Test No. 421: Reproduction/Developmental Toxicity Screening Test [electronic resource]

2015·0.46 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 421
Adopted:
28July2015
OECDGUIDELINEFORTESTINGOFCHEMICALS
Reproduction/DevelopmentalToxicityScreeningTest
INTRODUCTION
1. OECDGuidelinesfortheTestingofChemicalsareperiodicallyreviewedinthelightofscientific
progress.TheoriginalscreeningTestGuideline421wasadoptedin1995,basedonaprotocolfora
"PreliminaryReproductionToxicityScreeningTest"discussedintwoexpertmeetings,inLondonin1990
(1)andinTokyoin1992(2).
hG2.iugihd-eplriinoTerhsiitsyfoaTrctGtihveihtaysscirnbeieetenianitnegudpaadtnadtOeEtdeCswtDiitnighnoe1fn9d9po8octrteiontnrieeavldiisesenrdeuoxpictsortriinnregelTdeievssartnutGptueoinrddseplo(i3in)ne.tssT,aGnads4t0ao7dfeo(vlRelelopowepautnpeedwtoDToetsshete
28-DayOralToxicityStudyinRodents)forexample,wasenhancedin2008byparameterssuitableto
detectendocrineactivityoftestchemicals.TheobjectiveinupdatingTG421wastoincludesome
endocrinedisruptorrelevantendpointsinscreeningTGswheretheexposureperiodscoversomeofthe
sensitiveperiodsduringdevelopment(pre-orearlypostnatalperiods).
G3.eneratiTohneRseeplreocdtuecdtaidvdeitTiooxniaclietnydSotcurdiyn)e,dwiesrreupitenrclruedleedvainntTeGndp4o2i1ntbsa,saeldsoonpaartfeoafsiTbiGli4ty43st(uEdxytaednddreedssOinneg
scientificandtechnicalquestionsrelatedtotheirinclusion,aswellaspossibleadaptationsofthetestdesign
neededfortheirinclusion(4).
4. ThisGuidelineisdesignedtogeneratelimitedinformationconcerningtheeffectsofatestchemical
onmaleandfemalereproductiveperformancesuchasgonadalfunction,matingbehaviour,conception,
developmentoftheconceptusandparturition.Itisnotanalternativeto,nordoesitreplacetheexistingTest
Guidelines414,415,416or443.
INITIALCONSIDERATIONS
5. ThisScreeningTestGuidelinecanbeusedtoprovideinitialinformationonpossibleeffectson
reproductionand/ordevelopment,eitheratanearlystageofassessingthetoxicologicalpropertiesof
chemicals,oronchemicalsofconcern.Itcanalsobeusedaspartofasetofinitialscreeningtestsforexisting
chemicalsforwhichlittleornotoxicologicalinformationisavailable,asadoserangefindingstudyformore
esxtutdeyn,sitvheergeupirdoidnugctpiroinn/cidpelveesloapnmdenctoanlsisdteurdaietsi,onosrowuthleinnedotihnertwhiesOeEcConDsidGeurieddanrceleevDaontc.umIennctonnd°uc1t9inogntthhee
recognition,assessment,anduseofclinicalsignsashumaneendpointsforexperimentalanimalsusedin
safetyevaluations(5)shouldbefollowed.
6. Thistestdoesnotprovidecompleteinformationonallaspectsofreproductionanddevelopment.In
particular,itoffersonlylimitedmeansofdetectingpost-natalmanifestationsofpre-natalexposure,oreffects
thatmaybeinducedduringpost-natalexposure.Due(amongstotherreasons)totherelativelysmallnumbers
ofanimalsinthedosegroups,theselectivityoftheendpoints,andtheshortdurationofthestudy,this
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Youarefreetousethismaterialforpersonal,non-commercialpurposeswithoutseekingpriorconsent
fromtheOECD,providedthesourceisdulymentioned.Anycommercialuseofthismaterialissubjectto
writtenpermissionfromtheOECD.
421 OECD/OCDE
methodwillnotprovideevidencefordefiniteclaimsofnoeffects.Moreover,intheabsenceofdatafrom
otherreproduction/developmentaltoxicitytests,positiveresultsareusefulforinitialhazardassessmentand
contributetodecisionswithrespecttothenecessityandtimingofadditionaltesting.
