) 

OSTEOSCLEBOSIS 3 (6H) 

BODY CAVITIES
 

*ABDO«INAL CAVITY (47) (49) (48)
 

NECROSIS, FAT 1 (2%)
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

N O LESION REPORTED 8 7 7
 

AUTO/NECHOPSY/HISTO PERF 1 1
 

AOTOLYSIS/NO NECROPSY 3 1 2
 

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

76
 
TABLE 02. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED dl-MENTHOL IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

ANIMALS MISSING 1
 

ANIMALS NECROPSIED 49 47 48
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 49 47 48
 _•___.____«____.....__.._____•»_____________*» — — — — — — — •_•*••— — «.— _— «— *- — — — —— — — — — — •» — — — — — —
 

INTEGUMENTARY SYSTEM
 

*SKIN (49) (47) (48)
 

INFLAMMATION. NOS 1 (21)
 

RESPIRATORY SYSTEM
 

#LUNG (49) (47) (48)
 

PNEUMONIA. CHRONIC MORINE 3 (6X) 4 (9X) 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN (49) (47) (48)
 

HEMATOPOIESIS 1 (2*)
 

#THYMUS (23) (26( (29)
 

FOLLICULAR CYST, NOS 1 (3»)
 

CIRCULATORY SYSTEM
 

IMYOCARDIUM (49) (47) (48) 

FIBROSIS 1 (2X) 

DEGENERATION, NOS 1 (2X) 

DIGESTIVE SYSTEM
 

#LIVER (49) (47) (48)
 

INFLAMMATION, NOS 1 (2%)
 

FOCAL CELLULAR CHANGE 1 (2*)
 

ANGIECTASIS 1 (2%)
 

#PANCREAS (49) (47) (47)
 

£YSTIC_DUCTS 2_iitSl.
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

77
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

HEMORRHAG E 

INFLAMMATION ,

PEEIARTERITI S 

ATROPHY, NOS 

ANGIECTASI S 

 NOS 

1 (2%) 

1 (2%) 

1 (2X ) 

tSTOMAC H 

HYPEEKERATOSIS 

ACANTHOSI S 

(48) 

1

1

 (2%) 

 (2X) 

(47 ) 

#SMAL L INTESTINE 

DIVEPTICOLU M 

(49) (47) 

1 (2X) 

#COLON 

PARASITIS M 

(148) (46) 

URINAR Y SYSTEM 

IKIDNE Y (49) (47) 

INFLAMMATION , CHRONIC 1 (2*) 

tURINAR Y BLADDER (47) (45) 

INFLAMMATION , NOS 

ENDOCRINE SYSTEM 

IPITUITAR Y (33) (35) 

ANGIECTASIS 

#ADRENA L (48) (46) 

ANGIECTASIS 1 (2%) 

REPRODUCTIV E SYSTEM 

&UTERU S (48) (46) 

HYDROMETRA 6 (13%) 5 (11%) 

INFLAMMATION , NOS 1 (2X) 

tUTERUS/ENDOMETRIU M (48) (46) 

HYPERPLASIA, CYSTIC 17 (35X) 27 (59X) 

tOVAR Y (47) (47) 

CYSTA NOS JJLliHL U-ll&L 

# NDHBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

HIGH DOSE 

1 (2%) 

1 (2X) 

(48) 

(48) 

(48) 

1 (2*) 

(48) 

(47) 

1 (2%) 

(39) 

1 (3S) 

(48) 

(48) 

2 (4«) 

(48) 

23 (48X) 

(48) 

10_J21S1, 

78
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

FOLLICULAR CYST, NOS
 

PAROVARIAN CYST
 

INFLAMMATION, NOS
 

NERVOUS SYSTEM
 

#BHAIN
 

HYDROCEPHALUS, NOS
 

SPECIAL SENSE ORGANS
 

*EYE
 

SYNECHIA, POSTERIOR
 

CATARACT
 

PHTHISIS BULBI
 

*EYE/LACRIMAL GLAND
 

INFLAMMATION, NOS
 

*HARDERIAN GLAND
 

HYPEPPLASIA, NOS
 

MUSCOLOSKELETAL SYSTEM
 

*KNEE JOINT
 

OSTEOARTHRITIS 

BODY CAVITIES
 

*PERITONEUM
 

INFLAMMATION, NOS
 

*MESENTERY
 

NECROSIS, FAT
 

A L L OTHER SYSTEMS 

NONE 

SPECIAL MORFHOLOG Y SUMMAR Y 

NO_LESION_REPORTED

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

1 (2X) 3 (6X) 

