Chronic toxicity studies [electronic resource]

2009·0.52 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 452
Adopted:
7September2009
OECDGUIDELINEFORTHETESTINGOFCHEMICALS
ChronicToxicityStudies
INTRODUCTION
1. OECDGuidelinesfortheTestingofChemicals(TGs)areperiodicallyreviewedinthelight
oGufisdceileintniefi4c5p2rowgarsessa,docphtaendgiinng19a8s1s.essDmeevnetlporpamcetinctesofanadreavniismeadlTwGelf4a5r2ewcaonssicdoenrsaitdieornes.dTnehceesosrairgiynailn
o(3r)de(r4)t.oTrheefleucptdraetcienngtodfevTeGlo4p5m2enhtassibnetehnecfairerlideodfoauntiimnalpawraelllfealrewiatnhdrerveigsuiloantsoroyfrtehqeuiTreesmteGnutisde(1l)in(e2s)
451,CarcinogenicityStudiesand453,CombinedChronicToxicity/Carcinogenicitystudies,withthe
objectiveofobtainingadditionalinformationfromtheanimalsusedinthestudyandprovidingfurther
detailondoseselection.ThisTestGuidelineisdesignedtobeusedinthetestingofabroadrangeof
chemicals,includingpesticidesandindustrialchemicals.
2. Themajorityofchronictoxicitystudiesarecarriedoutinrodentspecies,andthisTest
Guidelineisintendedthereforetoapplyprimarilytostudiescarriedoutinthesespecies.Shouldsuch
tNsotogunde-itRehsoerdbeewnitrtsehqu(ti5h)roesdmeaiynountaollnis-noredobdeeinnatpOpslEpieeCcdiD,esw,TitGthhe4ap0pr9pi,rnocpRirepilpaeetsaetaenmdoddiDpfroioscceaetdi9uo0rne-ssd,aoyaustOloriuantleldiTnioenxditchiiintsytGhueSitdOuedlEyiCneiD,n
GuidanceDocumentNo.116ontheDesignandConductofChronicToxicityandCarcinogenicity
Studies(6).
i3n.halation.TheTthhereechmoaiicneroouftetsheofraodumtienisotfraatdimoinniussterdatiinonchrdoenpiecntdosxicointytshteudipehsysaircealoraal,nddercmhaemliacnald
cihnafroarcmtaetriisotnicosnocfhotihceetoefstrosuutbesotfanecxepoasnudrethisepprroevdiodmediniannttheroOuEteCDofGeuxipdoasnucreeDoofchuummeannts.NoA.dd1i1t6io(n6)a.l
4. ThisGuidelinefocusesonexposureviatheoralroute,theroutemostcommonlyusedin
chronictoxicitystudies.Whilelong-termchronictoxicitystudiesinvolvingexposureviathedermal
orinhalationroutesmayalsobenecessaryforhumanhealthriskassessmentand/ormayberequired
undercertainregulatoryregimes,bothroutesofexposureinvolveconsiderabletechnicalcomplexity.
Suchstudieswillneedtobedesignedonacase-by-casebasis,althoughtheGuidelineoutlinedherefor
theassessmentandevaluationofchronictoxicitybyoraladministrationcouldfonnthebasisofa
ptccslhrouieonbntssioutacclsaaostlnloeccdeafisnoiadrntbeytipdhnatehthaOhdelEoeailstCnioihogDgannylaaoGptnfuaidirlo/danoomanr(egn6tecd)ereer(rts7meD,)arolmaectnuscsd.ttmuuedddOeinieEretssmC,aoiDwlnnivrtoGaohlucuvtuirietdensesagpn(eiec6cn)xet.hpaotTilossaGutrariev4eoacn1ivo2limtaaem(bs8telt)heniednagoanintnd(hi7atTo)lh,nGaestsi4afhod1onomr3uirltno(driu9ets),ebat.tretamTotesginGpeotetnc4hpi1eefor0rifi(cwoa1tidle0tsls)h,yt
shouldbeconsultedinthecaseoftestingcarriedoutbythedermalroute.
f5r.omrepeTahteedcehxropnoiscurteoxoicvietryastcuodnyspidreorvaibdleesipanrftoromfattihoenloinfetshpeanpoosfsitbhleeshpeeaclitehshuaszeadr.dsThliekesltyutdoyawriilsle
provideinformationonthetoxiceffectsofthesubstance,indicatetargetorgansandthepossibilityof
©OECD,(2009)
Youarefreetousethismaterialforpersonal,non-commercialpurposeswithoutseekingpriorconsent
tforowmritttheenOpEeCrmDi,sspiroonvifdreodmtthheesOoEuCrDce.isdulymentioned.Anycommercialuseofthismaterialissubject
452 OECD/OCDE
accumulation.Itcanalsoprovideanestimateoftheno-observed-adverseeffectlevelwhichcanbe
usedforestablishingsafetycriteriaforhumanexposure.Theneedforcarefulclinicalobservationsof
theanimals,soastoobtainasmuchinformationaspossible,isalsostressed.
