Carcinogenesis Studies of Eugenol

7/24/2019 Carcinogenesis Studies of Eugenol

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NATIONAL

TOXICOLOGYPROGRAM

Technical

Report

Series

No. 223

CARCINOGENESIS STUDIES

OF

EUGENOL

(CAS

NO.

97-53-0)

IN

F344/N RATS

AND

B6C3F1 MICE

(FEED STUDIES)

U.S. DEPARTMENT

OF

HEALTH

AND

HUMAN

SERVICES

PublicHealthService

National Institutes of Health


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NATIONAL TOXICOLOGY PROGRAM

TheNational

Toxicology

Program

(NTP),

established in 1978, develops

andevaluatesscientificinformationaboutpotentiallytoxicandhazardous

chemicals. This knowledgecan beused forprotecting thehealth ofthe

American people

and for the

primaryprevention

of

chemicallyinduced

disease. Bybringingtogether therelevantprograms,

staff,

and resources

from

th e

U.S. Public Hea lth Service , DH HS,

the

NationalToxicology

Program hascentralizedandstrengthenedactivitiesrelatingtotoxicology

research, testingandtestdevelop men t/valid ationefforts,andthedissemi

nationoftoxicological informationtothepublicand

scientific

communi

tiesandtothe research and regulatory agencies.

The NTP i s c ompri se d o f

f ou r

char te r DHH S agenc ies: the Nationa l

Cancer Institute,N ational Institutes

of

Health;

the

National Institute

of

Envi ronmenta l Hea lth Sciences, Na tiona l Ins ti tu te s o f Hea lth; the

National Center

for

Toxicological Research, Food

and

DrugAdministra

tion; and the Na tiona l Inst itute for Occupational Safety a nd Heal th ,

Centers for DiseaseControl. In

July 1981,

the

Carcinogenesis Bioassay

Testing Program, NCI,

w as

transferred

to the

NIEHS.


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NTPTECHNICALREPORT

ON THE

CARCINOGENESIS

STUDIES

OF

EUGENOL

(CAS

NO.

97-53-0)

IN

F344/N RATSANDB6C3F1MICE

(FEEDSTUDIES)

NATIONAL

TOXICOLOGYPROGRAM

Box

12233

Research TrianglePark

North

Carolina

27709

December

1983

NTP-80-068

NIHPublication

No.

84-1779

NTP TR223

U.S.DEPARTMENTOFHEALTHANDHUMANSERVICES

PublicHealth

Service

National Institutes

of

Health


5/165

NOTE

TOTHE

READER

This isoneina seriesof experimentsdesigned to determine whetherselectedchemicalsproduce

cancer in animals. Chemicals selected for testing intheNTP carcinogenesis program a re chosen

primarilyonthebasesofhumanexposure,levelofproduction,andchemicalstructure.Selection perse

is

notan

indicator

ofa

chemical'scarcinogenicpotential.Negativeresults,

in

which

the

testanimals

do

not have a greater incidence of cancer than controlanimals, do no t necessarily mean that a test

chemical is not a carcinogen, inasmuch as the experiments are conducted under alimited setof

conditions. Positive results demonstrate that atestchemicaliscarcinogenic foranimalsunde rthe

conditions

o f the

test

and

indicate thatexposure

to the

chemical

hasthe

potential

for

hazard

to

humans.Thedetermination oftheriskto humansfromchemicalsfoundtobecarcinogenic in animals

requires

awideranalysiswhichextendsbeyondthepurview of thisstudy.

This studywasinitiated bytheNationalCancerInstitute'sCarcinogenesisTestingProgram,now

part ofthe National Institute ofEnvironmental HealthSciences,National Toxicology Program.

Comments

and

questionsabout

the

NationalToxicologyPro gramTechnicalReports

on

Carcino

genesisStudiesshouldbedirected totheNationalToxicology Program,located atResearchTriangle

Par k,N C27709(919-541-3991)oratRoom 835B,WestwoodTow ers,5401WestbardAve.,Beth esda ,

MD 20205(301-496-1152).

Although everyeffort

is

made

to

prepare

the

TechnicalReports

as

accurately

as

possible,mistakes

mayoccur.Readersarerequested to communicate anymistakestotheDeputyDirector,NTP(P.O.

Box 12233,Research Triangle Park, NC 27709),so that correctiveaction maybetaken. Further,

anyonewhoisawareof relatedongoing orpublishedstudies notmentionedinthisreport isencouraged

to

makethisinformation known

tothe

NTP.

TheseNTP Technical Reports are availablefor sale

from

the National Technical Information

Service, U.S. Department of Commerce,5285Port RoyalRoad,Springfield,VA 22161(703-487

4650).

Singlecopies

of

thiscarcinogenesisstudiestechnicalreport

are

availablewithoutcharge(andwhile

supplies last)from the NTP PublicInformation Office, National Toxicology Program,P.O.Box

12233,

ResearchTriangle Park,

NC

27709.

Eugenol

2


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TABLE OF CONTENTS

Page

Abstract 7

Contributors 8

Reviewers 9

Summary of Peer ReviewComments 10

I.

Introduction .....

11

II .

Materials

a nd

Methods

15

Chemical Analyses

16

Preparation of Test Diets 16

Source a nd Specifications of Test Animals 16

AnimalMaintenance 17

Short-Term Studies 17

Single-Dose Studies 17

Fourteen-Day Studies 19

Thirteen-Week Studies 20

Two-Year Studies 22

Clinical Examinations and Pathology 23

Data

Recordingand StatisticalMethods 23

III. Results 25

Rats 26

Two-Year Studies 26

Body

Weightsand ClinicalSigns 26

Survival 26

Pathology

and

StatisticalAnalyses

of

Results

29

Mice

32

Two-YearStudies 32

BodyWeightsand ClinicalSigns 32

Survival 32

Pathology and Statistical Analysesof Results 35

IV .

Discussion a nd Conclusions 39

V. References 43

TABLES

Table 1 Specificationsand Sources of Materials Usedfor AnimalMaintenance 17

Table 2 Survivaland Mean BodyWeightsofRats AdministeredaSingle

Dose

of Eugenolb y Gavage 18

Table3 Survivaland Mean BodyWeightsofMiceAdministeredaSingle Dose

of Eugenolby Gavage 18

Table4 Survivaland MeanBodyWeightsofRats Fed Diets Containing

Eugenol f or 14

Days

19

Table 5 Survivaland Mean BodyWeightsofMiceFedDiets Containing

Eugenol f or 1 4Days 20

Table 6 Survivaland MeanBodyWeightsofRats Fed Diets Containing

Eugenol f or 1 3Weeks 21

Table7 Survivaland MeanBodyWeightsofMice Fed Diets Containing

Eugenol f or 1 3Weeks 21

Table8 ExperimentalDesignofTwo-Year Feeding StudieswithEugenol

in Rats and Mice 22

Table9 MeanBodyWeights(Relativeto Controls) ofRats FedDiets

Containing Eugenol for Two Years 26

Table 10 Incidenceso fMale RatswithAlveolar/Bronchiolar Adenoma

or Carcinoma 29

3

Eugenol


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Table

11

Incidences

of

Rats withThyroidTumors

30

Table 12 Incidences of Female Rats with Tumors ofthe Uterus 31

Table

13 Incidences of

FemaleRats

wi th

MammaryGlandFibroadenoma

31

Table 14 M ean B ody Weights (Relative to Controls)ofM ice Fed Diets

Containing Eugenol for Two Years 32

Table 15 Incidences of Micewith Liver Tumors 36

Table 16 IncidencesofMaleMice

with

FollicularCellAdenom as

of the

Thyroid

37

FIGURES

Figure 1 G rowth C urves for Rats Fed Diets Containing Eugenol 27

Figure2 SurvivalCurvesfor R a tsFed DietsC ontainingEugenol 28

Figure 3 Grow thCurvesfor Mice Fed Diets Containing Eugenol 33

Figure4 Survival Curvesfor Mice Fed

Diets

Containing Eugenol 34

Figure5 Infrared

AbsorptionSpectrum

o f

Eugenol(Lot

No .

36483)

150

Figure 6 Infrared Absorption S pectrum of Eugenol (Lot No. 26068) 151

Figure 7 Nuclear

MagneticResonanceSpectrum

of

Eugenol(Lot

N o .

36483)

152

Figure8 Nuclear

MagneticResonanceSpectrum

of

Eugenol(Lot

No.

26068)

154

APPENDIXES

AppendixA Summaryofthe Incidence ofNeoplasms in

Rats

Fed Diets


TableA1 Summaryofthe Incidenceof Neoplasms in MaleRats Fe d Diets

ContainingEugenol 48

Table A2 Summary ofthe Incidence ofNeoplasms inFemale

Rats

Fed

Diets


Table A3 Ind iv idual AnimalTumor

Pathology

inMaleRatsinthe

2-YearStudy

of

Eugenol

58

Table

A4 Ind iv idualAnimalTumor Pathology in FemaleRats in the

2-Year

Study ofEugenol 64

Appendix

B

Sum mary

ofthe

Incidence

of

Neoplasms

in

Mice

Fed

Diets

ContainingEugenol 71

Table

Bl

Summary

of the

Incidence

of

Neoplasms

in

MaleMice

Fed

Diets


Table B2 Sum maryofthe IncidenceofNeoplasmsinFemaleMiceFed Diets

Containing

Eugenol 76

Table B3 IndividualAnimalTumor Pathology in Male Mice inthe

2-Year

Study

of

Eugenol

80

Table B4 Individual AnimalTumor Pathology inFemale Micein the

2-Year Study of Eugenol 86

AppendixC Summ aryofthe Incidenceo fNonneoplasticLesions in Rats

Fe d DietsContainingEugenol 93

Table Cl Summ aryofthe IncidenceofNonneoplastic LesionsinMale Rats

Fed Diets Containing Eugenol 94

Eugenol

4


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Table C2

Su m m ary

of the

Incidence

of

Nonneoplast ic Lesions

in

Female Rat s

Fe d Die ts C onta in ing Eugenol 99

Appendix D Summary of the Incidenceof Nonneoplas ti c Les ions in Mice

Fed Diets Containing Eugenol 105

Table Dl Summary of the Incidenceof Nonneoplas t ic Lesions in Male Mice

Fe d

Diet s Conta in ing Eugenol

106

Table

D2

Sum m ary

o f t he

Incidence

of

N onneoplast ic Lesions

in

Female Mice

F ed Diets C ontaining Eugenol I l l

A p p end i x E

Feed

Consumpt ion by Rat s and Mice Receiv ing Eugenol 117

Table

El Feed

Consumption

by

Male Rats ReceivingEugenol

118

119

120

121

Appendix F His tor ica l Incidences of L iver Neoplasms inUnt rea ted

123

TableFl Historical

Incidence

ofLiverNeoplasmsinUntreated

124

Table F2 H i st or ica l I nc id en ce of LiverNeoplasms in Unt rea ted

124

125

126

130

133

136

Table

E2 Feed

Consumption

by

Female Rats ReceivingEugenol

T ab le E 3 Feed Co n su m p ti on b y M ale M i ce Rece iv in g E u gen ol

Table E4 Feed Consumption by Female

Mice Receiving Eugenol

Control

B6C3F!

