OECD/OCDE 493 Adopted; 28July2015 OECDGUIDELINEFORTHETESTINGOFCHEMICALS Performance-BasedTestGuidelineforHumanRecombinantEstrogenReceptor (hrER)InVilroAssaystoDetectChemicalswithERBindingAffinity GENERALINTRODUCTION Performance-BasedTestGuideline 1. ThisPerfonnance-BasedTestGuideline (PBTG)describesthemethodolog>'forhuman recombinantinvitroassaystodetectsubstanceswithestrogenreceptorbindingaffinity(hrERbinding assays).Itcomprisestwomechanisticallyandfunctionallysimilartestmethodsfortheidentificationof mesettrhoogdesnrinceaeepetoorrd(ain.ee.eEwRiat)hbthiendperrisncainpdlesshfoourlvdaflaiedialtiitoatnestehtefodretvheilnopthmeenOtEoCfDneGwuisdiamnilcaerDooremuomdeifnited(GtDes)t ontheValidationandInternationalAeceptanceofNeworUpdatedTestMethodsforHazardAssessment (P1B).TTGhearef:ullyvalidatedreferencetestmethods(Annex2andAnnex3)thatprovidethebasisforthis • TheFreyberger-Wilson(FW)InVitroEstrogenReceptor(ER)BindingAssayUsingaFull LengthHumanRecombinantERa(2),and • TheChemicalEvaluationandResearchInstitute(CERI)InVitroEstrogenReceptorBinding AssayUsingaHumanRecombinantLigandBindingDomainProtein(2). mPeertfhoordmsafnocretshetasnadamredsha(zPaSr)d(e3n)daprocinatvaainldaballeltoowffaocriltiitmateelythaemdeenvdemleonptmeonftthainsdPvBaTliGdastoiotnhaotfnseimwilsairmitleasrt testmethodscanbeaddedtoanupdatedPBTG.However,similartestmethodswillonlybeaddedafter reviewandagreementthatperformancestandardsaremet.ThetestmethodsincludedinthisTest Guidelinecanbeusedindiscriminatelytoaddresscountries’requirementsfortestresultsonestrogen receptorbindingwhilebenefitingfromtheMutualAcceptanceofData. 1 ©OECD,(2015) Youarefreetousethismaterialforpersonal,non-commercialpurposeswithoutseekingpriorconsent fromtheOECD,providedthesourceisdulymentioned.Anycommercialuseofthismaterialissubjectto writtenpermissionfromtheOECD. 493 OECD/OCDE BackgroundandprinciplesofthetestmethodsincludedinthePBTG 2G.uidelinesTheforOEtCheDsicnriteieantiendgaahingdh-ptreisotriintgy-oafctipvoitteyntiinal199e8ndtoocrrienveisediesxirsutpitnign,ganchdetmoicdaelvse.loTphneewO,ETCesDt conceptualframew-ork(CF)fortestingandassessmentofpotentialendocrinedisruptingchemicalswas revisedin2012.TheoriginalandrevisedCFsarcincludedasAnnexesintheGuidanceDocumenton fdeSientvsdaecnordcliarebvrieednldiess,emidneecaThtcehahisnstilesGPvmue(BlisTd)ce/Golpriarnateerhssewpaoflynoe(rdvsie)Eln.vga2lt,TuohawiathsidiniPcgfhBfCeThrieGennmctiilsucldeafveloesrsli‘fon'ofirvnbiEtivronioltdororgoceirccaieaspnlsteaocyroDsmibpsiplrnreudoxpivitntiigydo.intneTs(gt4h)edm.aeETtthaRheoabdCbisonFduditecnsogismgepanlrseeiscdsateytessod identifyligandsforthehumanestrogenreceptoralpha(ERa). 3d.emonstratTehde.rEelRevbainncdeingofastshaeysinarevidterosigEnRedbtioniddienngtifaysscahyemtiocablisoltohgaitchaalvefutnhcetipoontsenthiaasltboedeinsrculpetatrhley estrogenhormonepathway,andhavebeenusedextensivelyduringthepasttw'Odecadestocharacterise ERtissuedistributionaswellastoidentifyERagonists/antagonists.Theseassaysreflecttheligand- receptorinteractionwhichistheinitialstepoftheestrogensignalingpathwayandessentialfor reproductionfunctioninallvertebrates. 