FINALREPORTStudyTitleINDUCTIONOFANTIOXIDANT-RESPONSE-ELEMENTDEPENDENTGENEACTIVITYANDCYTOTOXICITY(USINGMTT)INTHEKERATINOCYTEARE-REPORTERCELLLINEKERATINOSENSTestArticlesResearchFragranceChemicalTM11-0078ResearchFragranceChemicalTM10-0210ResearchFragranceChemicalTM10-0367ResearchFragranceChemicalTM09-0536ResearchFragranceChemicalTM11-0230ResearchFragranceChemicalTM08-033809-003505-0293AuthorsKimberlyNorman,Ph.D.,DABTMonicaKrcha,B.S.StudyCompletionDate26June2014PerformingLaboratoryInstituteforInVitroSciences,Inc.30W.WatkinsMillRoad,Suite100Gaithersburg,MD20878StudyNumber13AL98-AM03,AN30-AN31.170001LaboratoryProjectNumber7560Page1of16TABLEOFCONTENTSTABLEOFCONTENTS................................................................................................................2SIGNATUREPAGE.......................................................................................................................3TESTARTICLERECEIPT.............................................................................................................4INDUCTIONOFANTIOXIDANT-RESPONSE-ELEMENTDEPENDENTGENEACTIVITYANDCYTOTOXICITY(USINGMTT)INTHEKERATINOCYTEARE-REPORTERCELLLINEKERATINOSENSINTRODUCTION...............................................................................................................6MATERIALSANDMETHODS........................................................................................?APPENDIXASP170001(PROTOCOL)..............................................................................................1-17PROTOCOLATTACHMENT1.........................................................................................1APPENDIXB(DOSE-RESPONSECURVES).....................................................................B1-B3Project7560,FinalReport2SIGNATUREPAGEINDUCTIONOFANTIOXIDANT-RESPONSE-ELEMENTDEPENDENTGENEACTIVITYANDCYTOTOXICITY(USINGMTT)INTHEKERATINOCYTEARE-REPORTERCELLLINEKERATINOSENSInitiationDate:CompletionDate:Sponsor:Sponsor'sRepresentative:TestingFacility:ArchiveLocation:StudyDirector:LaboratoryManager:LaboratorySupervisor:Project7560,FinalReport7January201426June2014InternationalFlavors&FragrancesInc.800RoseLaneUnionBeach,NJ07735PaulRibeiro,Ph.D.,JoanChengPh.D.,DABTInstituteforInVitroSciences,Inc.30W.WatkinsMillRoad,Suite100Gaithersburg,MD20878InstituteforInVitroSciences,Inc.Gaithersburg,MD20878NathanR.Wilt,B.S.AllisonHilberer,M.S.3;Ultl.-lC,1UJ!tfDateTESTARTICLERECEIPTIIVSTestSponsor'sPhysicalStorageArticleReceiptDateDesignationDescriptionConditions*Number13AL98ResearchFragranceclearcolorless25Nov20132°C-8°CChemicalTM11-0078non-viscousliquid13AL99ResearchFragranceclearcolorless25Nov20132°C-8°CChemicalTM10-0210non-viscousliquid13AMOOResearchFragranceclearcolorless25Nov20132°C-8°CChemicalTM10-0367non-viscousliquid13AM01ResearchFragranceclearcolorless25Nov20132°C-8°CChemicalTM09-0536non-viscousliquid13AM02ResearchFragrancewhitepowder25Nov20132°C-8°CChemicalTM11-023013AM03ResearchFragrancecloudywhite25Nov2013roomChemicalTM08-0338semi-viscousliquidtemperature13AN3009-0035clearcolorless14Jan2014roomnon-viscousliquidtemperature13AN3105-0293clearcolorless14Jan2014roomnon-viscousliquidtemperature* -ProtectedfromexposuretolightProject7560,FinalReport4INDUCTIONOFANTIOXIDANT-RESPONSE-ELEMENTDEPENDENTGENEACTIVITYANDCYTOTOXICITY(USINGMTT)INTHEKERATINOCYTEARE-REPORTERCELLLINEKERATINOSENSProject7560,FinalReport5INTRODUCTIONTheInductionofAntioxidant-Response-ElementDependentGeneActivityintheKeratinocyteARE-ReporterCellLineKeratinoSensskinsensitizationassayisahigh-throughputcell-basedinvitrotesttoscreenfortheskinsensitizationpotentialofchemicals.TheKeratinoSenscells(Givaudan,Switzerland)werepropagatedasareportercellline.TheKeratinoSenscellsaretransfectedHaCaTkeratinocytesthatincludea56-base-pairinsertioncontainingtheAREsequencefromtheAKR1C2gene,aSV40promoter,theluciferasegeneinthevectorpGL4fromPromega,andastableinsertionallowingforcellstobeselectedinthepresenceofGeneticin(G418).ThesignallingpathwaywiththerepressorproteinKeap1andthetranscriptionfactorNrf2,whichbindstotheantioxidantIelectrophileresponseelement(AREIEpRE),wasshowntobeavaluablecellularendpointtodetectskinsensitizersinvitro1·2•TheinductionofluciferasedirectlyindicatestheactivationofARE-dependentgenes.Additionally,thecytotoxicityofatestarticlewasassessedusingcellviabilityendpointswithMTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide)andNeutralRedvitaldyes.CytotoxicitywasdeterminedbymeasuringtherelativeconversionofMTTandNeutralReduptakeintestarticle-treatedculturescomparedtothesolventcontrol.Theexperimentaldesignofthisstudyconsistedofthreedefinitiveassaystodeterminethemaximalinduction(lmax),theconcentrationformaximalgeneinduction(Cimax),theEC1.5value(concentrationforaninductionof50%abovesolventcontrols),andameanIC50(concentrationleadingto50%viabilityascomparedtosolventcontrols)forthetestarticles.Foreachdefinitiveassay,theKeratinoSenscellswereculturedinquintuplicateplatesfor24hours,treatedwiththetestarticlesfor48hours,andassessedforluciferaseinduction(3plates)andcytotoxicity(2plates).TheproceduresthatwereperformedinthisassaywereamodificationoftheprocedurespreviouslydescribedbyNatsch,etal.(2008)andwereperformedsimilartothoseproceduresperformedbytheInstituteforInVitroSciences,Inc.intheKeratinoSensring-study3.TheInductionofAntioxidant-Response-ElementDependentGeneActivityintheKeratinocyteARE-ReporterCellLineKeratinoSensAssaywasperformedtodeterminetheskinsensitizationpotentialofthetestarticles,suppliedbyInternationalFlavors&FragrancesInc.Thelaboratoryphaseofthisstudywasconductedfrom8January2014to31January2014attheInstituteforInVitroSciences,Inc.(IIVS).1Natsch,A.andEmter,R.(2008)Skinsensitizersinduceantioxidantresponseelementdependentgenes:Applicationtotheinvitrotestingofthesensitizationpotentialofchemicals.ToxicologicalSciences.102(1):p.110-119.2Natsch,A.,Emter,R.,andEllis,G.(2009)Fillingtheconceptwithdata:Integratingdatafromdifferentinvitroandinsilicoassaysonskinsensitizerstoexplorethebatteryapproachforanimal-freeskinsensitizationtesting.ToxicologicalSciences.107(1):p.106-121.3Natsch,A.,BauchC.,Foertsch,L.,Gerberick,F.,Norman,K.,Hilberer,A.,Inglis,H.,Landsiedel,R.,Onken,S.,Reuter,H.,Schepky,A.,andEmter,R.(2011)Theintra-andinter-laboratoryreproducibilityandpredictivityoftheKeratinoSensassaytopredictskinsensitizersinvitro:Resultsofaring-studyinfivelaboratories.ToxicologyinVitro.25(3):733-774Project7560,FinalReport6MATERIALSANDMETHODSReceiptofSkinSensitizationAssayReagentsReagentsusedfortheskinsensitizationbioassaywereinspecteduponreceiptandstoredaccordingtothemanufacturer'sinstructions.TheDMEM(Dulbecco'sModifiedEagleMedium),liquidwithGlutaMAXTMI,1000mg/LD-Glucose,SodiumPyruvate(Gibco)andGeneticin(G418)(Gibco)werestoredat2-8°C.TheFetalBovineSerum(FBS)(AtlantaBiologicals)wasstoredat-20±5°C.BatchesofAssayMediumwerepreparedbysupplementingDMEMwith9.1%FBS.BatchesofMaintenanceMediumwerepreparedbysupplementingDMEMwith9.1%FBSand500G418.Batchesof1%FBSDMEMwerepreparedbysupplementingDMEMwith1%FBS.TheDulbecco'sPhosphateBufferedSalinewithoutcalciumandmagnesium(CMF-DPBS)(Gibco)andEDTApowder(Sigma)werestoredatroomtemperature.BatchesofCMF-DPBScontaining0.05%EDTA,usedforcellpassaging,werepreparedbysupplementingtheCMF-DPBSwitha10%EDTAsolution.The0.05%Trypsin/EDTAsolution(Gibcollnvitrogen),lOXstocksolutionofMTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide)(Sigma),andDimethylsulfoxide(DMSO)(Sigma)werestoredat-20±5°C.ThelyophilizedsubstrateoftheONE-GloTMassaykit(Promega)andtheONE-GloassaybufferTMwerestoredat-20±5°C.TheONE-GloTMreagentwaspreparedbyaddingtheappropriatevolumeofreconstitutionbuffertothelyophilizedsubstrate.The3.3mg/mLNeutralRedstocksolution,preparedfromNeutralRedpowder(Sigma)atIIVS,wasstoredat2-8°C,protectedfromlight.TheDulbecco'sPhosphateBufferedSaline(DPBS)(QualityBiological)containingcalciumandmagnesium,usedfortheNeutralRedendpoint,wasstoredat2-8°C.UponreceiptofcryopreservedKeratinoSenscells(Givaudan),thevialofcellswasstoredinaliquidnitrogenfreezer.Theremainingreagentswerestoredatroomtemperature.CellThawingProcedureAcryovialofthecryopreservedKeratinoSenscellswasthawedinawaterbathatapproximately37°C(inabeakercontaining70%ethanol)forasbriefatimeaspossible.Oncethawed,thecryovialwasimmediatelydecontaminatedwith70%ethanolandplacedinalaminarflowhood.Thecellswereaddedtoasterileconicaltubeanddilutedbyslowlyadding10mLpre-warmedAssayMedium.Thecellswerethencentrifugedatapproximately200xgfor5minutesatroomtemperature.The supernatantwasaspirated,thepelletwasresuspendedinapproximately15mLoffreshpre-warmedAssayMedium,andthentransferredintoaT75tissuecultureflask.Theflaskwasthenincubatedat37±1°C,90±10%humidity,and5.0±1%C02inair(standardcultureconditions),untiltheKeratinoSenscellsreached-60%to90%confluence.RoutineCulturingoftheKeratinoSensCellsWhentheculturesreached-60to90%confluence,theywereremovedfromtheflaskbytrypsinization.Themediumwasaspiratedandthecellsheetrinsedtwicewithapproximately10mLofCMF-DPBScontaining0.05%EDTA.OnemLoftrypsin/EDTAwasaddedtocoverthecellsheet.Theflaskwasthenplacedintotheincubatorandincubatedatstandardcultureconditionsfor6-8minutes,oruntilthecellsbecamedislodged.Whenmorethan50%ofthecellsbecamedislodged,theflaskwasrappedsharplyagainstthepalmofthehand.Thenapproximately7mLofMaintenanceMediumwasaddedtoeach7 5cm2flasktoobtainasinglecellsuspensionandcellspassagedatappropriatedensities.TheKeratinoSenscellswereroutinelypassagedevery2-4days.Project7560,FinalReport7