M ic e

Male B6C3F, M ic e

Female B6C3F] Mice

A p p en d ix G A n a ly s is o f P r im ary T u m o rs i n F344 Ra ts and B6 C3 F] M i ce

T abl e G l A n a ly s is of P r im ary T u m o rs i n M a le Ra ts

T abl e G 2 A n a ly si s o f P r i m ary T u m o rs i n Fem a l e Ra t s

T abl e G 3 A n a ly si s o f P r im ary T u m o rs i n M a l e M ice

T ab le G 4 A n a ly s is o f P r i m ary T u m o rs i n Fem al e M ice

Appendix

H

M u tagenesi s Res ults

for

Eugenol

and

Methyl Eugenol

in

Salmonella 139

Appendix

I

C y to ge ne ti c R e s ul ts

for

E u g en o l

in

Chinese Hamster Ovary (CHO)

Table 11 C y to ge ne ti c

Effects

of Eugenol in Chinese Hamster Ovary (CHO)

Appendix J A nalysis of E ugenol (Lot Nos. 36483 and 26068)

Appendix K. Stab il ity Analys is o f Eugenol in Formulated Diet s

Appendix L Analyses o f Formulated Diet s fo r Concent ra tionsof

Table H1 Resu lt so f M utagenic ity Tes ts o fE ugenol in Salmonella 140

Table

H2

Results

of

Mutagenici tyTests

of

MethylEugenol

in

Salmonella

141

Cells

143

Cells 144

Midwest Research Ins t i tu te 145

Midwest

Research Institu te 155

EugenolSouthern Research Ins t i tu te

157

T ab le L I A n al ys es of Fo rm u la t ed D i et s 1 59

5 Eugenol


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Eugenol

6


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CARCINOGENESIS STUDIES

OF

EUGENOL

EUGENOL

(1-allyl-3-methoxy-4-hydroxybenzene)

{CASNO.

97-53-0)

ABSTRACT

Carcinogenesis studiesofeugenol(>99% pure),awidelyusedflavoradditiveandchemicalinterme

diate, wereconducted byfeedingdiets containing 6,000 or 12,500ppm ofeugenolto groupsof50

female

F344/N ratsand byfeedingdietsconta ining3,000 or6,000ppmtogroups of50maleF3 44/N

rats

and B6C3Fi mice ofeachsexfor 103weeks.Groupsof40ratsand50 miceofeachsexserved as

controls.Doselevelsselected

forthetwo

yearstudieswerebased

on

thirteen-week(91-day)studies

in

which dietaryconcentrations

for the s ix

groups ranged f rom

0 to

12,500ppm. Otherthan

a

-10%

difference from

controls inbod yweightsinthe 12,500ppm malerats,no chemicallyrelatedgrossor

histopathologic effects wereobserved.

Inthetwo-yearstudies,withtheexceptionofthehighdosefemaleratsandfemalemice,finalbody

weightsofthetreatedgroupswerecomparabletotheirrespectivecontrols.Nosignificant

differences

in

survivalwere

apparent

foranyofthe

eight

groups

receiving eugenol

andforthe

appropriatecontrols.

Foodconsumptionamonggroupswasnotdifferent incomparisonwithcontrolsrats:males >97%,

females >91%; mice:males>94%, females >90%.

There wereno significant observable

differences

between treated and controlgroupsofrats for

either

nonneoplastic (toxic) lesions or neoplasms that could beattributedto eugenol. Increasesin

tumor incidences werediagnosed for lowdose male rats withalveolar/bronchiolaradenomas or

carcinomas (combined), f o r C-cell adenomas o f the thyroidgland in lowdosefemalerats, and for

endometrial stromal polyps oftheuterusinhighdose

female

rats.Fibroadenomas ofthemam mary

glandweredecreased

in

dosedgroups

of

femaleratscomparedwithcontrols.None

of

these

differences

were

considered to beassociated withthedietaryadmin istrationofeugenol.

In male mice,thelowdoseanimals had anincreasedincidence(P


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CONTRIBUTORS

Th e

carcinogenesisstudies

of

eugenol

were

conducted

at

Southern ResearchInst ituteunder

a

subcontract toTracerJitco,Inc.,theprimecontractorfortheCarcinogenesisTestingProgram. The

2-yearstudieswerebegun inAprilandJune1977formiceandrats,respectively,andended inApriland

June1979.

Principal

Contributors

at

Southern

Research

Institute

2000NinthAvenueSouth

Birmingham,Alabama

35255

(Conducted

bioassayand evaluatedtissues)

JoanB.Belzer RubyH.

James,

B.S.

Animal

Care and Chemical Chemist

Administration

IsaacBrown J. David Prejean, Ph.D.

AnimalCare and Chemical PrincipalInvestigator

Administration

Daniel R. Farnell, D.V .M .,Ph.D. RogerB.Thompson, D.V.M.

Pathologist Pathologist

Principal

Contributors

at

Tracer Jitco

1776

East

Jefferson

Street

Rockville,M aryland 20852

Cipriano Cueto, Ph.D. Stephen

S.

Olin, Ph.D.

Director Bioassay Program Program Associate Director

Carolyn E.Dean,B.S. William D.Theriault, Ph.D.

Production Editor Reports Manager

Paul Hildebrandt,D.V.M . Joseph D.Tomaszewski, Ph.D.

Pathologist Chemist

Abigail

C.

Jacobs, Ph.D. John

W.

Warner, M.S.

Bioscience Writer

Statistician

James

R.

Joiner, Ph.D.

Statistician

Principal

Contributorsat the

National Toxicology Program

NationalInstitute

of

EnvironmentalHealthSciences

ResearchTrianglePark

Box

12233

North

Carolina

27709

(Evaluated

experiment,interpretedresults,

and

reported

findings)

JamesHuff , Ph.D. (Chemical Manager) E.E. McConnell, D.V.M.

J. Fielding Douglas, Ph.D. JohnA.Moore, D.V.M.

Charles K.Grieshaber, Ph.D. Sherman F.Stinson, Ph.D.

Larry

G.

Hart, Ph.D. Raymond

W.

Tennant, Ph.D.

Joseph K.Haseman, Ph.D. Jerrold M.Ward, Ph.D.

The

pathologyreport

and

selectedslides

were

evaluated

o n

April

18,

1980

bytheNTP

Pathology

Working

Groupcomposedof:

Dr.J.

Ward (NTP)

Dr. G.Reznik (NCI)

Dr.M.

Stedham(Tracer Jitco)

Dr.D.G oodman (Clement Associates)

Eugenol

8


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REVIEWERS

National

Toxicology Program Boardof Scientific Counselors'

TechnicalReportsReviewSubcommittee

Margaret Hitchcock,Ph.D. (Chairperson)

John B.Pierce Foundation Laboratory

NewHaven,Connecticut

AliceS. Whittemore, Ph.D.* Curtis Harper, Ph.D.

Stanford UniversitySchool

AssociateProfessor

of

Pharmacology

of Medicine University ofNorth Carolina

Palo Alto, California Chapel Hill,North Carolina

Ad Hoc

Subcommittee Panel

of

Experts

Norman Breslow, Ph.D.*

UniversityofWashington

Seattle,Washington

Joseph

H.

Highland, Ph.D.

(PrincipalReviewer)

Environmental DefenseFund

Washington, D.C.

FrankM irer, Ph.D.

InternationalUnion

United AutoWorkers

Detroit, Michigan

SheldonD.M urphy, Ph.D.

Professor ofToxicology

Universityof

Texas Medical

School

Houston, Texas

SvendNielsen,D.V.M., Ph.D.

Professor

of Pathology

TheUniversity of Connecticut

Storrs,

Connecticut

*Unable toattend 18 February, 1981meeting

Bernard A.Schwetz,

Ph.D.,

D.V.M

(PrincipalReviewer)

Toxicology ResearchL aboratory

Dow

ChemicalU.S.A.

Midland,Michigan

Roy

Shore,

Ph.D.

New

YorkUniversityMedicalCenter

Ne wYork,New York

JamesSwenberg,D.V.M.,P h.D.

Chiefof

Pathology

ChemicalIndustryInstitute

of

Toxicology

Research Triangle

Park,

North Carolina

GaryM.Williams,M.D.

Chief ofExperimentalPathology

American

HealthFoundation

Valhalla,

New

York

9

Eugenol


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SUMM ARY OF PEER REVIEW COMMENTS ON THE

CARCINOGENESIS STUDIESOF

EUGENOL

On18Feb rua ry 1981,thiscarcin ogen esisstud iestechnicalreport oneugenolunderwentpeerreview

and was

approved

bythe

NationalToxicologyProgram Board

of

ScientificCounselors' Technical

Review Subcommittee

and

Associated

Panel of

Experts

atan

open meeting held

in

B uilding 31C,

National Institutes

o f

Health,Bethesda,Maryland.