6. Theobjectivesofstudiescoveredbythistestguidelineinclude:
*Theidentificationofthechronictoxicityofachemical;
Theidentificationoftargetorgans;
*Characterisationofthedose-responserelationship;
Identificationofa no-observed-adverse-cffcctlevel(NOAEL)orpointofdeparturefor
establishmentofaBenchmarkDose(BMD);
Thepredictionofchronictoxicityeffectsathumanexposurelevels;
Provisionofdatatotesthypothesesregardingmodeofaction(6).
INITIALCONSIDERATIONS
7. Intheassessmentandevaluationofthetoxicologicalcharacteristicsofachemical,all
availableinformationonthetestsubstanceshouldbeconsideredbythetestinglaboratorypriorto
conductingthestudy,inordertofocusthedesignofthestudytomoreefficientlytestforchronic
toxicitypotentialandtominimizeanimalusage.Informationthatwillassistinthestudydesign
includestheidentity,chemicalstructure,andphysico-chemicalpropertiesofthetestsubstance;any
informationonthemodeofaction;resultsofanyinvitroorinvivotoxicitytests;anticipateduse(s)
andpotentialforhumanexposure;available(Q)SARdataandtoxicologicaldataonstructurally-
relatedsubstances;availabletoxicokineticdata(singledoseandalsorepeatdosekineticswhere
available)anddataderivedfromotherrepeatedexposurestudies.Thedeterminationofchronic
tdooxsieci2t8y-sdhaoyuladndo/nolry9b0e-dcaaryrtioexdiocuittyatfetsetrs.iniAtiaplhiansfeodrmtaetsitionngoanpptorxoiaccihtythoacshbroeneincotbotxaiicnietydtfersotimngrespheoautledd
beconsideredaspartoftheoverallassessmentofthepotentialadversehealtheffectsofaparticular
chemical(11)(12)(13)(14).
8. Thestatisticalmethodsmostappropriatefortheanalysisofresults,giventheexperimental
designandobjectives,shouldbeestablishedbeforecommencingthestudy.Issuestoconsiderinclude
whetherthestatisticsshouldincludeadjustmentforsurvivalandanalysisintheeventofpremature
terminationofoneormoregroups.Guidanceontheappropriatestatisticalanalysesandkeyreferences
tioniGnutiedranantcieonDalolcyuamcecnetptNeod.s3t5atiostnictahlemaentahloydsissaarnedgievveanluiantiGounidoafnccheroDnoiccutmoxeinctitNyoa.nd11c6ar(c6i)n,oagenndicailtsyo
studies(15).
t9.heOECDInGcuoinddaunccteinDgoacuchmreonnticNtoo.xic1i9tyonstutdhye,rtehceoggnuiitdiionng,parsisnecsispmleensta,nadncdonussiedeorfatciloinnsicaolutsliignnesdaisn
humaneendpointsforexperimentalanimalsusedinsafetyevaluation(16),inparticularparagraph62
thereof,shouldalwaysbefollowed.Thisparagraphstatesthat“/nstudiesinvolvingrepeateddosing,
whenananimalshowsclinicalsignsthatareprogressive,leadingtofurtherdeteriorationin
condition,aninformeddecisionastowhetherornottohumanelykilltheanimalshouldbemade.The
decisionshouldincludeconsiderationastothevalueoftheinformationtobegainedfromthe
continuedmaintenanceofthatanimalonstudyrelativetoitsoverallcondition.Ifadecisionismadeto
leavetheanimalontest,thefrequencyofobservationsshouldbeincreased,asneeded.Itmayalsobe
ptohsespiablien,owridtihsoturtesas,dvoerrsreeldyuacfefetchteitnegstthdeospeu.r”poseofthetest,totemporarilystopdosingifitwillrelieve
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OECD/OCDE 452
10. Detailedguidanceonanddiscussionoftheprinciplesofdoseselectionforchronictoxicity
aInntderncaatricoinnaolgeLniifceityScisteundcieessIcnastnitbuteefpouubnlidcaitnioGnsuid(1a7n)ce(18D)o.cumTehnetcNoroe.d1o1s6e(s6e)l,ecatsionwelstlraatsegytwios
dependentontheprimaryobjectiveorobjectivesofthestudy(paragraph6).Inselectingappropriate
doselevels,abalanceshouldbeachievedbetweenhazardscreeningontheonehandand
characterisationoflow-doseresponsesandtheirrelevanceontheother.Thisisparticularlyrelevantin
thesituationwhereacombinedchronictoxicityandcarcinogenicitystudy(TG453)istobecarried
out(paragraph11).
