Test No. 493 [electronic resource] : Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity

2015·1.8 MB·und

by  OECD

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.
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©OECD,(2015)
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fromtheOECD,providedthesourceisdulymentioned.Anycommercialuseofthismaterialissubjectto
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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,
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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).
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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
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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.
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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.
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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.
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^“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
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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;
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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
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