Dr.Schwetz,asaprincipalreviewer forthereportonthe carcinogenesisstudies of eugenol,agreed

with

the

conclusion thateugenol

wasnot

carcinogenic

for

F344rats

of

either

sexand

thatthere

was

some,

althoughequivocal,evidence

for

increasedlivertumors

in

male

and

femaleB6C3F]mice.

He

said thatthedatainthe reportonthedepression in weightgain in femalesof bothspeciesshouldbe

more quantitative. Infemalemicetherewasadose-relatedtrend intheincidences ofhepatocellular

adenomas andcarcinomas. Hesuggestedinclusionoftherange of thesetumors in groups of control

mice. Thus, th e r ange of values in historical control groups would be helpful in interpretingthe

importance

ofthe 6 and 12

percent incidences

of

hepatocellular

carinomasin

femalemice(see page

124).

Dr.Jo hn Doull,onbehalfoftheFlavoringExtract Manu facturersAssociationandthe Research

Institute

fo r Fragrance Materials , said the s tu dy w as wellconducted a nd th e conclusions were

supported by the

data.

Hequestioned theunknown effects ofimpurities, particularly inonelotof

eugenol;the

variation

in

weight

ofthe

rats

atthe

beginning

ofthe

two-yearstudies;

andtheuseof

ziram

in

thesameroomwiththeratsbeingfed eugenol-containingdiets.

As

asecond principal reviewer,Dr.Highland disagreedwiththeconclusion

that

thefindingsinmice

wereequivocal

for

carcinogenicity.

H e

said

the

increasedlivertumorincidence

in

malemicesupported

by the

results

in

femalemicewere evidence

of

carcinogenicity.

He

suggested that

th e

equivocal

judgmentseemstoresultfromthewiderangeofcontrolincidencesinmalesforthesetumorsinthetest

laboratory.Dr.Haseman,NTP,commentedthatthemeanlivercombinedtumorrateinmalecontrol

mice

was32percent(range24to39percent)fortheninemostrecentcarcinogenesisstudies inthetest

laboratory where

the

eugenolstudies wereperformed (dataupdated

asof

April 1983).

Dr.

Highland

saidhewasconcernedthat wegiveaconsistentevaluation,since,dependingon whichsetso fcontrol

data a r eused,onecouldarrivea t an equivocalresultforalmost anystudy.Yet,evenusing the32

percent figure,the incidence oflivertumors inthe mice receiving thelowdose ofeugenol wasstill

elevated

relative

tothe

controls.

Drs.Sw enbergand Hitchcockstated thattheimp ortantpointinsupport oftheconclusioninthe

reportwasthelackofdoseresponse.Dr.Williamsproposed

that

theincreasedincidenceinlowdose

mice might

bedueto

eugenol's

acting

asa

promoter.

As

support,

he

cited

a

study

bythe

Millers

(Universityof

Wisconsin)

in

whicheugenolproduced

no

livertumors

i n

CD-I malemicewhi lesafrole

induceda78percentincidence.[In 1983,M illeretal. reported a 15percentlivertumor incidencein

untreated male CD-1 mice

and3

percent

in

females

at 12

months.]

Dr.

Schwetzrepliedthat

the

result

could

be interpreted as supportingtheequivocaljudgmentinthecurrentstudy.Dr.Williamsasked

thatthereferencetotheMiller'sstudybecitedand,also,astatementbeincludedtonotethatcloveoil,

themajor ingredient inmanymouthwashes, is85-90percent eugenol. Therewasfurther discussion

about the lack ofdose responseinthe results formalemice,and, also,concerninga compromise

wording

fortheconclusionalthough no unanimitywasachievedamong thereviewers.

Dr. Schwetzmoved

that

thereport onthecarcinogenesisstudies ofeugenol beacceptedwiththe

statement that these resultsare considered equivocal. Dr. Swenbergseconded themotion and the

technical

report on eugenolwasapproved by avoteof 6to3.

Eugenol

10


14/165

I. INTRODUCTION

11 Eugenol


15/165

I. INTRODUCTION

EUGENOL

(1-allyl-3-methoxy-4-hydroxybenzene)

(CAS

NO. 97-53-0)

Eugenol (1 -al lyl-3-methoxy-4-hydroxyben-

Acute Toxici ty

zene), a colorless or

y ell ow is h

o ily liq uid

Th e

ora l s ingle dose LDso

of

eugenol

is2.7

extracted f romclove,pimento,bayleaf,andcin

g/kg inOsborne-Mendelrats,3.0g/kg in mice

namonoils,isused

primarily

asaflavoringagent

(strainand sexnot given)(Jenner eta l . , 1964) ,

a nd f ra gr an ce ( Op d yk e, 1975; B alsa m an d

a nd 1 .9 g / k g in a lb in o r at s (sex n ot s ta te d)

Sagarin, 1972).

Oil of

c love, containing 85%

(Sober

et

al., 1950).

95%eugenol,isthemajorsourceofthischemical

(Ki rk-Othmer , 1970) . In 1978,425,000 pounds

Metabolism

of

eugenol wereproduced i n the United States

When

14

C-eugenol(450mg/kg)wasadminis

(USITC, 1979).

tered to m ale W is ta r ra ts by intraperi toneal

injection, radioactivity

was

distributed

to

most

Uses

organs (Weinberg

et

al., 1972).

The

major por-

Eugenolisapprovedforuseasa foodadditive

t ion (percent unstated)of theradioactive mate-

by

the U.S .Food and DrugA dm in is tra tio nand rial recovered fro m tissues was unaltered

is

onthe list of substances"generallyrecognized

14

C-eugenol. By 24hours, approximately 1%of

assafe"(CFR, 1974).TheADI(acceptabledaily

the injected

14

C had been exhaled as

carbon

intake) for hum anshasrecentlybeenrevisedto

dioxide. Trace radioact iv i ty was found in a ll

0-2.5mgeuge nol /kgbw(IPC S,

1982).

Theaver-

tissues

examined 100hoursafteradm inistration.

ag ema x imum uselevelsinbeverages,icecream,

Delaforge

et al.

(1980) h av e sh ow n that

bakedgoods, gelat insandpuddings, andchew-

as

o ther r elatedugenol

(as

w ell a lkenylben

inggumsrangefrom 1.4to500 ppm,withlevels

zenes)undergoes biotransformation through

an

in p roce ssed me a t p r oduct s being

as

h ig h

as

epoxide-diolmetabolicpathway.Eugenolepox

2,000

ppm

(Fur ia and Bellanca, 1971).Eugenol

ide and a l ly lca techol epoxide and the corres

isa lsoused asa local anaesthetic in temporary

pondingdihydrodiols(dihydrodihydroxyeuge

dentalfillings

an d

cements(Kirk -O thm er,1965;

nol and dihydrodihydroxya lly lca techol ) were

1975),

as

f ung i cide

in

.S . P h ar m ac o pe ia ,

a

detectedintheurineofmaleWistar

rats

given a

Pharmaceuticals and cosmetics(Kirk-Oth mer,

single

in t raper itoneal in jec tion

of 200

m g / k g

1966),

asan

attractant

f o r

Japanesebeetles(Ber

eugenol

in

corn oil .

The

a l lylcatechol metabo

oza et

a l. , 1975; Farm Che mic als H a ndbook ,

litesconstitutethemajormetabolitesofeugenol,

1977), as a d en atu ran t for alcohol (K irk

safrole, and eugenol methyl ether (Delaforgeet

Othm er, 1965),andasas tartingmaterialinthe

al., 1980).

synthesis

of

3-methyl-4-hydroxybenzaldehyde,

commonly

kno w n

as

vani l lin (Kirk-Othmer ,

Genetic Toxicity

1970).

Eugenol was not mutagenic for

Salmone lla

Pharmacologically, eugenol

has

beenreported

typhimurium TA1964, T A 1535 , TA1532,

to

exhibitantisepticproperties, analgesic action

TA1531, TA1530, TA100, and T A9 8, w i tho r

(localandgeneral),spasmolyticandmyorelaxant

without

metabolic act ivation (Delaforgeet a l .,

activities,

parasympathetic effects(salivarygland

1977; Dorange

et

al., 1977;Green

and

Savage,

secretion), and d ire ct

peripheral

vasodilation 1978;Swanson

et

al., 1979;Eder

et

al., 1980).

At

(Dallmeier and Carlini, 1981). concentrations

up to333

ng/plate eugenol

was

Eugenol

12


16/165

I.

IN TR O D U C TI O N

not mutagenic in Salmonella

TA98, TA100,

TA1535,

orTA1537,withorwithoutexogenous

metabolic activation.The9,000xgm icrosomal

fraction

was

o bt ai ne d

f rom

A ro cl or 1254

induced Sprague-Dawley ra t o r Syr ian golden

hamster

liver

(AppendixH,Tables HIandH2).

Samples werep reincubatedprior

to

p lat ing

in

triplicate,

and

eachseries

was

repeated.Lelenget

al . (1982)reported slightincreases in revertants

forSalmonellaTA98(326.0versus224.7)at

500

geugenol/platewithoutactivation

but not

for

s trains

TA100,TA1535,

TA1537,

TA1538.

Greater increases were seen wi th microsomal

ac tivat ion in TA1537 a t 10, 50, 150, and 500

g/plate, but notwithTA98,TA100, TA1535,

an d TA1538. In view ofthesemarginaldifferen

ces in numbers of

revertants

and

considering

othernegative

f indings

thesereported increases

should not be

taken

as

evidence

ofa

mutagenic

response.

The 2 ', 3 '-oxide of eugenol was a lso tes ted

because

th is chemical

w a s

identifiedfollowing

incubation

of

eugenolwi th femalemouseliver

microsomes (Swanson e t a l. , 1978) as well as

with ep ithelia l l iver cell cultures (Delaforge e t

al., 1977).Eugenol-2',3'-oxide wasmutagen icin

Salmonella TA1535, with

or

without

activation

(Delaforge et a l ., 1977; Dorange et al ., 1977;

Swanson

et

al . , 1979).U nder

the

preincubation

p ro to co l d esc rib ed a bo ve , n ei th er m eth yl

eugenol

(93-15-2)(Appendix

H) nor

isoeugenol

(97-51-1) w a s

mutagenic

f o r Salmonella typhi

murium

s tr ains

TA98,

TA100, TA1535, and

TA1537.