11. Consideration should be given to carrying out a combined chronic toxicity and
carcinogenicitystudy(TG453),ratherthanseparateexecutionofachronictoxicitystudy(TG452)
andcarcinogenicitystudy(TG451).Thecombinedtestprovidesgreaterefficiencyintermsoftime
andcostcomparedtoconductingtwoseparatestudies,withoutcompromisingthequalityofthedatain
eitherthechronicphaseorthecarcinogenicityphase.Carefulconsiderationshouldhoweverbegiven
totheprinciplesofdoseselection(paragraphs9and20-25)whenundertakingacombinedchronic
toxicityandcarcinogenicitystudy(TG453),anditisalsorecognisedthatseparatestudiesmaybe
requiredundercertainregulatory'frameworks.
12. DefinitionsusedinthecontextofthisTestGuidelinecanbefoundinGuidanceDocument
No.116.
PRINCIPLEOFTHETEST
13. Thetestsubstanceisadministereddailyingraduateddosestoseveralgroupsofexperimental
animals,normallyforaperiodof12months,althoughlongerorshorterdurationsmayalsobechosen
dependingonregulatory'requirements(seeparagraph33).Thisdurationischosentobesufficiently
longtoallowanyeffectsofcumulativetoxicitytobecomemanifest,withouttheconfoundingeffects
ofgeriatricchanges.Deviationsfromexposuredurationof12monthsshouldbejustified,particularly
inthecaseofshorterdurations.Thetestsubstanceisnormallyadministeredbytheoralroutealthough
testingbytheinhalationordermalroutemayalsobeappropriate.Thestudydesignmayalsoinclude
oneormoreinterimkills,e.g.at3and6months,andadditionalgroupsofanimalsmaybeincludedto
accommodatethis(seeparagraph19).Duringtheperiodofadministrationtheanimalsareobserved
closelyforsignsoftoxicity.Animalswhichdieorarekilledduringthetestarenecropsiedand,atthe
conclusionofthetest,survivinganimalsarekilledandnecropsied.
DESCRIPTIONOFTHEMETHOD
Selectionofanimalspecies
14. ThisGuidelineprimarilycoversassessmentandevaluationofchronictoxicityinrodents(see
paragraph2)althoughitisrecognisedthatsimilarstudiesinnon-rodentsmayberequiredunder
certainregulatoryregimes.Thechoiceofspeciesshouldbejustified.Thedesignandconductof
chronictoxicitystudiesinnon-rodentspecies,whenrequired,shouldbebasedontheprinciples
outlinedinthisGuidelinetogetherwiththoseinOECDTG409,RepeatedDose90-dayOralToxicity
Study7inNon-Rodents(5).Additionalinformationonchoiceofspeciesandstrainisprovidedin
GuidanceDocumentNo.116(6).
15. InthisGuideline,thepreferredrodentspeciesistherat.althoughotherrodentspecies,e.g.
themouse,maybeused.Ratsandmicehavebeenpreferredexperimentalmodelsbecauseoftheir
relativelyshortlifespan,theirwidespreaduseinpharmacologicalandtoxicologicalstudies,their
susceptibilitytotumourinduction,andtheavailabilityofsufficientlycharacterisedstrains. Asa
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452 OECD/OCDE
consequenceofthesecharacteristics,alargeamountofinformationisavailableontheirphysiology
andpathology.Younghealthyadultanimalsofcommonlyusedlaboratorystrainsshouldbeemployed.
Thechronictoxicitystudyshouldbecarriedoutinanimalsfromthesamestrainandsourceasthose
usedinpreliminarytoxicitystudy(ies)ofshorterduration.Thefemalesshouldbenulliparousandnon-
pregnant.
Housingandfeedingconditions
16. Animalsmaybehousedindividually,orbecagedinsmallgroupsofthesamesex;individual
housingshouldbeconsideredonlyifscientificallyjustified(19)(20)(21).Cagesshouldbearranged
insuchawaythatpossibleeffectsduetocageplacementareminimised.Thetemperatureinthe
e3x0p%eriamnedntparlefaenriamballyrnootomexscheoeudld7b0e%2o2t°hCer(±th3a°nC)d.urAilntghoruogohmthceleraenliantgi,vethheumaiidimtyshsohuoludldbebe50at-6l0ea%s.t
Lightingshouldbeartificial,thesequencebeing12hourslight,12hoursdark.Forfeeding,
conventionallaboratorydietsmaybeusedwithanunlimitedsupplyofdrinkingwater.Thedietshould
meetallthenutritionalrequirementsofthespeciestestedandthecontentofdietarycontaminants
includingbutnotlimitedtopesticideresidues,persistentorganicpollutants,phytocstrogcns,heavy
metalsandmycotoxins,thatmightinfluencetheoutcomeofthetest,shouldbeaslowaspossible.
Analytical information onthenutrientanddietarycontaminant levels should be generated
periodically,atleastatthebeginningofthestudyandwhenthereisachangeinthebatchused,and
shouldbeincludedinthefinalreport.Analyticalinfomiationonthedrinkingwaterusedinthestudy
shouldsimilarlybeprovided.Thechoiceofdietmaybeinfluencedbytheneedtoensureasuitable
admixtureofatestsubstanceandtomeetthenutritionalrequirementsoftheanimalswhenthetest
substanceisadministeredbythedietaryroute.