1 (2%) 

1 (2X) 

(49) (47) (47) 

1 (2%) 

(49) (47) (48) 

1 

1 

(2%) 

(2X) 

1 <2X) 

(49) (47) (48) 

1 (2X) 

(49) (47) (48) 

1 (2X) 

(49) (47) (48) 

1 (2%) 

(49) (47) (48) 

1 (2*) 

(49) (47) (48) 

2 {«*) 

1 1. 

t NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIED 

79
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMA L MISSING/NO NECROPSY

AOTO/NECROPSY/HISTO PERF

AUTOLYSIS/NO NECROPSY

 1 

3

 1 

2 

t NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* SOMBER OF ANIMALS NECROPSIED 

80
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS ADMINISTERED dl-MENTHOL IN THE DIET
 

81
 
82
 
Table El. Analyses of the Incidence of

Administered dl-Menthol

Matched


Topography : Morphology Control


All Sites: Sarcomab 3/49 (6)


P Valuesc>d N.S.


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 105


CD Hematopoiet ic System: 

Monocytic Leukemia*5 14/49 (29)


P Value sc«d N.S.


Relative Risk^
 

Lower Lirnit
 

Upper Limit
 

Weeks to First Observed Tumor 81


 Primary

 in the

 Tumors in Male Rats 

Qieta 

 Low
 

 Dose
 

 3/50 (6)
 

 N.S.
 

0.980
 

0.137
 

6.989
 

 91
 

 14/50 (28)
 

 N.S.
 

0.980
 

0.487
 

1.976
 

 76
 

High
 

Dose
 

4/50 (8)
 

N.S.
 

1.307
 

0.233
 

8.508
 

98
 

11/50 (22)
 

N.S.
 

0.770
 

0.353
 

1.639
 

80
 
Table El.


(continued)
 

Topography: Morphology


Hematopoietic System: All
 

Leukemias or Lymphomasb


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


CD
 

Liver: Hepatocellular Carcinoma^


P Values0 >d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


 Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered dl-Menthol in the Diet3
 

High
 

Dose
 

12/50 (24)
 

N.S.
 

0.840
 

0.397
 

1.752
 

2/50 (4)
 

N.S.
 

Infinite
 

0.290
 

Infinite
 

105
 

Matched
 

 Control
 

 14/49 (29)
 

 N.S.
 

 81
 

 0/49 (0)
 

 N.S.
 

—
 

Low
 

Dose
 

14/50 (28)
 

N.S.
 

0.980
 

0.487
 

1.976
 

76
 

3/50 (6)
 

N.S.
 

Infinite
 

0.590
 

Infinite
 

97
 

70 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered dl-Menthol in the Dieta
 

(continued)
 

Topography: Morphology


Pituitary: Chromophobe Adenoma'3


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Adrenal: Pheochromocytoma'3


oo
 

P Values0 »d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 5/48 (10)
 

 N.S.
 

 103
 

 7/49 (14)
 

 N.S.
 

 67
 

Low
 

Dose
 

7/49 (14)
 

N.S.
 

1.371
 

0.403
 

5.119
 

105
 

7/50 (14)
 

N.S.
 

0.980
 

0.317
 

3.032
 

104
 

High
 

Dose
 

2/46 (4)
 

N.S.
 

0.417
 

0.041
 

2.404
 

105
 

7/50 (14)
 

N.S.
 

0.980
 

0.317
 

3.032
 

97
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered dl-Menthol in the Diet3
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Thyroid: Follicular-Cell
 

Adenoma or Carcinoma" 1/49 (2) 1/48 (2) 3/48 (6)
 

P Values0 >d N.S. N.S. N.S.
 

