441, adopted 7 September 2009 [electronic resource] : Hershberger bioassay in rats : a short-term screening assay for (anti)androgenic properties

2009·0.64 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 441
Adopted:
7September2009
OECDGUIDELINEFORTHETESTING OFCHEMICALS
HershbergerBioassavinRats:AShort-termScreeningAssayfor(Anti)AndrogenicProperties
INTRODUCTION
n1.ewguideTlhienesOEfoCrDthienitsicarteeednainhgigahn-dpritoersittiyngacotfivipto>t-enintia1l99e8ndtoocrreivniesediesxriuspttienrgsgu(i1)d.elOinneesealnedmteontdeovfeltohpe
activitywastodevelopaTestGuidelinefortheratHershbergerBioassay.Afterseveraldecadesofuseby
thepharmaceuticalindustry’,thisassaywasfirststandardizedbyanofficialexpertcommitteein1962asa
screeningtoolforandrogenicchemicals(2).In2001-2007,theratHershbergerBioassayhasundergonean
extensivevalidationprogrammeincludingthegenerationofaBackgroundReviewDocument(23),
compilationofadetailedmethodspaper(3),developmentofadissectionguide(21)andtheconductof
extensiveintra-andinterlaboratorystudiestoshow'thereliabilityandreproducibilityofthebioassay.
Thesevalidationstudieswereconductedw'ithapotentreferenceandrogen(testosteronepropionate(TP)),
tvvopotentsyntheticandrogens(trenboloneacetateandmethyltestosterone),apotentantiandrogenic
pharmaceutical(flutamide),apotentinhibitorofthesynthesis(finasteride)ofthenaturalandrogen
(dihydrotestostcrone-DHT),severalweaklyantiandrogenicpesticides(linuron,vinclozolin,procymidone,
p,p'DDE),apotent5areductaseinhibitor(finasteride)andtw'Oknownnegativechemicals(dinitrophenol
andnonylphenol)(4)(5)(6)(7)(8).ThisTestGuidelineistheoutcomeofthelonghistoricalexperience
withthebioassayandtheexperiencegainedduringthevalidationtestprogrammeandtheresultsobtained
therein.
2. TheHershbergerBioassayisashort-terminvivoscreeningtestusingaccessorytissuesofthe
malereproductivetract.Theassayoriginatedinthe1930'sandwasmodifiedinthe1940’stoinclude
androgen-responsivemusclesinthemalereproductivetract(2)(9-15). Inthe1960s,over700possible
androgensw'ereevaluatedusingastandardizedversionoftheprotocol(2)(14),anduseoftheassayfor
bothandrogensandantiandrogenswasconsideredastandardmethodinthe1960s(2)(15). Thecurrent
bioassayisbasedonthechangesinweightoffiveandrogen-dependenttissuesinthecastrate-peripubertal
malerat.Itevaluatestheabilityofachemicaltoelicitbiologicalactivitiesconsistentwithandrogen
agonists,antagonistsor5a-reductascinhibitors.Thefivetargetandrogen-dependenttissuesincludedinthis
TestGuidelinearetheventralprostate(VP),seminalvesicle(SV)(plusfluidsandcoagulatingglands),
levatorani-bulbocavemosus(LABC)muscle,pairedCowper’sglands(COW)andtheglanspenis(GP).In
thecastrate-peripubertalmalerat,thesefivetissuesallrespondtoandrogenswithanincreaseinabsolute
weight.Whenthesesametissuesarestimulatedtoincreaseinweightbyadministrationofapotent
referenceandrogen,thesefivetissuesallrespondtoantiandrogenswithadecreaseinabsoluteweight.The
primarymodelfortheHershbergerbioassayhasbeenthesurgicallycastratedpcripubcrtalmale,whichwas
validatedinPhases1,2and3oftheHershbergervalidationprogram.
3. TheHershbergerbioassayservesasamechanisticinvivoscreeningassayforandrogenagonists,
a‘nOdEroCgDenCaonntcaegpotnuiasltsFarnadm5eaw-orrckducftorasteheinhTiebsittionrgsaanndditAsssapepslsimceantitonofshEonudlodcrbienseeeDnisirnupttheincgonCtheexmtiocfaltsh”e
©OECD,(2009)
YtpheoerumOiaEsrsCeiDfo,rneefprrtooomvuitsdheeedtOhtihEseCmDsao.tuerricaelifsordupleyrsmoennatli,onneodn.-cAonmymecrocmimaelrcpiuarlpousseesowfitthhiosutmasteeerkiianlgisprsiuobrjcecotnsteonwtriftrtoemn
441 OECD/OCDE
(Annex2).InthisConceptualFrameworktheHershbergerBioassayiscontainedinLevel3asaninvivo
assayprovidingdataaboutasingleendocrinemeehanism,i.e.(anti)androgenieity. Itisintendedtobe
includedinabatteryofinvitroandinvivoteststoidentifysubstanceswithpotentialtointeractwiththe
endocrinesystem,ultimatelyleadingtohazardandriskassessmentsforhumanhealthortheenvironment.
4. Duetoanimalwelfareconcernswiththecastrationprocedure,theintact(uncastrated)stimulated
w'eanlingmalew'assoughtasanalternativemodelfortheHershbergerBioassaytoavoidthecastration
step.Thestimulatedw'eanlingtestmethodwasvalidated(24);however,inthevalidationstudies,the
weanlingversionoftheHershbergerBioassaydidnotappeartobeabletoconsistentlydetecteffectson
baiennncdelrfuoidgtesednb-iudntetpahelisnsodTemenasttvoGprurgioadvneildwieeniei.gnhftoHsromwfaertvoiemonrw,oenraekoctohagennrtiism-ioandngdertsohagotefnisatcstauitostne,hemitadiyossepavsraoitvleisadtbeelde.nointThGoeunrileydfaoanrnceie,maDitlowcwaueslmfeannrotet
115.