7. Theresultsobtainedbytheendocrinerelatedparametersshouldbeseeninthecontextofthe“OECD
CCoonncceeppttuuaallFFrraammeewwoorrk,ktfhoereTnehsatnicnegdaTnGd4A2s1seissscmoennttainoefdEinndloecvreiln4eaDsiasrnuipntivnigvoCahsesmaiycaprlosv”id(i6n)g.dIantathoins
adverseeffectsonendocrinerelevantendpoints.Anendocrinesignalmightnothoweverbeconsidered
sufficientevidenceonitsownthatthetestchemicalisanendocrinedisruptor.
8. ThisGuidelineassumesoraladministrationofthetestchemical.Modificationsmayberequiredif
otherroutesofexposureareused.
9. BeforeuseoftheTestGuidelineonamixtureforgeneratingdataforanintendedregulatory
purpose,itshouldbeconsideredwhether,andifsowhy,itmayprovideadequateresultsforthatpurpose.
Suchconsiderationsarenotneeded,whenthereisaregulatoryrequirementfortestingofthemixture.
10. DefinitionsusedaregiveninAnnex1.
PRINCIPLEOFTHETEST
s1h1o.uldbeTdhoesetedstfocrheamimcianliimsuamdmionfifsotuerrewdeienkgsraadnudatuepdtdoosaensdtoinscelvuedrianlggtrhoeupdsayofbmeafolreesascnhdefdeumlaeldesk.illMa(ltheiss
includesaminimumoftwoweekspriortomating,duringthematingperiodand,approximately,twoweeks
post-mating).Inviewofthelimitedpre-matingdosingperiodinmales,fertilitymaynotbeaparticular
sensitiveindicatoroftesticulartoxicity.Therefore,adetailedhistologicalexaminationofthetestesis
essential.Thecombinationofapre-matingdosingperiodoftwoweeksandsubsequentmating/fertility
observationswithanoveralldosingperiodofatleastfourweeks,followedbydetailedhistopathologyofthe
malegonads,isconsideredsufficienttoenabledetectionofthemajorityofeffectsonmalefertilityand
spermatogenesis.
12. Femalesshouldbedosedthroughoutthestudy.Thisincludestwoweekspriortomating(withthe
objectiveofcoveringatleasttwocompleteoestrouscycles),thevariabletimetoconception,thedurationof
p1r5.egnancyandatleastthirteendaysafterdelivery,uptoandincludingthedaybeforescheduledkill.
13. Durationofstudy,followingacclimatisation andpre-dosingoestrouscycleevaluation,is
dependentonthefemaleperformanceandisapproximately63days,[atleast14dayspremating,(upto)14
daysmating,22daysgestation,13dayslactation].
14. Duringtheperiodofadministration,theanimalsareobservedcloselyeachdayforsignsoftoxicity.
Animalswhichdieorarekilledduringthetestperiodarenecropsiedand,attheconclusionofthetest,
survivinganimalsarekilledandnecropsied.
DESCRIPTIONOFTHEMETHOD
Selectionofanimalspecies
ThisTestGuidelineisdesignedforusewiththerat.IftheparametersspecifiedwithinthisTG421
areinvestigatedinanotherrodentspeciesadetailedjustificationshouldbegiven. Intheinternational
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validationprogramforthedetectionofendocrinedisruptersonTG407,theratwastheonlyspeciesused.
Strainswithlowfecundityorwell-knownhighincidenceofdevelopmentaldefectsshouldnotbeused.
Healthyvirginanimals,notsubjectedtopreviousexperimentalprocedures,shouldbeused.Thetestanimals
shouldbecharacterisedastospecies,strain,sex,weightandage.Atthecommencementofthestudythe
weightvariationofanimalsusedshouldbeminimalandnotexceed20%ofthemeanweightofeachsex.
Wherethestudyisconductedasapreliminarystudytoalong-termorafull-generationstudy,itis
preferablethatanimalsfromthesamestrainandsourceareusedinbothstudies.
