The ChemScreen project to design a pragmatic alternative approach to predict reproductive toxicity of chemicals

The ChemScreen project to design a pragmatic alternative approach to predict reproductive toxicity of chemicals

Bart van der Burg

, Eva Bay Wedebye

, Daniel R. Dietrich

, Joanna Jaworska

, Inge Mangelsdorf

, Eduard Paune

, Michael Schwarz

, Aldert H. Piersma

, E. Dinant Kroese

aBioDetectionSystemsBV,Amsterdam,TheNetherlands

bDanishTechnicalUniversity,Lyngby,Denmark

cUniversityofKonstanz,Konstanz,Germany

dProcter&Gamble,Brussels,Belgium

eFraunhofer-ITEM,Hannover,Germany

fSimpple,Tarragona,Spain

gUniversityofTübingen,Tübingen,Germany

hNationalInstituteforPublicHealthandtheEnvironment,Bilthoven,TheNetherlands

iUtrechtUniversity,Utrecht,TheNetherlands

jTNO,Zeist,TheNetherlands

Keywords:

Invitro Insilico Integratedtesting Readaccross Reproductivetoxicity Endocrinedisruption

a b s t r a c t

Thereisagreatneedforrapidtestingstrategiesforreproductivetoxicitytesting,avoidinganimaluse.

TheEUFrameworkprogram7projectChemScreenaimedtofillthisgapinapragmaticmannerprefer- ablyusingvalidatedexistingtoolsandplacetheminaninnovativealternativetestingstrategy.Inour approachwecombinedknowledgeoncriticalprocessesaffectedbyreproductivetoxicantswithknowl- edgeonthemechanisticbasisofsucheffects.Weusedinsilicomethodsforprescreeningchemicals forrelevanttoxiceffectsaimingatreducedtestingneeds.Forthosechemicalsthatneedtestingwe havesetupaninvitroscreeningpanelthatincludesmechanistichighthroughputmethodsandlower throughputassaysthatmeasuremoreintegrativeendpoints.Insilicopharmacokineticmoduleswere developedforrapidexposurepredictionsviadiverseexposureroutes.Thesemodulestomatchinvitro andinvivoexposurelevelsgreatlyimprovedpredictivityoftheinvitrotests.Asafurtherstep,wehave generatedexampleshowtopredictreproductivetoxicityofchemicalsusingavailabledata.Wehave executedformalvalidationsofpanelconstituentsandalsousedmoreinnovativemannerstovalidate thetestpanelusingmechanisticapproaches.Weareactivelyengagedinpromotingregulatoryaccep- tanceofthetoolsdevelopedasanessentialsteptowardspracticalapplication,includingcasestudiesfor read-acrosspurposes.Withthisapproach,asignificantsavinginanimaluseandassociatedcostsseems veryfeasible.

1. Introduction

Thecomplexityofthesystemofriskassessmentofchemicals doesnotallowrapidevaluationofbasictoxicityprofilesofchemi- cals.Becauseofthis,thetoxicologicalpropertiesofmostindustrial chemicalsthatareincommonusearelargelyunknown[1].New legislationsuchas REACHaims toend this unacceptablesitua- tionbymodernizingandstreamliningchemicalriskassessment[2].

∗Correspondingauthor.Tel.:+31204350750;fax:+31204350757.

E-mailaddress:bart.van.der.burg@bds.nl(B.vanderBurg).

However,thisapproachisunlikelytobesuccessfulwithoutincor- poratingalternative,integratedtestingstrategiesinwhichcostly andtime consuminganimaltestsarereplacedtoalargeextent byrapidandcost-effectivealternativetestingmethods.Aninte- gratedtestingstrategy (ITS)isamethodtocombinetestresults fromdifferentsources, includingnon-testinformation (existing data,insilicoextrapolationsfromexistingdataormodelling)to giveacombinedtestresult[3]Inclusionofinsilicoandinvitrotest resultsandpre-existingdatainprincipleallowsmorerapideval- uationoftoxicologicalpropertiesofchemicals.Suchmethodsare particularlyneededforreproductivetoxicitytestingofchemicals.

Reproductivetoxicityassessmentisimportantforbothmanand

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-291169 Erschienen in: Reproductive Toxicology ; 55 (2015). - S. 114-123

https://dx.doi.org/10.1016/j.reprotox.2015.01.008

theenvironmentandusesrelativelylargenumbersofexperimental animalsandtime-andresourceintensivetestingprocedures[4,5].

Unfortunately,thereareveryfewifanyalternativemethodsthat areabletocoverthiscomplexity.TheChemScreenprojectaimed tofillthisgapandselectsuitabletestsandplacetheminamore generalalternativetestingstrategy.

Becauseofthecomplexityoftheprocessofmammalianrepro- duction intact organisms are often regarded to be essential in assessingreproductivetoxicity ofcompounds.Eventhen,it has beenshownthatlargespeciesdifferencesexistandinterspecies extrapolation of developmental toxicity typically usually is not muchhigherthan60%betweensinglespecieswhenusingapical endpointsin animals[6].Nevertheless,resultsfromtheFrame- workprogram(FP)6programReProTectveryclearlyshowedthat an in vitro test battery covering only part of thereproductive cycleprocessescanprovideverypromising resultswithrespect toreproductivetoxicitytesting [7].It hasbeenarguedthatthe use of in vitro tests that assess mechanisms of toxicity may formthebasisfora newparadigmintoxicitytestingand could alsoimprovethepossibilitytoextrapolatebetweenspeciessince pathwaysof toxicity share many similarities betweendifferent species [8,9]. This mechanism-based approach of toxicity test- ingwasoneofthecornerstonesoftheChemScreenprogram.In 2005wealreadydevelopedapanelofmechanism-basedCALUX assays to assess hormonal activityof compounds [10], a panel which has shown tobe highly predictive for suchactivities in experimentalanimals[11,12].Someofthesemechanisticassays alsoformedapartofthepromising batteryoftestsusedinthe ReProTect[7].