4. TheinteractionofestrogenswithERscanaffecttranscriptionofestrogen-controlledgenesand inducenon-genomiceffects,whichcanleadtotheinductionorinhibitionofcellularprocesses,including tPheorsteurbnaetcieosnsaorfynfoorrmcaelllesprtorloigfeenriactiosny,stneomrsmamlayfethaalvdeevtheeloppomteenntti,alantdotrreipgrgoedrucatdivveersfeunecftfieocnts(5o)n(n6o)rm(7a)l. development (ontogenesis), reproductive health and the integrity ofthe reproductive system. InappropriateERsignalingcanleadtoeffectssuchasincreasedriskofhormonedependentcancer, impairedfertility,andalterationsinfetalgrowthanddevelopment(8). 5li.gandbinIdninvigtrsoitbeitnhdaitngreagsuslaatyessatrheebgaesneedotrnanascdriirpetcitoni.ntTehraectkieoynocfomapsounbesnttancoeftwhiethhuamsapencirfeiccormebcienpatnotr eecsostmtrpraeodtgiieotnlo)rr)et.oceTpbetiosnrtdcahwleipmthihaca(tlhhserEtERhaaRt)pibonisnstdehisesngaprahesissgeahnycaefmfeioanfistuyirnfecosrrettahhseeinEagbRiclciotnoycmeponefttreaatwriiaotdnhisotlohafeberalaeddtieoslltiagbcaehnledemdi(lc[ia’glHaJn1(d7i.Pae-t. alow-erconcentrationascomparedwiththosechemicalswithloweraffinityforthereceptor.Thisassay- cinotnesriascttsioonfpatrwaomemtearjsorancdomdpoocnuemnetnst:EaRssapteucriaftiicointy,bifnodlilnogwedexbpyeraimceonmtpettoiticvhearabcitnedriinsgeerxepceerpitmore-nltigtahnadt characterisesthecompetitionbetweenatestchemicalandaradiolabeledligandforbindingtotheER. 6. ValidationstudiesoftheCERIandtheFWbindingassayshavedemonstratedtheirrelevanceand reliability-fortheirintendedpurpose(2). 7. DefinitionsandabbreviationsusedinthisTestGuidelinearedescribedinAnnex1. Scopeandlimitationsrelatedtothereceptorbindingassays 8. Thesetestmethodsarebeingproposedforscreeningandprioritisationpurposes,butcanalso provideinformationforamolecularinitiationevent(MIE)thatcanbeusedinaweightofevidence approach.TheyaddresschemicalbindingtotheERaligandbindingdomaininaninvitrosystem.Thus, 2 ©OECD,(2015) OECD/OCDE 493 resultsshouldnotbedireetlyextrapolatedtothecomplexsignalingandregulationoftheintactendocrine systeminvivo. 9. Bindingofthenaturalligand,17(5-estradiol,istheinitialstepofaseriesofmoleculareventsthat baecintndidovicanrtgiensettohdetihsterruapEntsRicoarnipl(tiEigoDan)n,odfabtliatnrhdgoeiutnggghedtnoheemsraeainnadrciuslottcihomenarsteimldeye,crhecadunloimnisenmastoeftshtrwhoieutghkheaywphhmiyecscihhoalEonDgiisccmaaslncoohcfacnEurgR,eim(n9e)cd.liuTadhtiuensdg (i)interactionswithsitesofERaotherthantheligandbindingpocket,(ii)interactionswithother receptorsrelevantforestrogensignaling,ERpandG-proteincoupledestrogenreceptor,otherreceptors andenzymaticsystemswithintheendocrinesystem,(iii)hormonesynthesis,(iv)metabolicactivation hanodr/moorneisnacftriovmattihoenboofdyh.orNmoonneeso,ft(hv)etdeissttmriebtuhtoiodnsoufndheorrtmhoisnePsBTtoGtaadrdgreetsstitshsueesse,maonddes(voif)acctlieoanr.anceof 10. ThisPBTGaddressestheabilityofsubstancestobindtohumanERaanddoesnotdistinguish betw'eenERaagonistsorantagonists.Thisassaydoesnotaddresseitherfurtherdownstreameventssuch asgenetranscriptionorphysiologicalchanges.Consideringthatonlysinglesubstanceswereusedduring