Housingandfeeding
16. Allproceduresshouldconformtolocalstandardsoflaboratoryanimalcare.Thetemperaturein
theexperimentalanimalroomshouldbe22°C(+3°).Althoughtherelativehumidityshouldbeatleast30%
andpreferablynotexceed70%otherthanduringroomcleaning,theaimshouldbe50-60%.Lightingshould
beartificial,thephotoperiodbeing12hourslight,12hoursdark.Forfeeding,conventionallaboratorydiets
maybeusedwithanunlimitedsupplyofdrinkingwater.Thechoiceofdietmaybeinfluencedbytheneedto
ensureasuitableadmixtureofatestchemicalwhenadministeredbythismethod.
17. Animalsshouldbegrouphousedinsmallgroupsofthesamesex;animalsmaybehoused
individuallyifscientificallyjustified.Forgroupcaging,nomorethanfiveanimalsshouldbehousedper
cage.Matingproceduresshouldbecarriedoutincagessuitableforthepurpose.Pregnantfemalesshouldbe
cagedindividuallyandprovidedwithnestingmaterials.Lactatingfemaleswillbecagedindividuallywith
theiroffspring.
18. Thefeedshouldberegularlyanalysedforcontaminants.Asampleofthedietshouldberetained
untilfinalisationofthereport.
Preparationoftheanimals
19. Healthyyoungadultanimalsarerandomlyassignedtothecontrolandtreatmentgroups.Cages
shouldbearrangedinsuchawaythatpossibleeffectsduetocageplacementareminimized.Theanimalsare
uniquelyidentifiedandkeptintheircagesforatleastfivedayspriortothestartofthestudytoallowfor
acclimatisationtothelaboratoryconditions.
Preparationofdoses
2a0d.ministraTttioinsarreeccoonmsmiedenrdeeddmotrhaetaptphreoprtieastte.chWehmeicnalthebeoraladrmoiuntiestiserseedlecotreadl,lytheuntelsetsscheomtihcearlriosuutseusalloyf
administeredbygavage;however,alternatively,testchemicalsmaybeadministeredviathedietordrinking
water.
21. Wherenecessary,thetestchemicalisdissolvedorsuspendedinasuitablevehicle.Itis
recommendedthat,whereverpossible,theuseofanaqueoussolution/suspensionbeconsideredfirst,
followedbyconsiderationofasolution/emulsioninoil(e.g.comoil)andthenbypossiblesolutioninother
vehicles.Forvehiclesotherthanwaterthetoxiccharacteristicsofthevehicleshouldbeknown.Thestability
andhomogeneityofthetestchemicalinthevehicleshouldbedetermined.
PROCEDURE
Numberandsexofanimals
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22. Ttisrecommendedthateachgroupbestartedwithatleast10malesand12-13females.Females
willbeevaluatedpre-exposureforoestrouscyclicityandanimalsthatfailtoexhibittypical4-5daycycles
willnotbeincludedinthestudy;therefore,extrafemalesarerecommendedinordertoyield10females
pergroup.Exceptinthecaseofmarkedtoxiceffects,itisexpectedthatthiswillprovideatleast8pregnant
femalespergroupwhichnormallyistheminimumacceptablenumberofpregnantfemalespergroup.The
objectiveistoproduceenoughpregnanciesandoffspringtoassureameaningfulevaluationofthepotentialof
thetestchemicaltoaffectfertility,pregnancy,maternalandsucklingbehaviour,andgrowthanddevelopment
oftheFioffspringfromconceptiontoday13post-partum.
Dosage
23. Generally,atleastthreetestgroupsandacontrolgroupshouldbeused.Doselevelsmaybebased
oninformationfromacutetoxicitytestsoronresultsfromrepeateddosestudies.Exceptfortreatmentwith
thetestchemical,animalsinthecontrolgroupshouldbehandledinanidenticalmannertothetestgroup
subjects.Ifavehicleisusedinadministeringthetestchemical,thecontrolgroupshouldreceivethevehiclein
thehighestvolumeused.
24. Doselevelsshouldbeselectedtakingintoaccountanyexistingtoxicityand(toxico-)kineticdata
available.Itshouldalsobetakenintoaccountthattheremaybedifferencesinsensitivitybetweenpregnant
andnon-pregnantanimals.Thehighestdoselevelshouldbechosenwiththeaimofinducingtoxiceffectsbut
notdeathorseveresuffering.Thereafter,adescendingsequenceofdoselevelsshouldbeselectedwithaview
todemonstratinganydosagerelatedresponseandno-observed-adverseeffects(NOAEL)atthelowestdose
level.Twotofourfoldintervalsarefrequentlyoptimalforsettingthedescendingdoselevelsandadditionof
afourthtestgroupisoftenpreferabletousingverylargeintervals(e.g.morethanafactorof10)between
dosages.