INITIALCONSIDERATIONSANDLIMITATIONS
5in.hibit,reAspnedcrtoivgeelny,aggeonniesttsraansncdriapnttiaognonciosnttsroalcltedasbylitghaendrsecfeoprtotrh.eIanndardodigteinonr,ecseopmteorcahnedmimcaalysaicnthiivbaittethoer
conversionoftestosteronetothemorepotentnaturalandrogendihydrotestosteroneinsomeandrogen
targettissues(5a-reductaseinhibitors).Suchsubstanceshavethepotentialtoleadtoadversehealth
hazards,includingreproductiveanddevelopmentaleffects.Therefore,theregulatoiy'needexiststorapidly
assessandevaluateachemicalasapossibleandrogenagonistorantagonistor5a-rcductascinhibitor.
Whileinformative,theaffinityofaligandforanandrogenreceptorasmeasuredbyreceptorbindingor
transcriptionalactivationofreportergenesinvitroisnottheonlydeterminantofpossiblehazard.Other
determinantsincludemetabolicactivationanddeactivationuponenteringthebody,substancedistribution
totargettissues,andclearancefromthebody.Thisleadstotheneedtoscreenthepossibleactivityofa
chemicalinvivounderrelevantconditionsandexposure.Invivoevaluationislesscriticalifthechemical’s
characteristicsregardingAbsorption-Distribution-Metabolism-Elimination(ADME)areknown.
Androgen-dependenttissuesrespondwithrapidandvigorousgrowthtostimulationbyandrogens,
particularlyincastrate-peripubertalmalerats.Rodentspecies,particularlytherat,arealsow'idelyusedin
toxicitystudiesforhazardcharacterization. Therefore,theassayversionusingthecastratedperipubertal
ratandthefivetargettissuesinthisassayareappropriatefortheinvivoscreeningofandrogenagonists
andantagonistsand5a-reductaseinhibitors.
6. ThisTestGuidelineisbasedonthoseprotocolsemployedintheOECDvalidationstudyw'hich
havebeenshowntobereliableandreproducibleinintra-andinterlaboratorystudies(4)(5)(6)(7)(8).
BothandrogenandantiandrogenproceduresarepresentedinthisGuideline.
7. AlthoughtherewassomevariationinthedoseofTPusedtodetectantiandrogensintheOECD
HershbergerBioassayvalidationprogrammebythedifferentlaboratories(0.2versus0.4mg/kg/d,
subcutaneousinjection)therewaslittledifferencebetweenthesetwoprotocolvariationsintheabilityto
detectweakorstrongantiandrogenicactivity.However,itisclearthatthedoseofTPshouldnotbetoo
highastoblocktheeffectsofweakandrogenreceptor(AR)antagonistsorsolow'thattheandrogenic
tissuesdisplaylittlegrowthresponseevenwithoutantiandrogencoadministration.
8. Thegrowthresponseoftheindividualandrogen-dependenttissuesisnotentirelyofandrogenic
origin,i.e.compoundsotherthanandrogenagonistscanalterthew'eightofcertaintissues.However,the
growthresponseofseveraltissuesconcomitantlysubstantiatesamoreandrogen-specificmechanism.For
example,highdosesofpotentestrogenscanincreasetheweightoftheseminalvesicles;however,theother
androgcn-dcpcndcnttissuesintheassaydonotrespondinasimilarmanner.Antiandrogenicchemicalscan
actcitherasandrogenreceptorantagonistsor5a-reductaseinhibitors.5a-reductaseinhibitorshavea
©OCDE,(2009) 2
.
OECD/OCDE 441
variableeffect,becausetheconversiontomorepotentdihydrotcstostcroncvariesbytissue.Antiandrogens
ttuihssaestduietnsohiabdsiitfcfo5earme-pnrateirdaeutdcettbaoeseta,wepleoitnkeenAftiRnAamsRteedraiinadteta,geodhnaiavsnted,m5loiark-eerefpdlruuotcnatmoaiusdneec.meedTdheiifasftedecitdfsfmieonrdetnehcseevoiefnntatrciatslisoupner.orsIetnsaptaeodndtsihteainocnao,nthtbheeer
androgenreceptorisevolutionarilyrelatedtothatofothersteroidhormones,andsomeotherhormones,
whenadministeredathigh,supraphysiologicaldosagelevels,canbindandantagonizethegrov\th-
promotingeffectsofTP(13).Further,italsoisplausiblethatenhancedsteroidmetabolismanda
consequentloweringofscrumtestosteronecouldreduceandrogen-dependenttissuegrowth.Therefore,any
paopspirtoiavceh,ouitnccolmuediningtihnevHietrrsohbaessragyesr,BsiuocahssaasytshheouAldRnoarnmdalelsytrboegeenvarleucaetpetdorus(iEnRg)abwienidgihntgoafsseavyisdeannced
correspondingtranscriptionalactivationassays,orfromotherinvivoassaysthatexaminesimilarandrogen
targettissuessuchasthemalepubertalassay,15-dayintactadultmaleassay,or28-dayor90-dayrepeat
dosestudies.