thevalidation,theapplicabilitytotestmixtureshasnotbeenaddressed.Thetestmethodisnevertheless theoreticallyapplicabletothetestingofmulti-constituentsubstancesandmixtures.BeforeuseoftheTest Guidelineonamixtureforgeneratingdataforanintendedregulatorypurpose,itshouldbeconsidered whether,andifsow'hy,itmayprovideadequateresultsforthatpurpose.Suchconsiderationsarenot needed,whenthereisaregulatoryrequirementfortestingofthemixture. i1n1.combinaTthieoncelwlitfhreemerteacbeopltiocrseynsztyemmeshsayvsetenmso.inHtorwi'nesivcerm,etiatbmoilgihctcabpeabpiolsistiyblaendtothienycowr'peorerantoetmveatlaibdoaltiecd activityinastudydesignbutthiswouldrequirefurthervalidationefforts. t1h2a.tcancChhaenmgiecatlhsetphHatomfatyhedeansastauyrebutfhfeerp,romtaeiynn(oi.te.breecteepsttoerdporrotmeiany),osnulcyhbaestseusrtfeadctaatntcoonrcecnhtermaitciaolnss devoidofsuchinteractions.Otherwise,theconcentrationrangethatcanbetestedintheassaysforatest chemicalislimitedbyitssolubilityintheassaybuffer. tc1el3sa.stseidfiiendFbaoosrthEiRnoffboirtnhmdeaetrifsuolnlaaynldvpa6ulriapdsoastneeosdn.-tbeTsiatnbdlmeeertsh1bopadrssoevddiedusepcsorintbhepedubtliesnitsthrheeidssulrPteBspoTrfGtos.r,tOihnfeclt2uh4deisnsegubsisuntbasvnticaternosceasts,hsaat1y7wseafrroeer tEchlReasstdirafatinacsacstruiiompntmsiaoroniaflsaealdlcttiihnveaTtaisobunlbesatna1d,n/ctoehrsetruheepwu'ttaoesr1oa0tlr"mo‘opMsh,tica1na0ds0s%eaayca(hg9r)seu(eb1m0se)tnat(n1cb1e)etw(w'1ea2se)nc(o1tr3hr)eec(tt1wl4oy)t(ce1ls5at)s.smiIefnitehrdoefdaessreaonnncetEhtReo bsuibnsdtearncoersntoens-tbeidndienr.thSeupEplRembeinntdairnygitensftormmeattihoondsondutrhiinsggtrhoeupvaolfidastuibosntanscteudsieassiwselplrovasideadddiitniontahle PerformanceStandardsforthehrERbindingassay(3),Annex2(Tables1,2and3). ©OECD,(2015) ' 493 OECD/OCDE FWanTdabClEeR1I:hCrlaEsRsiBfiicnadtiinognAosfssauybsstwaintchescoamspEarRisboinndweirtshoerxnpoenc-tbeidnrdeesrpsonwsheentestedinthe SubstanceName CRANS REexspepcotnesde CoRncaenngtera(FtMiW)onAssCalyassification CoRnacenngterCa(tEMiR)oInAsCslaayssification ChMCeElmaSiscsHal ProductClass 1 np-Estradiol 50-228- Binder Ixl0'‘-lxl0'^ Binder 1x10”'-1x10” Binder Steroid PhaVremtAaegcreeiunnttaircyal, 2 Norcthynodrel 68-523- Binder 3x10”-30x10'’ Binder 3x10”-30x10” Binder Steroid PhaVremtAaegcreeiunnttaircyal, 3 Norethindrone 68-422- Binder 3x10”-30x10” Binder 3x10”-30x10” Binder Steroid PhaVremtAaegcreeiunnttaircyal, 4 Di-w-butylphthalale 84-274- biNnodne-r* Ixl0"’-lxl0” BiNnodenr-^ lxl0-‘“-1x10-^ BiNnodenr- (Hcyycdlrioc)c,arEsbtoenr IPnCltahesertmmicieidcziaealrt,e 5 DES 56-153- Binder lxl0'“ 1x10' Binder 1x10”"-1x10” Binder Hy(dCPryhoceclniaocrl)b,on PhaVremtAaegcreeiunnttaircyal, 6 17a-ethynylestradiol57-663- Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder Steroid PhaVremtAaegcreeiunnttaircyal, 7 Meso-IIexeslrol 84-216- Binder IxlO’" 1x10-’ Binder 1x10-'“ IxlO'^ Binder Hy(dPCryhoceclniaocrl)b,on PhaVremtAaegcreeiunnttaircyal, 8 Genistein 47426--0 Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder (HhyeFtdlerarovooccynacorlibidco)n, NaturalProduct 9 Equol 