In

C hi ne se h am st er o va ry cells, e ug en ol

inducedbothchromosomeaberrationsands is

te r

c hr om a ti d e xc ha ng es ( A pp e nd ix

I). T he

aberrations

were

observed

a ft er a ctiv atio n,

whereasexchangeswerefoundwitho rwithout

microsomal inf luence.

Carcinogenicity

Eugenol, a k n o w n

tobacco

leaf phenol, was

reportedtobeaweakpromoter ofskin tumori

genesis

initiatedby7,12-dimethylbenz(a)anthra

cene ( D M B A )

in

female IC R /Ha Swis smice .

After

63

weeks ,

3of 14

micepretreatedwith

150

g D M B A and then pain tedwith5 mgeugenol

three

t imes

per

week

had

papillomas,compared

with no

p ap il lomas

i n 9

mice pre trea ted wi th

D M B A

a lo ne

a n d fo llowed b y 0 . 1 m l

acetone

(solvent) , and

none

in 13

m ice pa in ted

w i th

eugenola lone(VanDuuren et al., 1966).

Thestructurally relatedcom pound safrole(1

allyl-3,4-methylenedioxybenzene)

has

beenfound

to

cause increased incidences

of

hepatomas

in

(C57BL/6xCSH/Anf)F1miceofeithersexand

in fe ma le (C 57 BL /6 * A K R )F 1 m ice w he n

administered by gavage o r i n feed ( Innes et al.,

1968). When safrole

wasfedin

diets, increased

incidences oflivertumors(74%w ere hepatocel

lular carcinomas

or

cholangiocarcinomas) were

detected in male and female Osborne-Mendel

rats

(Longetal., 1963),andincreased incidences

of

hepatocellular carcinomas were observed in

male CD-1 mice(Borchert

et

al., 1973).S afrole

has

also beenfound

to bea

livercarcinogen

in

Balb/c mice (Lipsky

et

al. , 1979;Lipsky

e t

al.,

1980).

In arecentreportofaseriesofpublicationson

the

c ar cinogenic act iv ity

of

a lkenylbenzene

derivatives related

to

s af ro le

and

e st rago le ,

results on thecarcinogenesis testing ofeugenol

an d

methyleugenolw eredescribed

by

Miller

et

al .

(1983).

In

thesestudieseugenolgivenduring

thepreweaningperiodtoCD-1micebystomach

tube

(2.5 mol/g twiceweeklyforfiveweeksto

maleandfemales)orbyintraperitonealinjection

(once weekly for fourw eeks, tota l dose =9.45

mo l /g tomales)didnotcauseanyhepatocarci

nogenicactivityafter

14

(oral)

o r 12

(injection)

monthsofobservation.Themetaboliteeugenol

2',3'-oxidewaslikewiseinactivewhentested by

th e intraperitoneal route. These protocols have

proved sensitive for thedetection of chemically

induced hepa ti c neop lasms (Broche rt

et

a l.,

1973; Dr inkwa te r

et

a l. , 1973; Eps te in

et

a l .,

1970;Milleretal., 1979;M illeretal., 1983; Roe,

1975).

Tw o

g roups

of 30

femaleCD-I mice

ate

diets

conta in ing 0 .5% eugenol (5 ,000 ppm) for 12

monthsfollowedbyagraindietwithouteugenol

for 6

months;

onegroup also

received 0.05%

phenobarbital

i n t he

drinkingwater

forthe full

18months.Neithergroupdevelopedhepatomas.

Noneofthedietcontrolsand2ofthe phenobar

bital

contro ls developed hepatomas (Miller

e t

al., 1983).

In a dermal experiment, eugenol-2 ',3 '-oxide

was

applied top ical ly

to

g ro up s

of 4 0

female

CD-1 mice4days/wee kfor 6weeks(45 mol/

week) fo llowed by local sk in exposure twice

weekly

to croton oil(0.15mlofa0.6%solution

in acetone)

for

another

34

weeks.

Attheendof

the

40-week study, eugenol-2',3'-oxide induced

skin

tumors

in

16/40(40%)w ith

0.9

tumors

per

mouse versustheacetone controls having3/40

(7%)

with0.1permouse.Thetumorswereepid

ermalpapillomasandkeratoacanthomas

(Miller

et al., 1983).

13

Eugenol


17/165

I.

INTRODUCTION

Methyleugenol

and 1

'-hydroxymethyleugenol

weretestedbytheintraperitoneal

injection

route

inmaleB6C3F1mice.Chemicals

were adminis

teredondays1,8,15,and22.Attheendofthe18

month study, thenumb erof "hepatomas/bear

ing

mice"

for methyleugenol (total dose=4.75

mol)

was 56/58 (96%) wi th 3 .2 hepatoma s/

mouse

and for

1'-hydroxymethyleugenol (total

dose

=

2.85 mol)

was

4 1/4 4 (93% )

w ith

3.5/mouse, both comparedw ithtrioctanol con

trols having 2 4/58 (41% ) and 0 .5 /m ouse

(P


18/165

II. MATERIALS ANDMETHODS

CHEMICALANALYSES

PREPARATION OF

TESTDIETS

SOURCEAND SPECIFICATIONSOFTESTANIMALS

ANIMAL

MAINTENANCE

SHORT-TERM

STUDIES

Single-Dose Studies

Fourteen-Day Studies

Thirteen-Week

Studies

TWO-YEAR

STUDIES

ClinicalExaminations

and

Pathology

DataRecording

and

StatisticalMethods

15

Eugenol


19/165

II . MA TERIAL S AND METHODS: CHEM ICAL ANAL YSES

CHEMICAL

ANALYSES

U.S.P.extra gradeeugenol(alsosold

as

food

grade)

wasobtainedintwobatches

from

Givau

da n

Corporation

(Clifton,NJ). LotN o.36483

was

usedfor theshort-term studiesandthefirst

52

weeksofthetwo-yearstudies.LotNo.26068

wasused for the

f inal

52 weeksofthetwo-year

studies. Bothlotswere>99% pure.

Purity

and

identi ty analyses performed

at

Midwest

ResearchInstitutewereconsistent

with

thestructure(AppendixJ).Resultsofthin-layer

chromatography indicated

one

homogeneous

component. Results

of

vapor-phase chromato

graphywithonesystemindicatedasinglehomo

geneous pe ak f or L ot N o. 26068, b ut tw o

impurities,

eachwith

an area

0.1%

ofthe

area

of

the ma jo r peak, were observed for Lot No.

36483. Wh en

a

second vapor-phase chromato

graphy

system

was

used ,

an

impurity with

an

area 0.09%

of the

a rea

o f the

major peak

was

detectedinLotNo.26068.

Four

smallimpurities

in

LotNo.

36483weredetected

by

high-pressure

liquid

chromatography.

The

impuritieswere

not

further characterized(AppendixJ).

Both batches of chemical wereperiodically

analyzed t h roughou t th e studies by Southern

ResearchInstituteusingvapor-phase chromato

graphy(MidwestResearch Institute, Systems

1

and 2)and infrared spectroscopy. The results

from theseanalysesindicated

no

change

in the

compos it ion o f t he te st mater ia l du ring the

studies.

Thechemicalwasstoredat

20-24C

during

theshort-term studiesand thereafterat 5C.

PREPARATION OF

TEST DIETS

Sampledietmixturescontaining 100,000ppm

eugenol were analyzed

at

Midwest Research

Institute.Eugenolinfeedwasfoundtobestable

for

2

weeks

at

temperatures

as

high

as

45C

(Appendix

K) .

Test diets wereprepared

by

mixing

Wayne

L ab B lo x m eal (T able 1 ) an d e ug en ol in a

Patterson-Kelly twin-shell laboratory blender

for 15minutes. Eugenol wasadded tothemeal

through a liquiddispersion bar. Thetest diets

were stored at 5 C fo r 1weekfollowedbyno

morethan

1

week

at 21-23C.

Dosed feed samplesfrom

the

short-term

and

two-yearstudieswereanalyzed.

Inthe

two-year

studies,themeanconcentration of eugenolin26

random ly selected dosed feed samples contain

inga

target level

of

6,000

ppmwas6,014568

ppm.

The

meanconcentration

of

eugenol

in22

samples containing atarget levelof3,000 ppm

was2,799281ppmand in

eightsamplescon

taining a target level of 12,500 ppm w as

13,037947 ppm (AppendixL).

SOURCE AND SPECIFICATIONS OF TEST ANIMALS

The

male

and

fem ale F344/N rats

and

B6C3F] miceused inthe 14-day, 13-week,and

two-year studies were obtained from

the NCI

Frederick Cancer

Research Center (Frederick,

Maryland).

The

F344/N rats

and

B6C3F[

C57BL/6N x C3H /HeN MTV~)miceused in

thesestudieswereproduced unde rstrictbarr ier

conditions. Breeding starts

f or t he

foundation

colony at the production facility originated at

th e

National Insti tutes

of

Health Repository.

Animalsshipped

for

thesestudieswereprogeny

ofdefinedm icrobiallyassociated parentswhich

weretransferred fromisolatorstobarriermain

tainedrooms.Animalswereshipped

tothe

test

ing

laboratorya t 4-5

weeks

of age.

Upon

receipt, the animalswereisolated for

7-8 da ys and e xa min ed for the p re sence o f

parasites

or

otherdiseases.

Inallofthe

studies,

theanimalswereassignedrandomly

by

species

andsextocagesandthenthecageswereassigned

randomly to

dosed

and

controlgroups.

The

rats

and

micewere

6-7

weeks

o ld a t the

beginning

of

eachstudy.

Eugenol

16


20/165

II.