WiththisinmindtheChemScreenprojecthasbeendesigned, aiming at further simplification of the screening battery by increaseduseofpreferablyhighthroughputmechanisticscreening assays[13].Weaimedtogeneratearapidscreeningsystem,that isrelativelysimple,cost-effective,andcanbeimplementedprefer- ablywithinthetight timescheduleoftheREACHprogram.The toolshouldbeflexibleandadaptableforapplicationsbeyondthis specificprogram,suchasforsafedesignpurposes,orprioritiza- tion.Tofillthegapofsuitablealternativemethodsforreproductive toxicitytestingweusedanovelhighthroughputapproachcom- bining insilico/invitromethods. Inthis approach wecombined knowledge of critical processes affected by reproductive toxi- cantswithknowledge onthemechanisticbasis of sucheffects.

Toreachourgoalsthefollowingobjectiveswereformulated,cor- respondingtothemajorChemScreenworkpackages (WPs;see Fig.1):

1.Establishinsilicoprescreeningmethodsprioritizinginvitrotox- icitytesting.

2.Establishdatabasesandinsilicomethodstoidentifypotential reproductivetoxicants.

3.Establishmentofsensitiveparametersandmediumthroughput invitroassays.

4.Establish a high throughputmechanistic pathway screen for reproductivetoxicants.

5.Establishmethodstopredictinvivoreprotoxicity.

Furthermoreampleattentionwaspaidtovalidationandappli- cation of thetest methods developed. The ChemScreen project startedatJanuary1,2010.Theprogramaimedtocollaboratively generateaninnovativetestingstrategycombininguniqueexper- tiseoftheparticipants.Toattainthislevelofinteraction,frequent meetings, and workshops were held, and a highprofile Scien- tificAdvisoryBoardwasinstalled tohelpguidethis process.In theinternationaladvisoryboardmajorstakeholders(JRC/ECVAM, OECD, US EPA, Industry, ECETOC) were represented. Beginning 2010theChemScreenprojectenteredanimportanttransatlantic

Fig.1.GraphicalrepresentationofChemScreen’smajorcomponents.Thedottedlines indicateareaswherealternativemethodsarerelativelywelldevelopedorunder constructioninvarious(FP)programsinwhichpartnersparticipate.Allworkpack- ages(WPs)areindicated,exceptWP6(ICTtools),WP7(dissemination)andWP8 (management).

collaborationwiththeUSEPANationalCenterforComputational Toxicology (NCCT), and the Texas Indiana Virtual Star Center (TIVSC).Agreementsonscientificcollaboration,data-andchemical sharingwereestablished.Herewewillpresentasummaryofobjec- tivesanddiscussobtainedresultswithintheworkpackages,and theoverallpicturethatemergesfromthis.Specificmaterialsand methodologiescanbefoundinthereferencedindividualpapers withinthisissue.

2. Resultsanddiscussion

2.1. Insilicoprescreeningmethodsprioritizinginvitrotoxicity testing

AcrucialaspectoftheREACHprogramisprioritization[2].With, asitnowturnsout,about50,000chemicalsneedinganupdateof toxicologicalinformationwithinadecade,thepotentialamountof safetytestingisenormous,whichrequiresthatchemicalsofhighest concernareidentifiedforprioritizedtesting.Anelaboratequantita- tivestructure-activityrelationship(QSAR)moduleaimedtoallow predictionofthe“leadinghealth effect”asrequestedinREACH, avoidingunnecessarytestingofcompoundsforwhichtheleading healtheffecthasbeenestablished.Forexample,compoundswith mutagenicpropertieswillnotbeevaluatedforreproductivetoxic- itysincefurtherinformationonitspotentialcarcinogenicityisof higherprioritywhenconsideringleadinghealtheffects.Therefore, inthispartoftheprojectweconcentratedontheestablishment andselectionofinsilicoprescreeningmethodstocategorizechem- icals, using QSARs. This wasdone for major classes of toxicity prioritizedinREACH(carcinogenesis,mutagenesisandreproduc- tive(CMR))toxicity[2]. Positivepredictionsforcarcinogenicity,

Fig.2. Predictions usingQSAR analysisusingpre-screeningtools for human health effectsapplied to over 70,000 REACHpre-registrationchemicals[Wedebye etal.,2015].

mutagenicityand reproductivetoxicity canbeappliedto avoid unnecessaryanimaltestingofcompounds.Asnotestingforrepro- ductiveeffectsshouldbeperformedinREACHonknowngenotoxic carcinogensor germ cellmutagens withappropriate risk man- agementmeasuresimplemented,asstatedintheREACHannexes VIII-X,predictionsforgenotoxiccarcinogensandgermcellmuta- genscanfurthermorebeappliedtoavoidtestingforreproductive toxicity.TomaketheQSARscreeningascomprehensiveaspossi- bleitwasbasedonasmanyaspossibleofthe143,835chemicals, whichwerepre-registeredbeforeDecember2008.Thepredictions werebasedonstructureinformationgeneratedbytheComputa- tionalToxicologyGroupwithintheJointResearchCenter(JRC)for 80,413pre-registeredsubstances(PRS),ofwhich70,983chemicals weresuitable forgenerationofQSAR predictionsin theapplied QSARsoftware[14].ResultsareshowninFig.2showthatmore than25%ofthechemicalshavestructuralalertsofgenotoxiccar- cinogensandgermcell mutagens,which willexcludethem for beingtestedforreproductivetoxicity.Aspartoftheinvitropre- screen,it wasalsoenvisagedtodevelopanexposuremoduleto identifychemicalsthatwillhavenooranegligiblesystemicavail- ability (and anticipating that gonads and embryos will not be exposed)afterexposureviadifferentroutes.Tothateffect,liter- aturesearcheshavebeenperformedtoinvestigateononehand whetherspecificchemicalpropertiescanbeidentifiedtopredict internalexposureafterexposureviatheoral,respiratoryandder- malroutes,and ontheotherhandwhich(publiclyavailable)in silicotoolsexistleadingtoreliablepredictionofnegligibleinter- nalexposure.Basedonthecurrentknowledge,nocut-offsbased onphysicochemical properties couldbe identifiedfor thethree mainexposureroutesbeloworabovewhichinternalexposureis negligible[Buistand Verweij,unpublishedresults].REACHdoes providephysicochemicalcriteriatowaivetoxicitytestingviathe respiratoryroute,butthesecriteriacannotwaivetestingviaother routesofexposure.Therefore,itwasconcludedthatinclusionof thesecriteriainamoduletopredictinternalexposureaimedat excludingreproductivetoxicitytestingbasedonphysicochemical criteriaisnotaviableoption.Instead,thepharmacokineticmodels willnowbeusedexclusivelyinextrapolationofinvitrotoinvivo data.