9553-13- Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder PhMyettoaebsotlriotgeen NaturalProduct 10 hByudtryolbxuvptbayler-na4zb-oeant(en) 94-826- Binder IxlO’" 1x10-’ Binder 1x10-'°-IxlO'^ Binder Paraben Preservative 11 No(nmyilxptuhreen)ol 841855-32- Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder Alkylphenol ICntoemrpmeoduinadte 12 o.p’-DDT 0728-96- Binder 1x10“ 1x10” Binder 1x10”"-1x10” Binder Organochlorine Insecticide 13 Corticosterone 50-622- biNnodne-r* 1x10“-1x10” Non-binder 1x10”"-1x10” Non-Binder Steroid NaturalProduct 14 Zearalenone 19729-244- Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder (HhyetdLerarocotccyoacnrlebioc)n. NaturalProduct 15 Tamoxifen 12095-410- Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder Hyd(rCoyccalircb)on, PhaVremtAaegcreeiunnttaircyal, 16 dihydrote5sat-osterone 51281--6 Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder NonSptehreoniodl,ic NaturalProduct 17 BisphenolA 80-705- Binder 1x10“-1x10” Binder 1x10”"-1x10” Binder Phenol InCtheermmiedciaalte 18 4-rt-heptylphenol 15908-74- Binder 1x10“-1x10” Equivocal 1x10”"-1x10” Binder Alkylphenol Intermediate ©OECD,(2015) OECD/OCDE 493 19 (ChKloerpdoenceone) 5104-30- Binder 1x10“-IxlO’ Binder lxl0‘“-IxlO*’ Binder (HHyadlroogceanrabtoend,) Pesticide 20 Benz(a)anthracene 56-355- BNionnde-r 1x10'^®-1x10'^Non-Binder’’ IxlO''”-1x10'^ Non-Binder’’ HyAdrroomcaatribcon Intermediate 21 Enterolactone 78741-793- Binder 1x10“-1x10’ Binder 1x10'“-IxlO"’ Binder Phytoestrogen NaturalProduct 22 Progesterone 57-083- bNmodne-r* 1x10“-IxlO'* Non-Binder 1x10“-1x10^ Non-Binder Steroid NaturalProduct 23Octyltriethoxysilane 27954-31- bNionnde-r 1x10“-1x10’ Non-Binder 1x10"”-1x10“ Non-Binder Silane MSoudriffaiceer 24 Atrazine 12941-29- biNnodne-r* 1x10“-IxlO” Non-Binder 1x10”-1x10“ Non-Binder Hectoemrpocoyucnldic Herbicide ^’^*CTtLbalDlhahiemuaebmeebrsosinisireutigansofctuogebiooofscnratueaidshnstroeiedlistvcuoivecubnuoadlsinrlaliwsvitsina,odestgaisybsftftiei<iotnhc.omoeianltentFFxissocooltWonrlaunbudotsoabyhfirsl.esaesdsttdaieeaoiy-tnr-nrtiw(-lue-sbnOwsbeusi’uet.ott^>Rb\hdDMedsBfep(epeehrxF.hrtvpgtheee.whancdaaltctluaseeeartidtbetc(ieh2lcd()eali(,rDsatsDsBySaisB)FefiFdci)fiete)indcicaroasswatneoasiasamsos‘nneIe.eotVqeln.iuCsa-nBlitbb.aveori3sdoranascadda,iatisfeblooir'rlacaiiwaneogctasrdaoisndod‘VdnubeIbri.daboniAsifdn)teneg.e4rdds-'ittnohni-snegnhupteba3retps/sett5tt-yahivlnlenaacpglbehhiouicdrapgnatahotttelicoosoortannilsOcees'es"to‘n'uneutdcMqsricuieanisntbtv.gierooacctntaahsileuo’suCnepoE(rttRh1oI‘e0n'soau^nsb-)ssbatiayanndandecnU/redon’rdh5eaab/rdny6 3/5labsresultedinanaverageclassificationofequivocal.Closerinspectionrevealedthatthiswasduetochemicalsolubility ^ldaaeDsicxeumtpmriiueotvirsnaaniesttgmdtiieroonnanitstnth(aeit1ltnh7hgavic)tasolt(pnih1idrda8nieat)ttvt.eieirtono-hEnntvenseazldsifiytnotdmurhadavtetytiih,tpioerrncobFoedsmWentuesuzcttd(atayrainb.o)oodganleTnCinhotciEuhfcRsraaa,Icactfcueitatlvnhsilaeesvtabiiytciwosyonan.rdsrrioeenfcpgrtotehrccucetlileavassd'ssuesib.fifsofitrieacdnattchaieisosnwasofuuonbrlosdtnta-hninboscitensdbuee(b1rsa6tn)(atini.ieccs.ieppnairetsigemadaatrii‘invnletoy)hne-dbbecaiepsnledelneddfrr’eoenentwhhpureupEnboRlniubsshiieetndsddimunlengitdtaeearbsraostlatuyihrscee