9. Experienceindicatesthatxenobioticandrogensarerarerthanxenobioticantiandrogens.The
expectationthenisthattheHershbergerbioassaywillbeusedmostoftenforthescreeningof
antiandrogens.However,theproceduretotestforandrogenscould,nevertheless,berecommendedfor
steroidalorsteroid-likechemicalsorforchemicalsforwhichanindicationofpossibleandrogeniceffects
wadavserdseerievfefdectfsroamssmoceitahtoeddswictohnt(aainnteid)ainndrLoegveenlic1porrof2ileosfmthaeycboencoebpsteuravledfrianmLeewvoerlk5(aAsnsnaeysx,2l)e.adSiinmgiltaortlhy,e
needtoassesswhetherasubstanceoperatesbyanendocrinemodeofaction.
10. Itisacknowledgedthatallanimal-basedproceduresshouldconformtolocalstandardsofanimal
care;thedescriptionsofcareandtreatmentsetforthbelowareminimalperformancestandards,andwillbe
sOuEpeCrDse(d1e7d).bylocalregulations.Furtherguidanceofthehumanetreatmentofanimalsisgivenbythe
11. Asinanybioassayusingexperimentalanimals,carefulconsiderationsshouldbegiventothe
necessitytocarryoutthisstudy.Basicallytheremaybetworeasonsforsuchadecision:
• h(aingthi)aenxdproogseunriecitpyotinenitniavlitro(Laesvsealys(1Levoefl2t)hesupCpoorntcienpgtuianlvestFirgaamteiown'sorwkh)ethoerrsiuncdhiceaftfieocntssmafoyr
occurinvivo;
• effectsconsistentwith(anti)androgenicityinLevel4or5invivotestssupportinginvestigationsof
thespecificmodeofaction,e.g.,todeterminew'hethertheeffectswereduetoan(anti)androgenic
mechanism.
12. DefinitionsusedinthisTestGuidelinearegiveninAnnex1
PRINCIPLEOFTHETEST
13. TheHershbergerBioassayachievesitssensitivitybyusingmaleswithminimalendogenous
androgenproduction.Thisisachievedthroughtheuseofcastratedmalesprovidedanadequatetimeafter
castrationforthetargettissuestoregresstoaminimalanduniformbaselineweightisallowed.Thus,when
screeningofpotentialandrogenicactivity,therearelowendogenouslevelsofcirculatingandrogens,the
hypothalamic-pituitary-gonadaxisisrenderedunabletocompensateviafeedbackmechanisms,the
Wabhileintysocfretehneintgisosfuepsotteontiraelspaonntdi-aisndmraogxeinmiiczeacdt,iviatnyd,athmeorsetarctoinnsgisttiesnstuetiwsesuieghwteivgahrtiagbaiilintycainsbmeinaicmhiizeevde.d
whenthetissuesarestimulatedbyareferenceandrogen.Asaresult,theHershbergerBioassayrequires
only6animalsperdosegroupwhereasotherassayswithintactpubertaloradultmalessuggestusing15
malesperdosegroup.
©OCDE,(2009)
441 OECD/OCDE
14. Castrationofpcripubcrtalmaleratsshouldbedoneinanappropriatemannerusingapproved
anesthetiesandaseptietechnique.Analgesicsshouldbeadministeredonthefirstfewdaysfollowing
surgerytoeliminatepost-surgicaldiscomfort.Castrationenhancestheprecisionoftheassaytodetectweak
androgensandantiandrogensbyeliminatingcompensatoiyendocrinefeed-backmechanismspresentinthe
intactanimalthatcanattenuatetheeffectsofadministeredandrogensandantiandrogensandbyeliminating
thelargeinter-individualvariabilityinsemmtestosteronelevels.Hence,castrationreducesthenumbersof
animalsrequiredtoscreenfortheseendocrineactivities.
15. Whenscreeningforpotentialandrogenicactivity,thetestsubstanceisadministereddailybyoral
gavageorsubcutaneous(sc)injectionforaperiodoftenconsecutivedays.Testsubstancesare
gardomuipn.istTehreedatnoimaalmsianriemnuemcroopfstiwedoatprperaotxmienmtatgerloyup2s4hoofuerxspeafrtiemrenthteallaasntidmoasles.uAsisntgatoisnteicadlolsyesilgenvieflicpaenrt
increaseintwoormoretargetorganweightsofthetestsubstancegroupscomparedtothevehiclecontrol
gArnodurpogeinndsi,caltieksetthraetnbtohleonteestthsautbcstaannncoetbisep5oasi-trievdeucfoerdphoatvenetimaolraendprroogneonuincceadctievfifteycts(SoenetphaeraLgAraBpCh6a0n)d.
GPversusTP.butalltissuesshoulddisplayincreasedgrowth.
o1r6a.lgavagWehoernssucbreeuetnainnegoufsoripnojteecnttiioanlfoarntaiapnedrrioogdcnoifctaecnticvointsy,ectuhteivteestdasyusbsitnanecoenciesrtadwmiitnhisdtaeirleydTdPaidloysbeys
m(0g./2kgo/rd0.o4fmTgP/kcgo/udl)dbbyesucseidnjaecstiboont.hIwtewraesedfefetcetrimvieniendtihnetdheetevcatliiodnatoifonanptrioagndrraomgmeensthaantd.eitthheerref0o.r2e,oron0l.y4
onedoseshouldbeselectedforuseintheassay.Graduatedtestsubstancedosesareadministeredtoa
marieninemcurmopsoifetdhraepeptrroexaitmmaetnetlygr2o4uphsouorfsexapfeterrimtehnetallastandiomsael.sAussitnagtiostniecadlloysesilgenvieflicpaenrtgdreocurpe.asTeheinatnwi'mOalosr
moretargetorganweightsofthetestsubstanceplusTPgroupscomparedtotheTPonlycontrolgroup
indicatesthatthetestsubstanceispositiveforpotentialantiandrogenicactivity(Seeparagraph61).