2k5i.dneyefIfencttsh,eeptcr.e)seonrceotohferobcshearnvgeedsgtehnatermalaytoxnioctitbye(et.ogx.icrerdeuscpeodnsbeosd(ye.gw.eirgehtd,ucleidvefro,odheairntt,akel,unlgivoerr
enlargement),observedeffectsonendocrinesensitiveendpointsshouldbeinterpretedwithcaution.
Limittest
26. Ifanoralstudyatonedoselevelofatleast1000mg/kgbodyweight/dayor,fordietaryordrinking
wateradministration,anequivalentpercentageinthediet,ordrinkingwaterusingtheproceduresdescribed
forthisstudy,producesnoobservabletoxiceffectsandiftoxicitywouldnotbeexpectedbasedupondata
fromstructurallyrelatedsubstances,thenafullstudyusingseveraldoselevelsmaynotbeconsidered
necessary.Thelimittestappliesexceptwhenhumanexposureindicatestheneedforahigheroraldoselevel
tobeused.Forothertypesofadministration,suchasinhalationordermalapplication,thephysicalchemical
propertiesofthetestsubstancesoftenmaydictatethemaximumattainableconcentration.
Administrationofdoses
27. Theanimalsaredosedwiththetestchemicaldailyfor7daysaweek.Whenthetestchemicalis
administeredbygavage,thisshouldbedoneinasingledosetotheanimalsusingastomachtubeorasuitable
intubationcannula.Themaximumvolumeofliquidthatcanbeadministeredatonetimedependsonthesize
osoflutthieontesstwhaenrimea2l.mlT/h1e00voglbuomdeyswheoiuglhdtnmotayexbceeeusded1.mElx/c1e0p0tgfobroidrryitwaetiingghto,rceoxrcreopstivienttehstecchaesmeicoaflasqwuheiocuhs
willnormallyrevealexacerbatedeffectswithhigherconcentrations,variabilityintestvolumeshouldbe
minimizedbyadjustingtheconcentrationtoensureaconstantvolumeatalldoselevels.
q2u8a.ntitiesFoofrthteesttescthcehmeimciaclaladimnivnoilsvteedreddovnioattihnteerdfieerteowritdhrinnokrimnaglwnautterri,tiiotniosriwmaptoerrtbaanltantcoe.enWshureentthhaetttehset
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chemicalisadministeredinthedieteitheraconstantdietaryconcentration(ppm)oraconstantdoselevelin
termsoftheanimals'bodyweightmaybeused;thealternativeusedshouldbespecified.Foratestchemical
administeredbygavage,thedoseshouldbegivenatsimilartimeseachday,andadjustedatleastweeklyto
maintainaconstantdoselevelintermsofanimalbodyweight.
Experimentalschedule
29. Dosingofbothsexesshouldbeginatleast2weekspriortomating,aftertheyhavebeen
acclimatisedforatleastfivedaysandfemaleshavebeenscreenedfornormaloestrouscycles(ina2weeks
pre-treatmentperiod).Thestudyshouldbescheduledinsuchawaythatoestrouscycleevaluationbegins
soonaftertheanimalshaveattainedfullsexualmaturity.Thismayvaryslightlyfordifferentstrainsofratsin
differentlaboratories,e.g.SpragueDawleyrats10weeksofage,Wistarratsabout12weeksofage.Dams
withoffspringshouldbekilledonday13post-partum,orshortlythereafter.Thedayofbirth(viz.when
parturitioniscomplete)isdefinedasday0post-partum.Femalesshowingno-evidenceofcopulationare
killed24-26daysafterthelastdayofthematingperiod.Dosingiscontinuedinbothsexesduringthemating
period.Malesshouldfurtherbedosedafterthematingperiodatleastuntiltheminimumtotaldosingperiod
of28dayshasbeencompleted.Theyarethenkilled,or,alternatively,areretainedandcontinuedtobedosed
forthepossibleconductionofasecondmatingifconsideredappropriate.