Preparationofanimats
17. Healthyanimals,whichhavebeenacclimatedtolaboratoryconditionsforatleast7daysand
havenotbeensubjectedtopreviousexperimentalprocedures,shouldbeused.Inthecaseofrodents,
dosingoftheanimalsshouldbeginassoonaspossibleafterweaningandacclimatisationand
preferablybeforetheanimalsare8weeksold.Thetestanimalsshouldbecharacterisedastospecies,
ssterxaion,fasonuirmcael,ssuesxe,dwesihgohutldabnedmaigen.imAatltahendcnoomtmeexncceeemde±nt20of%thoefstthuedym,etahnewweeiigghhttvoafrialaltitohnefaonriemaaclhs
withinthestudy,separatelyforeachsex.Animalsshouldberandomlyassignedtothecontroland
treatmentgroups.Afterrandomisation,thereshouldbenosignificantdifferencesinmeanbody
weightsbetweengroupswithineachsex. Iftherearestatisticallysignificantdifferences,thenthe
randomisationstepshouldberepeated,ifpossible.Eachanimalshouldbeassignedaunique
identificationnumber,andpermanentlymarkedwiththisnumberbytattooing,microchipimplant,or
othersuitablemethod.
PROCEDURE
Numberandsexofanimals
18. Bothsexesshouldbeused.Asufficientnumberofanimalsshouldbeusedsothatattheend
ofthestudyenoughanimalsineverygroupareavailableforthoroughbiologicalandstatistical
evaluation.Forrodents,atleast20animalspersexpergroupshouldnormallybeusedateachdose
level,whilefornon-rodentsaminimumof4persexpergroupisrecommended.Instudiesinvolving
mice,additionalanimalsmaybeneededineachdosegrouptoconductallrequiredhaematological
determinations.
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Provisionforinterimkills,satellitegroupsandsentinelanimals
19. Thestudymaymakeprovisionforinterimkills(atleast10animals/sex/group),e.g.at6
months,toprovideinformationonprogressionoftoxicologicalchangesandmechanisticinformation,
ifscientificallyjustified.Wheresuchinfomiationisalreadyavailablefrompreviousrepeatdose
toxicitystudiesonthesubstance,interimkillsmaynotbescientificallyjustified.Satellitegroupsmay
alsobeincludedtomonitorthereversibilityofanytoxicologicalchangesinducedbythechemical
underinvestigation;thesewillnormallyberestrictedtothehighestdoselevelofthestudyplus
control.Anadditionalgroupofsentinelanimals(typically5animalspersex)mayalsobeincludedfor
monitoringofdiseasestatus,ifnecessary,duringthestudy(22).Ifinterimkillsorinclusionofsatellite
orsentinelgroupsareplanned,thenumberofanimalsincludedinthestudydesignshouldbeincreased
bythenumberofanimalsscheduledtobekilledbeforethecompletionofthestudy. Theseanimals
shouldnormallyundergothesameobservations,including bodyweight,food/waterconsumption,
haematologicalandclinicalbiochemistrymeasurementsandpathologicalinvestigationsastheanimals
inthechronictoxicityphaseofthemainstudy,althoughprovisionmayalsobemade(intheinterim
killgroups)formeasurementstoberestrictedtospecific,keymeasuressuchasneurotoxicityor
immunotoxicity.
Dosegroupsanddosage
20. GuidanceonallaspectsofdoseselectionanddoselevelspacingisprovidedinGuidance
DocumentNo.116(6).Atleastthreedoselevelsandaconcurrentcontrolshouldbeused,except
wherealimittestisconducted(seeparagraph27).Doselevelswillgenerallybebasedontheresultsof
shorter-termrepeateddoseorrangefindingstudiesandshouldtakeintoaccountanyexisting
toxicologicalandtoxicokineticdataavailableforthetestsubstanceorrelatedmaterials.
h2i1g.hestdoUsnelelsesvellismihtoeudldbynotrhmeaplhlyysibcealc-hcohscemnictaolindaetnutriefyotrhbeioplroignicciaplaleftfaercgtestoofrgtahensteasntdsutbosxtiacnceef,fetchtes
whileavoidingsuffering,severetoxicity,morbidity,ordeath.Whiletakingintoaccountthefactors
outlinedinparagraph22below,thehighestdoselevelshouldbechosentoelicitevidenceoftoxicity,
asevidencedby,forexample,depressionofbodyweightgain(approximately10%).
t2h2e.doseHporowveivdeirn,gdeevpiednednceentoofntotxhieciotbyjemctaiyvesbeofchthoesesnt,udey.g.(,seiefpaardaogsreapehli6c)i,tsaatnopaddvoesreseloewfefrectthaonf
concernthatnonethelesshaslittleimpactonlifespanorbodyweight.Thetopdoseshouldnotexceed
1000mg/kgbodyweight/day(limitdose,seeparagraph27).