# Subculture

of KeratinoSens

Cells

into

96- Well Plates The KeratinoSens

cells
were subcultured

into transparent

Costar 96-well

plates

or white-walled

Perkin Elmer plates when

the flasks
were

-60

to 90% confluent.

The flasks
were rinsed

and trypsinized

as previously described.

The cells were resuspended

in

5 mL

of Assay Medium

per flask.

The concentration

of cells

in suspension

was determined

using

a Coulter Counter.

A cell suspension

of
1.0

x

10

5 

# cells/mL

in Assay Medium

was prepared.

One hundred

**~L**



# of the cell suspension

was added

to all but one well designated

as

a blank

well (H12) (See Figure

1). The stock

cell suspension

was mixed
often

to ensure

a uniform distribution

of cells into each well. Three white-walled

and two transparent

plates
were seeded

for the test article replicate

set for each assay.

The plates

were incubated

for approximately

24 hours

at standard
culture conditions.

The plate cultures seeded

into

the clear plates
were examined

under

a phase contrast microscope

and evaluated

for uniform seeding

and confluence

prior

to treating

the cells with

the test article

or positive control. Solubility Determination The Sponsor specified

that

the test articles

were soluble

in DMSO,

and therefore
DMSO was selected

as the solvent

to dissolve

and dilute

the test articles.

For each

test article,

the lOOX top stock prepared

in DMSO

was described

as

a clear colorless non-viscous

solution. MTT Direct Reduction

Test The ability

of the test articles

to directly reduce
MTT

was assessed

at the same
time

of test article treatment

in the definitive

assays.

A 1.0 mg/mL

MTT solution

was prepared

by dissolving

a 10 mg/mL

stock solution

of MTT

into warm MTT
Addition Medium. Approximately

100

**~L**



# of the lOOX

test article concentration

in DMSO

was added

to

1 mL

of the MTT solution

and then incubated in the dark

at 37°C

for one

to three hours.

One hundred

**~L**



# of

a negative control

(e.g. DMSO)

was tested concurrently.

If 

# the MTT solution

color turned blue/purple,

the test articles

were presumed

to have reduced

the MTT.

The
test article, Research Fragrance Chemical

TM 11-0230,

was found

to directly reduce
MTT.

The

test articles, Research Fragrance Chemical

TM 11-0078, Research Fragrance Chemical

TM 10-0210, Research Fragrance Chemical

TM 10-0367, Research Fragrance Chemical

TM 09-0536, Research Fragrance Chemical

TM 08-0338, 09-0035,

and 05-0293,

did not directly reduce MTT. Controls

Each assay plate included

a range

of doses

of the positive control, Cinnamic Aldehyde (Sigma).

A 100X concentration

of positive control

was prepared

by weighing approximately 84.6

mg

of Cinnamic Aldehyde

into

a prelabeled

conical

tube.

Ten mL

of DMSO

were added

to prepare

a 64,000

**~M** 

# dilution.

The 64,000

**~M** 

# dilution

was further diluted

1:100

in DMSO

to prepare

a 6400

**~M** 

# 100X stock concentration.

The final

1X concentrations

of the positive control were

64,
32,

16,

8, and

4 **~M.**


# The solvent control

for the test articles

and

the positive control

was DMSO

(to

a final concentration

of 1%)

in 1% DMEM.