2.2. Establishdatabasesandinsilicomethodstoidentify potentialreproductivetoxicants

Anextstepinourstrategywastofurtherexpandthepossibili- tiesofidentifyingpotentialreproductivetoxicantswithoutfurther testing.Databases wereexpandedthat categorizetheeffects of knownreproductive toxicants. These databases can be usedto derive so-called read across information that allows extrapola- tionfromaknowntoxicanttootherrelateduntestedmolecules.

Thiscan be complemented with otherin silico,or in vitro test information onthesecompounds. We expandeddatabases that containinformationrelevanttoreproductivetoxicityintheRep- Dose(repeateddose)[15]andFeDTex(fertilityanddevelopmental toxicity)databases[16].First,reproductivetoxicitystudieswere identifiedfrompeerreviewed publications.Allavailablestudies onreproductivetoxicitywereselectedfordataentry.Inparticu- lar,theamountofchemicalsinFeDTexweremorethandoubledin thecourseofChemScreen,mountinguptoover300.Theexpanded databaseswereusedtoidentifyredundantdatacomingfromani- malstudiesandthuspossibilitiestoreducetesting.Evaluatingthe responsivenessofthedifferentgenerations,acomparisonofthe doselevelsattheNo-Observed-EffectLevel(NOEL)wasperformed.

ThesedatasuggestatendencyofF0andF1beingmostlyequally sensitiveinone-andmulti-generationalstudies,whereasF2seems tobelessindicativeforNOELderivation[16].Thisconfirmsearlier workon2-generationstudydatabasesbyPiersmaetal.[17].

AsignificantoverlapwithRepDosewascreatedtoallowcom- parisonofinvivodatawiththesamechemicalandevaluatepossible predictivityofrepeateddosetoxicityforreproductivetoxicity.Tar- getorgansandeffectsobservedwithinrepeateddosetoxicitywere exploredforanyrelationshiptoeffectsoccurringinreproductive toxicitystudies.Whetherandhowminimaleffectconcentrationsof repeateddosetoxicitycanbeextrapolatedtoreproductivetoxicity appearedtodependonthechemicalclassofthecompoundsstud- ied.Forexample,noneoftherepeateddosestudiesonchlorinated phenols(includingphenolasparentalsubstance)providedanindi- cationforreproductivetoxiceffects.,Themajoreffectsobserved afterrepeatedexposurewerebodyweightlossrelatedtoreduced availabilityofenergyandincreasedcatabolismaswellaschanges

inliverweightandpathology.Thereproductivetoxicityobserved mightalsoberelatedtoinsufficientenergysupplyduetouncoupled oxidativephosphorylationresultingingrowthretardationoreven increasedprenatalmortality.Themechanismof toxicityis thus likelytobethesameinrepeateddoseaswellasreproductivetoxic- itybutwithoutmechanisticknowledgeitisdifficulttopredictsuch effectsbasedofrepeateddosetoxicityobservationsonly.Foralco- holicsolvents,phthalatesandglycoletherscomparableconclusions havebeendrawn[Batkeetal.,unpublishedresults].

UsingthestructuraldataofthePRS(seeSection1)areproduc- tivetoxicityscreenwascarriedoutaswell,howeveronlycoveringa smallpartofreproductivetoxicity(teratogeniceffectsbyalimited numberofmechanisms).Thelimitedamountofcompoundsavail- ablefortrainingandvalidationincombinationwithmanydifferent mechanismsandstructuralalertshamperdevelopmentofgener- allyapplicableQSARmodelsinthisfield[18].

Severaltoolsweredevelopedthatcanassisttopredictingenvi- ronmental reproductive toxicity of chemicals on a mechanistic basisaimingatmorestraightforwarddatainterpretation[Dietrich et al., unpublished results]. By focusing on molecular mecha- nismsconservedinvariousspecies(e.g.variousreceptor-mediated processes) possibilities wereexplored toextrapolate molecular screeningdatatoeffectsinaquaticorganisms(particularlyfish)and thusforecotoxicologicaleffects.Forthis,extrapolationmethods havebeendesigned toestablishifthereareconserved molecu- larmechanismsinaquaticorganismsresponsivetotheactionof differentchemicals. Asone of thefirst stepsanexpansion and restructuringof thelargeEDUKON ecotoxicology database[19]

hasbeencarriedoutaswellasaninventoryofpossiblechemo- biological interactions thatcan berelated todistinct molecular mechanisms.Allrelevanttechnicalprerequisitestoscreenandto usetheEDUKON DBasintended withintheprojectdescription werefulfilled,andtheintegrationofapproximately1400newdata setsof peer-reviewedstudies intotheDB wasaccomplishedin accordancewiththeintentiontointegratethenewest scientific findingsintotheDBandtosetittothecurrentstateofscience.

Theheterogeneityofthedatainvolvingmanyspeciesandeffects togetherwiththecomplexitiesofreproductivetoxicity,however, hamperedthegenerationoffirmconclusionsontheapplicability ofmechanisticinsightsinenvironmentalriskassessment[Dietrich etal.,unpublishedresults].