N23O.AELoDrosoethelrevienltsenadnedddooustecolmeevelofstphaecisntgudmy,aye.gbeasBelMeDcte(dseteopeasrtaabglriasphha25d)osaet-trheesploonwseestanddosea
level.Factorsthatshouldbeconsideredintheplacementoflowerdosesincludetheexpectedslopeof
thedose-responsecurve,thedosesatwhichimportantchangesmayoccurinmetabolismormodeof
toxicaction,whereathresholdisexpected,orwhereapointofdepartureforlow-doseextrapolationis
expected.
24. Thedoselevelspacingselectedwilldependonthecharacteristicsofthetestsubstance,and
cannotbeprescribedinthisGuideline,buttwotofourfoldintervalsfrequentlyprovidegoodtest
performancewhenusedforsettingthedescendingdoselevelsandadditionofafourthtestgroupis
oftenpreferabletousingverylargeintervals(e.g.,morethanafactorofabout6-10)betweendosages.
Ingeneraltheuseoffactorsgreaterthan10shouldbeavoided,andshouldbejustifiedifused.
5 ©OCDE,(2009)
452 OECD/OCDE
25.
AsoutlinedfurtherinGuidanceDocumentNo.116(6),pointstobeconsideredindose
selectioninclude:
* Knownorsuspectednonlinearitiesorinflectionpointsinthedose-response;
* Toxicokinetics,anddoserangeswheremetabolicinduction,saturation,ornonlinearity
betweenexternalandinternaldosesdoesordoesnotoccur;
Precursorlesions,markersofeffect,orindicatorsoftheoperationofkeyunderlying
biologicalprocesses;
• Key(orsuspected)aspectsofmodeofaction,suchasdosesatwhichcytotoxicity
begins to arise, hormone levels are perturbed, homeostatic mechanisms are
overwhelmed,etc.;
• Re.egg.,ioinnsthoefrtahnegedoosfet-hreeasnptoincsiepatceudrvBeMwDheorreapsaurtsipceucltaerdlythrroebsuhsotlde;stimationisneeded,
• Considerationofanticipatedhumanexposurelevels.
26. Thecontrolgroupshallbeanuntreatedgrouporavehicle-controlgroupifavehicleisused
inadministeringthetestsubstance.Exceptfortreatmentwiththetestsubstance,animalsinthecontrol
groupshouldbehandledinanidenticalmannertothoseinthetestgroups.Ifavehicleisused,the
controlgroupshallreceivethevehicleinthehighestvolumeusedamongthedosegroups.Ifatest
substanceisadministeredinthediet,andcausessignificantlyreduceddietaryintakeduetothe
reducedpalatabilityofthediet,anadditionalpair-fedcontrolgroupmaybeuseful,toserveasamore
suitablecontrol.
27. Ifitcanbeanticipated,basedoninformationfrompreliminarystudies,thatatestatonedose
level,equivalenttoatleast1000mg/kgbodyweight/day,usingtheproceduresdescribedforthis
study,isunlikelytoproduceadverseeffectsandiftoxicitywouldnotbeexpectedbasedupondata
nfercoemsssatrryu.ctAurallilmyitreolfat1e0d00sumbsgt/akngcesb,odtyhewneiaghfutl/ldasytumdyayusaipnpglytherxeecedpotswehleenvehlsummaanyenxoptosbuerecoinnsdiidcearteeds
theneedforahigherdoseleveltobeused.
Preparationofclosesandadministrationoftestsubstance
28. Thetestsubstanceisnormallyadministeredorally,viathedietordrinkingwater,orby
gavage. AdditionalinformationonroutesandmethodsofadministrationisprovidedinGuidance
DocumentNo.116(6).Therouteandmethodofadministrationisdependentonthepurposeofthe
rsotuudtye,atnhdempehytshiocdalo/fcheexmpiocsaulreproopferhtuimeasnosf.tAhertaetsitonsaulbestsahnocue,lditbsebiporaovvaiildaebdilfiotrytahnedcthhoesepnrerdooumtienaanndt
methodofadministration,hitheinterestsofanimalwelfare,oralgavageshouldnormallybeselected
onlyforthoseagentsforwhichthisrouteandmethodofadministrationreasonablyrepresentpotential
humanexposure(e.g.,pharmaceuticals).Fordietaryorenvironmentalchemicalsincludingpesticides,
administrationistypicallyviathedietordrinkingwater.However,forsomescenarios,e.g.,
occupationalexposure,administrationviaotherroutesmaybemoreappropriate
29. Wherenecessary,thetestsubstanceisdissolvedorsuspendedinasuitablevehicle.
Considerationshouldbegiventothefollowingcharacteristicsofthevehicleandotheradditives,as
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appropriate:effectsontheabsorption,distribution,metabolism,orretentionofthetestsubstance;
effectsonthechemicalpropertiesofthetestsubstancewhichmayalteritstoxiccharacteristics;and
effectsonthefoodorwaterconsumptionorthenutritionalstatusoftheanimals.Itisrecommended
that,whereverpossible,theuseofanaqueoussolution/suspensionbeconsideredfirst,followedby
considerationofasolution/emulsioninoil(e.g.,comoil)andthenbypossiblesolutioninother
vehicles.Forvehiclesotherthanwater,thetoxiccharacteristicsofthevehicleshouldbeknown.