30. Dailydosingoftheparentalfemalesshouldcontinuethroughoutpregnancyandatleastupto,and
including,day13post-partumorthedaybeforesacrifice.Forstudieswherethetestchemicalisadministered
bgeystianthiaolna,taionndodrosbiyngtshehoduelrdmbaelrreo-uitnei,tidaotesdinagsssohoounladsbpeoscsoinbtleinauneddnaottlleaatsetrutphatno,PaNnDd4i.ncluding,day19of
31. AdiagramoftheexperimentalscheduleisgiveninAnnex2.
Matingprocedure
32. Normally,1:1(onemaletoonefemale)matingsshouldbeusedinthisstudy.Exceptionscanarise
inthecaseofoccasionaldeathsofmales.Thefemaleshouldbeplacedwiththesamemaleuntilevidenceof
copulationisobservedortwoweekshaveelapsed.Eachmorningthefemalesshouldbeexaminedforthe
presenceofspermoravaginalplug.Day0ofpregnancyisdefinedasthedayonwhichmatingevidenceis
confirmed(avaginalplugorspermisfound).Incasepairingisunsuccessful,re-matingoffemaleswith
provenmalesofthesamegroupcouldbeconsidered.
Littersize
33. Onday4afterbirth,thesizeofeachlittermaybeadjustedbyeliminatingextrapupsbyrandom
selectiontoyield,asnearlyaspossible,fourorfivepupspersexperlitterdependingonthenormallitter
sizeinthestrainofratsused.Bloodsamplesshouldbecollectedfromtwoofthesurpluspups,pooled,and
usedfordeterminationofserumT4levels.Selectiveeliminationofpups,e.g.baseduponbodyweight,or
anogenitaldistance(AGD)isnotappropriate.Wheneverthenumberofmaleorfemalepupsprevents
havingfourorfiveofeachsexperlitter,partialadjustment(forexample,sixmalesandfourfemales)is
tbaycepclieocpawtlaltbyhleer.ceutIlafliintnehgdertweaoragureletd(i8bnseourfufs1ie0cidpeunfptosr/pluibtpltsoeor)id,ncpaorlellfieetctrteeirnoctnoefhwoiarlvlseebretuwgmoivTse4unrptalosusrseespmsuompevsne,tstt.wwooWhfoeefmnatlhleeitptpueurppsssifztoehradtProNaprDse
4bloodsamplesinordertoretainmoremalepupsfornippleretentiononPND13;however,retained
femalepupsineachlittershouldnotdropbelow2ifthiscanbeavoided.
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34.
Iflittersizeisnotadjusted,twopupsperlitteraresacrificedonday4afterbirthandblood
samplesaretakenformeasurementofserumthyroidhormoneconcentrations.Ifpossiblethetwopupsper
littershouldbefemalepupstoreservemalepupsfornippleretentionevaluations.
Inlifeobservations
Clinicalobservations
35. Throughoutthetestperiod,generalclinicalobservationsshouldbemadeatleastonceaday,and
morefrequentlywhensignsoftoxicityareobserved.Theyshouldbemadepreferablyatthesametime(s)
eachday,consideringthepeakperiodofanticipatedeffectsafterdosing.Pertinentbehaviouralchanges,signs
ofdifficultorprolongedparturitionandallsignsoftoxicity,includingmortality,shouldberecorded.These
recordsshouldincludetimeofonset,degreeanddurationoftoxicitysigns.
Bodyweightandfood/waterconsumption
36. Malesandfemalesshouldbeweighedonthefirstdayofdosing,atleastweeklythereafter,andat
termination.Duringpregnancy,femalesshouldbeweighedondays0,7,14and20andwithin24hoursof
parturition(day0or1post-partum)andatleastday4and13post-partum.Theseobservationsshouldbe
reportedindividuallyforeachadultanimal.
37. Duringpre-mating,pregnancyandlactation,foodconsumptionshouldbemeasuredatleastweekly.
Themeasurementoffoodconsumptionduringmatingisoptional.Waterconsumptionduringtheseperiods
shouldalsobemeasuredwhenthetestchemicalisadministeredviadrinkingwater.