Each plate included

a set

of

6 DMSO solvent control
wells. Project

7560, Final Report

8TestingConcentrationsThetestarticles,ResearchFragranceChemicalTM11-0078,ResearchFragranceChemicalTM10-0210,ResearchFragranceChemicalTM10-0367,ResearchFragranceChemicalTM09-0536,ResearchFragranceChemicalTM11-0230,ResearchFragranceChemicalTM08-0338,09-0035,and05-0293,hadmolecularweightsof,182.1g/mol,194.2g/mol,184.2g/mol,192.2g/mol,148.0g/mol,194.2g/mol,200.2g/mol,and156.2g/mol,respectively,andweredilutedbasedonmolarity.The100Xstockdilutionwaspreparedtoatopconcentrationof200,000Thefinal1Xtestedconcentrationswere2000,1000,500,250,125,62.5,31.3,15.6,7.81,3.91,1.95,and0.978DefinitiveAssaysThetestarticlesweretestedinthreedefinitiveassays.Eachdefinitiveassayincludedasetof4plates(3forgeneinduction,1forcytotoxicityassessment).Eachplatecontaineduptoseventestarticleswitharangeof12dosingconcentrationsforeachtestarticle.Eachplatealsoincluded5wellsdesignatedforthepositivecontrol(testedoverarangeof5dosingconcentrations),6wellsdesignatedastheDMSOsolventcontrol,and1wellwhichwasleftblank(SeeFigure1).Eachdefinitiveassaywasperformedindependently:1)Theplatesforeachdefinitiveassaywereseededusingseparateflasksofcellsbydifferentindividuals;2)Aseparate4XAssayMasterplatewaspreparedforeachdefinitiveassaybydifferentindividuals;3)Threedifferentindividualsdosedeachset(5totalplates)ofdefinitiveassayplates;and4)SeparatebatchesofMTTandneutralredwerepreparedforeachdefinitiveassay.Afteratleast22hoursofincubationofthecells,theAssayMediumwasremovedfromthecells.Theplatesweredecantedandgentlyblottedonsterilepapertowels.Onehundredandfiftymicrolitersoffreshpre-warmed1%DMEMwereaddedtoallwells,includingtheblank.Theplateswerereturnedtotheincubatoruntilthedosingwasinitiated.Foreachtestarticle,twelvedecreasingdoseswereselectedfortheassay(testarticleswerediluteduptoafinalconcentrationof200,000inDMSO).Forthepositivecontrol,5decreasingdoseswereprepared.Foreachsetof7testarticles,thepositivecontrol(5doses),andthesolventcontrol,a100XDMSOmasterplatewasmade,followedbya4XMasterPlate.Whenaddedtothe150of1%DMEMalreadyineachwell,theadditionofthe504Xdosebroughtthefinaldoseontheplatesto1X.Thepreparationofthe100XDMSOMaster,4XAssayMaster,and1XplatesisoutlinedinFigure2.Project7560,FinalReport9ABcDEFGHFigure1.96-wellplatemap1 2 3 4 5T1C12TlCllTlC10T1C9TlC8T2C12T2CllT2C10T2C9T2C8T3C12T3CllT3C10T3C9T3C8T4C12T4CllT4C10T4C9T4C8T5C12TSCIIT5C10T5C9T5C8T6C12T6CllT6C10T6C9T6C8T?C12T7CIIT7C10T7C9T7C8NCNCNCNCNCTl-T7-TestArticles6 78910TlC7T1C6T1C5TlC4TlC3T2C7T2C6T2C5T2C4T2C3T3C7T3C6T3C5T3C4T3C3T4C7T4C6T4C5T4C4T4C3TSC1T5C6T5C5TSC4TSC3T6C1T6C6T6C5T6C4T6C3T7C7T7C6T7C5T7C4T7C3NCPCPCPCPC(0.4)(0.8)(1.6)(3.2)C1_12-Testarticleconcentrations(12=lowestconcentration,1=highest)NC-SolventControl(DMSO)PC-PositiveControl(6.4-0.4mM)blnk-Blank(containsnocells)1OOXMasterPlatePreparation1112TlC2TlC1T2C2T2C1T3C2T3CIT4C2T4C1TSC2TSCIT6C2T6C1T?C2T7C1PCblnk(6.4)The100XDMSOMasterPlatewaslabelledwiththeappropriatetestarticleandcontrolconcentrationsaccordingtothestandardplatemap(seeFigure1).The100Xtopstockconcentration(200,000ofthetestarticleswaspreparedandthenseriallydilutedtomakethe11remainingdoses.The100Xtopstockforthepositivecontrol(6400waspreparedandthenseriallydilutedtomakethe4remainingdoses.ThewellsdesignatedforthesolventcontrolreceivedDMSO.Thewelldesignatedasblankwasleftempty.4XMasterPlatePreparationThecorresponding4XMasterPlatewassimilarlylabeledandthe96-wellplatewaspre-filledwith300of1%DMEMineachwell.Theprepareddilutionsfromthe100XDMSOMasterPlatewerefurtherdiluted25-foldbyadding12.5each100Xsample(testarticleconcentration,positivecontrolconcentration,orsolventcontrol)tothe4XMasterPlate.TestArticleTreatmentTheresulting4XMasterPlatewasthenusedtodosethereplicateassayplates(transparentorwhite-walled)containingcells.Afinal1Xconcentrationwasachievedforeachdosebyremoving50fromeachwellofthe4XMasterPlateandaddingthedosetothecorrespondingwellinthe1Xplates(alreadycontaining150of1%DMEMineachwell).Theblankwellreceived50of1%DMEM.Whenalloftheplatesweredosed,theplatesweresealedwithaplatesealertoavoidevaporationofvolatilecompoundsandtoavoidcross-contaminationbetweenwells.Theplateswerethenincubatedatstandardcultureconditionsfor48±1hours.Project7560,FinalReport10Figure2.TreatmentofKeratinoSens:platepreparationoverview1:25dilution12.51:4dilutionI.x.x.x.x.x.xxX.x.x.x.x.x.xxX.X.X.XXXYYXXIIIx..X.X.XIIIIIIIX..A.X.XIXXXXT.XIIXXIXIIIXXXXXXXIIIXXIII.I_IIx.X.XXIIIIIIX.XXXIXIIIT0.9820000.9820000.982000XXyXy.x.x.x.xxxxx.x.x.I.X..X.MTT/CvtotoxPlate(lX)LuciferasePlates(1X)VisualObservationsAfterapproximately48hoursofpost-treatmentincubation,visualobservationsofthecultureswereperformedforatleastoneofthecytotoxicityplatesandrecorded.Visualobservationswereperformedtoconfirmtheresultsofthecytotoxicityendpoint.TreatmentTermination&LuciferaseInductionDeterminationAfter48±1hoursofexposure,eachwhite-walledcultureplatewasremovedfromtheincubatorandallowedtoequilibratetoroomtemperatureforatleast30minutes.Onceatroomtemperature,thetreatmentmediumwasdecantedfromeachplate.Thecultureswererinsedwith250ofCMF-DPBS(roomtemperature),theCMF-DPBSrinsatewasdecantedfromthewells,andtheplatesweregentlyblottedontopapertowels.Project7560,FinalReport11FiftymicrolitersofCMF-DPBSwasaddedtoeachwellfollowedbyfiftymicrolitersofONE-GloTMReagent.Theplatesremainedatroomtemperatureinthedarkforatleast5minutesbeforebeingreadbytheluminometer.Theplateswerereadwithin30minutesofadditionoftheONE-GloTMReagent.TheluminescensedeterminationofeachplatewasperformedbyaBertholdDetectionSystemsluminometerinitiatedfromanIBM-PChostingtheWindows-basedSimplicityTMsoftware.Thelightintensityineachwellwasmeasuredat565nmintheformofrelativelightunits(RLUs).TreatmentTermination:CytotoxicityUsingtheMTTEndpointA0.59mg/mLMTTsolutionwaspreparedin1%DMEMandusedwithin2hours.After48±1hours,theclear96-wellplatesdesignatedfortheMTTendpointweredecantedandgentlyblottedonpapertowels.Norinsingwasperformed.TwohundredIlLof1%DMEMcontaining0.59mg/mLMTTwasaddedtoeachwell.Theplatewasincubatedwithaplatesealatstandardcultureconditionsforapproximately4hrs.Afterapproximately4hours,theMTTsolutionwasdecanted,theplatewasblotted,and200IlLof10%SLSwasaddedtoeachwell.Theplatewascoveredwithaplatesealandincubatedatstandardcultureconditionsovernight.Aftertheovernightincubation,theplatewasplacedonaplateshakerandshakenforatleast20minutesatroomtemperature.Theabsorbanceat570nm(OD570)ofeachwellwasmeasuredwithaMolecularDevicesVmaxplatereader.DataAnalysisForeachsetoftestarticles,acopyofthestandarddatafile,providedbyGivaudan,wasmade(i.e.3trials,eachwith4platespertrial-3platesfortheluciferaseinductionand1plateforMTTorNRUcytotoxicity).RawdatafromtheluminometerwastransferreddirectlyfromtheluminometersoftwareintothedesignatedExcel®spreadsheetforluminescenceanalyses.RawdatafromtheVmaxwastransferredintothedesignatedExcel®spreadsheetforcytotoxicityanalyses.Thedatafileautomaticallycalculatedthegeneinductionandthewellswithstatisticallysignificantinductionoveragiventhreshold(defaultvaluesetto1.5=50%enhancedgeneactivity).FurthermorethemaximalinductionCimax),theconcentrationformaximalgeneinduction(Cimax)andtheECvalue(concentrationforinductionabovethreshold),bothwithlinearandloglinearextrapolation,wascalculatedsimilartotheLLNA(LocalLymphNodeAssay).Relativesurvival(viability)wasobtainedbycomparingtheamountofMTTconversionorneutralreduptakebytestarticletreatedgroupscomparedtotheassociatedsolventtreatedgrouponthesameplate.AnIC50valuewasdeterminedfortheMTTandneutralredcytotoxicityplates.Asummaryoftheresultsfromthe3definitiveassayswascalculated.Theluciferaseinductionandcytotoxicitydatawereplottedonthegraphs.Fortheluciferaseinduction,thefoldgeneinduction(ascomparedtothesolventcontrols)wasplottedoverthetestarticleconcentration.Forthecytotoxicity,the%viability(ascomparedtothesolventcontrols)wasplottedoverthetestarticleconcentration.Project7560,FinalReport12CriteriaforDeterminationofaValidDefinitiveAssayThehistoricalacceptancerangeforthepositivecontroliscurrentlyunderdevelopmentattheTestingFacility.However,theresultsofthethreedefinitivetrialsforeachplatewereassessedusingsimilarcriteriaoutlinedinthevalidationringtrial4.Thoseacceptancecriteriaincluded:1)variabilityinDMSOsolventcontrolwellsforeachdefinitiveassaywas<20%;and2)thepositivecontrolproducedastatisticallysignificantinductionabove1.5foldbelow64ineachdefinitiveassay.EvaluationofTestResultsAtestarticlewaspredictedtohavesensitizationpotentialif:1)TheEC1.5valuefellbelow1000atleast2of3repetitions;2)Atthelowestconcentrationwithageneinductionabove1.5,cellularviabilitywasgreaterthan70%;and3)Therewasapparentoveralldoseresponsewhichwassimilarbetweenrepetitions.4Natsch,A.,BauchC.,Foertsch,L.,Gerberick,F.,Norman,K.,Hilberer,A.,Inglis,H.,Landsiedel,R.,Onken,S.,Reuter,H.,Schepky,A.,andEmter,R.(2011)Theintra-andinter-laboratoryreproducibilityandpredictivityoftheKeratinoSensassaytopredictskinsensitizersinvitro:Resultsofaring-studyinfivelaboratories.ToxicologyinVitro.25(3):733-774Project7560,FinalReport13RESULTSANDDISCUSSIONThetestarticlesweretestedinthreedefinitiveassays.Eachdefinitiveassayincludedasetof4plates(3forgeneinduction,1forcytotoxicityassessment).Thetestarticlesweretestedat12concentrationsrangingfrom2000to0.975Thepositivecontrol,CinnamicAldehyde,wastestedat5concentrationsrangingfrom4to64AsummaryoftheEC1.5(concentrationforaninductionof50%abovesolventcontrols)andIC50(concentrationleadingto50%viabilityascomparedtosolventcontrols)resultsofthedefinitiveassaysarepresentedinTable1.Additionalluciferaseinductioninformation(whichwasnotusedforthecurrentpredictionmodel)thatincludestheImax(themaximalfoldinduction)andtheClmax(theconcentrationatwhichthemaximalfoldinductionoccurs),ispresentedinTable2.AsummarygraphrepresentingtheluciferasefoldinductionandthecellviabilityoverconcentrationforeachtestarticleisincludedinAppendixB.TheInductionofAntioxidant-Response-ElementDependentGeneActivityintheKeratinocyteARE-ReporterCellLineKeratinoSensassaywasusedtoassesstheskinsensitizationpotentialofthetestarticles.Atestarticlewaspredictedtohavesensitizationpotentialif:1)TheEC1.5valuefellbelow1000inatleast2of3repetitions;2)Atthelowestconcentrationwithageneinductionabove1.5,cellularviabilitywasgreaterthan70%;and3)Therewasanapparentoveralldoseresponsewhichwassimilarbetweenthethreedefinitiveassays.Accordingtothecurrentpredictionmodel,thetestarticle,ResearchFragranceChemicalTM11-0230,waspredictedtobeaskinsensitizer.Thetestarticles,ResearchFragranceChemicalTM11-0078,ResearchFragranceChemicalTM10-0210,ResearchFragranceChemicalTM10-0367,ResearchFragranceChemicalTM09-0536,ResearchFragranceChemicalTM08-0338,09-0035,and05-0293,werepredictedtobenon-sensitizers.Althoughthecurrentpredictionmodeldoesnotincludeguidanceonpotency,earlydataindicateacorrelationbetweentheEC1.5valueandpotency;thelowertheEC1.5valuethemorepotentthesensitizer,thehighertheEC1.5valuethelesspotentthesensitizer.Celltoxicitywasmeasuredusingtwoviabilityassaysinordertoaccountforanydirectinteractionswithviabilitydyereagentandthetestarticle(suchasdirectMTTreductionbyatestarticle).Itisimportanttoaccuratelymeasurecellviabilitybecauseitisacriterioninthepredictionmodel.WhenevaluatingsomestrongirritantsintheKeratinoSensassay,suchassodiumlaurelsulfate,itwasfoundthattheycausedgeneinductionabovethe1.5thresholdeventhoughtheywerenonsensitizers;howeverattheconcentrationatwhich thesestrongirritantscausedgeneinductionabovethe1.5thresholdtheyhadcausedsignificantcelldeath(greaterthan30%celldeath)andlikelythisirritation/toxicitywasresultinginactivationofthegeneresponse.Project7560,FinalReport14Table1EC1.5andICsoSummaryTableIIVSTestEC1.5MeanICsoPotential1\AssayDateArticleSponsor'sDesignationvalue(,..,M)Sensitizer?+Number(,_.,M)13AL98ResearchFragranceChemical>2000299.20NO*TM11-007813AL99ResearchFragranceChemical>2000110.10NO*TM10-02108Jan201413AMOOResearchFragranceChemical>2000100.73NO*TM10-036713AM01ResearchFragranceChemical>200032.82NO*TM09-0536CinnamicPositiveControl7.83>64YESAldehyde13AM02ResearchFragranceChemical35.33579.4YESTM11-023013AM03ResearchFragranceChemical>2000209.1NOTM08-033829Jan201413AN3009-0035>2000192.8NO*13AN3105-0293>2000153.2NOCinnamicPositiveControl7.03>64YESAldehydeNOTE:WhenanEC1.5orIC50valuewasnotobtained,theresultswerepresentedasgreaterthanthehighestdosetested."-TheEC1.5valueistheconcentrationforgeneinductionabovethethreshold(1.5fold)ascomparedtotheDMSOsolventcontrols+ -Atestarticleispredictedtohavesensitizationpotentialif:1)TheEC1.5valuefallsbelow1000jlg/mLinatleast2outof3independenttrials2)Atthelowestconcentrationwithageneinductionabove1.5,cellularviabilitywasgreaterthan70%3)Therewasanapparentoveralldoseresponsesimilarbetweenrepetitions*ThesetestarticlesarenotsensitizerspertheKeratinoSenspredictionmodel.Althoughinsomeoftherepetitionswithineachindependenttrialtheinductionvaluewasgreaterthan1.5,theoverallinductionwasnotstatisticallysignificant.Theinductionabove1.5wasobservedatdoseswhichwerecytotoxic(ascanbeobservedinthegraphspresentedonthefollowingpages).Project7560,FinalReport15Table2SummaryResultsImaxandCimaxIIVSTestMaximalCone.forAssayDateArticleSponsor'sInductionMaximalGeneNumberDesignation1Induction(Imax)2(Cimax)13AL98ResearchFragranceChemical1.28NDTM11-007813AL99ResearchFragranceChemical1.04NDTM10-02108Jan201413AMOOResearchFragranceChemical1.16NDTM10-036713AM01ResearchFragranceChemical1.08NDTM09-053613AM02ResearchFragranceChemical2.40125TM11-023013AM03ResearchFragranceChemical1.11ND29Jan2014TM08-033813AN3009-00351.24ND13AN3105-02931.18ND1-LuciferasemaximalfoldinductionascomparedtotheDMSOsolventcontrols.AnImaxvalue1.5producedanEC1.5value.2-Concentrationwheremaximalfoldinductionoccurred.Whenthefoldinductiondidnotcrossthe1.5foldthreshold,theClmaxwaspresentedasND.ND-Notdeterminedsincethemaximalinductionwas<1.5foldProject7560,FinalReport16APPENDIXA0