Relative Riskf 1.021 3.063
 

Lower Limit 0.013 0.257
 

Upper Limit 78.494 157.336
 

Weeks to First Observed Tumor 105 105 91
 

00
 

Thyroid: C-cell Carcinoma'3 4/49 (8) 0/48 (0) 1/48 (2)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf 0.000 0.255
 

Lower Limit 0.000 0.0.05
 

Upper Limit 1.100 2.457
 

Weeks to First Observed Tumor 81 105
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

(continued)
 

Topography; Morphology
 

Thyroid: C-cell Adenoma or
 

Carcinoma'3
 

P Values0 »d
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

oo
 

Mammary Gland: Fibroadenoma"
 

P Values0 >d
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Administered dl-Menthol in the Diet3
 

Matched Low
 

Control Dose
 

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

N.S. N.S.
 

0.255
 

0.005
 

2.457
 

81 105
 

1/49 (2) 4/50 (8)
 

N.S. N.S.
 

3.920
 

0.407
 

188.989
 

105 97
 

High
 

Dose
 

1/48 (2)
 

N.S.
 

0.255
 

0.005
 

2.457
 

105
 

2/50 (4)
 

N.S.
 

1.960
 

0.106
 

113.312
 

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

Administered dl-Menthol in the Diet3
 

(continued)
 

Topography; Morphology
 

Preputial Gland: Carcinoma, NOS"
 

P Values0>d
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

00 Testis: Interstitial-Cell Tumorb
 

00
 

P Values0 »d
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low 

Control Dose 

2/49 (4) 2/50 (4) 

N.S. N.S. 

0.980 

0.074 

13.058 

105 105 

45/49 (92) 47/50 (94) 

P = 0.046 N.S. 

1.024 

0.912 

1.130 

78 72 

High
 

Dose
 

4/50 (8)
 

N.S.
 

1.960
 

0.296
 

20.886
 

105
 

50/50 (100)
 

N.S.
 

1.089
 

0.985
 

Infinite
 

70
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered dl-Menthol in the Diet3
 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

All Sites: Mesothelioma, NOS,
 

or Malignant Mesothelioma'3 2/49 (4) 5/50 (10) 1/50 (2)
 

F Values0 >d N.S. N.S. N.S.
 

Relative Risk/ 2.450 0.490
 

Lower Limit 0.424 0.008
 

Upper Limit 24.778 9.103
 

Weeks to First Observed Tumor 94 76 105
 

oo
 

vO
 a

Dosed groups received 3,750 or 7,500 ppm.
 

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

G

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


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

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

the comparison of that dosed group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

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

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

group.
 
 Primary Tumors in

 in the Diet3 

 Low
 

 Dose
 

 0/49 (0)
 

 N.S.
 

0.000
 

0.000
 

1.696
 

 5/49 (10)
 

 N.S.
 

0.510
 

0.147
 

1.510
 

89
 

 Female Rats 

High
 

Dose
 

0/48 (0)
 

N.S.
 

0.000
 

0.000
 

1.730
 

— —
 

6/49 (12)
 

N.S.
 

0.612
 

0.198
 

1.708
 

Table E2.

Topography: Morphology
 

Lung: Alveolar /Bronchiolar
 

Adenoma or Carcinoma*3
 

P Valuesc»d
 

Relative Risk^
 

Lower Limit
 

Upper Limit
 

Weeks	 to First Observed Tumor
 

\o 

o	 Hematopoietic System: 

Monocytic Leukemia*3 

P Value sc'd
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks	 to First Observed Tumor
 

 Analyses of the Incidence of

Administered dl-Menthol

Matched


Control


3/50 (6)


P = 0.040(N)


105
 

10/50 (20)


N.S.


94
 95 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered dl-Menthol in the Dieta
 

(continued)
 

Matched Low
 

Topography: Morphology Control Dose
 

Hematopoietic System: All Leukemia^ 10/50 (20) 6/49 (12)
 

P Values0 >d N.S. N.S.
 

Relative Riskf 0.612
 

Lower Limit 0.198
 

Upper Limit 1.708
 

Weeks to First Observed Tumor 94 89
 

\o Pituitary: Chromophobe Adenoma'2 28/48 (58) 25/47 (53)
 M
 

d
 P Values0 » N.S. N.S.
 