Oestrouscycles
38. Oestrouscyclesshouldbemonitoredbeforetreatmentstartstoselectforthestudyfemaleswith
regularcyclicity(seeparagraph22).Vaginalsmearsshouldalsobemonitoreddailyfromthebeginningof
thetreatmentperioduntilevidenceofmating.Ifthereisconcernaboutacutestresseffectsthatcouldalter
oestrouscycleswiththeinitiationofdosing,laboratoriescanexposetestanimalsfor2weeks,thencollect
vaginalsmearsdailytomonitoroestrouscycleforaminimumoftwoweeksduringthepre-matingperiod
withcontinuedmonitoringintothematingperioduntilthereisevidenceofmating.Whenobtaining
vaginal/cervicalcells,careshouldbetakentoavoiddisturbanceofmucosa,whichcouldinduce
pseudopregnancy(7)(8).
Offspringparameters
39. Thedurationofgestationshouldberecordedandiscalculatedfromday0ofpregnancy.Eachlitter
shouldbeexaminedassoonaspossibleafterdeliverytoestablishthenumberandsexofpups,stillbirths,live
births,runts(pupsthataresignificantlysmallerthancorrespondingcontrolpups)andthepresenceofgross
abnormalities.
40. Livepupsshouldbecountedandsexedandlittersweighedwithin24hoursofparturition(day0or
1post-partum)andatleastonday4and13post-partum.Inadditiontotheobservationsdescribedin
paragraph35,anyabnormalbehaviouroftheoffspringshouldberecorded.
44.1P.upbodTyheweAigGhDtsohfoeualcdhbpeucpolslheocutleddboenmteheasduaryedthoenAtGheDsiasmmeepaossutrnaetdalanddaythbeeAtwGeDensPhoNuDld0btehnroorumghalPizNeDd
tpoupasmsehaosuulrdeboefcpouupntseidze,onprPefNeDrab1l2yotrhe13cuasbererocootmomfebnoddeydwieniOgEhtC(D9).GTDhe15n1u(m1b0e).rofnipples/areolaeinmale
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Clinicalbiochemistry
42. Bloodsamplesfromadefinedsitearetakenbasedonthefollowingschedule:
-fromatleasttwopupsperlitteronday4afterbirth,
-fromalldamsandatleasttwopupsperlitteratterminationonday13,and
-fromalladultmales,attermination,
Allbloodsamplesarestoredunderappropriateconditions.Bloodsamplesfromtheday13pupsandtheadult
malesareassessedforserumlevelsforthyroidhormones(T4).FurtherassessmentofT4inbloodsamples
fromthedamsandday4pupsisdoneifrelevant.Asanoptionotherhormonesmaybemeasuredifrelevant.
Pupbloodcanbepooledbylitterforthyroidhormoneanalyses.Thyroidhormones(T4andTSH)should
preferablybemeasuredas‘total’.
43. Thefollowingfactorsmayinfluencethevariabilityandtheabsoluteconcentrationsofthe
hormonedeterminations:
timeofsacrificebecauseofdiurnalvariationofhormoneconcentrations
methodofsacrificetoavoidunduestresstotheanimalsthatmayaffecthormoneconcentrations
- testkitsforhormonedeterminationsthatmaydifferbytheirstandardcurves.
44. Plasmasamplesspecificallyintendedforhormonedeterminationshouldbeobtainedata
comparabletimeoftheday.Thenumericalvaluesobtainedwhenanalysinghormoneconcentrationsdiffer
withvariouscommercialassaykits.
Pathology
Grossnecropsy
45. Atthetimeofsacrificeordeathduringthestudy,theadultanimalsshouldbeexamined
macroscopicallyforanyabnormalitiesorpathologicalchanges.Specialattentionshouldbepaidtotheorgans
ofthereproductivesystem.Thenumberofimplantationsitesshouldberecorded.Vaginalsmearsshouldbe
examinedinthemorningonthedayofnecropsytodeterminethestageoftheoestrouscycleandallow
correlationwithhistopathologyofovaries.
46. Thetestesandepididymides(aswellaslevatoraniplusbulbocavemosusmusclecomplex,
Cowper’sglandsandglanspenis)ofallmaleadultanimalsshouldbeweighed.
47. Deadpupsandpupskilledatday13post-partum,orshortlythereafter,should,atleast,becarefully
examinedexternallyforgrossabnormalities.Particularattentionshouldbepaidtotheexternalreproductive
genitalswhichshouldbeexaminedforsignsofaltereddevelopment.Atday13thethyroidfrom1maleand1
femalepupperlittershouldbepreserved.