hrERBINDINGTESTMETHODCOMPONENTS EssentialTestMethodComponents 14. ThisPBTGappliestomethodsusinganERreceptorandasuitablystrongligandtothereceptorthat canbeusedasamarker/tracerfortheassayandcanbedisplacedwithincreasingconcentrationsofatest chemical.Bindingassayscontainthefollowingtwomajorcomponents: 1)saturationbindingand2) competitivebinding.Thesaturationbindingassayisusedtoconfirmthespecificityandactivity^ofthe receptorpreparations,whilethecompetitivebindingexperimentisusedtoevaluatetheabilityofatest chemicaltobindtohrER. Controlsubstances 15. Thebasisfortheproposedconcurrentreferenceestrogenandcontrolsshouldbedescribed. Concurrentcontrols(solvent(vehicle),positive(ERbinder;strongandweakaffinity),negative(non- binder)),asappropriate,serveasanindicationthatthetestmethodisoperativeunderthetestconditions andprovideabasisforexperiment-to-experimentcomparisons;theyareusuallypartoftheacceptability criteriaforagivenexperiment(1). Fullconcentrationcurvesforthereferenceestrogenandcontrols(i.e. weakbinderandnon-binder)shouldbeusedinoneplateduringeachrun.Allotherplatesshouldcontain:1) ahigh-(approximatelyfulldisplacementofradiolabeledligand)andmedium-(approximatelytheIC50) concentrationeachofE2andweakbinderintriplicate;2)solventcontrolandnon-specificbinding,eachin triplicate. ©OECD,(2015) 493 OECD/OCDE StandardQualityControlProcedures 16. Standardqualitycontrolproceduresshouldbeperformedasdescribedforeachassaytoensure aetivereceptors,tbecorreetchemicalconcentrations,toleranceboundsremainstablethroughmultiple replications,andretaintheabilitytoprovidetheexpectedER-bindingresponsesovertime. DemonstrationofLaboratoryProficiency 17. PriortotestingunknownchemicalswithanyofthetestmethodsunderthisPBTG,each lceasobtnorfroiagrtemonrsyapnescdhifociuoclnidttryodlaesnmdo(nwsacettraiakvtietbyinpodrfeorftihaceinedEnRcnyopnri-nebpiaunrsdaietnri)go.nt,Aheanhtdiesstctoormimpceeattlhidtoiadvteabbyabsienpdeiwrnifgtohramsriesnsaguylstssawtifutorhrattithohenerareesffseearryeesnnccteeo estrogenandcontrolsgeneratedfrom3-5independentexperimentsconductedondifferentdaysshouldbe establishedbythelaboratory'.Theseexperimentswillbethefoundationforthereferenceestrogenand historicalcontrolsforthelaboratoryandwillbeusedasapartialassessmentofassayacceptabilityfor futureruns. 18. Theresponsivenessofthetestsystemwillalsobeconfirmedbytestingtheproficiencysubstances tlPeihxeseprtefeccdoltraiesmndsaenTscafeoborlfSectEha2eR.nmdTiabchriadenlsdslifinsoctgrootmf(hmie.operE.nolRfsyitbcriaionsensdngoiccnyigtaosatusebwsd'saetywaasiknt)c(he3)saE.niRdTshbaeniosnsnedu-ibbsnsiuegnbtdsaetocratfsnivtcihe(teisy.,era.erefexnehecrigeobanmitctimeveearssc)usi.buaislttlaaFbynolceraevsareiaalpncarghboelvepio,rdfoerfedpipocritieneesnntecchnyeyt substance,concentrationstestedshouldcovertherangeprovidedinTable2.Atleastthreeexperiments shouldbeperformedforeachsubstanceandresultsshouldbeinconcordancewithexpectedchemicalactivity. Eachexperimentshouldbeconductedindependently(i.e.withfreshdilutionsofreceptor,chemicals,and reagent),withthreereplicatesforeachconcentration. Proficiencyisdemonstratedbycorrectclassification (positive/negative)ofeachproficiencysubstance.Proficiencytestingshouldbeperformedbyeachtechnician whenlearningthetestmethods. 