Relative Riskf 0.912
 

Lower Limit 0.616
 

Upper Limit 1.348
 

Weeks to First Observed Tumor 85 69
 

High
 

Dose
 

6/49 (12)
 

N.S.
 

0.612
 

0.198
 

1.708
 

95
 

19/43 (44)
 

N.S.
 

0.757
 

0.481
 

1.178
 

81
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

(continued)
 

Topography: Morphology


Adrenal: Pheochromocytomab


P Values0 «d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma
 

or Carcinomab


V Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

AdTlinistered dl-Menthol in the Diet3 

Matched Low High 

 Control Dose Dose 

 0/50 (0) 1/49 (2) 3/49 (6) 

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

Infinite Infinite 

0.055 0.614 

Infinite Infinite 

98 105 

 2/48 (4) 3/47 (6) 2/46 (4) 

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

1.532 1.043 

0.184 0.078 

17.658 13.866 

105 105 86 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered dl-flenthol in the Diet3 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Mammary Gland: Adenocarcinoma, NOS^ 1/50 (2) 3/49 (6) 0/49 (0) 

P Values0 » d N.S. N.S. N.S. 

Relative Riskf 3.061 0.000 

Lower Limit 0.256 0.000 

Upper Limit 157.341 19.032 

__ 

Weeks to First Observed Tumor 94 105 

Mammary Gland: Fibroadenoma^ 20/50 (40) 10/49 (20) 7/49 (14) 

P Values0 >d P = 0.003(N) P ­ 0.028(N) P - 0.004(N) 

Relative Riskf 0.510 0.357 

Lower Limit 0.240 0.142 

Upper Limit 1.015 0.789 

Weeks to First Observed Tumor 98 95 95 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats 

Administered dl-Menthol in the Diet3 

[continued) 

Matched Low High 

Topography; Morphologag yy Control Dose Dose 

Uterus/Endometriufl Endorae trial 

Stromal Polypb 7/50 (14) 6/49 (12) 8/48 (17) 

P Valuesc»d N.S. N.S. N.S. 

Relative Riskf 0.875 1.190 

Lower Limiwi tt 0.261 0.409 

Upper Limini tt 2.820 3.557 

Weeks to First Obs fed Tumor 92 98 62 

VO
 

a

Dosed groups received 3,750 or 7,500 ppm.
 

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

c

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


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

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

the comparison of that dosed group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

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

957, confidence interval of the relative risk between each dosed group and the control 

group. 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE ADMINISTERED dl-MENTHOL IN THE DIET
 

95
 
96
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered dl-Menthol in the Diet3
 

Topography: Morphology


Integumentary System: Fibroma
 

of the Subcutaneous Tissue15


P Values0 >d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


vO
 

Integumentary System: Fibrosarcoma
 

of the Subcutaneous Tissueb


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 4/47 (9)
 

 N.S.
 

 98
 

 8/47 (17)
 

 N.S.
 

 89
 

Low
 

Dose
 

1/49 (2)
 

N.S.
 

0.240
 

0.005
 

2.309
 

104
 

6/49 (12)
 

N.S.
 

0.719
 

0.222
 

2.182
 

94
 

High
 

Dose
 

6/48 (13)
 

N.S.
 

1.469
 

0.373
 

6.658
 

90
 

4/48 (8)
 

N.S.
 

0.490
 

0.115
 

1.695
 

89
 
Table Fl.


Topography: Morphology


Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma'5


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


vo 

00 Hematopoietic System: Lymphoraa'5

P Valuesc»d

Relative Risk^ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

 Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered dl-Menthol in the Diet3
 

Matched Low
 

 Control Dose
 

 6/46 (13) 7/49 (14)
 

 M.S. N.S.
 

1.095
 

0.341
 

3.661
 

 104 94
 

 6/47 (13) 6/49 (12)
 

 N.S. N.S.
 

0.959
 

0.276
 

3.338
 

74 84
 

High
 

D.qse
 

6/48 (13)
 

N.S.
 

0.958
 

0.276
 

3.330
 

89
 

10/48 (21)
 

N.S.
 