MATERIALSANDMETHODS:ANIMALMAINTENANCE

ANIMAL MAINTENANCE

The

rats

and

micewerehoused

fiveper

cage

in

suspended

solid-bottom polycarbonate cages

(Table

1)

covered withReemay spun-bonded

polyesterfilters and Dupont style#2024

filters.

Hardwood chipbeddingwaschangedtwiceper

week,and

feed

hoppers (stainlesssteelfor

rats

and g lazed clay for mice) were changed and

washed

once

per

week.

Cages

werewashedtwice

perweekinatun nelcagedishwasherat 82C.

An automatic wateringsystem supplied

tap

water.

Feed wasavailable

adlibitum.

Animal

roomsweremaintained a t21-23C andhumid

ity

was 30%-50%. Incoming air w as filtered

through fiberglass roughing

filters.

R oom

a ir

was changed

15

t imes

p er

hour. Fluorescent

lightingwasprovided 12 hoursperday.

TABLE

1. SPECIFICATIONS AND SOURCES OF MATERIALS USED F O R A N I M A L

M A I N TE N A N C E

Item

Bedding

Cages

Feed

Watering

System

Cage

Filters

Cage

and R ac k W as h

in g

Compound

Specifications

Beta

chips

Solid bot tom, polycarbonate

Wayne Lab

B lox

meal

Edstrom Automat ic

Reemay spun-bonded

polyester

Dupont #2024

M W C C ompound

Source

Nor theas te rn P roducts, Inc.

(Warrensburg ,

N Y )

Lab Products , Inc.

(Garfield ,

N J )

Allied

Mills,Inc.

(Chicago,

I L)

Edstrom Industries

(Water ford , W I)

Snow Fil trat ion

(Cincinnat i , O H )

Vestal Laboratories

(S t. Louis , MO)

SHORT-TERM

STUDIES

Singledoseoral

and

14-dayrepeateddose

feed

wereadministered150to3,000mg/kgeugenolin

studies wereconducted usingF344/N rats and

a 1%

solution

of

carboxymethylcellulose

in

B6C3Fi mice

to

determine toxicity, potential

saline

by

gavage.Survivinganimalswerekilled

target

organs, andtheconcentrations ofeugenol

on day 16.

Deaths occurred

in 1 /5

female

rats

tobe

used

inthe

13-weekstudies.

receiving2,000mg/kg,1/5malemice

adminis

tered

750

mg/kg,

and 2/5

male mice

and 5/5

female

mice administered

3,000

mg/kg . One

Single-Dose

Studies

dea th occur red in the g roup of female

rats

Inthe singledose oral toxicitystudy,groups administered 250 mg/kg as a result of gavage

of

five males and five females of each species

error (Tables 2and3).

17

Eugenol


21/165

TABLE2. SURVI VALAN D MEAN BODY WEIGHTSOF RATS ADMINISTERED ASINGLE DOSE OF

EUGENOL BY GAVAGE(a)

Mean

Body

Weights(grams)

Dose

(b)

Survival (c)

(nig/

kg)

(day

of

death)

Initial

Final Change (d)

Males

150

5/5

92

5.8

147 5.

4

55

0.8

250

5 /5

87

6.5

150

8.

1

63

2.2

500

5/5

89

7.6

150

7.9

61

3.5

1

,000

5/5 86

8.3

140 +

12.

1

54

4.6

2,000

5/5

75

5.1 131

5,

2 56

3.7

Females

150

5/5

74

3.9

108 .3,

1

33

1.3

250

4/ 5 ( e)

80

3.3

114+

2.

7 34

2.1

500 5/5 83

5.6

113

6,

6 30

2.0

1

,000 5/5 734.6 114

9.

0

41

1.9

2

,000

4/5(2)

78

3.5

107

2.

7

29

1.4

(a)

Untreated controlswere no t included in

this

test.

(b)

In 1%

solution

of


in

saline.

(c) N u mb ersurviving/numberper group.

d)

Meanweightchangeof thegroup standard errorof the mean.

(e) Accidentaldeath by gavage error.

TABLE3 . SURV IVAL AND

M E A N

BO DY WE IGHTS OF MICE ADMINISTE RED A SINGLE DOSE OF

EUGENOL

BY

G A V A G E(a)

Mean Body Weights (grams)

Dose

(b) Survival

(c)

(mg/kg)

(day

of

death) Initial

Final

Change

(d )

Males

180

5/5

20 +0.9

251.0

50 .4

375

5/5 19 1.0 241.3 5 0 .5

750

4/5( 6) 21 1.1 26

0.5

5 0 .9

1,500 5/5 19 0 .9 23

0.8

4 0 .4

3,000

3/5(1,2)

19 1.0

23

0.9

41.2

Females

180

5/5 15+ 0.5

19

0.4

4+0.5

375 5/5

160.6

200.4

4 0 .4

750

5/5

16 0.7 20

0.5

4+0.7

1,500

5/5

16

+ 0.4

19

0.5

3 0.4

3,000 0/5(1,1,2,2,2)

16 0 .3

(a)

Untreatedcontrolswere

not

included

in

thistest.

(b) In 1%

solution

of


in

saline.

(c)

Number surviving/number

per

group.

d)

Meanweightchange

of the

group

standard error

of the

mean.

Eugenol

18


22/165

II.

MAT ERIALS AND METHO DS: SHO RT-TERM STUDIES

Fourteen-Day Studies

In the four teen-day s tudies, groups of

f ive

males and five females of each species were

administered 6,000

to

100,000

ppm

eugenol

in

feed

for 14days (Tables 4 and 5). No control

group was used. All surviving animals were

killed

on day 15. One offive

malerats

and all

female

rats

that received 100,000

ppm

died.

A

dose-associated decrease

in

mean bodyweight

gain

was

observed

for

bothmale

and

femalerats

ator

above25,000ppm.Maleratsthatreceived

100,000

ppm

los t weight. Deaths occurred

in

threeof

five

malemicethatreceived50,000ppm

eugenol and in a ll male and femalemice that

received

100,000

ppm.

A dose-associated

decrease

in

meanbodyweightgain

was

observed

for

both ma le a nd fema le mice. We ight loss

occurredinmalemicethatreceived12,500ppm

and in all

micethat received25,000

or

50,000

ppm.

TABLE

4 .

SU R V I V A L

A ND

M E A NB ODY WE I G H T S

O F

RAT S

FE D

DIETS

CONTAINING

EUGENOL

Dose

(ppm)

Males

6,000

12,500

25,000

50,000

100,000

Females

6,000

12,500

25,000

50,000

100,000

FOR 14 DAYS

(a)

Survival

(b)

Mean BodyWeights(grams)

(dayof

death)

Initial

Final

Change

(c)

5/5 82 + 2.3

1282.6

+46 + 2.3

5/5

916 .6

1335.0

+422.6

5/5

92

4.5

128 5.9

+36 + 3.1

5 /5 90 6.5

103 + 8.2

+133.3

4/5(9)

98

5.8

72 3.8

-26

5.1

5/5

89 3.0

1213.4

+323.1

5/5

853.3

118

1.5

+33 2.7

5/5 79

4.2

101

3.4

+22

1.2

5/5

741.8

82

3.0

+ 8 2.2

0/5(7,8,8,9,10)

77

3.6

(a)

Untreatedcontrols were

not

included

in

thistest.

(b ) Number surviving/number

per

group.

(c) Meanweight change of the g r oupstandarderror of the mean.

19

Eugenol


23/165

TABLE 5. S U R V I V A LAND M E A N BODY WEIGHTS OFMICEFEDDIETSCONTAINING EUGENO L

FOR 14 D A Y S (a)

Dose

(ppm)

Males

6,000

12,500

25,000

50,000

100,000

Females

6,000

12,500

25,000

50,000

100,000

Survival

(b )

(day

of

death)

5/ 5

5 / 5

5/5

2/5(10 ,10 ,15)

0 /5 (11 ,11,12

12,13)

5 / 5

5/5

5 / 5

5/ 5

0 /5 (7,7,

7,7,8)

MeanBody

Weights

(grams)

Initial

Final

Change

( c)

19 0 .7

2 2 + 1 . 0

+3+ 1 .0

21

0 . 6

20 1.0

-1 + 0 . 9

20

0.5 17+ 1.0

-31.2

200.4 13

0 .9

-7 +1.0

21

0 . 5

17 0 .6

18

+ 0.5

+1

+ 0 .2

17

+ 0.6

18+ 0.4

+1

+ 0 .2

17

+ 0.4 15 + 0 .7

-2 1.0

17 + 0.5

12 + 0.4 -50.7

18 0 .4

(a)

Un t r e a te d c o n tr o ls we re

n o t

i nc luded

in

thistest.

(b ) N u m b e r s u r v iv i n g /n u m b e r

per

g roup.

(c) Meanw eightchange of the group s t andard e r ror of the mean.

Thirteen-Week

Studies

Thesestudieswereconducted

t o

evaluate

the

cumulativetoxicityofthetestmaterial,toiden

tify organs

affected,and todetermine themost

a ppropr ia te doses for the two-ye ar s tudies .

Weightgaindataandresultsofhistopathologic

examination wereusedind eterminingthecon

centrations

t o b e

used

in the

two-yearstudies.

Diets containing0, 800, 1,500, 3,000, 6,000, o r

12,500

ppm

e ugenol we re

f ed fo r 13

weeks

to

groups o f 10male and 10femalerats(Table 6),

and

g rou ps

of 10

male

and 10

female m ic e

received

dietswith

0,

400, 800, 1,500,3,000,

o r

6,000

p pm

(Table

7 ).

Observations

fo r

clinical

signs or

mortality were made twice daily

and

animalswereweighedweekly.Attheendofthe

91-day study, survivorswerekilled,necropsies

were

performedonallanimals,andtissues

from

the

controls

and the highest

dose

group

were

taken forhistopathologic analysis.

Finalbodyweightswere10%lessformale

rats

receiving

12,500

ppm

when compared

to

con

trols; weights

of

femalerats

at the

12,500

ppm

diet arylev elwere6%less.No comp ound -related

histopathologic

effects

w ere observed. No

deaths

occurred

among

therats.