DESCRIPTIONOFTHEMETHOD
Selectionofspeciesandstrain
17. TherathasbeenroutinelyusedintheHershbergerBioassaysincethe1930s. Althoughitis
biologicallyplausiblethatboththeratandmousew'oulddisplaysimilarresponses,basedupon70yearsof
experiencewiththeratmodel,theratisthespeciesofchoicefortheHershbergerBioassay.Inaddition,
sinceHershbergerBioassaydatamaybepreliminaiytoalong-termmultigenerationalstudy,thisallows
animalsfromthesamespecies,strainandsourcetobeusedinbothstudies.
18. Thisprotocolallowslaboratoriestoselectthestrainofrattobeusedintheassaywhichshould
generallybethatusedhistoricallybytheparticipatinglaboratory.Commonlyusedlaboratoryratstrains
maybeused;however,strainsthatmaturesignificantlylaterthan42daysofageshouldnotbeusedsince
castrationofthesemalesat42daysofagecouldprecludemeasurementofglanspenisweights,whichcan
onlybedoneaftertheprepuceisseparatedfromthepenileshaft.Thus,strainsderivedfromtheFisher344
ratshouldnotbeused,exceptinrarecases.TheFisher344rathasadifferenttimingofsexual
developmentcomparedwithothermorecommonlyusedstrainssuchasSpragueDaw'leyorWistarstrains
(16).Ifsuchastrainistobeused,thelaboratoiy'shouldcastratethemataslightlyolderageandbeableto
demonstratethesensitivityofthestrainused.Therationaleforthechoiceofratstrainshouldbeclearly-
statedbythelaboratory.Wherethescreeningassaymaybepreliminarytoarepeateddoseoralstudy,a
reproductiveanddevelopmentalstudy,oralong-termstudy,preferablyanimalsfromthesamestrainand
sourceshouldbeusedinallstudies.
©OCDE,(2009) 4
OECD/OCDE 441
Housingandfeedingconditions
19. Allproceduresshouldconformtoalllocalstandardsoflaboratoryanimalcare. These
descriptionsofcareandtreatmentareminimumstandardsandwillbesupersededbymorestringentlocal
regulations,whenpresent. Thetemperatureintheexperimentalanimalroomshouldbe22°C(withan
approximaterange±3°C).Therelativehumidityshouldbeaminimumof30%andpreferablyshouldnot
exceedamaximum70%,otherthanduringroomcleaning. Theaimshouldberelativehumidityof50-
60%.Lightingshouldbeartificial.Thedailylightingsequenceshouldbe12hourslight,12hoursdark.
20. Grouphousingispreferabletoisolationbecauseoftheyoungageoftheanimalsandthefactthat
ratsaresocialanimals. Housingoftwoorthreeanimalspercageavoidscrow'dingandassociatedstress
thatmayinterferewiththehormonalcontrolofthedevelopmentofthesexaccessory'tissue.Cagesshould
bethoroughlycleanedtoremovepossiblecontaminantsandarrangedinsuchawaythatpossibleeffects
duetocageplacementareminimized. Cagesofapropersize(-2000squarecentimeters)willprevent
overcrowding.
21. Eachanimalshouldbeidentifiedindividually(e.g.,earmarkortag)usingahumanemethod.
Themethodofidentificationshouldberecorded.
22. Laboratory'dietanddrinkingwatershouldbeprovidedadlibitum. Laboratoriesexecutingthe
HershbergerBioassayshouldusethelaboratory'dietnormallyusedintheirchemicaltestingw'ork.Inthe
validationstudiesoftheBioassay,noeffectsorvariability'wereobservedthatwereattributabletothediet.
Thedietusedwillberecordedandasampleofthelaboratory'dietshouldberetainedforpossiblefuture
analysis.
PerformanceCriteriaforandrogen-dependentorganweights
23. Duringthevalidationstudy,therewasnoevidencethatadecreaseinbodyweightaffected
increasesordecreasesinthegrowthoftissuew'eightsfortargettissues(i.e.,thatshouldbeweightedinthis
study).
24. Amongthedifferentstrainsofratusedsuccessfullyinthevalidationprogramme,androgen-
dependentorganweightsarelargerintheheavierratstrainsthaninthelighterstrains.Therefore,the
HershbergerBioassayperformancecriteriadonotincludeabsoluteexpectedorganweightsforpositiveand
negativecontrols.
p2(l5aoT.bawoberlrae,tot1r)hi.BeeseHcTesahrhuesoshueCbleVdtrhsgeeexaCrraoemeBfiidfnoeiaercsiisvetaenhydteopfCferVroVsfamorirtfahmretaoinmoOcneEt(ChcCerDVit)cevorafniloatirradoaralettiigbosrnasosuuseetpduhdaoiasennsda.mnatiIhxnneivetmhrhiusegemhcraCesdlVeoastoeivfoanltnsureheegisaapttfwmioevrinettheoasugcttrhactoitousimptseiscsuta,eol
determineifthemaximumCVperformancecriteriahavebeenexceeded.