llVS Study Number: 13AL98-AM03, AN30-AN31.170001 IIVS Protocol

No. SP170001

12/5/13

1 of
17 1.0 2.0 3.0
INDUCTION

OF ANTIOXIDANT-RESPONSE-ELEMENT DEPENDENT

GENE ACTIVITY

AND CYTOTOXICITY

(USING
MTT)

IN THE KERATINOCYTE ARE-REPORTER

CELL LINE KERATINOSENS PURPOSE The purpose

of this study

is to evaluate

the skin sensitization

potential

of

a test article

as measured

by its induction

of luciferase

in the KeratinoSens

cells,

a keratinocyte

reporter cell line which contains

an antioxidant response element (ARE). Luciferase induction directly indicates activation

of ARE-dependent

genes. Measurement

of the induction

of this signalling pathway

in

a reporter

cell line provides

a high-throughput

cell-based

in vitro

test

to screen

for the skin sensitization

potential

of novel chemicals. Luciferase induction

will

be determined

through

the bioluminescent

activity

of the conversion

of ATP

and

a luciferin substrate

into light

due

to the enzymatic

activity

of luciferase. Additionally,

the cytotoxicity

of each

test article

will

be determined

using

cell viability endpoints

with MTT (3-[4,5- dimethylthiazol-2-yl]-

2,5- diphenyltetrazolium

bromide). Cytotoxicity

will

be determined

by measuring

the relative conversion

of MTT

in the test article treated cultures

as compared

to the solvent control. SPONSOR 2.1 Name:

International

Flavors

& Fragrances

Inc. 2.2 Address:

800 Rose Lane Union Beach,

NJ 07735 2.3 Representatives:

Paul Ribeiro,
Ph.D.,

Joan Cheng Ph.D., DABT IDENTIFICATION

OF TEST SUBSTANCES

AND ASSAY CONTROLS 3.1

Test Article:

See Protocol Attachment

1 3.2 Assay Controls:

Positive: Cinnamic Aldehyde Solvent:

Assay Media containing

1% DMSO 3.3 Determination

of Strength,

Purity,

etc. 3.3.1

For GLP studies

only,

the Institute

for

In Vitro Sciences,

Inc. (IIVS)

will attempt

to secure documentation

of the analytical

purity

and composition of the test article

and
the stability

and strength

of the dosing solutions

from the Sponsor.

If the Sponsor

is unable

to provide

such information,

IIVS will retain documentation

supporting
attempts

to obtain

this information with

the study

file and

an exception

will

be noted

in the Statement

of Compliance

in the Final Report. 3.3.2

IIVS
will

be responsible

for the documentation

of the analytical

purity

and composition

of the positive

and solvent controls

used

for the stock

orc

llVS Study Number: 13AL98-AM03, AN30-AN31.170001 llVS Protocol

No. SP170001

12/5/13

2of

17 working dilution

of the controls.

This may

be accomplished

by maintaining

a certificate

of analysis

from

the supplier. 4.0 TESTING FACILITY

AND KEY PERSONNEL 4.1 Name: 4.2 Address: 4.3 Study Director:

Institute

for

In Vitro Sciences,

Inc. 30 W. Watkins

Mill Road, Suite

100 Gaithersburg,

MD 20878 Kimberly Norman,

Ph.D., DABT 5.0 TEST SCHEDULE 6.0 7.0

5.1 Proposed Experimental

Initiation

Date:

9 December

2013 5.2 Proposed Experimental Completion

Date:

27 December

2013 5.3 Proposed
Report
Date:

21 February

2014 TEST SYSTEM The KeratinoSens

cells (Givaudan, Switzerland)

have been propagated

as

a reporter

cell line.
The KeratinoSens

cells

are transfected

HaCaT keratinocytes

that include

a 56-basepair insertion containing

the ARE sequence

from

the AKR1C2

gene,

a SV40 promoter, the luciferase

gene

in the vector pGL4 from Promega,

and

a stable insertion allowing

for cells

to be selected

in the presence

of Geneticin
(G418).

The signalling pathway

with

the repressor
protein

Keap

1 and
the transcription

factor Nrf2, which binds

to the antioxidant

I electrophile
response element

(ARE

I EpRE),

was shown

to be

a valuable cellular endpoint

to detect

skin sensitizers

in vitro (Natsch

& Emter,
2008; Natsch

et.al, 2009). The induction

of luciferase
directly indicates

the activation

of ARE-dependent

genes. EXPERThffiNTAL

DESIGN

AND METHODOLOGY The experimental

design

of this study consists

of three definitive

assays

to determine average maximal induction

of gene activity

(Imax),

the concentration

for average maximal induction

of gene activity (Cimax),

the average concentration

inducing luciferase

gene activity
>50% above solvent control values (EC1.5 value),

and
the average concentration leading

to 50% cytotoxicity

(IC

50

) for each

test article.

The luciferase

gene induction (using

the ATP luminescence

endpoint)

will

be used

to calculate

the Imax, Clmax,

and the EC
1.5 value.

The induction

of luciferase

will

be evaluated

to determine sensitization potential

of the test article.

The cytotoxicity

of the test articles

will

be evaluated

on the basis

of the NAD(P)H-dependent

microsomal enzyme reduction

ofMTT (Berridge,

et al., 1996)

by the KeratinoSens

cells.