Doses

selected

for the

two-year studies were3,000

an d

6,000

ppm for males and 6,000 and 12,500 ppm for

females.

No s ignif icant differences in body weights

were

observedamonggroupsofmice.Nodeaths

occurred among

the

mice

and no

dose-related

gross or histopathologic

effectswere

observed.

Dosesforthemiceforthechronicstudywereset

at 3,000 and

6,000

ppmforbothmaleand female

mice.

Eugenol

20


24/165

TABLE6 .

S U R V I V A L

A N D

MEAN BODY WEIGHTS

OF

RATS

F E D

DIETS CONTAI NING EUGEN Ol

FOR 13

WE E K S

Mean Body Weights(grams) FinalBodyW eights

Dose Relative to

Controls

(pp m) Survival

(a) Initial

Final

Change

(b)

(Percent) (c)

Males

0

10/10

69 3.8 334 5 .4

+265

3 . 4

800 9 /9 66 2.6

3304.9 +264

3 .6 - 1

1,500 10/10

682.6

3245.2

+2565.4 - 3

3,000

10/10

68

3.4

32 4

6.1

+2565.4

- 3

6.000

10/10

632.4 3093.8

+246

3 . 4 - 7

12,500

10/10

68 3 .1

30 03 .9

+232

4.4 -10

Females

0

10/10

71 1.8 190 1.9

+119 1.8

800 10/10

682.9

188

+ 2 .4

+1202.5 1

1,500

10/10

71

2 . 1 188 3 .4

+

1 1 7 2 . 3

-1

3,000

10/10

65 + 1 0

18 4

2 .4 +119 2 .5

-3

6,000

9/9

691.8

184

2.0

+ 1 1 5 2 . 7

3

12,500

10/10

661.8 178 + 2.2

+

1122 .5

-6

(a ) N u mb e r surviv ing/numberpergroup.

(b )

M ean

weight

change

o f the

g r o u p

s tandard e r ro r

o f th e

mean.

(c) Weight r e l at ive

to con t ro ls=

Weight (Dosed Group) - Weight(Control Group)

x

100

Weight (Contro l Group)

TABLE 7. S U R V I V A L AND

MEAN BODY WEIGHTS

OF

MICE

F ED

DIETS CON TAIN ING

EUGENOL

FOR 13

WEEKS

Mean

Body Weights(grams)

FinalBody Weights

Dose

Relative to Controls

(ppm)

Survival

(a) Initial

Final

Change (b) (Percent) (c)

Males

0

10/10 210 .5 31 0 . 5 + 100 .5

400

10 / 10

220.6 320 .7

+

100.9 +3

800

10/10

220.6

33

0.5

+

1 10.6 +6

1,500

10/10

22

0.7

320.5

+100.4 +3

3,000

10/10

2 1

0 . 4

3 1 + 0 . 5

+100.6 0

6,000

10/10 22

0.5

31

+ 0 . 5 + 9

0 .5 0

Females

0

10/10 17 0 .3

24+0.4

+ 70 .3

400

10/10

18

0 .4 24

0.7 + 6

0 .4 0

800

10/10

17 0 .4

24

0.5

+

7

0 .4 0

_4

1.500

10/10

17

0 .4

23

0.5

+

60 .2

3,000

10/10 17

0 .4

230.5

+

60 .3

-4

6,000

IO /

1 0

17

+ 0.3 24 + 0.3 +

7 + 0 .3 0

(a ) N u m be r

su rv i v i n g / n u mb er

p e r

group.

(b ) M ean

weight

changesofthe g r o u p +s tandarderror o f th e mean .

(c) Weight relative

to con t ro ls

Weight (Dosed Group)- Weigh t (Contro l Group)

x 100

Weight (Contro l Group)

21

Eugenol


25/165

II.

MATERIALS

AND

METHODS: TWO-YEAR STUDIES

TWO-YEAR

STUDIES

The test groups, doses administered, and

the two

yearstudies,mice

fed

eugenol

and the

durations

ofthe

two-yearstudies

areshownin

controlswerehoused withmice

onfeeding

stud-

Table 8.For thefirst9m onthsofthetwo-year

iesofmannitolandziram.Thenthemicewere

studies, rats fedeugenoland thecontrolswere

movedtothe

room

in

which

the

ratswere

on

test

housed

in the

same room

as

rats

on feeding

with

eugenol.No other chemicalswerethenon

studies of mannitol (CAS No. 69-65-8) and

test in

thatroom.

ziram

(CAS

No.

137-30-4).

Forthefirst

year

of

TABLE 8. EXPERIMENTAL DESIGN OFTWO-YEAR FEEDING

STUDIES

WITH EUGENOLINRATS

AND MICE

Initial

Weeks

on

Study

Test No.of

Dose

Group

Animals (ppm) Dosed (a)

Not

dosed

Male Rats

Control (b)

40

0

0

105

Low

D ose

50

3,000

103

1

High Dose

50 6,000 103

1

Female Rats

Control (b)

40

0

0 105

Low

Dose

50 6,000 103

2

High Dose 50 12,500 103

1

Male

Mice

Control

(b)

50 0 0

105

Low

D ose

50

3,000

103

2

High

Dose

50

6,000

103

1

FemaleMice

Control

(b) 50

0 0

105-106

Low Dose

50

3,000

103

2

High

Dose

50

6,000

103

1

(a) ThestartdateswereJune3,1977,forratsand Ap ril12,1977,formice.Thekilldateswe reJun e 1,1979,for

ratsand April 10,1979,for mice.

(b) Control and dosed groups wereof the same s train, sex, and age rangeand

f ro m

the same source and

shipment. All animals of the same species shared the same room, and al l aspects of animal

care

and

maintenanceweresimilar.Animalswererandomized

to

dosed

and

controlgroups

as

described

in

Section

II.C.

Eugenol

22


26/165

II. MATERIALSAND METHODS:

TWO-YEAR

STUDIES

ClinicalExaminations

and

Pathology

Allanimalswereobservedtwicedailyform or

bidity

or

mortality.Clinicalsignswererecorded

monthly. Individual animals were weighed

weekly

for the first 13weeks,then monthlyto

week93,andevery2weeksthereafter.Themean

body weight of each group was calculated by

dividing

the

totalweight

ofa ll

animals

in the

groupbythenumberofsurvivinganimalsinthe

group.Moribundanimals andanimalsthatsur

vivedtotheendofthebioassaywerekilledwith

carbon dioxide.

Examinations forgrossly visiblelesionswere

performed

on

major tissues

or

organs. Tissues

were

preserved

in 10%

neutral buffered for

malin, embedded in paraff in, sectioned, and

stained withhematoxylin

and

eosin.

The

follow

ing were examined microscopically : tissue

masses, abnormal lym phnodes,skin,mand ibu

lar

l ymph nodes, mammary g land , sa livary

gland,thighmuscle,sciaticnerve,bonemarrow,

costochondral junction (rib), thymus, larynx,

trachea,lungs

and

bronchi,heart,thyroid,para

thyroid, esophagus, stomach, duodenum,jeju

num, i leum, colon, mesenter ic lymph nodes ,

liver,

gallbladder (mice),pancreas,spleen,kid

neys, adrenals, bladder, seminalvesicles/pros

tate/testesor ovaries/uterus,nasalcavity,brain,

pituitary,and spinalcord.

Necropsieswereperformedonallanimalsnot

excessivelyautolyzedorcannibalized.Thus,the

numberofanimalsfromwhichparticularorgans

or tissueswereexamined microscopicallyisnot

necessarilyequaltothenumberofanimals that

wereplaced

on

study

in

eachgroup.

Neoplasticnodules

ofthe

liverwereclassified

according to the recommendations of Squire

and Levitt(1975)and theNation alAca demyof

Sciences(1980).

When

the

pathology examination

was

com

ple ted, the slides, ind iv idual animal data

records, and summary tables were sent to a n

independentqualityassurancelaboratory.Indi

vidualanimalrecords

and

tableswerecompared

for accuracy,slidesand tissuecountswereveri

fied,

and histotechniques were evaluated. A ll

tumordiagnoses,

all

targettissues,

andall

tissues

from

a

randomlyselected

10

percent

ofthe

ani

malswereevaluatedbyanexperienced patholo

gist.

Slides of all target t issues and those on

whichthe

original

andquality

assurance

pathol

ogistsdisagreedweresubmittedtotheChairper

sonofthePathologyWorkingGroup(PWG)for

evaluation . Repr esenta tiveslidesselectedbythe

PW G

Chairperson werereviewedblindly

bythe

PW G pathologists, who reached a consensus

and compared their findingswiththe original

diagnoses. When disagreements occurred,

the

PW G

sent

the

appropriate slides

and

theircom

ments

to the

original pathologist

for

review.

(This procedure

has

beendescribed,

in

part,

by

Wardet

al., 1978,

andby

Maronpot

and

Boor-

man,1982).

The

finaldiagnosisrepresents

a

con

sensus

of

contractor pathologists

and theNTP

PathologyWorking Group.

DataRecording

and

StatisticalAnalyses

Data f rom

thisexperiment wererecorded in

the

Carcinogenesis BioassayDataSystem(Lin

hart

et

al. , 1974).

The

d ata elements include

descriptive information

on the

chemicals, ani

mals,experimentaldesign,clinicalobservations,

survival,bodyweight,andindividualpathologic

results , as recommended bythe International

Union Against Cancer(B erenblum, 1969).

Probabilitiesof survivalwereestimatedbythe

product-limit procedure of Kaplan and Meier

(1958) and are

presented

in

this report

in the

form

ofgraphs. Animals werestatisticallycen

sored

asofthe

timethattheydied

of

otherthan

natural

causes or werefoundtobemissing;ani

malsdying

from

naturalcauseswerenotstatisti

callycensored.Statisticalanalysesforapossible

dose-related

effect

on

survivalused

the

method

of

Cox

(1972)

for

testing

two

groups

for

equality

and

Taron e's (1 975) e xte nsio ns

of

Cox's

methodsfortestingforadose-relatedtrend.

