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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
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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
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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
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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
Containing Eugenol 47
TableA1 Summaryofthe Incidenceof Neoplasms in MaleRats Fe d Diets
ContainingEugenol 48
Table A2 Summary ofthe Incidence ofNeoplasms inFemale
Rats
Fed
Diets
Containing Eugenol 53
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
Containing Eugenol 72
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
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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
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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
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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
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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
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I. INTRODUCTION
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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
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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
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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
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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
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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.
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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).
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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
carboxymethylcellulose
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
carboxymethylcellulose
in
saline.
(c)
Number surviving/number
per
group.
d)
Meanweightchange
of the
group
standard error
of the
mean.
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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.
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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.
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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
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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.
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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
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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.
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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
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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
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Figure1. Growth
Curves
for
R ats
FedDietsContainingEugenol
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Figure2. Survival urvesfor RatsFed Diets Containing Eugenol
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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
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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
Cochran-Armitage Trend 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
Cochran-Armitage Trend Test
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
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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
Cochran-Armitage Trend Test
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
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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
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Figure3. Growth urvesfor Mice FedDiets Contain