2.3. Establishsensitiveparametersandamediumthroughput invitroassaypanel

Theaimofthispartoftheprojectwasthedesignofarepro- ductivetoxicityscreen,consistingofanumberofinvitroassays thatarerepresentativeofthoseparametersinreproductivetox- icitythatarecrucialforreproductivehazardassessment.Oneof thestartingpointswasarathercomplexbatteryoftestsoriginally developedintheFP6ReProTectprojectusingprimarymaterialand cellsfromvarious sources(e.g.ratembryos,oocytes,spermato- cytes,etc)[7].Thefirstapproachin simplificationofthetesting battery was directed towards inclusion of integrative medium throughputassays thatcanberunin aroutinelaboratoryenvi- ronment,avoidingassayswithdifferentiated(primary)tissuesas presentintheReProTectbattery.Thiscanthenbecombinedwith morehighthroughputassays(seenextsection)therebyenhanc- ingthroughputandfurthersimplifyingtestprocedures[20].The nextstepwouldbetovalidatethis batteryand selectthemost promisingassaysneededtogiveproperpredictionsofreproductive toxicityofchemicals.Ourapproachinthatsenseisanintermedi- atebetweenthisReProTectapproachandthatofToxCast,which initiallyfocused ona much largervarietyof lessselective high throughputscreening(HTS)assays[21].Weexpectedthatasmaller batterywouldreducenoiseandallowmorestraightforwarddata

interpretation.Validationstepssubsequentlycanleadtoidentifi- cationofmissingendpoints,andthroughinclusionofadditional teststhisbatterycanbefurtherimprovedwithtime.

Atthestartoftheprojectweenvisionedthatonewaytosimplify testingschemesistheapproachofidentifyingcriticalendpoints forreproductivetoxicityusingliteratureand databasesearches, andfocusingondevelopmentoftestsfortheseendpoints.Sofar mostendpointsidentifiedwerequitegenericandnon-specificin nature,whichmaymeanthatscreeningpanelsshouldalsoinclude quitesimple,relativelynon-specifictests(cytotoxicity)inaddition to more mechanisticassays. We have therefore includedsepa- ratecytotoxicityendpointsinallourscreening assaysandhave extendedthenumberofmedium-andhighthroughputmechanis- ticscreeningassays.

Firstofall,progresswasobtainedwiththeoptimizationand amendmenttohigherthroughputofteststhataimedtobeincluded inthefinalbattery,namelyagenomics-basedimprovedembry- onicstemcelltest(EST),thezebrafishembryotoxicityassay(ZET) and steroidogenesis assays, in particular four CYP17/19 assays.

In theembryonic stem cell test,transcriptomics readouts were employedtoimprovethemechanisticreadoutofthesystemand improveitspredictivevalue.TheCYP17/19assayswerebeingopti- mizedandmicrosomesversuswholecellassayswerecompared fortheirpredictivevalue[22].Theembryonicstemcellassaywas appliedtothecategoryofphthalatesandpredictivitywithinthis class of compoundsona comparativebasis withexisting tran- scriptomicspatternsof otherphthalates wasshown[23,24].All above-mentioned tests were included in a first, relatively low throughputversionoftheChemScreenbattery[25].

2.4. Establishahighthroughputmechanisticpathwayscreenfor reproductivetoxicants

Amajorfocuswasontheestablishmentofmechanistictests andadaptingthemtohighthroughputscreeningmethodspredic- tiveofreproductivetoxicpotential,basedoninsightinmolecular mechanismsthatarerelevanttoreproductivetoxicity.Theseassays compriseapanelofhighlyspecifichumanCALUX^®reportergene assays,whichincludesaselectionoutofabout50differentassays.

ThispanelisbasedonthehumanU2-OScelllinewhicharemod- ifiedtoexpressreportergenesforseparatepathways[10,11].The highselectivityisreachedthroughtheuseofreportergeneswith syntheticpromotersconsistingofminimalpromoterelementscou- pledtomultimerised,highlyselectiveresponseelementsdriving theexpressionofthereportergeneluciferase.Whenrelevantto thepathwayofinterest,specificreceptorsareintroducedtoacti- vatethecognatereportergene.Thepanelofselectiveassayswas designedtocreateverylowlevelsoffalse-positives.Thepanelcon- tainsasinglereportergeneassayperpathway.Thisisdifferentin themuchlargerToxCastassaypanelthattypicallyincludesmulti- pleassaysperendpoint[26].

Asystematicapproachhasbeentakendevelopingthescreening panelusingverysensitiveandselectivereportergenes,covering a wide range of receptors and signaling pathways that poten- tially are involved in reproductive toxicity [20]. The approach takenaimedtoprovideseveraladvantages ontopofalow rate offalsepositivity.First,byprovidingamechanisticbasisregula- toryacceptanceshouldbefacilitated.Second,thismechanisticbasis willprovideinputfordecisionsonfurthertestrequirementsand riskassessment.Third,cost,speed,robustness,andquantification areadvantageousinthesetypesofassays.Finally,thisapproach facilitatespredictionsforbothhuman-andecotoxicologicalprop- erties ofchemicals,when differentprediction modelsare used.

Oneapproach takentodevelop additional reportergene assays forChemScreenistousecentralintracellularpathwaysthatare involved inregulatingtranscriptionin amammaliancell.These

pathwaysincludebesidesnuclearhormonereceptorsalsoreporter geneassaysforquitegenericresponsestotoxicants,includingcyto- toxicity,apoptosisandgenotoxicity.Theseresponsescanalsobe relevantinreproductivetoxicity,however.Acontrolcellline,called cytotoxCALUXhasbeengeneratedwhichconstitutivelyexpresses theluciferasegene[27].Tovalidatethisapproachofusinglimited setsofselectiveassaysitwasshownthatasingleselectiveand extensivelyvalidated CALUX assay(ERalphaCALUX)gave com- parableresultsasabattery ofToxCasttestsmeasuringestrogen receptorpathway-mediatedendpoints[VanderBurgetal.,unpub- lishedresults].

InadditiontothesehighlyselectiveCALUXHTSreportergene assays, other assays have been established in more complex, murineembryonicstem(ES)cellsbyintroducingreportersystems forsignalingpathwayscontrollingkeydifferentiationpathwaysin embryonicdevelopment(so-calledReProGloassays)[28].Despite severalattemptstointroducereportergenesotherthanthatmea- suringwnt-pathwayactivation,asusedintheoriginalReProGlo assay,noneofthemsucceeded[Uibeletal.,unpublishedresults].