The

incidence

o f

neoplastic

or

nonneoplastic

lesions

hasbeengivenastheratioofthenumber

of

animalsbearingsuchlesions

ata

specificana

tomicsite

tothe

number

of

animals

in

whichthat

site w as examined . In

most instances,

the

denominators included onlythose animals

for

which that sitewasexaminedmicroscopically.

However,

whenmacroscopicexam inationwas

required

todetectlesions(e.g.,skinormammary

tumors)priortomicroscopicsamplingorwhen

lesions

could have appeared at mul tiplesi tes

(e.g.,lymphomas),thedenominatorsconsistof

thenumbersofanimalsnecropsied.

For thestatistical analysisoftum orincidence

data,

two different m eth od s of adjusting for

intercurrent mortal ity were employed. Each

usedtheclassicalmethodsforcombiningcontin

gency

tablesdevelopedbyMantelandHaenszel

(1959). Tests

of

significanceincludedpairwise

23

Eugenol


27/165

II. MATERIALS AND METHODS: TWO-YEAR STUDIES

comparisons

of

high

and low

dosegroupswith

controls and tes ts for overal l dose-response

trends.

Thefirst metho dofanalysisassumedthatall

tum orsofagiventypeobservedinanimalsdying

beforetheendofthe

studywere"fatal,"i.e.,they

eitherdirectly or indirectly caused thedeathof

the

animal.According

to

thisapproach,

the

pro

portions

of

tumor-bearinganimals

inthe

dosed

andcontrolgroupswerecomparedateachpoint

intimeatwhichananima ldiedwithatumorof

interest.Thedenominators of theseproportions

were

the

totalnumber

of

animals

atriskin

each

group. These results, includingthe data fro m

animals

killed

attheendofthe

study,werethen

combined bythe Mantel-Haenszelmethods to

obtain an overall P-value. This method of

adjusting

for intercurrent mortality is the

l ife

table method of Cox (1972) and of Tarone

(1975).

Thesecondmethodofanalysisassumedthat

all tumors

o f a

giventypeobserved

in

animals

dyingbeforetheendofthestudywere"inciden

tal,"i.e.;theyweremerelyobserved

a t

autopsy

in

animals

dying

ofan

unrelatedcause.According

to this approach, the proportions of animals

found to have tumors in dosed and con trol

groupswerecompared ineacho f

five

timeinter

vals:0-52weeks, 53-78weeks ,79-92week s,week

93

totheweekbeforetheterminalkill,andthe

terminalk illperiod.Thedenominators ofthese

proportionswere

the

number

of

animalsactually

autopsied during

the

timeinterval.

The

individ

ual time interval comparisons werethencom

bined by the previously describedmethods to

obtaina singleoverallresult. Thecomputational

detailsofbothmethodsarepresentedinPetoet

al. (1980).

In addition to these tests , one other setof

statisticalanalyseswascarriedoutandreported

in the tables analyzing pr imary tumors ; the

Fisherexacttestforpairwise comparisonsand

theCochran-Armitagelineartrendtestfordose-

response trends (Armitage, 1971; Gart et al.,

1979).Thesetestswerebased

onthe

overallpro

portion oftumor-bearing animals.Allreported

P-values

are

one-sided.

For s tudies in whichthere is little,effect of

compound admin is tr ation on survival, the

resultsofthethreealternativeanalyseswillgen

erally be

similar.

When dif fering

results

are

obtained bythe three methods,the

final

inter

pretationofthedatawilldependontheextentto

whichthetumorunderconsiderationisregarded

asbeingthecause ofdeath.

Eugenol

24


28/165

III. RESULTS

RATS

TWO-YEAR

STUDIES

Body Weights and Clinical Signs

Survival

Pathology andStatistical Analyses of Results

MICE

TWO-YEAR

STUDIES

Body Weights and Clinical Signs

Survival

Pathology

and

Statistical Analyses

of

Results

25

Eugenol


29/165

III. RESULTS:

RATS

TWO-YEAR STUDIES

RATS

TWO-YEAR STUDIES

BodyWeightsandClinical

Signs

Meanbodyweightsformaleratsandlowdose

females were comparable amonggroups. For

high dose femalerats mean bodyweightswere

lowerthancontrolsthroughoutmostofthestud

ies(Table 9 andFigure 1). The

average daily

feed

consumptionper rat bylowdoseand high

dosemaleratswas98%and97%andforfemales

it

was94%and91%thatofthecontrols

(Appen

dixE).

Survival

Estimates of the probabilities ofsurvivalof

male

and

female

rats

administered eugenol

inthe

diet

atthe

concentrations

used

in

these

carcino

genesis studies and those of the controls are

shown

bythe

Kaplan

and

Meiercurves

inFigure

2.Nosignificantdifferenceswerefou ndbetween

any ofthegroups ofeithermaleorfemalerats.

In

male rats, 23/40 (58%)

o f the

controls,

26/50 (52%)ofthelowdose,and

37/50(74%)

of

the highdosegrouplived totheendofthestudy

at 105weeks.Inf emalerats, 30/40(75%)ofthe

controls,36/50(72%)

ofthelow

dose,

and

44/50

(88%)ofthehighdo segr oup livedtotheendof

the study a t 105weeks.

TABLE 9. MEAN BODY WEIGHTS

(RELATIVE

TO CONTROLS)OF RATS FE D DIETS CONTAINING

EUGENOL FOR TWO YEARS

Weeks

Vehicle

Control

LowDose

High

Dose

on

Study Av.Wt.

No.f

Av.Wt.

Wt.(percent

No.of Av.

t. Wt.percent

No.of

(grama) Survivors

(grams)

of

controls)

Survivors

(grams) ofcontrols)

Survivors

MALE

0 94 40 93

98.9 50 90

95.7

50

4

195 40 199

102.1

50 193 99.0 50

8 259

40

260 100.4 50

253

97.7

50

13

307

40 308 100.3 50 306 99.7 50

17 333 40 335

100.6

50

331

99.4

50

21

365

40 365 100.0 50

363

99.5

50

25

377 40 386 102.4

50

375

99.5 50

28

391

40

397

101.5 50

385

98.5 50

34

404 40 405 100.2

50

381 94.3 50

38

411

40

410 99.8 50

399

97.1 50

42

408

40

419 102.7 50 406 99.5 50

46 435 40

430 98.9

50

417

95.9

50

51

428

39

427

99.8

50

415

97.0 49

55 432

39

434

100.5

50 418 96.8

49

59

444

39

439 98.9

50

423

95.3 49

64

439

39

440 100.2

49

413

94.1

49

68

447 39

439

98.2

49

421 94.2

49

72

442 39 439

99.3

49

424 95.9 48

77

453

37

437 96.5

48

430 94.9

48

81

442

36 427

96.6 45 423

95.7

48

86

443

35 430

97.1

42

418

94.4

48

90

438

33 433

98.9

41

417

95.2

46

94

437

32 422

96.6 40 410 93.8 42

98 432 29 418

96.8 38

407

94.2

40

102

422

27

413

97.9

29 402 95.3

37

104

418

25 413

98.8

26

404

96.7 37

FEMALE

0

83

40 85

102.4

50 85

102.4

50

4

140

40 136

97.1

50 131

93.6

50

8 168

40 167 99.4

50 158

94.0 50

13

188

40

186

98.9 50 175 93.1 50

17

188

40

189

100.5

50

183 97.3

50

21

203

40

199

98.0

50

196 96.6

50

25 205

40

203 99.0

50

196

95.6

50

28 214

40

212 99.1

50

203 94.9 50

34

214

40 208 97.2 50 199

93.0 50

38 218

40

217

99.5 50 207

95.0

50

42 220

40 216 98.2

50

208

94.5

50

46 227

40 221

97.4 50 211 93.0

50

51

232 40 220

94.8

50

208

89.7

50

55

239

40

223

93.3

50 213 89.1 50

59

240

40

226

94.2 50

216 90.0 50

64 244 40 230 94.3

50 221 90.6 50

68

254

40

241

94.9

50

223 87.8

50

72

258

40

244

94.6 49 225

87.2

50

77 269

40

255

94.8

49

235

87.4

50

81

274 39 253 92.3 49 234 85.4

49

86 280

38

266

95.0

48 242

86.4

49

90

281

37 272

96.8

48 247

87.9

48

94 287 36 273 95.1

48

245 85.4

48

98

289

34 274

94.8

45

251

86.9

46

102

291 33 281

96.6 38 253 86.9 45

104

290

30

293

101.0 36

271 93.4

44

Eugenol

26


30/165

Figure1. Growth

Curves

for

R ats

FedDietsContainingEugenol

27 Eugenol


31/165

Figure2. Survival urvesfor RatsFed Diets Containing Eugenol

Eugenol

28


32/165

III. RESULTS: RATS TWO-YEAR

STUDIES

Pathology

and

Statistical

Analyses

of

Results

Histopathologicfindingsonneoplasmsinrats

aresumm arizedinAppendixA,TablesA l and

A2;

Appen dixTables A3andA4givethesurvi

val

an d

tumor s ta tus

f o r

individual male

and

female

rats. Findings on nonneoplastic lesions

aresummarizedinAppendixC,Tables Cl and

C2.AppendixG,TablesGl andG2containthe

statisticalanalysesofthoseprimarytumorsthat

occurred

with an

incidence

ofat

least

5%inone

of

the

groups.

The

statisticalanalysesused

are

discussed

inChapter II (Data

Recording

and

Statistical Methods). Significant increases or

decreases

in the

occurrence

of

particular neo

plasmsare presentedbelow.

Lung:

Alveolar/bronchiolaradenomas

or

car

cinomasofthelunginmaleratsoccurred

with

an

increased

(P


33/165

TABLE

11 .