The amount

of reduced

MTT

will

be measured spectrophotometrically.

Cytotoxicity

data will

be presented

in the form

of relative survival (relative

MTT reduction)

versus

test article concentration. For each definitive

assay,

the KeratinoSens

cells
will

be cultured

in

at least quadruplicateIIVS Study Number: 13AL98-AM03, AN30-AN31.170001 IIVS Protocol

No. SP170001

12/5/13

3 ofl7 plates

for
24 hours, treated

with

the test article

for

48 hours,

and assessed

for luciferase induction

(3 plates)

and MTT cytotoxicity

(1 plate).

The procedures

that will

be performed

in this assay

are

a modification

of the procedures previously described

by Natsch,

et al. (Natsch

& Emter, 2008)

and will

be performed similarly

to those procedures performed

by IIVS

in the KeratinoSens
ring-study
(Natsch

et al, 2011). 7.1 Media

and Reagents (suggested
vendors

in parenthesis) 7 .1.1 Assay Medium: Dulbecco's Modified

Eagle Medium(DMEM)

containing GlutaMAXTM

I, 1000 mg/L D-Glucose,

Sodium Pyruvate (Gibco) supplemented

with: 9.1% fetal bovine serum (FBS) (Hyclone) 7 .1.2 Maintenance

Medium: Dulbecco's Modified

Eagle Medium(DMEM) containing GlutaMAXTM

I, 1000 mg/L D-Glucose,

Sodium Pyruvate supplemented

with: 9.1% FBS
and
5.5 mL Geneticin

(G418) (Invitrogen) (final concentration

500 J,tg/mL) 7 .1.3

1% DMEM: Dulbecco's Modified

Eagle Medium containing GlutaMAXTM I, 1000 mg/L D-Glucose,

Sodium Pyruvate supplemented

with:

1% FBS 7.1.4 Dulbecco's Phosphate Buffered

Saline without Calcium

& Magnesium supplemented

with 0.05% EDTA 7 .1.5 0.05o/o Trypsin/0.02%

EDTA Solution (Gibco/Invitrogen) 7.1.6 Dulbecco's Phosphate Buffered

Saline without Calcium

& Magnesium (CMF-DPBS)

(Gibco) 7.1.7 Dimethyl Sulfoxide (DMSO) (Sigma) 7.1.8 Promega ONE-GloTM Luciferase

Assay System

(kit contains Luciferase Assay Buffer

and lyophilized Luciferase

Assay Substrate) (Promega) 7.1.9
MTT (3-[4,5- dimethylthiazol-2-yl]-

2,5- diphenyltetrazolium

bromide) (Sigma) 7.1.10 Extraction

solvent: Isopropanol

(Sigma)

[MTT Assay](IIVSStudyNumber:13AL98-AM03,AN30-AN31.170001IIVSProtocolNo.SP17000112/5/134of177.27.3RouteofAdministrationThetestarticleswillinitiallybepreparedatIOOXanddilutedto4X.The4XdilutionswillresultinaIXfinaldilutionafterdirectadditionof50ofeach4XtestarticletothecellculturescontainingI50ofI%DMEM.AlOOXDMSOMasterplateanda4X1%DMEMplatewillbepreparedasdescribedinSection7.9.1.Cellswillbeexposedtothevariousdilutionsofthetestarticlesforapproximately48±Ihoursintheincubatoratstandardcultureconditions(37±Iochumidifiedaircontaining5±I%C02).Iftestarticlesaremixtures,orhavenodefinedmolecularweighttheymaybedilutedona,...gtmL(w/v)basis,ratherthanbaseduponmolarityasspecifiedinProtocolAttachment1.Unlessotherwisespecifiedthesetestarticleswillbetestedconsideringaproformamolecularweightof200g/mol,thereforethelOOXstocksolutionwillbepreparedtoatopconcentrationof40mg/mLinDMSOorotherappropriatesolventanddilutedthesameasthe200mMtopconcentrationamongsttheDMSOMasterPlate,4X,andlXplates.TheIXconcentrationsforthetestarticlesdilutionsbasedonmolaritywillbe2000,IOOO,500,250,I25,62.5,31.3,I5.6,7.8I,3.9I,1.95,and0.975mM.TheIXconcentrationsforthetestarticledilutionsbasedonweight/volume(w/v)willbe400,200,IOO,50,25,I2.5,6.25,3.I3,1.56,0.78I,0.391,andO.I95Thestabilityofthetestarticle(s)underthestorageconditionsatthetestingfacilityandundertheactualexperimental conditionswillnotbedeterminedbyInstituteforInVitroSciences,Inc.(IIVS).ControlsPositivecontrol:CinnamicaldehydewillbeseriallydilutedinDMSOtoconcentrationsIOOXofthefinalconcentrationsfortheassay.TheIOOXDMSOMasterPlatestocksolutionswillbeaddedtothe4XMasterPlatecontainingI%DMEMtoa4Xconcentration.Additionofthe4XconcentrationstothewellsalreadycontainingI50I%DMEMwillresultinafinalIXconcentration.Thefinalconcentrationsofthepositivecontrolwillbe6.40,3.20,I.60,0.800,and0.400mM.Eachplatewillcontainaserialdilutionofthepositivecontrol.SolventControl:Eachplatewillinclude6vehiclecontrolwells(cellsexposedtoI%DMSOonly).Hanintermediatesolventisused(e.g.,water),theconcentrationoftheintermediatesolventwillbethesameasusedwiththetestarticletreatment.Eachplatewillhaveablankwell(HI2).c (

llVS Study Number: 13AL98-AM03, AN30-AN31.170001 llVS Protocol

No. SP170001

12/5/13

5 of
17 7.4
MTT Direct Reduction

Test The ability

of each

test article

to directly reduce MTT

will

be assessed.

A 1.0 mg/mL
MTT solution

will

be prepared

by dissolving

a 10 mg/mL

stock solution of MTT

into warm MTT Addition Medium.

The

1.0 mg/mL

MTT solution

will

be used within

2 hours

of preparation. Approximately

100
**~L**

of the test article

will

be added

to

1 mL

of the MTT solution

and then incubated

in the dark

at 37°C

for one

to three hours.

If the MTT solution

color turns blue/purple,

the test article

is presumed to have reduced

the MTT. Water insoluble

test materials

may show direct reduction (darkening)

only

at the interface between

the test article

and
the medium. 7.5 Solubility Compatibility

Testing 7.6

Prior

to testing

in the assay,

the solubility

of the test article

in DMSO

(or water,

if not soluble

in DMSO)

may

be assessed

as determined

by the Study Director, especially

if the solubility

of the test article

is unknown.

The preferred
solvent

is DMSO.

The test article

will

be diluted

in DMSO

at the highest

100X concentration

to be tested

in the definitive

assays.
Some

test articles

may cause precipitation

in DMSO

or water.

If the test article

is not soluble

in DMSO,

the test article

may

be tested

in sterile deionized

water.

If the test article forms

a homogenous

working suspension

in either DMSO

or water,

the Study Director may elect

to proceed

with testing. Note: Although

some chemicals

may

not

be fully soluble

at the maximum

dosage, the gene induction

may
still

be measured

in many cases (Natsch,

2009 SOP). Maintenance

of KeratinoSens

cell line Cryopreserved KeratinoSens

cells (Givaudan),

tested

for the presence

of mycoplasma contamination,

will

be stored

in the liquid nitrogen freezer.

The stock ampules

will

be thawed

and seeded

as needed.

The cells

can

be maintained for
up

to 25 passages. The cryopreserved KeratinoSens

cells will

be thawed

in

a water
bath

at approximately

37°C.

The cryovial

will

be gently swirled

in

a beaker containing 70% ethanol.

Once thawed,

the cryovial

will

be immediately decontaminated with 70% ethanol

and the contents

will

be transferred

to

a sterile centrifuge

tube and diluted

by slowly adding approximately

9.0 mL

of KeratinoSens

Assay Medium.

The cells will

be centrifuged

at 200

x

g for approximately

5 minutes

to remove

the DMSO. Following centrifugation,

the supernatant

will

be removed and the cells will

be resuspended

in approximately

15 mL

of Assay Medium. Cultures

will

be incubated

at 37

±

1 oc humidified

air containing

5

± 1% C0

2 (standard
culture conditions).

Cells

are initially maintained

in Assay Medium. They

are passaged

every

2-4 days

to maintain

a cell density

less than 80%

of confluence.

Cells

will

be maintained

in Maintenance

Medium
(Assay Medium supplemented

with G418)

after

the first passage

upon thawing.

These stock cultures

will

be used

to seed

the 96-well plates.c c

llVS Study Number: 13AL98-AM03, AN30-AN31.170001 IIVS Protocol

No. SP170001

12/5/13

6 of17 7.7 7.8

Cell Culture Procedures Routine passaging procedures:

The KeratinoSens

cells
will

be subcultured

when the flask(s)

are 60--90% confluent.

The medium

will

be aspirated

and
the cell sheet

will

be rinsed twice
with
-10 mL ofDPBS containing

0.05% EDTA

for approximately

2 minutes.

One
mL

of trypsin/EDT

A will

be added

to each flask

to cover

the cell sheet.

The flask
will

be incubated

at standard culture conditions

for 5-10 minutes

(or until they became dislodged).

When more
than 50%

of the cells become dislodged,

the flask
will

be rapped sharply against

the palm

of the hand. When more
than 90%

of the cells become dislodged,

the cells
will

be each re-suspended

in

7 mL Maintenance

Media.