6 ©OECD,(2015) - OECD/OCDE 493 Tahle2:ListofcontrolsandproficiencysnbstancesforthehrERcompetitivebindingassays.' No. SubstanceName CASRN' REexsppeocnstee^d’* cornacneTgneetsrt(aMt)ion chemiMceaSlHclass^ Productclass^ Controls(Referenceestrogen,weakbinder,non-binder) 1 17p-estradiol 50-28-2 Binder 1x10“-lxl0‘“ Steroid VPehtaerrmiancaeruytiacgaeln,t 2 NNoorreetthhyinnoddrroenle(or) 686-82-3-252-(4or) Binder 3xl0‘’-30xl0‘‘ Steroid VPehtaerrmiancaeruytiacgaeln,t 3 Octyltriethoxysilane 2943-75-1 Non-binder IxIO'"’-l.xlO'^ Silane Surfacemodifier Proficiencysubstances^ 4 Diethylstilbestrol 56-53-1 Binder Ixl0'”-lxl0-^ (cHyycldirco),caPrhbeonnol VPchtacrrmiancaieyu'tiacgaeln,t 5 17a-ethynylestradiol 57-63-6 Binder lxl0-"-lxl0-^ Steroid VPehtaerrmiancaeruytiacgaelnt 6 me.so-Hexestrol 84-16-2 Binder 1x10"“-lxl0‘‘ (cHyycldirco).caPrhbeonnol VPehtaerrmiancaeruytiacgaeln,t 7 Tamoxifen 10540-29-1 Binder 1x10“-lxl0"‘ Hyd(rcyocclairc)bon VPehtaerrmiancaeruytiacgaeln,t Heterocyclic 8 Genistein 446-72-0 Binder 1x10’“-1x10“ compound, Naturalproduct Flavonoid. 9 BisphenolA 80-05-7 Binder 1x10'“-1x10“ Phenol inCtheermmiedciaalte 10 Zcaralononc 17924-92-4 Binder 1x10“-1x10“ comHpeotuenrdo,cyTc.laicctone NaturalProduct 11 Butv'lparaben 94-26-8 Binder 1x10'“--1x10“ CarboPxhyelnioclacid. Preservative 12 Atrazinc 1912-24-9 Non-binder 1x10"“-1x10“ Hectoemrpocoyucnldic Herbicide 13 D(iD-BnF-)b'uty’lphthalate 84-74-2 Non-binder^ 1x10““-1x10"* HydrocarEsbtoenr(cyclic). ChemPilcasatliciinzetre,rmediate 14 Corticosterone 50-22-6 Non-binder IxlO'” 1x10-" Steroid Naturalproduct aproficiencysubstanceisnolongercommerciallyavailable,asubstancewiththesameERbindingclassification,comparable potency,andchemicalclasscanbeused. ©^AObEbrCevDia,tio(n2s0:1C5)ASRN=ChemicalAbstractsServiceRegistr7yNumber. . 493 OECD/OCDE ^“C^lEaRssbiifnidciantgionacatsivaintyEwRaasBbaisnedderuopronNotnh-ebIinCdCerVAduMriBnagctkhgerovaulniddaRteiovnieswtuDdoycfuomretnhtesCE(RBIRDa)ndfoFrWERhrBEinRdBiinngdainndgATsAsatyesst(m2e)t.hods (9)aswellasempiricaldataandotherinformationobtainedfromreferencedstudiespublishedandreviewed(10)(11)(12)(13) (^1S4u)b(s1t5a)n.ceswereassignedintooneormorechemicalclassesusingtheU.S.NationalLibraryofMedicine’sMedical h^SutSbtujpbe:sctwtawnwc.Heneslamdw.iennrieghs.asaso(ivMg/enmeSedHs)hi,n)t.oaonneionrtemronraetiopnraoldluyctrcelcaossgensizuesdingstthaendUa.rSd.izNeadtiocnlaalssLiifibcraatriyonofsMecdhiecmiene’(savHaaizlaabrldeousat:Substances D®baaDLstiPeamdBbiatoscneaonft(easbsvoetlaiuiunblsgiaelbudilpteaystafota:ohrn1t0t’ta'nhl*:its/Me/rtsnouabxbtenseccttaca.uonnnslctneeri.otilhnseinh1o0s.’ung’b-*osbvMt/i.acngndiTce-ehrhehitenausdsstieesbd.eahwentindmtohlcblgmascseansrx'ivflieiHmdeSauttDmoiBob)nceoonficnedsniot-lrnua-bbtluietovnaltopf1h01t’0h*’a'M'laM(t.ee.g(.DtBurFb)idaistya)ninons-obminedelarbohraastobreieens