I NCI DE NCE S

OF

RATS WITH C-CELL NEOPLASMS

OF THE

T H YRO I D G L AND

Males

C-Cell A d en oma

Overall

Incidence

Adjusted Incidence

Terminal Incidence

LifeTableTest

IncidentalTumor Tes t

Cochran-Armitage Trend Test

Fisher Exact Test

Weeks to F irs t Observed Tumor

C-Cell Carcinoma

Overall Incidence

Adjusted Incidence

Terminal Incidence

Life

Table Test

Incidental

Tumor Test

Cochran-Armitage Trend

Test

Fisher Exact Test

Weeks to F irs t Observed T u mo r

C-CellAdenomaorCarcinoma

Overall Incidence

Adjusted

Incidence

Terminal Incidence

Life Table Test

Incidental

T umor Test


Fisher Exact Test

Weeks to F irs t ObservedTu mo r

Females

C-Cell Adenoma

Overall

Incidence

Adjusted

Incidence

Terminal Incidence

Life TableTest

IncidentalT um or Tes t

Cochran-Armitage T ren d Test

F isher Exact Test

Weeks to F irs t Observed Tumor

C-Cell Carcinoma

Overall Incidence

Adjusted Incidence

Terminal Incidence

Life

Table

Test

IncidentalTumorTest

Cochran-Armitage Trend

Tes t

Fisher Exact Test

Weeks to First Observed

T u m o r

C-Cell

A d en oma o r C a rci n oma

Overall Incidence

Adjusted

Incidence

Terminal Inccidence

Life Table Test

Incidental T u m o rTest


FisherE xact Test

Weeks

to

Firs t Observed Tumor

Control

4 /40 (10%)

14.5%

2/25(8%)

P=0.030N

P=0.038N

P=0.037N

100

3/40(8%)

10.9%

2 /25 (8%)

P=0.254N

P=0.295N

P=0.3I3N

96

7/40(18%)

24.3%

4 /2 5 ( 1 6 %)

P=0.021N

P=0.030N

P=0.032N

96

Control

3/40 (8%)

10.0%

3/30(10%)

P=0.187N

P=0.253N

P=0.271N

105

4 /40 (10%)

12.8%

3 /30 (10%)

P=0.399N

P=0.441N

P=0.493N

103

7 /4 0 ( 1 8 %)

22.5%

6/30(20%)

P=0.149N

P=0.2I5N

P=0.254N

103

3,000

ppm

5/50(10%)

15.5%

3 /2 6 ( 1 2 %)

P=0.563

P=0.601N

P=0.634N

80

3 / 5 0 (6 %)

11.5%

3 /26 (12%)

P=0.633N

P=0.591N

P=0.550N

104

8/50(16%)

26.5%

6/26(23%)

P=0.572

P=0.530N

P=0.535N

80

6,000 ppm

11/49

(22%)

28.1%

8 /35(23%)

P=0.048

P=0.040

P=0.049

85

1/49(2%)

2.9%

1/35

(3%)

P=0.138N

P=0.

1 1 1 N

P=0.124N

105

12,49(24%)

30.7%

9/35

(26%)

P=0.269

P=0.271

P=0.296

85

6,000

ppm

0/50 (0%)

0.0%

0 /37 (0%)

P=0.029N

P=0.055N

P=0.036N

2/50(4%)

5.1%

1/37(3%)

P=0.346N

P=0.454N

P=0.395N

100

2/50(4%)

5.1%

1 /37 (3%)

P=0.027N

P=0.056N

P=0.039N

100

12,500ppm

2/50 (4%)

4.4%

2/45

(4%)

P=0.319N

P=0.319N

P=0.395N

104

4 /50 (8%)

8.9%

4/45(9%)

P=0.416N

P=0.490N

P=0.512N

104

6 /50 (12%)

13.3%

6/45

(13%)

P=0.217N

P=0.264N

P=0.330N

104

Eugenol

30


34/165

HI.

RESULTS: RATS

-

TWO -YEAR STUDIES

Uterus:Therewasa positivetrend (P/cO.05)

6/50,

12%;and 16/50,32%)(Table12).The32%

andamarginally(P=0.051)increasedincidence

incidence inthe high dosegroup isabove the

ofendometrialstromalpolyps oftheuterusin

historical averagefor thislab oratory (66/438,

female rats inthehighdosegroup(6/40,15%;

15%).

TABLE

12 .

INCIDENCES

OF

FEMALE RATS WITH TUMORS

OF THE

UTERUS

Control

6,000ppm

12,500

ppm

Uterus:Endometrial

Stromal

Polyp

or

Sarcoma

OverallIncidence

6 /40 (15%)

6/50(12%) 16/50(32%)

Adjusted Incidence 18.3%

15.2%

35.6%

TerminalInccidence

4 /30 (13%)

4/ 36(11%)

16/45(36%)

Life

Table Test

P=0.062

P=0.479N

P=0.121

Incidental

Tumor Test

P=0.031

P=0.369N P=0.077


P=0.022

Fisher ExactTest

P=0.456N

P=0.051

Weeks

to FirstObserved Tumor

94 98

104

Mammary G land :Fibroadenomasofthe mam

mary gland

in

female rats were decreased

(P


35/165

III. RESULTS: MICE - TWO-YEAR

STUDIES

TWO-YEAR STUDIES

Body Weights

and

ClinicalSigns

Mean bodyweightswerecomparable among

all groups except the 6,000 ppm femalemice,

which

were

14and 11

percent lowerthan con

trolsatweeks101and 104,respectively(Table14

an d

F ig ure

3). N o

compound-related clinical

signswereobserved.Theaveragedailyfeedcon

sumption permousebylowand highdosemice

was97%and94% thatofthecontrolsformales

and 95%and 90%forfemales(Appendix E).

Survival

No

s ignif icant di fferences

in

surviva l were

seen between

an y o f th e

g ro u ps

of

either sex;

survival ofthe high dose maleswassomewhat

lower

thanthat

inthe

othergroupsafterweek

38

and the

survival

in the low

dose female

group

TABLE 14. MEAN

BODY

WEIGHTS

(RELATIVE

T O

CONTROLS)

OF MICE FED

DIETSCONTAINING

EL'GENOL

FOR TWO

YEARS

Vehicle

Control

was

lowerafterweek

80.

Estimates

ofthe

proba

bil it ies of surviva l of male and female mice

administered eugenol in thedietat the concen

trations of thesestudiesandthoseofthe

control

group are sh ow n by the Kap la n and Meier

curvesin Figure4.

In

male

mice, 41/50 (82%) o f the

controls,

35/50(70%)ofthelowdose,and35/50(70%)of

the

highdosegrouplived

totheendofthe

study

at106weeks. Infemalemice,43/50(86%) ofthe

controls,40/50(80%)

ofthelow

dose,and45/50

(90%)

ofthe

highdosegroup lived

totheendof

the

studyat 106weeks.Fiveofthelowdose male

micew ereaccidentally killed during week13of

th e

study,

at

wh ichtimetheywerecensored from

the

statisticalanalysis

ofsurvival.

Weeks

on

Study

MALE

0

7

11

15

20

24

28

32

36

41

46

49

53

58

62

66

71

75

79

84

88

93

97

101

104

FE MAL E

0

7

ji

15

20

24

28

32

36

41

46

49

53

58

62

66

71

75

79

84

88

93

97

101

104

LowDose

Wt. (percent

of controls)

105.6

103.6

96.9

100.0

100.0

100.0

100.0

102.8

102.9

97.3

97.3

102.8

97.4

97.4

95.0

95.1

95.0

95.1

97.5

97.5

100.0

95.0

97.4

97.4

97.4

100.0

100.0

96.0

96.0

96.2

100.0

96.6

96.6

96.4

103.6

100.0

96.7

96.8

100.0

96.9

100.0

100.0

100.0

97.0

97.1

97.0

100.0

97.1

97.2

94.3

No.of

Survivors

50

50

50

45

45

45

45

45

45

45

45

45

45

45

45

44

43

41

41

41

39

39

38

38

36

50

50

50

50

50

50

50

50

50

49

49

49

49

49

49

49

48

48

48

47

44

43

43

41

40

Av.Wt.

(grams)

19

28

31

32

33

34

35

36

36

37

36

35

37

37

38

39

37

38

38

39

38

38

38

38

38

16

22

24

25

25

26

27

28

28

28

28

28

29

29

30

30

31

31

31

31

31

32

32

31

31

HighDose

Wt .(percent

of

controls)

105.6

100.0

96.9

100.0

97.1

97.1

97.2

100.0

102.9

100.0

97.3

97.2

97.4

94.9

95.0

95.1

92.5

92.7

95.0

97.5

97.4

95.0

97.4

97.4

97.4

100.0

100.0

96.0

100.0

96.2

96.3

93.1

96.6

100.0

100.096.6

93.3

93.5

93.5

93.8

96.8

91.2

91.2

93.9

91.2

93.9

91.4

91.4

86.1

88.6

No.of

Survivors

50

50

50

49

49

49

49

49

49

48

47

47

47

47

47

47

44

44

44

43

42

40

37

37

36

50

50

50

50

50

50

50

50

50

50

49

49

49

49

49

49

48

48

48

48

48

48

47

46

45

Av.Wt.

(grams)

18

28

32

32

34

35

36

36

35

37

37

36

38

39

40

41

40

41

40

40

39

40

39

39

39

16

99

25

25

26

27

29

29

28

28

29

30

31

31

32

31

34

34

33

34

33

35

35

36

35

No.of

Survivors

50

50

50

50

50

49

49

49

49

49

49

49

49

49

49

48

47

47

47

46

45

44

43

43

41

50

50

50

50

50

50

50

50

50

50

50

50

50

49

49

49

49

49

49

48

48

47

45

44

43

Av.Wt.

(grams)

19

29

31

32

34

35

36

37

36

36

36

37

37

38

38

39

38

39

39

39

39

38

38

38

38

16

22

24

24

25

27

28

28

27

29

29

29

30

31

31

31

34

34

32

33

32

35

34

35

33

Eugenol

32


36/165

Figure3. Growth urvesfor Mice FedDiets Contain

Documents

Carcinogenesis Studies of Eugenol