An appropriate

volume

of the trypsinized cells

in Maintenance

Medium

will

be added

to new flasks

for passaging.

The KeratinoSens

cells
will

be routinely passaged

every

2-5 days. 96-well

plate

cell procedures:

When

the flask(s)

are 60--90% confluent,

the KeratinoSens

cells
will

be prepared

for seeding

in the 96-well plates.

The medium

will

be aspirated

and the cell sheet

will

be rinsed twice

with
-10 mL

of DPBS containing

0.05% EDTA

for approximately

2 minutes.

One
mL

of trypsin/EDT

A will

be added

to each flask

to cover

the cell sheet.

The flask(

s) will be incubated

at standard culture conditions

for 5-10 minutes

(or until they become dislodged).

When more
than 50%

of the cells become dislodged,

the flask(s)

will be rapped sharply against

the palm

of the hand. When more
than 90%

of the cells become dislodged,

the cells will

be each re-suspended

in

5 mL Assay Medium (without G418).

The concentration

of cells
will

be determined

with

a coulter counter

or hemacytometer.

Cells

will

be diluted

to approximately

1.0

x 10

5 cells/mL

in Assay Medium. Subculturing KeratinoSens

Cells

Into 96-well
Plates Cultured

cells
that will

be used

in seeding

the 96-well
plates should

be fed fresh Growth Medium

the day before subculturing

to the plates.

Each definitive

assay will have

4 plates;

3 white-walled

plates

will

be used

for the luciferase endpoint, while

1 clear bottomed

plate
will

be used

for the cytotoxicity
endpoint.

These plates

are designated

as the

1X plates.

Each 96-well

plate
will

be uniquely numbered

and labeled

with

the seeding density,

cell type,

and date

of seeding.

Cell suspensions

of 1x10

5 cells/mL

in the Assay Medium

will

be prepared.

One hundred JA,L

of the cell suspension

will

be added

to the designated

wells

on the 96-well

plate (i.e.

all wells except

well H12, which

will

be left blank).

For example,

using

8 tips, one hundred

**~L**

of the cell suspension

will

be placed

into

the first

11 columns

of the 96-well
tissue culture microtiter

plate;

one

tip will

be removed

and 100
**~L** will

be added

to the remaining
column

of wells (A12-G12)

(1x10

5 cells/mL

= 1000 cells/well).

Using

a pipette,

100
**~L**

of Assay Medium

will

be placed

into

the well designated

as blank (H12).

The cells
will

be incubated

for 24±1 hours

at standard culture conditions.c

llVS Study Number: 13AL98-AM03, AN30-AN31.170001 llVS Protocol

No. SP170001

12/5/13

7 of17 7.9 Definitive

Assay Three definitive

assays

will

be performed

for each

test article.
Each plate

will consist

of up

to

12 concentrations

of up

to

7 test articles,

a solvent control

(6 wells),

a series

of

5 concentrations

of the positive control,

and

a single blank well. Each

test article concentration

will

be tested

in

a single
well. Each positive control concentration

will

be tested

in

a single
well. On the day following seeding,

visual observations

will

be performed

on the clear plate(s)

for appropriate

cell growth, uniform seeding,

and confluence.

Prior

to treatment,

the Assay Medium

was removed

from

the culture plates

by careful inversion

and blotting

of the plates

onto sterile paper towels.

The cells were

refed with

150
**~L**

of pre-warmed

(37°C)

1% DMEM (DMEM containing

1% FBS).
After re-feeding,

the plates
were incubated

at standard culture conditions until treatment. The test article(s)

will

be prepared

and treated

as described

below

in Section

7.9.1 (See also Figure

2). Test articles

will

be generally
diluted

in DMSO. Chemicals not soluble

in DMSO

may

be dissolved

and diluted

in sterile deionized

water,

and then DMSO

will

be added

to bring

the final concentration
ofDMSO

to 1%.

A 1 OOX DMSO master
plate, followed

by

a 4X Master

Plate containing

1% DMEM, will

be prepared

for each

set

of test articles,

the positive control,

and
the solvent control.

The preparation procedures

for each plate

are described

below;

and

a graphic overview

is outlined

in Figure

2. 7. 9.1 Preparation

of

1 OOX Master

Plate The 100X DMSO Master Plate(s)

will

be labelled

with

the appropriate

test article

and control concentrations

according

to the standard

plate map (see Figure

1). The 100X

top stock

will

be 200 mM

for neat chemicals, 40 mg/mL

for mixtures,

and
6.4 mM

for the positive control.

The serial dilutions

of the test article(s)

and the positive control

will

be prepared

in the 96-well

plate.

The dilutions

of the test articles

and positive control will

be prepared according

to their dilution schemes

and then added

to the appropriate

wells

on the 96-well

plate.

The highest

stock concentrations for the test articles

and
the positive control

will then

be serially diluted

in the DMSO master

plate

to create

the other

11 (or

4) dilutions. Note:

The wells designated

as solvent controls

will already contain 150

f.tL

of DMSO; therefore

no dilutions

will

be made.

The well designated

as blank

will remain empty. 7.9.2 Preparation

of 4X Master

Plate The corresponding

4X Master Plate(s)

with

1% DMEM

will

be similarly labeled

as the 100X DMSO Master
Plate.

300

**~L**

of 1% DMEM (DMEM without

G418 containing

1% FBS)

will

be added

to all applicable

test wells.cccIIVSStudyNumber:13AL98-AM03,AN30-AN31.170001IIVSProtocolNo.SP17000112/5/138of17Forwater-solubletestarticles:12.5J1L1%DMEMwillberemovedfromeachwellandreplacedwith12.5J1LDMSO(Totalvolumeremains300J.LL)TheconcentrationofDMSOinthe4Xplatewillbe4%afterthe12.5J1Ldosefromthe100xplateisadded).Thedilutionsfromthe100XDMSOMasterPlatewillbefurtherdiluted25-foldinthe4XMasterPlatewithMedia.12.5uLofeachsamplefromthe100XDMSOmasterplatewillbeaddedtothecorrespondingwellsof4XMasterPlatecontaining300of1%DMEM.Thewelldesignatedasblankwillcontain300of1%DMEM.7.9.3AdditionofTestArticletoCultures(Final1X)Theresulting4XMasterPlatewith1%DMEMwillthenbeusedtodistributedosesof50fromeachwelltothereplicateassayplatescontainingcells.Dosingdilutionswillbepreparedat4Xthedesiredfinalconcentrationsothatwhen50addedtothe150of1%DMEMalreadyintheeachwell,afinal1Xdosewillbeobtained.Aplatesealwillbecarefullyplaceddirectlyovertheculturewells.Thesealerwillbeassuredtoadheretoeachculturewell.The96-wellplatecoverwillbeplacedlooselyoverthesealedplateandwillbeincubatedwiththeplateunderstandardcultureconditions.Theplatecovershouldbeplacedlooselyatoptheplatesealtoavoiddeformingthesealerandcausingthesealertodetachfromculturewells.Aloosefitoftheplatelidisacceptable.Theplateswillbeincubated(withtheplateseals)for48±1hoursatstandardcultureconditions.The96welltemplate(Figure1)illustratestheplatesetupfortreatmentofthepositivecontrolandtestarticles.Thetestarticleconcentrations1-12correspondwiththeDoseIDnumbersfoundonthedilutionschemeforthespecifictestarticle.cITVSStudyNumber:13AL98-AM03,AN30-AN31.170001ITVSProtocolNo.SP17000112/5/13Figure1.PlatemapshowingfinalIXconcentration.Cone.("M)(r.tg/mL)ABcDEFGH0.9750.1951TIC12T2C12T3Ct2T4Ct2T5C12T6C12T7C12NC1.953.917.810.3910.7811.56234TIC11TIC10TIC9T2C11T2C10T2C9T3C11T3C10T3C9T4C11T4C10T4C9T5CuT5C10T5C9T6C11T6C10T6C9T7CuT7C10T7C9NCNCNCT1-T7-TestArticles15.631.362.53.136.2512.556 7TICsTIC1TIC6T2CsT2C7T2C6T3CsT3C7T3C6T4CsT4C7T4C6T5CsT5C7T5C6TQCsT6C7T6C6TICsT1C1T7C6NCNCPC(4)12525050025501008910TIC5TIC4TIC3T2C5T2C4T2C3T3C5T3C4T3C3T4C5T4C4 T4C3T5C5T5C4TSC3T6C5T6C4T6C3T7C5T7C4T7C3PC PCPC(8)(I6)(32)C1_12-Testarticleconcentrations(12=lowestconcentration,1=highest)NC-SolventControl(DMSO)PC-PositiveControl(64-4blnk-Blank(containsnocells)9of17100020002004001112TIC2TIC1T2C2T2C1T3C2T3C1T4C2T4C1TSC2TSC1T6C2T6C1T7C2T7CtPCblnk(64)Note:Platemapapplicabletotesting7testarticles.Whenlessthan7testarticleswillbetestedinanassay,thelocationofeachtestarticleontheplatewillbedesignatedinthestudyworkbook.Thetestarticleconcentrationsontheplatewillremainlowesttohighestfromthelefttorightsideoftheplate.(IIVSStudyNumber:13AL98-AM03,AN30-AN31.170001IIVSProtocolNo.SP17000112/5/1310of17Figure2.TreatmentofKeratinoSens:PlatePreparationOverview(dosesindicatedinmolarconcentrations)0.098mM-------------7200mM1:25dilution'12.5(<lLJnzflt-'lulr')1:4dilution0.98!lM2000!lMf0.98!lM2000!lMq0.98!lM2000!lM0.98!lM2000!lMXXJ...J..J..J...J..XXX.XxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxMTTCytotoxPlate(1X)LuciferasePlates(1X)llVS Study Number: 13AL98-AM03, AN30-AN31.170001 llVS Protocol

No. SP170001

12/5/13

11 of 17 7.9.2 Microscopic Evaluation After

at least

46 hours

of treatment,

visual observations,

using

the inverted or phase contrast microscope,

will

be performed

and recorded

for the clear-bottom cytotoxicity

plates

as indicated

in Table

1. Table

1: Visual observations

of Cytotoxicity

96-well plate(s) Note Code

Note Text

Note Code

Note Text 1

Normal

Cell Morphology

p

Precipitate observed

in well 2

Low Level

of Cell Toxicity

M

Media discolored

in well 3 Moderate

Level

of Cell Toxicity

N

No cells observed

in well 4

High Level

of Cell Toxicity

0 Precipitate

in wells observed

as oily droplets 7.9.3 Preparation ofPromega ONE-GloTM

Reagent The ONE-Glo

TM Reagent

will

be prepared

by reconstituting

the lyophilized Substrate

with

the appropriate

volume

of Assay Buffer.