2c6o.ntrolanTdhheigshtuddoyseshtorueladtmbeentrgerpoeuaptesdewxhceene:dt1h)etmharexeimorummsordeesoifgntahteedtefnorpoasgsoinbilsetiannddivaindtuaaglonCiVstsstiundtihees
inTable1and2)atleasttwotargettissuesweremarginallyinsignificant,i.e.,pvaluesbetween0.05and
0.10.
Tcaasbtlreate1:modMealxiinmtuhemOaElCloDwaVballeidCaVt.isonDSetutdeirems'i.nedfortheTargetSexAccessoryTissuesforthe
'ThethresholdCVforagiventissuewasidentifiedfromagraphofCVvalues-arrangedfromsmallest
sequentiallytolargest-forallmeansfromallexperimentsinthevalidationexerciseusingaspecificmodel(agonist
5 ©OCDE,(2009)
441 OECD/OCDE
Tissue Antiandrogeniceffects Androgeniceffects
Seminalvesicles 40% 40%
Ventralprostate 40% 45%
LABC 20% 30%
Cowper.sglands 35% 55%
Gianspenis 17% 22%
PROCEDURE
Regulatorycomplianceandlaboratoryverification
27. Unliketheutcrotrophicassay(TG440),ademonstrationoflaboratory'competenecpriortothe
initiationofthestudyisnotnecessary'fortheHershbergerassaybecauseconcurrentpositive(Testosterone
PropionateandFlutamide)andnegativecontrolsarerunasanintegralpartoftheassay.
Numberandconditionofanimals
28. Eaehtreatedandcontrolgroupshouldincludeaminimumof6animals.Thisappliestoboththe
androgenicandantiandrogenicprotocols.
Castration
29. Thereshouldbeaninitialacclimatizationperiodofseveraldaysafterreceiptoftheanimalsto
ensurethattheanimalsarehealthyandthriving.Sinceanimalscastratedbefore42daysofageorpostnatal
day(pnd)42maynotdisplaypreputialseparation,animalsshouldbecastratedonpnd42orthereafter,not
before. Theanimalsarccastratedunderanesthesiabyplacinganincisioninthescrotumandremoving
bothtestesandepididy'mideswithligationofbloodvesselsandseminalducts.Afterconfirmingthatno
bleedingisoccurring,thescrotumshouldbeclosedwithsutureorautoclips.Animalsshouldbetreated
withanalgesicsforthefirstfewdaysaftersurgery'toalleviateanypost-surgicaldiscomfort. Ifcastrated
animalsarepurchasedfromananimalsupplier,theageofanimalsandstageofsexualmaturityshouldbe
assuredbythesupplier.
Acclimatizationaftercastration
30. Theanimalsshouldcontinueacclimationtothelaboratory'conditionstoallowfortheregression
inthetargettissueweightsforaminimumof7daysfollowingcastration. Animalsshouldbeobserved
daily,andanyanimalswithevidenceofdiseaseorphysicalabnormalitiesshouldberemoved. Thus,
treatmentwithinitiationofdosing(onstudy)maycommenceasearlyaspnd49daysofage,butnotlater
thanpnd60.Ageatnecropsyshouldnotbegreaterthanpnd70.Thisflexibilityallowsalaboratory'to
scheduletheexperimentalworkefficiently.
omtahrgiotsanhnieatsnatsageloarynsiisessisatsy)a.irsdeTehndhotrewiafemtiadhetrdiaecrsmaehllooallrtydeivlCeualVrnygiewfrroeatlrsihmaarbnielnaetcdhr“eefbarprsreoeeamkcmpetaohdkeiiinnptngosgi”fneitfdwoearntCtVwti.hhfesiie-caahttnihttoeahnge“oobnifrinesacatrktepmhmoorieednnsetth”lso.lbodIefttCstwVhheeoeubnalysdtsotabhyeti,shneCmonteVeetxdhetouthhdraivtgsehsoaelmsftetohwroChutVaghsthe
arbitrary'.
©OCDE,(2009) 6
OECD/OCDE 441
Bodyweightandgrouprandomization
a3n1d.amonDgifgfreoruepnsceosfianniinmdailvsi.duaIlncbroeadsyinwgeitgihsstuseawreeiaghstouvarrcieaboilfitvya'riraebsiullittsyiinnatnissiunecrwe'aesiegdhtcsoebfoftihciweintthionf
variation(CV)anddecreasesthestatisticalpoweroftheassay(sometimesreferredtoasassaysensitivity).
Therefore,variationsinbodyweightshouldbebothexperimentallyandstatisticallycontrolled.
32. Experimentalcontrolinvolvesproducingsmallvariationsinbodyweightwithinandamongthe
Asttudsytugdryoucpso.mmFeirnsct,emuneunstuatlhleywsemiagllhtorvalrairagteioanniomfalasnismhaolusldubseedavsohioduelddannodtneoxtcpeleadce±di2n0th%esotfudtyhecomheoratn.
w(beoitghhtco(net.rgo.l1a7n5dgtr±ea3t5megntf)orbycarstarnadtoemdipzeerdipwu'beeirgthatldirsattsr)i.butSieocno,nsdo,tahnatimmaelasnsbhooudlydwbeeigahstsiogfneeadchtogrgoruopupiss
notstatisticallydifferentfromanyothergroup. Theblockrandomizationprocedureusedshouldbe
recorded.
33. Becausetoxicitymaydecreasethebodyweightoftreatedgroupsrelativetothecontrolgroup,the
bodyW'eightonthefirstdayoftestsubstanceadministrationcouldbeusedasthestatisticalcovariate,not
thebodyw'eightatnecropsy.