Informationshouldbeavailableonthestabilityofthetestsubstanceandthehomogeneityofdosing
solutionsordiets(asappropriate)undertheconditionsofadministration(e.g.diet).
30. Forsubstancesadministeredviathedietordrinkingwateritisimportanttoensurethatthe
quantitiesofthetestsubstanceinvolveddonotinterferewithnormalnutritionorwaterbalance.In
long-termtoxicitystudiesusingdietaryadministration,theconcentrationofthechemicalinthefeed
shouldnotnormallyexceedanupperlimitof5%ofthetotaldiet,inordertoavoidnutritional
imbalances.Whenthetestsubstanceisadministeredinthediet,eitheraconstantdietaryconcentration
(mg/kgdietorppm)oraconstantdoselevelintermsoftheanimal’sbodyweight(mg/kgbody
weight),calculatedonaweeklybasis,maybeused.Thealternativeusedshouldbespecified.
31. Inthecaseoforaladministration,theanimalsaredosedwiththetestsubstancedaily(seven
dayseachweek),normallyforaperiodof12months(seealsoparagraph33),althoughalonger
durationmayberequireddependingonregulatoryrequirements.Anyotherdosingregime,e.g.,five
dwiatyhstpheertweesetks,ubnseteadnscetofobreajtuslteiafsited6,hhoiutrhsepcearsedaoyf,d7erdmaaylsapdemriwniesetkr,ataisons,peacniifmiaeldsinarTeGno4r1m0al(l1y1)t,refaotread
periodof12months.Exposurebytheinhalationrouteiscarriedoutfor6hoursperday,7daysper
week,butexposurefor5daysperweekmayalsobeused,ifjustified.Theperiodofexposurewill
mnoarxmialmluymbeexpfoorsuarepedruiroadtioofns1m2amyonbtehs.adjIufsrtoeddenttosmpienciimeisseothseprectihesa-nspreactisfiacrediesxtrpeosss.edAnorsaet-ioonnlayl,e
shouldbeprovidedwhenusinganexposuredurationoflessthan6hoursperday.SeealsoTG412(8).
32. Whenthetestsubstanceisadministeredbygavagetotheanimalsthisshouldbedoneusinga
stomachtubeorasuitableintubationcannula,atsimilartimeseachday.Normallyasingledosewill
bmeaiandtmaiinnitshteerdeadiloyndcoesed-arialtye,bwyheardeminfiorsteexrianmgpliteasaacospmlpitoduonsdei(stwaicleocaaldaiyr)ri.taTnht,eimtamxaiymbuempvoossliubmleeotof
liquidthatcanbeadministeredatonetimedependsonthesizeofthetestanimal.Thevolumeshould
bekeptaslowaspractical,andshouldnotnormallyexceed1ml/lOOgbody'weightforrodents(23).
Variabilityintestvolumeshouldbeminimisedbyadjustingtheconcentrationtoensureaconstant
volumeatalldoselevels.Potentiallycorrosiveorirritantsubstancesaretheexception,andneedtobe
dilutedtoavoidseverelocaleffects.Testingatconcentrationsthatarelikelytobecorrosiveorirritant
tothegastrointestinaltractshouldbeavoided.
Durationofstudy
33. WhilethisTestGuidelineprimarilyisdesignedasa12monthchronictoxicitystudy,the
studydesignalsoallowsforandcanbeappliedtoeithershorter(e.g.6or9months)orlonger(e.g.18
or24months)durationstudies,dependingontherequirementsofparticularregulatoryregimesorfor
specificmechanisticpurposes. Deviationsfromanexposuredurationof12monthsshouldbe
justified,particularlyinthecaseofshorterdurations.Satellitegroupsincludedtomonitorthe
reversibilityofanytoxicologicalchangesinducedbythechemicalunderinvestigationshouldbe
maintainedwithoutdosingforaperiodnotlessthan4weeksandnotmorethanonethirdofthetotal
studydurationaftercessationofexposure.Furtherguidance,includingconsiderationofsurvivalinthe
study,isprovidedinGuidanceDocumentNo.116(6).
7 ©OCDE,(2009)
452 OECD/OCDE
OBSERVATIONS
34. Allanimalsshouldbecheckedformorbidityormortality,usuallyatthebeginningandend
ofeachday,includingatweekendsandholidays.Generalclinicalobservationsshouldbemadeatleast
onceaday,preferablyatthesametime(s)eachday,takingintoconsiderationthepeakperiodof
anticipatedeffectsafterdosinginthecaseofgavageadministration.