The prepared reagent

will

be allowed

to equilibrate

for

at least

15 minutes

at room temperature

(optimal temperature

for enzymatic
activity)

prior

to use. 7.9.4 Treatment Termination: Luciferase Endpoint

Assay After approximately

48 hours

of exposure,

each white-walled

culture

plate will

be removed

from

the incubator

and allowed

to cool

to room temperature

for

at least

30 minutes.

Once

at room temperature,

the treatment medium

will

be decanted

from each plate.

The cultures

will

be rinsed

with
250

J!L

of CMF-DPBS

(room temperature),

and
the DPBS rinsate

will

be decanted

from

the wells. Excess DPBS

will

be removed

by blotting

the plates gently

onto paper towels. Fifty microliters

of CMF-DPBS

will

be added

to each well. Fifty microliters

of Promega ONE-Glo

TM Reagent

will

be added

to each well and
the plates

will

be allowed

to incubate

at room temperature

in the dark for approximately

5 minutes before being
read

by the luminometer. The plates

will

be kept away from sunlight

or bright fluorescent

light before reading.
Bright

light may cause plate phosphorescence

resulting

in higher background

levels.
Plate phosphorescence

has

a half-life

of several minutes.

The plates

will

be read within

45 minutes

of addition

of the OneGlo reagent.

Each plate
will

be placed

onto

the plate
tray

in the Berthold Detection Systems luminometer,

and
the luminescence determination

will

be initiated

from

an ffiM-PC hosting

the Windowsbased SimplicityTM

software.

The light intensity

in each well will

bec c

IIVS Study Number: 13AL98-AM03, AN30-AN31.170001 IIVS Protocol

No. SP170001

12/5/13

12 of
17 7.10

measured

at 565 nm

in the form

of relative

light units (RLUs).The

raw data

for each plate reading

will

be saved

to

a unique

file

by the SimplicityTM

software,

and

a screenshot
printout

will

be made

of the raw data. 7.9.4 Treatment Termination: Cytotoxicity

Using

the MTT Endpoint A 0.59 mg/mL
MTT solution

will

be prepared

in 1% DMEM

and used within

2 hours. After

48
±1 hours,

the clear 96-well
plates designated

for the MTT endpoint

will

be removed

from

the incubator.

The test article dilutions

will

be decanted

from

the plates.

No rinsing

will

be performed. Two hundred

J..LL

of 1% DMEM containing

0.59 mg/mL

MTT

will

be added

to each well.
The plate
will

be incubated

with

a plate

seal

at standard culture conditions

for 4±0.3

hrs. After 4±0.3 hours,

the MTT solution

will

be decanted,

the plate

will

be blotted,

and 200

J..LL

of 10% SLS will

be added

to each well.

The plate
will be covered

with

a plate

seal and incubated

at standard culture conditions overnight. After

the overnight incubation,

the plate(s)

will

be placed

on

a plate shaker and shaken

for

at least

20 minutes

at room temperature.

The absorbance

at 570
nm (0Ds

7 o) of each well will

be measured

with

a Molecular
Devices Vmax
plate reader.

The plate reader

will

be turned

on

at least

15 minutes prior

to use,
and will

be calibrated according

to standard operating procedures.

The AUTO

MIX setting

will

be selected.

The reading

will

be saved

to

a designated

print

file and

a print screen

will

be made

of the raw data. Relative survival

will

be obtained

by comparing

the amount

of MTT reduction

by test article treated groups

to the MTT reduction

by the solvent treated group

on the same plate.

An

IC

50 value

will

be determined

for the MTT cytotoxicity

plate. Data Analysis For each

set

of chemicals,

a copy

of the standard Excel®

data analysis spreadsheet file, provided

by Givaudan,

will

be made

(i.e.

3 trials,
each with

4 plates

per trial3 plates

for the luciferase induction

and

1 plate

for MTT cytotoxicity). In the designated

Excel® spreadsheet

standard

file,

the fields which
need

to be filled

in will

be marked yellow.

On the 'Summary

sheet'

the compound identifiers

and
the plate identifier

will

be inserted.

On the sheet

'rep

1' the plate readout

of the triplicate analysis

of the first definitive

assay

will

be directly inserted

in the yellow

areas marked repetition

1.1,
1.2, and
1.3. The plate readout of the triplicate analysis

of the second definitive

assay

and third definitive

assay will

be added

to the sheets marked

'rep2'

and "rep3"

as described

for the first

for the first definitive

assay.

The cytotoxicity

plate readout results

from each repetition

will

be inserted

into

the sheets labeled 'Cytotoxicity

(1)-

(3), with

thec

llVS Study Number: 13AL98-AM03, AN30-AN31.170001 IIVS Protocol

No. SP170001

12/5/13

13 of 17 results

from

the first definitive

assay being inserted

into Cytotoxicity

(1) and

so on for cytotoxicity

results

for the second

and third definitive

assays. 7 .10.1 Cytotoxicity

Data Analysis The raw data values

for each well will

be imported

into

the Excel spreadsheet (provided

by Givaudan)

for cytotoxicity

data analysis.

The optical density

(OD) for the MTT endpoint

is 570
nm (0Ds

70

). The corrected

OD
570

, 550 value

of the DMSO solvent control,

each individual

test article concentration,

and
the positive control

will

be determined

by subtracting the raw ODs

1 o1sso value

of the blank

well (H12)
from their mean 0Ds7o/sso

values. Corrected ODs?o!sso

value

= Raw data 0Ds7o/sso

- raw 0Ds7o/sso

of blank

well The mean corrected
solvent control

0Ds

70

, 550 value

will

be determined

from

the

6 solvent control

wells (A12, B12, C12,

D12, E12,

and F12).

For each well,

the percent viability

will

be calculated

as the ratio

of the corrected

ODs

1 o1sso

of the test

or control sample to the corrected

ODs

1 o1sso

of the appropriate

solvent control.

Corrected

OD 5101550

of test

or control article cell viability

= --------------

X 100 Mean corrected

OD

570

, 550

of Solvent Control The Givaudan

Excel

file will calculate

an IC

50 value using

two concentrations, one

of which results

in viability

of <50%

and one >50%

of the solvent control. The summary

IC

50 which appears

on the 'Summary
Results'

page

is calculated

by determining

the mean

of the

IC

50 values
from each definitive

assay

. . · 7.10.2 Luciferase

Data Analysis Raw data from

the luminometer

will

be transferred

directly

from

the luminometer software

into

the designated

Excel® spreadsheet (provided

by Givaudan)

for luminescence

analyses.

Each definitive

assay

will include

a set
of

3 luciferase plates.
Since
each plate contains

a set

of solvent control

wells

and

a blank well, the corrected
RLUs6s values

and individual

well fold induction

for each concentration

of the test article

or positive control

will

be determined

using

its corresponding

plate solvent control

wells

and blank well. The corrected

RLU

565 value

of the DMSO solvent control,

each individual

test article concentration,

and
the positive control

will

be determined

by subtracting the raw RLUs6s

value

of the blank

well (H12) from their mean RLUs65· Corrected
RLUs65 value=

Raw data RLUs6s-

raw RLUs6s

of blank

well The
file will then automatically

calculate

the gene induction

and
the wells with statistically significant induction

over

a given threshold (default

value

set

to 1.5

= 50% enhanced

gene activity).

To calculate

the fold gene induction

(over

the controls),

the corrected

RLU

565 value

of each well will

be divided

by the mean corrected

RLU

5 6 5 value

of the solvent control

wells (which equals

1).c

IIVS Study Number: 13AL98-AM03, AN30-AN31.170001 llVS Protocol

No. SP170001

12/5/13

14 of 17 Corrected

RLU

565 value

of test article

or positive control concentration Fold Induction

= ---------------------- Corrected

mean RLU

565 value

of Solvent Control An average

fold gene induction

for each

test article

at each concentration

will

be determined

using

the

3 replicate

plates

(for each definitive

assay).

The average fold gene induction

at each concentration

will

be used

to calculate

the EC
1.5 value (concentration

for induction

1.5 fold above threshold)

for each definitive assay.

For each

test article

at each concentration

the values obtained

from repetitions

1,
2, and

3 are compared

to the average solvent control values

of each plate from repetitions

1, 2, and

3 using

an unpaired two-tailed

t-test.

An average fold induction

value

of greater

than

1.5 which

is statistically significant (significance

is defined

by

a p-value

of <0.5)

is used

to calculate

the EC1.5 value. The spreadsheet calculates

the EC1.5

both with linear

and log-linear extrapolation.