Dosage
34. Inordertoestablishwhetheratestsubstancecanhaveandrogenicactioninvivo,twodosegroups
ofthetestsubstancepluspositiveandvehicle(negative)controls(Seeparagraph3)arenormallysufficient,
andthisdesignisthereforepreferredforanimalW'elfarereasons.Ifthepurposeiseithertoobtainadose-
responsecurveortoextrapolatetolow'crdoses,atleast3dosegroupsarcneeded.Ifinformationbeyond
identificationofandrogenicactivity(suchasanestimateofpotency)isrequired,adifferentdosingregime
sahnodurlodgebneacgoonnsiisdte.reAd.mTionitemsutmfoorfa3nttieasntdgrroogeunpss,wtihtehtedsitffseurbesnttadnocseesisoafdmtihneitsetsetrcehdetmoigceatlhearnwd'iathpoasirteifveereanncde
anegativecontrol(Seeparagraph44)shouldbeused. Exceptfortreatmentwiththetestsubstance,
animalsinthecontrolgroupshouldbehandledinanidenticalmannertothetestgroupsubjects. Ifa
vehicleisusedinadministeringthetestsubstance,thecontrolgroupshouldreceivethevehicleinthe
highestvolumeusedw'iththetestgroups.
35. Alldoselevelsshouldbeproposedandselectedtakingintoaccountanyexistingtoxicityand
(fitrosxtictoa-k)ekiinnteoticcodnastidaearvaatiiloanbltehefoLrDth5e0taensdt/osrubasctuatnecetooxricrietlyatiendfmoartmeartiiaolns.inThoredehrigthoesatvodiodsedelaetvehl,ssheovuelrde
sufferingordistressintheanimals(17)(18)(19)(20)and,second,takeintoconsiderationavailable
informationonthedosesusedinsubchronicandchronicstudies.Ingeneral,thehighestdoseshouldnot
causeareductioninthefinalbodyweightoftheanimalsgreaterthan10%ofcontrolweight.Thehighest
tdooxsiecitsyhoourlddisbteresesitthoetrhe1)antihmealhsigahfetsetr1d0osceontsheactuteinvseurdeasysanoifmaadlminsuirsvtirvaatlioannudptthoatamisaxwiimtaholudtosseigoniffi1c0a0n0t
mg/kg/day(Seeparagraph36)or2)adoseinducing(anti)androgeniceffects,whicheverislower. Asa
screen,largeintervals,e.g.,onehalflogunits(correspondingtoadoseprogressionof3.2)orevenonelog
units,betweendosagesarcacceptable.Iftherearcnosuitabledataavailable,arangefindingstudy(See
paragraph37)maybeperformedtoaidthedeterminationofthedosestobeused.
Limitdoselevel
36. Ifatestatthelimitdoseof1000mg/kgbodyweight/dayandalow'crdoseusingtheprocedures
describedforthisstudy,failstoproduceastatisticallysignificantchangeinreproductiveorganw'eights.
7 ©OCDE,(2009)
441 OECD/OCDE
thenadditionaldoselevelsmaybeeonsidcredunneeessary. Thelimitdoseappliesexceptwhenhuman
exposuredataindicatetheneedforahigherdoseleveltobeused.
Considerationsforrangefinding
3a7p.propriatIefndeocseessgarroyu,psap[rmeoldiimfiineadryOEraCnDgegfuiinddeilnignesstufdoyraccauntebetocxaircriiteydtoeusttiwnigt(hTaGf4e2w0,anTiGma4l2s3t,oTseGle4c2t5)t]h.e
TheobjectiveinthecaseoftheHershbergerBioassayistoselectdosesthatensureanimalsurvivalandthat
arewithoutsignificanttoxicityordistresstotheanimalsaftertenconsecutivedaysofchemical
aOdEmiCnDistGruaitdiaonncuepDtoocuamleinmitt(d1o7s)emoafy1b0e00usmegd/dkegf/idniansgncloitneidcalinsipganrsaginrdaipchasti3v5eoafndtox3i6c.ityInorthdiisstrreessspetcottahne
animals.Iffeasiblewithinthisrangefindingstudyaftertendaysofadministration,thetargettissuesmay-
beexcisedandw'eighedapproximately24-hoursafterthelastdoseisadministered.Thesedatacouldthen
beusedtoassisttheselectionofthedosesinthemainstudy.
Referencesubstancesandvehicle
38. ThereferenceandrogenagonistshouldbeTestosteronePropionate(TP),CASNo57-82-5.The
referenceTPdosagemaybeeither0.2mg/kg-b\v/dor0.4mg/kg-bw/d.Thereferenceandrogenantagonist
sFhTouslhdoublodFbleutcaomaiddmcin(iFsTt)e,reCdAwSitNhoth1e3r1e1f-e8r4e-n7c.eTTPhedorseafgeer.enceFTdosageshouldbe3mg/kg-bw/d,andthe
39. Itisrecommendedthat,whereverpossible,theuseofanaqueoussolution/suspensionbe
consideredfirst.However,sincemanyandrogenligandsortheirmetabolicprecursorstendtobe
holyidvreopohilo)b.ic,Tetshtesmuobssttacnocemsmocannabpeprdoiascsholisvetdouinseaamsionliumtiaoln/asmuospuenntsioofni9n5o%ile(tc.hga.noclomo,rpoetahneurt,apspersoaprmieatoer
solventsanddilutedtofinalworkingconcentrationsinthetestvehicle. Thetoxiccharacteristicsofthe
solventshouldbeknowm.andshouldbetestedinaseparatesolvent-onlycontrolgroup. Ifthetest
substanceisconsideredstable,gentleheatingandvigorousmechanicalactioncanbeusedtoassistin
dissolvingthetestsubstance.Thestabilityofthetestsubstanceinthevehicleshouldbedetermined.Ifthe
testsubstanceisstableforthedurationofthestudy,thenonestartingaliquotofthetestsubstancemaybe
prepared,andthespecifieddosagedilutionsprepareddailyusingcaretoavoidcontaminationandspoilage
ofthesamples.