AllCALUXandReProGloassayshavenowbeenautomatedin 384and 96wellsformat,respectively,anda seriesofreference compoundshasbeenusedtodeselectlessinformativeassaysand identifymissingendpoints.Datastorageandanalysishasbeenset upandmorethan250compoundshavebeenscreenedintheCALUX panelandasubsetintheReProGloassay.Becauseoftheneedofa practicaltooltostoreandprocessthelargeamountofhighthrough- putdatathispartofthetoolwasgenerated.Itprovidesthefeatures requiredtostore,manageandquicklyanalyzeassaydatagenerated inhighHTSbioassaysallowingaccesstoprimarydata.Thistoolwas successfullyconstructedandinstalledandallCALUXHTSscreening datahavebeenuploaded.Finally,theCALUXHTSpanelendoge- nouslyexpresseslittlemetabolicactivitybutcanberunwithand withoutS9metabolicfractions[27].

Ourapproachfromthebeginningonwardsfocusedoninclusion ofalimitednumberoftestswithrelevantmodesofaction.Thisis differentfromtheToxCasttestpanelthatincludesawiderrange ofmechanismswithmultipletestspermechanisticendpoint[21].

Resultssuggestthatitmaynotbeneededtoincludea rangeof assaysforeachendpointtomakegoodpredictionsofinvivoactiv- ityofchemicals,aswasshownfortheestrogenreceptorreporter geneassays [20].Thisis consistentwiththeearliernotionthat transactivationofERsinoneassay,ERsubtypeorspeciescanbe extrapolatedtootherspeciesorsubtypesofERsforthepurposeof chemicalscreeninginthecurrentpracticeofriskassessmentrely- ingonrelativelyuninformativeanimaldata[29].Alsoinapanel theassays performedverywellpredictingreproductivetoxicity inmoregeneralterms[25]whichagainsuggeststhatarelatively simplescreeningpanelcansufficeforthispurpose.

2.5. Establishmethodstopredictinvivoreprotoxicity

Asanextstepinourtestingstrategywehavebeenestablishing integrativemethodstopredictinvivoreproductivetoxicitycon- centrationsusinginvitrobenchmark(threshold)concentrationsas astartingpoint,alsoreferredtoasreversedosimetry[30].Predic- tionofthecorrectinvivodoselevelatwhichadverseeffectscanbe expectedisoneofthekeyissues.Highthroughputpharmacokinetic modelshavebeensetupdescribingthebioavailabilityviarelevant routes(oral,dermal,inhalation)andthepharmacokineticsofthe mostrelevantreferencechemicalclasses.ArapidPBPKmodeling strategyhasbeendevelopedtotranslateinvitroconcentrationsto invivomaternaldoselevels([31];Fig.3).Insteadofbuildingcus- tommodelsforeachchemical(class)asplanned,itwaschosento developonegenericPBPKmodel frameworkdescribingallrele- vantcompartments,physiologicalprocessesandexposureroutes.

Thegenericmodelwithits(chemical-independent)anatomicaland

physiologicalparametervaluesinprincipleappliestoallchemicals.

Thepharmacokineticsofspecificcompoundshasbeenaddressed byprovidingchemical-specificparameters(relatingtopermeabil- ity,partitioningandclearance)thatcanbeobtainedbyeitherin silicopredictionsorroutineinvitromeasurements,asinputtothe genericmodel.Tothisend,analgorithm hasbeenimplemented topredictthebindingandpartitioningbehaviorofcompoundsin water,proteinandlipidphasesofblood(plasmaandredbloodcells) andtissues(interstitialfluidandcells).Thisalgorithmtakesasinput readilyobtainablechemicalproperties.Itcanalsobeusedtopredict bindingandpartitioningbehaviorinculturemediumifitspHand proteinandlipidfractionsareknown,andthustocorrectfordiffer- encesinunboundconcentrationsbetweeninvitroandinvivo.This genericframeworkismorecompatiblewiththerapidscreening characteroftheChemScreentoolasawhole,andallowspharma- cokineticpredictionofamuchlargernumberofcompoundsina relativelyshorttime[31].

In addition, to assess dermal exposure routes a specialized methodologyforinvitro—dermalinvivoextrapolationwasdevel- oped [32]. The method enables the calculation of an external exposuredoseleadingtosystemictoxicitybycombininginvitro experimentaldatarelevanttothetoxicantaffectedpathwayswith insilicopredictionsof skinpermeationand whole-body ADME.

Themethodisinlinewiththecurrentdevelopmentofmechanis- ticmethodsusedtopredictinvivotoxicityfromsetsofreference data,whichmaycombinedatafrominvivoexperiments,invitro cell-basedassaysand/orcomputationalresults.Duetoincomplete understandingofthemechanismsoftoxicityanddatalimitations, determinationoftherepresentativeconcentrationinvitrowillmost likelyneedtorelyonastatisticalapproachofchoosingthelowest valueofEC50distribution.Theuseoftheinternalsystemicconcen- trationexternaltoestimateasafedosehasbeencomparedtoan approachbasedoninvivodose(LOEL).ResultssuggestthatLOELs maybetooconservativeforuseinpredictionoftoxicityandrisk assessment[32].

2.6. Validation

Acceptance byregulatoryagencies is a major bottleneckfor applicationofanyalternativetesting strategy.Therefore,during theprojectampleattentionhasbeenpaidtovalidationandregu- latoryacceptanceofthetestmethodsdevelopedinChemScreen.

Thishasbeendoneinthecontextoftheringtrails,butalsoformal validationsusingtraditionalmethodshavebeenundertaken.Close contactshavebeenestablishedwithrelevantECVAMandOECD workinggroups,scientistsandregulators.Inselectingteststobe includedinthebattery,methodswerepreferredthatwereeither validatedorintheprocessofbeingvalidated,includingtheESTtest [33],thezebrafishembryotest[34],andreportergeneassaysfor estrogens[35,36]andandrogens[37].TheERalpha-andARCALUX assayalreadywereprevalidatedinthecontextoftheReProTectFP6 program,andsubsequentlyhavebeenthisissuetoEURL-ECVAM andOECDtoallowformalvalidation,andregulatoryacceptance [35,36,38,39].Sinceourbatteryoftestsofreportergeneassaysis verycomparabletotheselattertests,thevalidatedtestsmaybe usedas“validationanchors”inascreeningbattery,i.e.givingcon- fidenceintheotherbatteryconstituents.Itwouldalsobepossible todesignmoresimpleandrapidvalidationproceduresforthese additionalbatteryconstituentssincethetestproceduresofallthese testsessentiallyarethesame.