35. Detailedclinicalobservationsshouldbemadeonallanimalsatleastoncepriortothefirst
exposure(toallowforwithin-subjectcomparisons),attheendofthefirstweekofthestudyand
monthlythereafter.Theprotocolforobservationsshouldbearrangedsuchthatvariationsbetween
individualobserversareminimisedandindependentoftestgroup.Theseobservationsshouldbemade
outsidethehomecage,preferablyinastandardarenaandatsimilartimesoneachoccasion.They
shouldbecarefullyrecorded,preferablyusingscoringsystems,explicitlydefinedbythetesting
laboratory.Effortsshouldbemadetoensurethatvariationsintheobservationconditionsareminimal.
Signsnotedshouldinclude,butnotbelimitedto,changesinskin,fur,eyes,mucousmembranes,
occurrenceofsecretionsandexcretionsandautonomicactivity(e.g.,lacrimation,piloerection,pupil
size,andunusualrespiratorypattern).Changesingait,postureandresponsetohandlingaswellasthe
presenceofclonicortonicmovements,stereotypies(e.g.,excessivegrooming,repetitivecircling)or
bizarrebehaviour(e.g.,self-mutilation,walkingbackwards)shouldalsoberecorded(24).
36. Ophthalmologicalexamination,usinganophthalmoscopeorothersuitableequipment,
shouldbecarriedoutonallanimalspriortothefirstadministrationofthetestsubstance. Atthe
terminationofthestudy,thisexaminationshouldbepreferablyconductedinallanimalsbutatleastin
thehighdoseandcontrolgroups. Iftreatment-relatedchangesintheeyesaredetected,allanimals
shouldbeexamined. Ifstructuralanalysisorotherinformationsuggestsoculartoxicity,thenthe
frequencyofocularexaminationshouldbeincreased.
37. Forchemicalswherepreviousrepeateddose28-dayand/or90-daytoxicitytestsindicatedthe
potentialtocauseneurotoxiceffects,sensoryreactivitytostimuliofdifferenttypes(24)(e.g.,
auditory,visualandproprioceptivestimuli)(25),(26),(27),assessmentofgripstrength(28)andmotor
activityassessment(29)mayoptionallybeconductedbeforecommencementofthestudyandat3
monthperiodsafterstudyinitiationuptoandincluding12months,aswellasatstudytennination(if
longerthan12months).Furtherdetailsoftheproceduresthatcouldbefollowedaregiveninthe
respectivereferences.However,alternativeproceduresthanthosereferencedcouldalsobeused.
38. Forchemicalswherepreviousrepeateddose28-dayand/or90-daytoxicitytestsindicatedthe
potentialtocauseimmunotoxiceffects,furtherinvestigationsofthisendpointmayoptionallybe
conductedattermination.
Bodyweight,food/waterconsumptionandfoodefficiency
39. Allanimalsshouldbeweighedatthestartoftreatment,atleastonceaweekforthefirst13
weeks,andatleastmonthlythereafter. Measurementsoffoodconsumptionandfoodefficiency
shouldbemadeatleastweeklyforthefirst13weeksandatleastmonthlythereafter.Water
consumptionshouldbemeasuredatleastweeklyforthefirst13weeksandatleastmonthlythereafter
whenthesubstanceisadministeredindrinkingwater.Waterconsumptionmeasurementsshouldalso
beconsideredforstudiesinwhichdrinkingactivityisaltered.
©OCDE,(2009) 8
OECD/OCDE 452
Haematologyandclinicalbiochemistry
40. Instudiesinvolvingrodents,haematologicalexaminationsshouldbecarriedoutinatleast10
mloanlgeeranthdan1012femmoanltehsa)n,imusailnsgptehregsraomuep,anatim3a,ls6,tharnodug1h2oumt,onhtrhsm,icaes,wseatlellliatseaatnismtauldsymtearymibneatnieoende(idf
inordertoconductallrequiredhaematologicaldeterminations(seeparagraph18).Innon-rodent
studies,sampleswillbetakenfromsmallernumbersofanimals(e.g.4animalspersexandpergroup
indogstudies),atinterimsamplingtimesandatterminationasdescribedforrodents.Measurementsat
3months,eitherinrodentsornon-rodents,neednotbeconductedifnoeffectwasseenon
haematologicalparametersinaprevious90daystudycarriedoutatcomparabledoselevels.Blood
samplesshouldbetakenfromanamedsite,forexamplebycardiacpunctureorfromtheretro-orbital
sinus,underanaesthesia.
41. Thefollowinglistofparametersshouldbeinvestigated(30): Totalanddifferential
leukocytecount,erythrocytecount,plateletcount,haemoglobinconcentration,haematocrit(packed
cellvolume),meancorpuscularvolume(MCV).meancorpuscularhaemoglobin(MCH), mean
corpuscular haemoglobin concentration (MCHC), prothrombin time, and activated partial
thromboplastintime.OtherhematologyparameterssuchasHeinzbodiesorotheratypicalerythrocyte
morphologyormethaemoglobinmaybemeasuredasappropriatedependingonthetoxicityofthe
substance.Overall,aflexibleapproachshouldbeadopted,dependingontheobservedand/orexpected
effectfromagivensubstance.Ifthechemicalhasaneffectonthehaematopoieticsystem,reticulocyte
countsandbonemarrowcytologymayalsobeindicated,althoughtheseneednotberoutinely
conducted.