duringthepre-validationstudies. SolubilityTestingandConcentrationRangeFindingforTestChemicals 19. Apreliminary'testshouldbeeonduetedtodeteimincthelimitofsolubilityforeaehtestchemieal andtoidentifytheappropriateconeentrationrangetousewheneonductingthetest.Tlielimitofsolubilityof eTs1oahlcmevheMfntietnsoatclrocnclhtooernwomcelierc,naatllrboaanitsgsietowdonitbuthepesoeitinnegidhtttihi,aelnllyltoihgdmeeisttearesiorsafmlaiysdnoielslduhubtoiiiulnolintdtsyh,)en,sosttaaonrletdvxiencntgetheeaadtnptdr1heefmsumeMrnat.chxeeiromRcfauonmncglfeoiaucrdcmfieienpndetdsaeusrbnldteoeerrsctopairnnsecgsceaincpytoirntcasaotitniesdotinsntoi(otoeenf.dsg..a. Concentrationsinthesecondandthirdexperimentsshouldbeadjustedasappropriatetobettercharacterise theconcentration-responsecurve. TestRunAcceptabilityCriteria 20. Acceptanceorrejectionofatestrunisbasedontheevaluationofresultsobtainedforthe referenceestrogenandcontrolusedforeachexperiment.First,forplate1.thefullconcentrationcurvesfor thereferencecontrolsfromeachexperimentshouldmeetthemeasuresofperformancewithcurve-fit pCaErRaImeatnerdsF(We.ga.ss1aCy5s0(AannndeHxil2laslnodpc3)),baansdedtheuphiosntotrhicealrecsounlttrsolredpaotratefdrofmorthteirelarbeosrpaetcotriyvecopnrdoutcotcionlgstfhoertetshte. Allcontrols(referenceestrogen,weakbinder,andnon-binder)shouldbecorrectlyclassifiedforeach eAxpseurfifmiecnietn.tSreacnognedloyf,ctohnecceonnttrraotlisonosnoaflltshuebsteesqtuecnhtempilcataelssnheoeudldtobbeeuassesdesstoedclfeoarrclyondseifsitneenctyhweittohpploafteth1e competitivebindingcurve.Variabilityamongreplicatesateachconcentrationofthetestchemicalaswellas amongthethreeindependentrunsshouldbereasonableandscientificallydefensible. Theabilityto consistentlyconductthetestmethodshouldbedemonstratedbythedevelopmentandmaintenanceofa historicaldatabaseforthereferenceestrogenandcontrols.Standarddeviations(SD)orcoefficientsof variation(CV)forthemeansofreferenceestrogenandcontrolweakbindercurvesfittingparametersfrom multipleexperimentsmaybeusedasameasureofwithin-laboratoryreproducibility.Professionaljudgment shouldbeappliedwhenreviewingtheplatecontrolresultsfromeachrunaswellasforeachtestchemical. Inaddition,thefollowingprinciplesregardingacceptabilitycriteriashouldbemet: • DatashouldbesufficientforaquantitativeassessmentofERbinding • Theconcentrationstestedshouldremainwithinthesolubilityrangeofthetestchemical. Analysisofdata 21. Thedefineddataanalysisprocedureforsaturationandcompetitivebindingdatashouldadhereto ©OECD,(2015) 8 : OECD/OCDE 493 tahnealkyzeeydpursiinncgipalensonf-olrincehaarrarcetgerreisssiinognrmeocdeepltotrh-altigaacncdouinnttserfaocrtitoontsa.lTaynpdicnaolnl-ys,pecsiatfuircatbiionndinbgi.ndiAngcodrarteactiaroen forliganddepletion(e.g.Swillens,1995(19))maybeneededwhendeterminingBma.xandKd.Datafrom competitivebindingassaysarctopicallytransformed(e.g.percentspecificbindingandconcentrationoftest cnfiohtneplmaiirncaeaamlret(celurorgsvMea)nf)di.ttaiEnvsgitssiuomafalttweraserveoifetwolofogift(ahICof5wo0u)rwfepolralreatamhceehtbetiernstdHiiclnhlgeemdqiauctaaatlifoistnh.tohuFelodlglebnoeewridanetgteedarnmcioinnmieptdeitaulistaiinnvagelyasbniisn,adpitpnhrgeopccruuirravvteee shouldbeconducted.Insomecases,additionalanalysismaybeneededtoobtainthebestcurvefit(e.g. constrainingtopand/orbottomofcurve,useof10%rale,seeAtmex4andReference2(Section1II.A.2). 