An important element in the project from the beginning onwardswastheexecutionofdedicatedringtrails(alsoreferred toasfeasibilitystudies)totestandimprovethetestbatteryasa whole.Withinthebudgetaryconstraints,wechosetogoforrela- tivelysmalldedicatedtrailsratherthananunbiasedlargescreening withmanyassaysandmanychemicals.Thefirstringtrailusingthe

Fig.3.SchematicrepresentationofkeyelementsofthehighthroughputPBPKmodel.FordetailsseetextandBosgraetal.,2015.

completepreselectedtestbatteryshowedthattestingalimitedlist ofcompoundsforwhichsufficientinvivoinformationisavailable, canbeavaluablestepintheidentificationofkeyelementsinabat- teryaswellasmissingones[25].Correctpredictionsweremade for11outof12chemicalswhilechemicalthatwasmissedcould beexplained bythelackofrelevantmechanismofactionbeing presentinthetestingbattery.Interestingly,thepredictionsofthe highthroughputCALUXpanelalone,whencombinedwithPBPK modelingwasexactlythesame([25];Table1)

QSAR and related chemoinformatics tools alone clearly are insufficient for reliable predictions of reproductive toxicity of chemicals.Similarly,biologicalmethodsinvitroscreeningmeth- odshavetheirlimitations.Whencombined,predictionscangreatly improve[40].Therefore,weassessedfurtheroptionstocombine structuralpropertiesincombinationwithinvitrodatatoidentify substanceswithreproductivetoxicproperties[41].Byusingthe OECDToolbox[42]andexpertjudgmentweidentified89chemi- calcategories.Inaddition275chemicalswereidentifiedwithany effectonthereproductivesysteminmalesandfemaleanimals.We combinedtheseresultswithalargedatasetonestrogenandandro- genreceptor activityusing CALUXassays todetermineifthese hormonaleffectsarerelatedtothereproductiveaberrations.The invivoandinvitroeffectswerefoundnottobeconfinedtodistinct chemicalcategories,showingthelimitationsofactivitypredictions onchemicalstructureonly.Whilethechemicalclasseswerenot predictiveforaninvivoeffect,aclearcorrelationwasseenbetween theinvivoreproductiveaberrationsandactivityinthereporter geneassayonestrogenicactivity[41].

Afurtherapplicationthatwasexploredusingafeasibilitystudy was the possibility of read across using the ChemScreen bat- tery:whenanuntestedquerychemicalisstructurallysimilarto areproductivetoxicant,thebatterycouldbeusedtoconfirmthese reproductiveproperties,andtoavoidanyfurtherinvivostudies.

Ideally,thisbatteryshouldthenalsobecapabletocorrectlydis- tinctreproductivetoxicantsfromnon-reproductivetoxicants,even whenbothhaveaclearstructuralsimilarity.Thisstudyshowedthat thebatterywasabletomakethisdistinctionusingthreedifferent chemicalclassesasanexample.Thisprovidesimportantopportu- nitiesforapplicationsunderREACHwhereread-acrossprocedures arebeingconsideredasanimportantelementofintegratedtesting toavoidorreduceanimalexperimentation[43].Heretheweight ofevidenceofavailablerepeateddosetoxicitydataforbothsource and query chemical, theirstructural similarity,as wellas their batteryresultscouldbeusedtojustifywaivinganinvivostudy [42].Alternativelyoradditionally,linkagetoregulatoryapplication mayusecombinationofinvitrotestresultswithexistingrepeated dosestudydatapotentialreproductivetoxicantscouldbeidentified aheadofinvivostudies,allowingprioritizationsofanimaltesting [41].

2.7. Mechanisticvalidationandlinkagetoadverseoutcome pathways

Because of the strong interest in molecular screening approaches asdevelopedin ChemScreenand ToxCast,currently there are significant activities ongoing to improve linkage of

Table1

ResultsfromthefirstChemScreenringtrail(adaptedfromPiersmaetal.[25]).

comp oun d Toxi city in vivo

EST ZET

Cyp17 Cyp19 CALUX CALUX PBPK

Entire battery

Monoethylhexyl phthalate Sodium valproate D-mannitol Flusilazole Glufosinate ammonium Methoxy acetic acid

Retinoic acid Dioctyltin chloride Endosulfan Diethylstilbestrol Methylmercury chloride

Gray:positiveresult;white:negativeresult;EST:embryonicstemcelltest;ZET:zebrafishembryonictest;CALUX:CALUXHTSpanelofreportergeneassays;battery:

combinedpredictionsofalltests.

mechanisticin vitrotoolsto resultsin animalexperiments, e.g.

throughthedesignofso-calledAdverseOutcomePathways[44].

ChemScreen’sscientistshaveembarkedonthesenewandimpor- tantactivitiesthoughttobeessentialmissinglinksinapplicationof molecularscreeningtools.Wehavebeenactivelyengagedinpro- motingregulatoryacceptance ofthetoolsdeveloped,alsousing alternativevalidationmethodsthatprovidelinkagetothemecha- nismofactionofchemicalsleadingtoadversity.

Tothisend,inanextfeasibilitystudysetsofchemicalswere selectedthatcaninduceeithersexorgan(SO)deformitiesorneural tubedeformities(NTD).Ontheonehandthesewereusedtostudy thedifferentadverseoutcomepathwaysinvolvedinneuraltube defects[45],whileontheotherhandthesesetsofchemicalswere usedtofurthervalidateandspecifytheapplicationdomainsofthe differentelementsinthehighthroughputCALUXtestbattery[20].