Administrationofdoses
40. TPshouldbeadministeredbysubcutaneousinjection,andFTbyoralgavage.
41. Thetestsubstanceisadministeredbyoralgavageorsubcutaneousinjection.Animalwelfare
considerationsandthephysical/chemicalpropertiesofthetestsubstanceneedtobetakenintoaccount
w'henchoosingtherouteofadministration. Inaddition,toxicologicalaspectsliketherelevancetothe
humanrouteofexposuretothechemical(e.g.oralgavagetomodelingestion,subcutaneousinjectionto
modelinhalationordermaladsorption)andexistingtoxicologicalinformationanddataonmetabolismand
kinetics(e.g.needtoavoidfirstpassmetabolism,betterefficiencyviaaparticularroute)shouldbetaken
intoaccountbeforeextensive,long-termtestingisinitiatedifpositiveresultsareobtainedbyinjection.
42. Theanimalsshouldbedosedinthesamemannerandtimesequencefortenconsecutivedaysat
approximately24hourintervals.Thedosagelevelshouldbeadjusteddailybasedontheconcurrentdaily-
mdeaaysoufreesxpoofsubroed.yCwaerieghsth.ouTlhdebevotlaukmeneionfodrodseeranontdttoimeexcteheadtittheismaadmxiinmisutmerdeodseshdoeuslcdribbeedreicnopradreadgornapeha3c5h
toallowameaningfulinterpretationofthedata. Reductionofbodyweight,clinicalsigns,andother
findingsshouldbethoroughly-assessedinthisrespect.Fororalgavage,astomachtubeorasuitable
©OCDE,(2009) 8
OECD/OCDE 441
intubationcannulashouldbeused.Themaximumvolumeofliquidthatcanbeadministeredatonetime
dependsonthesizeofthetestanimal.Localanimalcareguidelinesshouldbefollowed,buttbevolume
shouldnotexceed5ml/kgbodyweight,exceptinthecaseofaqueoussolutionswhere10ml/kgbody-
weightmaybeused.Forsubcutaneousinjections,dosesshouldbeadministeredtothedorsoscapularand
orlumbarregionsviasterileneedle(e.g.23-or25-gauge)andatuberculinsyringe.Shavingtheinjection
siteisoptional. Anylosses,leakageattheinjectionsiteorincompletedosingshouldbereeorded.The
totalvolumeinjectedperratperdayshouldnotexceed0.5ml/kgbodyweight.
Specificproceduresforandrogenagonists
t4h3e.positivFeorcotnhterotle.stBfioorlaongdicraolgeanctaigvointyisctosn,stihsetevnethwiciltehiasntdhreogneegnataigvoenicsotnstrioslt,esatneddtbhyeaTdPm-itnriesatteerdigngroauptesits
substancetotreatmentgroupsattbeselecteddosesfor10eonsecutivedays.Theweightsofthefivesex
accessorytissuesfromthetestsubstancegroupsarecomparedtothevehiclegroupforstatistically
significantincreasesinweight.
Specificproceduresforandrogenantagonistsand5a-reductaseinhibitors
44. Forthetestforandrogenantagonistsand5a-reductaseinhibitors,theTP-treatedgroupisthe
negativecontrol,andthegroupcoadministeredwithreferencedosesofTPandFTisthepositivecontrol.
Biological activityconsistentwithandrogenantagonistsand5a-reductasc inhibitorsistestedby-
administeringareferencedoseofTPandadministeringthetestsubstaneefor10eonsecutivedays.The
weightsofthefivesexaecessory-tissuesfromtheTPplustestsubstancegroupsarecomparedtothe
referenceTP-onlygroupforstatisticallysignificantdecreasesinw-eights.
OBSERVATIONS
Clinicalobservations
4o5f.toxicityGeanreeraolbscelrivneidca.lOobbsseerrvvaattiioonnsssshhoouullddbbeemcaadrreieadtolueatstproenfceeraabldyayatatnhdemsoarmeeftriemqeu(esn)tleyawc-hhednaysiagnnds
consideringtheperiodofanticipatedpeakeffectsafterdosing.Allanimalsshould beobservedfor
mortality,morbidity-andgeneralclinicalsignssuchaschangesinbehaviour,skin,fur,eyes,mucous
membranes,occurrenceofsecretionsandexcretionsandautonomicactivity-(e.g.lacrimation,piloerection,
pupilsize,unusualrespiratory'pattern).
46. Anyanimalfounddeadshouldberemovedanddisposedofw'ithoutfurtherdataanalysis.Any-
mortalityofanimalspriortonecropsyshouldbeincludedinthestudyrecordtogetherwithanyapparent
reasonsformortality. Anymoribundanimalsshouldbehumanelyterminated. Anymoribundand
subsequentlyeuthanizedanimalsshouldbeincludedinthestudy-recordwithapparentreasonsfor
morbidity-.