The final EC1.5 value

for each

test article

will

be determined

by averaging

the mean EC1.5

of the

3 definitive

assays. Furthermore,

the maximal induction

(lmax)

will

be determined.

The Imax

for each

test article

run

in each definitive

assay

is simply

the largest average

fold induction

value

for that test article.

An Imax value

for each

test article

will

be calculated

in the 'Summary

page'

by simply taking

the arithmetic

mean

of the Imax values obtained

for each

test article
from each definitive

assay.

The concentration

for maximal

gene induction (Cimax)

will

be calculated

for each definitive

assay.

The Clmax

is simply

the concentration

at which

the largest average

fold induction

value, which

is statistically significant,

is achieved. The results

from

the different repetitions (definitive

assays)

will

be summarized

in the 'Summary'

file.
The data, including

the average

fold induction

and

IC

50 values

for each

test article

at each concentration

will

be automatically

plotted

in the graphs

on the 'Summary'

page

and

in the different repetition

sheets.

The Study Director

may manually
modify values

in the spreadsheet

as needed.

All manual modifications

will

be documented

in the final report. 8.0 CRITERIA

FOR DETERMINATION

OF

A VALID
TEST Average luciferase

gene induction

by the positive control

( cinnamic aldehyde)

must cross the

1.5 threshold

in

at least

one dose

in each definitive

assay. Furthermore,

each definitive assay

will

be accepted (considered

valid)

if the cinnamic aldehyde luciferase

gene induction

falls within

2 standard deviations

of the historical

mean (updated

every three months). Further determination

of the validity

of an assay

will

be

at the discretion

of the Study Director.

Each assay

will

be assessed

(but
not necessarily

deemed

a valid assay) using similar criteria

as outlined

in the Ring Trial (Natsch,

et al. 2011):

1) The standard deviation

of the viability

of the solvent controls

for the luciferase

plates

for

all of the definitive

assays

(9 total plates)

will

be <20%;

2) The cinnamic aldehyde

will

becc(llVSStudyNumber:13AL98-AM03,AN30-AN31.170001llVSProtocolNo.SP17000112/5/1315of179.0significantlypositiveandresultinanEC1.5valueless than64J..tM;and3)Thecinnamicaldehydewillproducea 2to8foldinductionat64J..tM.EVALUATIONOFTESTRESULTSThesensitizationpotentialofatestarticlewillbeevaluatedusingthesamecriteriaasoutlinedinRingTrial(Natsch,etal.,2011).Atestarticlewillbeconsideredtohavesensitizationpotentialif:1)TheEC1.5valuefallsbelow1000J..tM(or200J..tg/mL)inatleast2of3repetitions;2)Atthelowestconcentrationwithageneinductionabove1.5,cellularviabilityshouldbegreaterthan70%;and3)Anapparentoveralldoseresponseshouldbesimilarbetweenrepetitions.Thepredictionmod;elfortheKeratinoSensassaywasbasedonstudieswithneatchemicals,notextractsormixtures.Thepredictionmodelpresentedabovebasedon(wlv)concentrationsisbasedonthepredictionmodelestablishedforneatchemicals,butmaynotaccuratelyassessmixtures.10.0REPORT11.0AreportoftheresultsofthisstudywillbepreparedbytheTestingLaboratoryandwillaccuratelydescribeallmethodsusedforgenerationandanalysisofthedata.Acopyoftheprotocolusedforthestudy,anyamendmentsandanysignificantdeviation(s)fromtheprotocolwillappearasapartofthefinalreport.RECORDSANDARCHIVESAseparateworkingnotebookwillbeusedtorecordthematerialsandproceduresusedtoperformthisstudy.Uponcompletionofthefinalreport,allrawdata,reportsandspecimenswillberetainedinthearchivesforaperiodofeithera)5years,b)thelengthoftimespecifiedinthecontracttermsandconditions,orc)aslongasthequalityofthepreparationaffordsevaluation,whicheverisapplicable.12.0PROTOCOLAMENDMENTS13.0Whenitbecomesnecessarytochangetheapprovedprotocolforaspecificstudy,thechangeandthereasonforitshouldbeputinwritingandsignedbytheStudyDirectorassoonaspractical.Whenthechangemayimpactthestudydesignand/orexecution,verbalagreementtomakethischangeshouldbemadebetweentheStudyDirectorandSponsor.ThisdocumentisthenprovidedtotheSponsorandisattachedtotheprotocolasanamendment.REFERENCESNatsch,A.andEmter,R.(2008)Skinsensitizersinduceantioxidantresponseelementdependentgenes:Applicationtotheinvitrotestingofthesensitizationpotentialofchemicals.ToxicologicalSciences.102(1):p.110-119.JIIIr(I/IllVSStudyNumber:13AL98-AM03,AN30-AN31.170001IIVSProtocolNo.SP17000112/S/1316ofl714.0Natsch,A.,Emter,R.,andEllis,G.(2009)Fillingtheconceptwithdata:Integratingdatafromdifferentinvitroandinsilicoassaysonskinsensitizerstoexplorethebatteryapproachforanimal-freeskinsensitizationtesting.ToxicologicalSciences.107(1):p.106-121.Berridge,M.V.,Tan,A.S.,McCoy,K.D.,Wang,R.(1996)TheBiochemicalandCellularBasisofCellProliferationAssaysThatUseTetrazoliumSalts.Biochemica4:14-19.Natsch,A.,BauchC.,Foertsch,L.,Gerberick,F.,Norman,K.,Hilberer,A.,Inglis,H.,Landsiedel,R.,Onken,S.,Reuter,H.,Schepky,A.,andEmter,R.(201I)Theintra-andinter-laboratoryreproducibilityandpredictivityoftheKeratinoSens·assaytopredictskinsensitizersinvitro:Resultsofaring-studyinfivelaboratories.ToxicologyinVitro.25(3):733-774SPONSORREPRESENTATIVEDATEPaulRibeiro,Ph.D.,JoanCheng,Ph.D..(PrintorTypeName)lfiII·II.IIIllVS Study Number: 13AL98-AM03, AN30-AN31.170001 llVS Protocol

No. SP170001

12/5/13

1 of

1 PROTOCOL ATTACHMENT

1 IIVS TEST ARTICLE

SPONSOR DESIGNATION

MOLECULAR

WEIGHT 13AL98

Research Fragrance Chemical

TM 11-0078

182.1 g/mol 13AL99

Research Fragrance Chemical

TM 10-0210

194.2 g/mol 13AMOO

Research Fragrance Chemical

TM 10-0367

184.2 g/mol 13AM01

Research Fragrance Chemical

TM 09-0536

192.2 g/mol 13AM02

Research Fragrance Chemical

TM 11-0230

148.0 g/mol 13AM03

Research Fragrance Chemical

TM 08-0338

194.2 g/mol 13AN30

09-0035

200.2 g/mol 13AN31

05-0293

156.2 g/mol Additional

Testing Information:

: The test articles, 13AL98- AM03, AN30-AN31

are neat chemicals

and
the

1 OOX

top stock solution

will

be prepared

to

a concentration

of 200 mM

for each

test article. REGULATORY REQUIREMENTS: Will

this study

be conducted according

to GLPs?

[ ] YES

or

[X

] NO If YES, please indicate
which agency(ies) guidelines

are

to be followed: [ ] FDA;

[ ] EPA TSCA;

[ ] EPA FIFRA;

[ ] OECD;

[

] Other _____

_APPENDIXBc:0:wu3.00LUCIFERASEINDUCTIONANDVIABILITY:1401201001.508060.5"0:240200.00+---------r-------r------IC:a.....---+01.00s:::0u:::J"0.5"00c.2u3.001.500.00.a1.50.!::'C1.0010.00cone(uM)10.00cone(uM)100.001000.00-----induction•3AL98---140120100006040200100.001000.00lnOUCliOn1Y--B--Vitihility1::!AIQQ140120100806040200.00+-------..,..---------r--11.-..--fl!l----..01.0010.00100.001000.00cone{UM)-lrduc:ion13Afi.IOO---e-viablity13.1WCOBl:.0m"5>-::cCIS>:0"''Ss:;0:::7140120100-g1_5080"'CCs:;0:u604ULUo_ooo1.0010.00100.001000.00COIIlC(uM)-induction13Aiv10113AI'.11013.00140120100.g1.5080c"0:e604020+---------------r---------------.--------------+00.001.00010.000100.0001000.000:E>:£g:0>cone(uM)----+-induction13AM02--E!-viability13AM023.001401201:100.2u80::31.50'C:c,;;60>'Ce40200.0001.00010.000100.0001000.000cone(uM)--+-mducbon13MI03--s-viabihty13AM03B2

# s::: 0



# 140 120 1UU



# :E' 

# ! c

1 _ 50 

# +------------------t't-------;



# 80



# :c C'G 

# "C 

# ~

60



# > **~** 0 

# 40 20 

# o _ oo +--------,----------,r-------'_..;;::=-t9--•



# o 1 _ 000

10

_ 000

100_000

1000

_ 01)0 

# r----------, ---.-.... induction
13AN30 

# -e- viability 

# 13AN30



# cone (uM) 3.00

140 

# 120 t:



# 100 0 

# ts

80



# ~ 

# = 1.50 'C



# :c .5 "0



# 60



# s: 

# :2

**~"!** 

# 40 20 0.00

0 1.000

10.000

100.000

"1 000.000 

# cone (uM)

-+-inductcn



# 13AN31 -e-viabi

ity 13.AN31 

# B3