42. Clinicalbiochemistrydeterminationstoinvestigatemajortoxiceffectsintissuesand.
specifically,effectsonkidneyandliver,shouldbeperformedonbloodsamplesobtainedfromatleast
10maleand10femaleanimalspergroupatthesametimeintervalsasspecifiedforthehaematological
investigations,usingthesameanimalsthroughout.Inmice,satelliteanimalsmaybeneededinorderto
conductallrequiredclinicalbiochemistrydeterminations. Innon-rodentstudies,sampleswillbe
takenfromsmallernumbersofanimals(e.g.4animalspersexandpergroupindogstudies),at
interimsamplingtimesandatterminationasdescribedforrodents.Measurementsat3months,either
inrodentsornon-rodents,neednotbeconductedifnoeffectwasseenonclinicalbiochemistry
parametersinaprevious90daystudycarriedoutatcomparabledoselevels.Overnightfastingofthe
paanriammaeltse(rwsitshhothueldexbceeptiinovensotifgmaitecde)(p3r0)i:ortoglbulcoosoed,saumrpelain(guriesarneictoromgmeen)n,decdre1a.tTihnienef,oltlootwailngprloitsetion,f
albumin,calcium,sodium,potassium,totalcholesterol,atleasttwoappropriatetestsforhepatocellular
evaluation(alanineaminotransferase,aspartateaminotransferase,glutamatedehydrogenase,totalbile
acids)(31),andatleasttwoappropriatetestsforhepatobiliaryevaluation(alkalinephosphatase,
gammaglutamyltransferase,5'-nucleotidase.totalbilirubin,totalbileacids)(31). Otherclinical
chemistryparameterssuchasfastingtriglycerides,specifichormonesandcholinesterasemaybe
measuredasappropriate,dependingonthetoxicityofthesubstance. Overall,thereisaneedfora
flexibleapproach,dependingontheobservedand/orexpectedeffectfromagivensubstance.
43. Urinalysisdeterminationsshouldbeperformedonatleast10maleand10femaleanimals
pergrouponsamplescollectedatthesameintervalsasforhaematologyandclinicalchemistry.
Measurementsat3monthsneednotbeconductedifnoeffectwasseenonurinalysisinaprevious90
daystudycarriedoutatcomparabledoselevels.Thefollowinglistofparameterswasincludedinan
expertrecommendationonclinicalpathologystudies(30):appearance,volume,osmolalityorspecific
gravity,pH,totalprotein,andglucose. Otherdeterminationsincludeketone,urobilinogen,bilirubin,
andoccultblood.Furtherparametersmaybeemployedwherenecessarytoextendtheinvestigationof
observedeffect(s).
9 ©OCDE,(2009)
452 OECD/OCDE
44. Itisgenerallyconsideredthatbaselinehaematologicalandclinicalbiochemistryvariables
areneededbeforetreatmentfordogstudies,butneednotbedeterminedinrodentstudies(30).
However,ifhistoricalbaselinedata(seeparagraph50)areinadequate,considerationshouldbegiven
togeneratingsuchdata.
Pathology
Grossnecropsy
45. Allanimalsinthestudyshallnormallybesubjectedtoafull,detailedgrossnecropsywhich
includescarefulexaminationoftheexternalsurfaceofthebody,allorifices,andthecranial,thoracic
andabdominalcavitiesandtheircontents.Howeverprovisionmayalsobemade(intheinterimkillor
satellitegroups)formeasurementstoberestrictedtospecific,keymeasuressuchasneurotoxicityor
immunotoxicity(seeparagraph19).Theseanimalsneednotbesubjectedtonecropsyandthe
subsequentproceduresdescribedinthefollowingparagraphs.Sentinelanimalsmayrequirenecropsy
onacase-by-casebasis,atthediscretionofthestudydirector.
46. Organweightsshouldbecollectedfromallanimals,otherthanthoseexcludedbythelatter
partofparagraph45.Theadrenals,brain,epididymides,heart,kidneys,liver,ovaries,spleen,testes,
thyroid(weighedpost-fixation,withparathyroids),anduterusofallanimals(apartfromthosefound
moribundand/orintercurrentlykilled)shouldbetrimmedofanyadherenttissue,asappropriate,and
theirwetweighttakenassoonaspossibleafterdissectiontopreventdrying.Inastudyusingmice,
weighingoftheadrenalglandsisoptional.
47. Thefollowingtissuesshouldbepreservedinthemostappropriatefixationmediumforboth
thetypeoftissueandtheintendedsubsequenthistopathologicalexamination(32)(tissuesinsquare
bracketsareoptional):
©OCDE,(2009) 10