22. Meetingthe acceptabilitycriteria(paragraph20)indicatestheassaysystemis operating properly,butitdoesnotensurethatanyparticulartestwillproduceaccuratedata.Replicatingthecorrect resultsofthefirsttestisthebestindicationthataccuratedataw'ereproduced. GeneralDataInterpretationCriteria 23. ThereiscurrentlynouniversallyagreedmethodforinterpretingERbindingdata.However,both qausasleistsamtievnets(eo.fg.hrbEinRd-emre/dnioant-cbidnadcctri)viatynds/hoorulqduabnetibtaatsievdeo(ne.eg.mpliorgicIalC5d0a,tRaelaantdivseouBninddsicnigenAtfiffiicnijtuydg(mReBnAt).,etc.) TestReport 24. The test report should include the following information: Testmethod: testmethodused; Control/Reference/Testchemical - source,lotnumber,limitdateforuse.ifavailable - stabilityofthetestchemicalitself,ifknown; - solubilityandstabilityofthetestchemicalinsolvent,ifknown. - measurementofpH,osmolalityandprecipitateintheculturemediumtowhichthetestchemicalw'as added,asappropriate. Mono-constituentsubstance - physicalappearance,watersolubility,andadditionalrelevantphysicochemicalproperties; - chemicalidentification,suchaslUPACorCASname,CASnumber,SMILESorInChlcode, structuralformula,purity',chemicalidentityofimpuritiesasappropriateandpracticallyfeasible,etc. Multi-constituentsubstance,UVBCsandmixtures: - characterisedasfaraspossiblebychemicalidentity(seeabove),quantitativeoccurrenceandrelevant physicochemicalpropertiesoftheconstituents. Solvent/Vehicle: - characterisation(nature,supplierandlot); - justificationforchoiceofsolvent/vehicle; - solubilityandstabilityofthetestchemicalinsolvent/vehicle,ifknown; ©OECD,(2015) 9 0 493 OECD/OCDE Receptors: - sourceofreceptors(supplier,catalogNo.,lot,speciesofreceptor,activereceptorconcentration providedfromsupplier,certificationfromsupplier) " characterizationofreceptors(includingsaturationbindingresults):Kd,Bmax, “ storageofreeeptors - radiolabeledligand: - supplier,catalogNo.,lot,specificactivity Testconditions: - solubilitylimitationsunderassayconditions: - compositionofbindingbuffer; " concentrationofreceptor; " concentrationoftracer(i.e.radiolabeledligand); - concentrationsoftestchemical; - percentvehicleinfinalassay; - incubationtemperatureandtime; - methodofbound/freeseparation; - positiveandnegativecontrols/referencesubstances; - criteriaforconsideringtestsaspositive,negativeorequivocal; Acceptabilitycheck: - actualIC50andHillslopevaluesforconcurrentpositivecontrols/referencesubstances; Results: - rawandbound/ffeedata; - denaturingconfirmationcheck,ifappropriate; -- iRfBitAexiasntds/,otrhIeCl5o0wevsatlueefsf,ecatsivaeppcroonpcreinattrea;tion(LEC); -- cstoantciestnitcraaltainoanl-yrseessp,onifseanrye,lattoigoentshheipr,wwihtheraempeoasssiubrlee;oferrorandconfidence(e.g.SEM,SD,CVor95% Cl)andadescriptionofhowthesevalueswereobtained; D-iscaupspsliicoantoiofnthoefr1esu%ltsr:ule Conclusion ©OECD,(2015) 10