AnalysisoftheresultsoftheCALUXhighthroughputpanelshows thatitisverysuitabletoidentifynotonlyendocrineactivecom- pounds,butalsocanpredictchemicallyinducedSOdeformations withover80%accuracy[20].Thisisconsistentwiththestudyof Lewinetal.[41],showingaclearcorrelationbetweentheinvivo reproductiveaberrationsandactivityinthereportergeneassayon estrogenicactivity.

Whenusingmolecularscreeningtoolsitshouldbenotedthat couplingtheseresults1:1toapicalendpointsinexperimentalani- malscannotbereadilydone.Inthefirstplace,theremaybemultiple mechanismsleadingtothesameendpoint,whilethesamemech- anismmaybeinvolvedindifferentendpointtoxicitieswhentime andtissuedistributionisdifferent.Inaddition,asingletestonly givespartofthepuzzleandonlybatteriessuchastheChemScreen batterycanbeeffectiveinreplacinganimalstudies.Thevalidation ofsuchabatteryisachallenge,inparticularintheabsenceofgold standard,causedbyrelativelypoorpredictivityofanimaldatafor

humans[6].Aninterestingapproach inthislightwastocluster apicalreproductivetoxiceffectsofchemicalswhencomparingthis withdataofmolecularscreeningassays,resultinginastrongcorre- lationofrelevantCALUXassayswiththoseclusteredapicaleffects [20,41].Thisverylikelywillchallengethedesignofverysimple linearAOPsand suggestthat networkanalysisand/orclustered AOPelementsmaybeneededtolinkmechanisticteststoapical endpointsinanefficientmanner.

InthiscontextitisinterestingtonotethattheReProTecttest- ing battery was able to provide predictions ongender-specific effectsofthechemicals,andsometimeseventhetargetorgan[7].

Thiscould bedone becauseof inclusion of tests basedon pri- marymaterialofmaleandfemalereproductiveorgans.Noattempts weremadetopredictgender-specificeffectswiththeChemScreen battery,but it islikely tobemore difficultbasedontheapical testsincludedandthemechanistictestsbecauseoftheirlimited genderspecificity.EventheresultsofCALUXassays thatassess sexhormonereceptoractivationwherefoundtoberelevantfor predictingreproductiveeffectsinbothmaleandfemalerodents [41],whichisconsistentwithexpressionandfunctionofARand ERin both sexes. Therefore, although mechanistic tests canbe highlypredictiveforinvivotoxicity,exactpredictionoftheapi- calendpointsthatwillbeaffectedinanimalswillingeneralnotbe straightforward.Thesameproblems,however,occurinextrap- olatingbetweenanimalspecies,life stagesand organtoxicities.

Mechanistictestsdohavetheopportunitytomoredirectlylink tohumanriskassessment,however.Thiscanleadtobetterhuman riskassessment.Forinstance,estrogensignalingiscomplexand aberrantactivationisassociatedwithseveralhumandiseasesprop- ertiesnottestedinstandardanimaltests[46,47].Inthefuturemore mechanism-based risk assessment using tests providing more directmechanisticinformationonhumantissue-specificreceptor

activity and associated disorders may become an interesting opportunity.

2.8. ApplicationoftheChemScreentoolbox

AnimportantaimoftheEuropeanframeworkprogramsisdis- seminationofresultsandtheirapplication,preferablyleadingto newbusinessopportunitiesofsmallandmediumenterprises.To reachthisweworkedoncouplingdifferenttoolsanddatabasesin ordertodesign integratedtestingstrategiesthatcanbeusedin chemicalriskassessment.Thefollowingmodulesweredefined:

2.8.1. Module1.Insilicoprescreeninganddatacollectionmodule Thismoduleincludesinsilicoprescreening(QSAR)methodsto categorizechemicalsintomajorclassesoftoxicityprioritizedin REACH.Predictionsforcarcinogenicity,mutagenicityareapplied, whichincaseofpositivitywilldriveriskassessmentinthisdirec- tion avoiding reproductive toxicity testing according toREACH guidelines.Italsousesestablisheddatabasesandliteraturetofind anytestingresultsofthechemicalitselfandusechemoinformatics tofindstructuralanalogueswithreproductivetoxicitydatatobe usedassourcechemicalsinareadacrossapproach.

2.8.2. Module2.Invitroscreeningmodule

Thetest battery establishedwithinChemScreen canprovide confirmation ofalready predictedreproductive toxicity fromin module1,orgiveafirstalertofpotentialreproductivetoxicityinthe absenceofpredictionsfromtheseinsilicomethods.Thisscreening modelalready givesreliablepredictionsforstrongreproductive toxicants.Clearly,thispromisingscreeningmodulecanbefurther improvedwhenmoremechanismsofreproductivetoxicityareelu- cidated,allowingagreaterrelianceonthehighthroughputassays attheexpenseofthelowerthroughputones.

2.8.3. Module3.Kineticmodule

TheestablishedPBPKtoolslinkinvitroandinvivoplasmacon- centrations,and extrapolatethese toassociatedoral, dermalor potentiallyinhalationdoses.Thesearetobeappliedforvalidating andinterpretingmodule2results,i.e.toclassifychemicals,priori- tizechemicalstobetested,andforhelpingtodesignlimitedinvivo studiesforselectedchemicalsincaseconsiderednecessary.

2.8.4. Module4.Dataintegrationandriskassessment

AllelementsoftheChemScreenintegratedtestingstrategyhave beensetupandarein anadvancedstateofbeingcoupledand integrated. The efficacy of this integration in terms of making decisionsontheneedofsubsequentinvivoanimaltests,heavily dependsonthepurposeoftheinvestigation,e.g.whetheritisfor safedesignofnewchemicalsorforregistrationofexistingchem- icals.Forinstance,inthecontextoftheREACHtestingprogram readacrosscanbeaviableoption,e.g.toreducethereproductive toxicitytestingrequirementforchemicals(i.e.OECD421,or422) producedatmorethan10tons.Thecostsofthisinvitrotestingis around10timeslowerthantheinvivotesting,whichinitscheap- estversioncostsaround55,000Euro[48].Expertjudgmentstillis neededindifferentsituationstointerprettheoutcomes,e.g.tover- ifytheapplicabilitydomainsofthemodelsused,bothwithregard tostructuralaswellasbiologicalcharacteristics.Currently,more caseexamplesarebeingruntofurthervalidatetheseintegrated approaches.