48. Theovaries,testes,accessorysexorgans(uterusandcervix,epididymides,prostate,seminal
vesiclespluscoagulatingglands),thyroidandallorgansshowingmacroscopiclesionsofalladultanimals
shouldbepreserved.Formalinfixationisnotrecommendedforroutineexaminationoftestesand
epididymides.AnacceptablemethodistheuseofBouin'sfixativeormodifiedDavidsonsforthesetissues
(11).Thetunicaalbugineashouldbegentlyandshallowlypuncturedatthebothpolesoftheorganwitha
needletopermitrapidpenetrationofthefixative.
Histopathology
49. Detailedhistologicalexaminationshouldbeperformedontheovaries,testesandepididymides
(withspecialemphasisonstagesofspermatogenesisandhistopathologyofinterstitialtesticularcellstructure)
oftheanimalsofthehighestdosegroupandthecontrolgroup.Theotherpreservedorgansincludingthyroid
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frompupsandadultanimalsmaybeexaminedwhennecessary.Thethyroidweightcouldbedetermined
afterfixation.Trimmingshouldalsobedoneverycarefullyandonlyafterfixationtoavoidtissuedamage.
Tissuedamagecouldcompromisehistopathologyanalysis.Examinationsshouldbeextendedtotheanimals
ofotherdosagegroupswhenchangesareseeninthehighestdosegroup.TheGuidanceonhistopathology
(11)detailsextrainformationondissection,fixation,sectioningandhistopathologyofendocrinetissues.
DATAANDREPORTING
Data
50. Individualanimaldatashouldbeprovided.Additionally,alldatashouldbesummarisedintabular
form,showingforeachtestgroupthenumberofanimalsatthestartofthetest,thenumberofanimalsfound
deadduringthetestorkilledforhumanereasons,thetimeofanydeathorhumanekill,thenumberoffertile
animals,thenumberofpregnantfemales,thenumberofanimalsshowingsignsoftoxicity,adescriptionof
thhiestsoipgantshoolfogtioxciaclitcyhaonbgseesr,veadn,dianlcllruedlienvgatntimleitotefrodnasteat.,Adutraabtuiloanr,saundmmsaevreyrirteypoorftafnoyrtmoaxtictheaftfehcatss,ptrhoevetynpteosboef
veryusefulfortheevaluationofreproductive/developmentaleffectisgiveninAnnex3.
5ar1e.oflimDitueedtvoatlhueelfiomritmeadndyimeenndspiooinnstso,fetshpeecstiuadlyl,ysrteaptirsotdiucacltiavnealeynsdepsoiinnttsh.eIfforstmatoifsttiecsatlsafnoarl"yssiegsniafriecaunscee"d
tprhieonrtthoetmheetshtaordtocfhotsheenstsuhdoyu.lSdtabteisatipcparloparniaaltyesifsoorfthAeGdDistarnibdutniiopnploefrtehteenvtairoinabslheouelxdambienebda,seadnodnbeindsievliedcutaeld
pupdata,takinglittereffectsintoaccount.Whereappropriate,thelitteristheunitofanalysis.Statistical
analysisofpupbodyweightshouldbebasedonindividualpupdata,takinglittersizeintoaccount.Because
ofthesmallgroupsize,theuseofhistoriccontroldata(e.g.forlittersize),whereavailable,mayalsobe
usefulasanaidtotheinterpretationofthestudy.
Evaluationofresults
52. Thefindingsofthistoxicitystudyshouldbeevaluatedintermsoftheobservedeffects,necropsy
andmicroscopicfindings.Theevaluationwillincludetherelationshipbetweenthedoseofthetestchemical
andthepresenceorabsence,incidenceandseverityofabnormalities,includinggrosslesions,identifiedtarget
organs,infertility,clinicalabnormalities,affectedreproductiveandlitterperformance,bodyweightchanges,
effectsonmortalityandanyothertoxiceffects.
53. Becauseoftheshortperiodoftreatmentofthemale,thehistopathologyofthetestesand
epididymidesshouldbeconsideredalongwiththefertilitydata,whenassessingmalereproductiveeffects.