1.632
 

0.587
 

5.035
 

95
 
Table Fi. Analyses of the Incidence of Primary Tumors in Hale Mice 

Administered dl-Menthol in the Diet3 

[continued) 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Hematopoietic System:
 

Lymphoma or Leukemia^ 6/47 (13) 8/49 (16) 10/48 (21)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf 1.279 1.632
 

Lower Limit 0.422 0.587
 

Upper Limit 4.143 5.035
 

Weeks to First Observed Tumor 74 84 95
 

All Sites: Hemangiosarcoma^ 2/47 (4) 2/49 (4) 3/48 (6)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf 0.959 1.469
 

Lower Limit 0.072 0.176
 

Upper Limit 12.769 16.939
 

Weeks to First Observed Tumor 84 76 92 
__ __ 

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

Administered dl-Menthol in the Diet3
 

(continued)
 

Matched Low
 

Topography: Morphology Control Dose
 

Liver: Hepatocellular Carcinoma'5 8/47 (17) 8/49 (16)
 

P Values0 »d N.S. N.S.
 

Relative Riskf 0.959
 

Lower Limit 0,342
 

Upper Limit 2.692
 

Weeks to First Observed Tumor 89 71
 

o
 

o
 Thyroid: Follicular-Cell
 

Adenoma or Carcinoma'3 0/43 (0) 0/46 (0)
 

P Values0 »d N.S. N.S.
 

Relative Riskf
 

——
 Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

High
 

Dose
 

14/48 (29)
 

N.S.
 

1.714
 

0.745
 

4.262
 

91
 

2/44 (5)
 

N.S.
 

Infinite
 

0.290
 

Infinite
 

80
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered dl-Menthol in the Oieta
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Harderian Gland: Adenoma, NOSb 0/47 (0) 1/49 (2) 3/48 (6)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf Infinite Infinite
 

Lower Limit 0.051 0.590
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 104 104
 —
 a

Dosed groups received 2,000 or 4,000 ppm.
 

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

c

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


Arraitage test when P < 0,05; otherwise, not significant (N.S.) is indicated. Beneath the
 

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

the comparison of that dosed group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

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

'The 95X confidence interval of the relative risk between each dosed group and the control
 

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

Administered dl-Menthol in the Oieta
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma*3 1/49 (2) 3/47 (6) 5/48 (10)
 

P Values0 >d N.S. N.S. N.s.
 

Relative Risk^ 3.128 5.104
 

Lower Limit 0.262 0.602
 

Upper Limit 160.605 236.011
 

Weeks to First Observed Tumor 104 97 104
 

Hematopoietic System: All Lymphomas*3 20/49 (41) 14/47 (30) 12/48 (25)
 

P Values0 >d N.S. N.S. N.S.
 

Relative Riskf 0.730 0.613
 

Lower Limit 0.391 0.311
 

Upper Limit 1.329 1.159
 

Weeks to First Observed Tumor 81 61 88
 
__ 

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

Administered dl-Menthol in the Dieta
 

(continued)
 

Topography: Morphology


All Sites: Hemangiosarcomab


P Valuesc«d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Liver: Hepatocellular Carcinoma'5


P Values0 »d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

 Control
 

 3/49 (6)
 

 N.S.
 

 102
 

 1/49 (2)
 

 N.S.
 

104
 

Low
 

Dose
 

0/47 (0)
 

N.S.
 

0.000
 

0.000
 

1.730
 

3/47 (6)
 

N.S.
 

3.128
 

0.262
 

160.605
 

96
 

High
 

Dose
 

1/48 (2)
 

N.S.
 

0.340
 

0.007
 

4.060
 

69
 

3/48 (6)
 

N.S.
 