Ourtestbatteryandintegratedtestingconceptwillbedissem- inatedfurther,andtrainingandsupportfacilitiesareofferedusing theworld-wideexpertiseavailabletodisseminatebioassays.The coordinatoratBioDetectionSystemsisthecontactpersonforthese activities(contactdetails;seewww.chemscreen.eu).

3. Conclusionsandfuturedirections

ThecombinedeffortsoftheChemScreenconsortiumhaveledto amodularintegratedassessmentstrategywhichincludesmethods forinsilicoprescreening,invitroscreening,methodstoextrapolate invitroresultstoinvivopredictions,andintegrativeonestocom- binethesedatawithpreexistingknowledgeonthetestchemical orrelatedchemicals.Thisworkconfirmsandextendstheearlier findingoftheFP6ReProTect project[7]that arelativelysimple batteryoftestscanbeusedsuccessfullytopredictreproductive toxicityofchemicalsseeTable1.Significantadvanceshavebeen madeparticularlyinthefurthersimplificationofthetestbattery, whilestillbeingabletoidentifyreproductivetoxicchemicalseffi- ciently,eitherinisolation[25],orinagroupingcontext[41,43].

Wefoundthatevenwitharelativelysmallnumberoftests,either apicaltestscombinedwithmechanistictestsoranrelativelysmall numberofmechanistictestspredictivitiesrangingfrom74to94%

canbereached[20,25],whichiscomparabletothatobtainedwith muchlargerToxCastscreeningpanels[21],theReProTectbattery [7],orthezebrafishELStests[49],ahumanembryonicstemcell test[50].Thisisremarkable,sincetheconcordancebetweendif- ferenttestspecies,likerabbitandrathasbeenestimatedtobenot morethan60%[6].Itseemsthatthesevalidationresultsmaybe biasedtosomeextent,e.g.becauserelativelystronglyactingrepro- ductivetoxicchemicalsareoftenusedinthevalidation studies.

Therefore,tocreatemoreconfidencewehavealsodevelopedand appliedothermeansofvalidatingtestsusingmechanisticknowl- edgeonthetestsandusingselectedchemicalstotestwhetherthe predictedadverseoutcomesmatchwiththemechanismsincluded inthebattery[20].Moredetailedclassificationschemesofchemi- cals,linkedtobothchemicalandbiologicalpropertiesasdeveloped byWu etal.[40]maybeveryhelpfulinfurthervalidationand improvementofthetestbattery,throughidentificationofchemical classesormechanismsmissedbythebattery.

AlthoughtheChemScreenbatteryshowedaverygoodperfor- mance identifyingstrongreproductivetoxicants, it isuncertain howitwillperformwithchemicalsthathavealessexplicitactivity profile.Thisisrelevant,sincethemajorityofindustrialchemicals arelikelytohaverelativelynon-specificmodesofaction[51].There isnoreasontobelievethatthesechemicalswillbemissedbythe battery,sincerelevantpathwaysseemavailable.Alsopartofthe reproductivetoxicchemicalsusedinourfirstfeasibilitystudydo notactthroughspecificmechanisms,andneverthelessarepicked upthroughrelativelynon-specificcytotoxicityendpointsinourtest battery[25].

Clearlynotallpathwaysofreproductivetoxicityareknownat present,andthepanelofmechanisticassaysmaythereforebemiss- ingcompoundsactingvialesscommonmechanismsofaction.On theotherhand,itseemsalsolikelythatchemicalsoftenaddress multiplepathwaysoftoxicity,therebyreducingtheneedtohave allpossiblemechanismofactionincludedinafinalbattery.This issupportedbytheverygoodperformanceofourbattery,even when compared withmuch largerbatteries [21] or more inte- grativelowerthroughputbatteriesandtests[7,49,50].Atpresent thecurrent combination of apicaland HTStestsseems a prag- maticandeffectivechoicethatcanbefurtherimprovedwhenmore mechanismsofreproductivetoxicitybecomeknown.Forregula- toryacceptance,itmayalsobehelpfultoincludetestdataofthese moreapicaltestssinceinitiallytheymaygiveadditionalconfidence inthetestresultsandassistwithclassificationandlabeling,par- ticularlyintheabsenceofanimaldata.Itshouldbenotedthatthe majorityofREACHchemicalsareexpectedtoberelativelyinertand nottobereproductivetoxic.Becauseofthis,forregulatoryapplica- tionawidecoverageofpossiblemechanismsofactionoftoxicants isneeded,butwithoutgivingmanyfalse-positivesthatmaylead toadditional testing requirement.Our battery of veryselective

mechanistictestsincombinationwithapicaltestsandavailable animaltestdataseemsinthisrespectaneffectivetestingstrategy.

Withincreasedexperienceandconfidenceininvitrotestingthe weightoninvitrotestingandextrapolationtohumanratherthan animaldatawillbecomeincreasinglyimportant.

Conflictofinterest

Theauthorsdeclarethattherearenoconflictsofinterest.

Acknowledgments

Theworkdescribed in this manuscript couldnothave been exertedwithouttheeffortsof alladditionalChemScreen partic- ipantsnotspecificallymentioned. Wewouldparticularlyliketo sincerelythankouradvisoryboardmembersDrsDavidDix,Robert Chapin,MariaBondesson,MarkCronin,andNeilCarmichael for theirsupportandvaluablecontributiontothesuccessoftheChem- Screenproject.WearealsoverygratefulforthecontributionofDrs BurkhardFlick,ThomasSobanskiandSandervanderLindentothe finalsymposiumandapaneldiscussionthatprovidedelementsof thisreview.Thisworkwascarriedoutwithfinancialsupportfrom theCommissionoftheEuropeanCommunities,thecollaborative projectChemScreen(GA244236).

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