TS-K784YDAttachment 9PAGE1OF8STUDYSPONSOR:CBISTUDYTYPE:TESTITEM:PROJECTNUMBER:INTRODUCTIONAstudywasperformedtoassesstheskinsensitizationpotentialofthetestitemintheCBNCastrainmousefollowingtopicalapplicationtothedorsalsurfaceoftheear.Thisstudywasdesignedtobecompatiblewiththeproceduresindicatedbythefollowinginternationally·acceptedguidelinesandrecommendations:•OECDGuidelinefortheTestingofChemicalsNo.429"SkinSensitization:LocalLymphNodeAssay"(adopted22July2010)•MethodB42SkinSensitization(LocalLymphNodeAssay)ofCommissionRegulation(EC)No.440/2008Theassayhasundergoneextensiveinter-laboratoryvalidationandhasbeenshowntoreliablydetecttestitemsthataremoderatetostrongsensitizers.ExperimentalStartingDate:ExperimentalCompletionDate:18June201408July2014TestitemcharacterizationdataaretheresponsibilityoftheSponsor.AllrawdatawillberetainedinL..jc_s_t_________________.METHODSFollowingapreliminaryscreeningtestinwhichnoclinicalsignsoftoxicitywerenotedataconcentrationof100%,thisconcentrationwasselectedasthehighestdoseinvestigatedinthemaintestoftheLocalLymphNodeAssay.Threegroups,eachoffouranimals,weretreatedwith50J.lL(25J.lLperear)oftheundilutedtestitemorthetestitemasasolutionindimethylsulphoxideatconcentrationsof50%or25%v/v.Afurthergroupoffouranimalswastreatedwithdimethylsulphoxidealone.TS-K784YDAttachment9ReportPage2RESULTSPreliminaryScreeningTestClinicalobservations,bodyweightandmortalitydataaregiveninTable1andlocalskinirritationisgiveninTable2.TheearthicknessmeasurementsandmeanearthicknesschangesaregiveninTable3.MainTestTheradioactivedisintegrationsperminuteperlymphnodeandthestimulationindexaregiveninTable4.IndividualclinicalobservationsandmortalitydatafortestandcontrolanimalsaregiveninTable5andtheindividualbodyweightsandbodyweightchangefortestandcontrolanimalsaregiveninTable6.TheStimulationIndexexpressedasthemeanradioactiveincorporationforeachtreatmentgroupdividedbythemeanradioactiveincorporationofthevehiclecontrolgroupareasfollows:Concentration(%v/v)inStimulationIndexResultdimethylsulphoxide251.54Negative501.84Negative1001.85NegativeCONCLUSIONThetestitemwasconsideredtobeanon-sensitizerundertheconditionsofthetest.ThisstudywasconductedinafacilityoperatingtoGoodLaboratoryPracticewithintheUKnationalGLPmonitoringprogramme,butthestudyreporthasnotbeenauditedbytheQAUnit.NoformalclaimofGLPcomplianceismadeforthisstudy.Thisreportisanaccuraterecordofthestudyanditsoutcome.APoolesStudyDirectorDate:TS-K784YDTABLESReportAttachment9Page3Table1ClinicalObservations,BodyWeightandMortalityData-PreliminaryScreeningTestBodyWeightDayAnimal(g)ConcentrationNumber123DayDayPre-PostPostPre-Post45616DoseDose Dose Dose DoseDose100%S-117160000000000=NosignsofsystemictoxicityTS-K784YDAttachment9ReportPage4Table2LocalSkinIrritation-PreliminaryScreeningTestLocalSkinIrritationConcentrationAnimalDay1Day2Day3 Day4DaySDay6Numberleftrightleftrightleftrightleftrightleftrightleftright100%S-1000000000000TS-K784YDReportAttachment9Page5Table3MeasurementofEarThicknessandMeanEarThicknessChanges-PreJiminaryScreeningTestEarThicknessMeasurement(mm)AnimalDay1Day3ConcentrationNumberDay6pre-dosepostdoseleftrightleftrightleftright100%S-10.210.200.230.220.220.20overallmean(mm)0.210.230.21overallmeanearthickness9.762.44change(%)nana==Notapplicable;,TS-K784YDAttachment9ReportPage6Table4IndividualDisintegrationsperMinuteandStimulationIndicesConcentration(%v/v)indpmdpm/NodeaStimulationIndexbResultdimethylsulphoxideVehicle26789.793348.72nana2541186.345148.291.54Negative5049302.476162.811.84Negative10049580.076197.511.85Negativedpm=Disintegrationsperminutea=Disintegrationsperminute/nodeobtainedbydividingthedisintegrationsperminutevalueby8(totalnumberoflymphnodes)b=StimulationIndexof3.0orgreaterindicatesapositiveresultna=NotapplicableTS-K784YDAttachment9ReportPage7Table5IndividualClinicalObservationsandMortalityDataConcentrationDay1Day2Day3(%v/v)inAnimalDay4DaySDay6dimethylNumberPre-PostPostPre-PostsulphoxideDoseDoseDoseDoseDoseDose1-10000000001-2000000000Vehicle000000000l-40000000000000000002-2000000000252-30000000002-40000000000000OFIOFIOFIOF!OF!3-20000OFIOF!OFIOFIOFI500000OF!OF!OFIOFIOFI3.40000OF!OF!OF!OF!OFI0000OF!OFIOF!OFIOFI0000OF!OF!OF!OF!OF!1004-30000OF!OF!OFIOF!OFI4-40000OF!OFIOFIOF!OFI0=NosignsofsystemictoxicityFl=FurlossTS-K784YDAttachment9ReportPage8Table6IndividualBodyWeightsandBodyWeightChangeConcentrationBodyWeight(g)(%v/v)inAnimalNumberBodyWeightdimethylDay1Day6Change(g)sulphoxide1-117181l-22119-2Vehicle1-32119-21-41917-22-1192122-218180252-3181912-4202003-1191903-218191503-31817-13.4181914-1181914-2182021004-3191904-418180