3.063
 

0.257
 

157.336
 

104
 
Table F2. Analyses of the Incidence oE Primary Tumors in Female Mice
 

(continued)
 

Topography: Morphology


Pituitary: Chromophobe Adenomab


P Valuesc>d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Thyroid: Follicular-Cell
 

o
 

-e- Adenoma or Carcinoma'5


P Values0 »d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Administered dl-Menthol in the Oieta
 

Matched Low High
 

 Control Dose Dose
 

 3/33 (9) 0/35 (0) 1/39 (3)
 

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

0.000 0.282
 

0.000 0.006
 

1.546 3.322
 

—


 104 

_ 

104
 

 3/43 (7) 3/46 (7) 1/46 (2)
 

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

0.935 0.312
 

0.132 0.006
 

6.634 3.707
 

 104 104 76
 
Table F2, Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered dl-Menthol in te Dieta
 

(continued)
 

tiatched Low High
 

Topography: Morphology Control Dose Dose
 

Mammary Gland: Adenocarcinma, NOS" 1/49 (2) 3/47 (6) 2/48 (4)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Riskf 3.128 2.042
 

Lower Limit 0.262 0.110
 

Upper Limit 160.605 117.915
 

Weeks to First Observed Tumor 104 97 92
 

g aDosed groups received 2,000 or 4,000 ppm.
 

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

c

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


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

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

the comparison of that dosed group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

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

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

group.
 
106
 
APPENDI X G 

ANALYSIS OF FORMULATED DIETS FOR 

dl-MKNTHOL CONCENTRATION 

107 
108
 
APPENDIX G
 

Analysis of Formulated Diets for
 

dl-Menthol Concentration
 

Duplicate 10-g dosed feed samples were extracted with 20 ml of
 

carbon disulfide, and aliquots of the extract analyzed by gas
 

chromatography (thermal conductivity detector). Spiked samples
 

containing 0.2, 0.4, and 0.8% dl-menthol were worked up simultaneously with each set of dosed feed samples. The average
 

recoveries from these spiked samples were greater than 90%.
 

Theoretical No. of Sample Analy- Coefficient of Range 

Concentrations Samples tical Mean Variation (%) (% in diet) 

(% in diet) (% in diet) 

0.375 9 0.34 15.23 0.23-0.40*
 

0.75 9 0.70 14.69 0.53-0.85*
 

0.20 6 0.19 13.81 0.16-0.23*
 

0.40 6 0.36 13.71 0.28-0.41*
 

See Section II, B, Dietary Preparation for discussion of these data.
 

109
 
110
 
Review of the Bioassay of dl-Menthol* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

August 31, 1978
 

The Clearinghouse on Environmental Carcinogens was established
 

in May, 1976, in compliance with DHEW Committee Regulations and the
 

Provisions of the Federal Advisory Committee Act. The purpose of the
 

Clearinghouse is to advise the Director of the National Cancer Institute
 

(NCI) on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed. The
 

members of the Clearinghouse have been drawn from academia, industry,
 

organized labor, public interest groups, State health officials, and
 

quasi-public health and research organizations. Members have been
 

selected on the basis of their experience in carcinogenesis or related
 

fields and, collectively, provide expertise in chemistry, biochemistry,
 

biostatistics, toxicology, pathology, and epidemiology. Representatives
 

of various Governmental agencies participate as ad hoc members. The
 

Data Evaluation/Risk Assessment Subgroup of the Clearinghouse is
 

charged with the responsibility of providing a peer review of reports
 

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

the bioassay of dl-Menthol for carcinogenicity.
 

The primary reviewer agreed with the conclusion in the report that
 

dl-Menthol was not carcinogenic in rats or mice, under the conditions of
 

test. After a brief description of the experimental design, the primary
 

reviewer said that the study was adequate to serve as the basis for the
 

negative findings. Based on the results of the study, he said that dl-


Menthol would not appear to pose a carcinogenic risk to humans. However,
 

he indicated that the negative findings could not be extrapolated to
 

evaluate the potential human hazard of menthol in tobacco products.
 

The secondary reviewer agreed with the conclusion that dl-Menthol
 

was not carcinogenic, under the conditions of test. He indicated that
 

the study was well conducted and that the data suggested the compound
 

did not pose a risk to man.
 

There was no objection to a recommendation that the report on the
 

bioassay of dl-Menthol be accepted as written.
 

Members present were:
 

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

Joseph Highland, Environmental Defense Fund
 

111
 
(Verald K. Rowe, Dow Chemical USA, submitted a written review)
 

Michael 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.
 

9 U.S . GOVERNMEN T PRINTIN G OFFICE : 1978-281-217/326 7 

112
 
DREW Publication No. (NIH) 79-1348