Theuseofhistoricalcontroldataonreproduction/development(e.g.,forlittersize,AGD,nippleretention,
serumT4levels),whereavailable,mayalsobeusefulasanaidtotheinterpretationofthestudy.
54. Forqualitycontrolitisproposedthathistoricalcontroldataarecollectedandthatfornumerical
datacoefficientsofvariationarecalculated,especiallyfortheparameterslinkedwithendocrinedisrupter
detection.Thesedatacanbeusedforcomparisonpurposeswhenactualstudiesareevaluated.
Testreport
55. Thetestreportshouldincludethefollowinginformation:
Testchemical:
-source,lotnumber,limitdateforuse,ifavailable
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-stabilityofthetestchemical,ifknown.
Mono-constituentsubstance:
- physical appearance, water solubility, and additional relevant physicochemical
properties;
-chemicalidentification,suchasIUPACorCASname,CASnumber,SMILESorInChl
code,structuralformula,purity,chemicalidentityofimpuritiesasappropriateand
practicallyfeasible,etc.
Multi-constituentsubstance,UVBCsandmixtures:
-characterisedasfaraspossiblebychemicalidentity(seeabove),quantitativeoccurrence
andrelevantphysicochemicalpropertiesoftheconstituents.
Vehicle(ifappropriate):
justificationforchoiceofvehicleifotherthanwater.
Testanimals:
species/strainused;
number,ageandsexofanimals;
source,housingconditions,diet,etc.;
individualweightsofanimalsatthestartofthetest.
justificationforspeciesifnotrat
Testconditions:
rationalefordoselevelselection;
detailsoftestchemicalformulation/dietpreparation,achievedconcentrations,stability
andhomogeneityofthepreparation;
detailsoftheadministrationofthetestchemical;
conversionfromdiet/drinkingwatertestchemicalconcentration(ppm)totheactualdose
(mg/kgbodyweight/day),ifapplicable;
detailsoffoodandwaterquality;
detaileddescriptionoftherandomisationproceduretoselectpupsforculling,if
culled.
Results:
bodyweight/bodyweightchanges;
foodconsumption,andwaterconsumptionifavailable;
toxicresponsedatabysexanddose,includingfertility,gestation,andanyothersignsof
toxicity;
gestationlength;
toxicorothereffectsonreproduction,offspring,post-natalgrowth,etc.;
nature,severityanddurationofclinicalobservations(whetherreversibleornot);
numberofadultfemaleswithnormalorabnormaloestrouscycleandcycleduration;
numberoflivebirthsandpost-implantationloss;
ApuGpDboofdyallwpeuigphst(adantdabodyweightondayofAGDmeasurement)
nippleretentioninmalepups,
thyroidhormonelevels,day13pupsandadultmales(anddamsandday4pupsif
measured)
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421 OECD/OCDE
numberpupswithgrosslyvisibleabnormalities,grossevaluationofexternalgenitalia,
numberofrunts;
timeofdeathduringthestudyorwhetheranimalssurvivedtotermination;
numberofimplantations,littersizeandlitterweightsatthetimeofrecording;
bodyweightatsacrificeandorganweightdatafortheparentalanimals;
necropsyfindings;
detaileddescriptionofhistopathologicalfindings;
absorptiondata(ifavailable);
statisticaltreatmentofresults,whereappropriate.
Discussionofresults.
Conclusions.
Interpretationofresults
56. Thestudywillprovideevaluationsofreproduction/developmentaltoxicityassociatedwith
cadomnidnuicsttrfautritohneroifnvreespteiagtaetdiodnossaensd(psreoevpidaersaggruaipdhsanc5eainndth6)e.dIetsicgonulodfpsruobvsiedqeueanntisntduidiceast.ioOnEoCfDtheGuniedeadncteo
DOoEcCuDmeGnuti4d3anscheouDlodcbuemceonntsu1l0t6edofnorHiasitdoilnogtiheciEnvtaelruparettiaotnioonfoEfnrdeopcrroidnuectainodnaRenpdrdoedvuecltoipvmeeTnetsatlsriesnuRltosde(1n2t)s.
(m1a1y)pbreohveildpefsulinffoorrtmhaitsiToGn.onthepreparationandevaluationof(endocrine)organsandvaginalsmearsthat
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©OECD,(2015)