Bodyweightandfoodconsumption
t4c7ro.enastumemnetdAil.dcl.u,rawin'nihgmeantlhtsehestrhaeonauitlmmdaelnsbtearpweeeraiilgoldhoecmadtaedydaiiblneytotmgoeraotushpuesr.enedAasrpeeasrntoe0pa.tg1ieogn,bayl-stmwae-raetsiinugghriejnmugestntthp,eritfohereedtaeomrosi.unnitTtihaoetfioffnoooooddf
consumptionresultsshouldbeexpressedingramsperratperday.
9 ©OCDE,(2009)
441 OECD/OCDE
Dissectionandmeasurementoftissueandorganweights
48. Approximately24hoursafterthelastadministrationofthetestsubstance,theratsshouldbe
euthanizedandexsanguinatedaccordingtothenormalproceduresoftheconductinglaboratory',and
necropsycarriedout.Themethodofhumanekillingshouldberecordedinthelaboratoryreport.
49. Ideally,thenecropsyordershouldberandomizedacrossgroupstoavoidprogressiondirectlyup
ordowndosegroupsthatcouldaffectthedata.Anyfindingatnecropsy,i.e.,pathologicalchanges/visible
lesionsshouldbenotedandreported.
50. Thefiveandrogen-dependenttissues(VP,SV,LABC,COW,GP)shouldbeweighted. These
tissuesshouldbeexcised,carefullytrimmedofexcessadheringtissueandfat,andtheirfresh(unfixed)
weightsdetermined.Eachtissueshouldbehandledwithparticularcaretoavoidthelossoffluidsandto
avoiddesiccation,whichmayintroducesignificanterrorsandvariabilitybydecreasingtherecorded
weights.Severalofthetissuesmaybevery'smallordifficulttodissect,andthiswillintroducevariability.
wTdiihsetsrheecfstotiraoenn,daiisrtdaivsdaiiismlspaeboclrtetiaofnnrtoptmrhoatctheepdeuOrrEseosCnDsforc(a2tr1hr)ye.'sienCgtairoseusftuuetlsh.terAadiinssistneagcntadicaocrndoorofdpitenhrgeattsioenxtghaepcrcSoeOcsePsdougrruyeitdi(esSswOuiPels)lammriaennfiuamamilizleifaoarr
potentialsourceofvariationinthestudy. Ideallythesameprosectorshouldberesponsibleforthe
dissectionofagiventissuetoeliminateinter-individualdifferencesintissueprocessing.Ifthisisnot
possible,thenecropsyshouldbedesignedsuchthateachprosectordissectsagiventissuefromall
treatmentgroupsasopposedtooneindividualdissectingalltissuesfromacontrolgroup,whilesomeone
elseisresponsibleforthetreatedgroups.Eachsexaccessory'tissuesshouldbeweighedwithoutblottingto
thenearest0.1mg,andtheweightsrecordedforeachanimal.
P5r1e.viouswSoevrekrahlasofintdhiecatitsesdueasrmanagyeboefvcoeerfyfiscmiaelnltoorfvdiafrfiiactuilotntso(dCiVssse)ctt,haatnadpptheiasrswitloldiinftfreordbuacseevdaruipaboinlitthye.
parsotfhieciVePncaynodfCthOeWlSabohraavtoeryb.eenInoabsfeerwvecdasweist,hilnaragepadritfifceurleanrcleasboirnattohrey'a.bsoluteweightsofthetissuessuch
52. Liver,pairedkidney,andpairedadrenalweightsareoptionalmeasurements.Again,tissues
shouldbetrimmedfreeofanyadheringfasciaandfat.Thelivershouldbeweighedandrecordedtothe
nmega.resTthe0.l1igv,era,nkdidtnheeypaainrdedadkriednnaelyssaarnednoptaiorneldyadirnefnlaulesncsehdobulydabnedrwoegiegnhs;edthaenydarlescooprrdoevditdoetuhseefnuelariensdtic0e.1s
ofsystemictoxicity.
53. Measurementofserumluteinisinghormone(LH),follicularstimulatinghormone(FSH)and
tmveiesattaaobsnotelarniotsnimeano(dfTr)toegsiestnoisoctptemireoonncaehl,a.nloiSwsc'mer.ruimnLgTHsellreeuvveemllsslepvaerrlocsv.iudsWeefsiutlihnotfuootrdmteahtteeiroTnmidanabetoa,uitfsutthhceehatabenislteitfsyfuebocstftmaainngcahenttiainapdnpudecraeorsgtelonivbteeor
notonlyreduceorganweights,butalsotoaffecthypothalamic-pituitaryfunction,whichinlongterm
studiescaninducetestistumors.FSHisanimportanthormoneforspermatogenesis.SerumT4andT3also
arcoptionalmeasuresthatwouldprovideusefulsupplementalinformationabouttheabilitytodisrupt
thyroidhonnonchomeostasis.Ifhormonemeasurementsarctobemade,theratsshouldbeanesthetized
priortonecropsyandbloodtakenbycardiacpuncture,andthemethodofanesthesiashouldbechosenwith
caresothatitdoesnotaffecthormonemeasurement.Themethodofserumpreparation,thesourceof
rreacdoirodiemd.muLnHoaslesvaeylsosrhooutlhderbemreeapsourtreedmeasntngkiptes,rmtlheofansaelryatmi,caalndprTocsedhuoruelsd,alasnodbethreeporrestueldtsassnhgoupledrmble
ofserum.
©OCDE,(2009) 10