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DevelopmentofstructuralalertsfortheinvivomicronucleusassayinrodentsaaaRomualdoBenigni,CeciliaBossa,OlgaTcheremenskaiabandAndrewWorthaIstitutoSuperiorediSanita’,EnvironmentandHealthDepartment,Rome,ItalybInstituteforHealth&ConsumerProtection,EuropeanCommission-JointResearchCentre,Ispra,ItalyEUR23844EN-2009 ThemissionoftheIHCPistoprovidescientificsupporttothedevelopmentandimplementationofEUpoliciesrelatedtohealthandconsumerprotection.TheIHCPcarriesoutresearchtoimprovetheunderstandingofpotentialhealthrisksposedbychemical,physicalandbiologicalagentsfromvarioussourcestowhichconsumersareexposed.EuropeanCommissionJointResearchCentreInstituteforHealthandConsumerProtectionContactinformationAddress:TP582E-mail:andrew.worth@ec.europa.euTel.:+390332789566Fax:+390332786717http://http://ecb.jrc.ec.europa.eu/qsar/http://ec.europa.eu/dgs/jrc/LegalNoticeNeithertheEuropeanCommissionnoranypersonactingonbehalfoftheCommissionisresponsiblefortheusewhichmightbemadeofthispublication.AgreatdealofadditionalinformationontheEuropeanUnionisavailableontheInternet.ItcanbeaccessedthroughtheEuropaserverhttp://europa.eu/JRC52274EUR23844ENISSN1018-5593Luxembourg:OfficeforOfficialPublicationsoftheEuropeanCommunities©EuropeanCommunities,2009ReproductionisauthorisedprovidedthesourceisacknowledgedPrintedinItaly ABSTRACTInvivomutagenicityandcarcinogenicitystudiesareposingahighdemandfortest-relatedresources.Amongthesestudies,themicronucleustestinrodentsisthemostwidelyused,asfollowuptopositiveinvitromutagenicityresults.Arecentsurveyofthe(Q)SARmodelsformutagenicityandcarcinogenicityhasindicatedthatno(Q)SARmodelsforinvivomicronucleusareavailableinthepublicdomain.Therefore,thedevelopmentandextensiveuseofestimationtechniquessuchas(Q)SARs,read-acrossandgroupingofchemicals,promisestohaveahugeanimalsavingpotentialforthisendpoint.Inthisreport,wedescribetheidentificationofstructuralalertsfortheinvivomicronucleusassay,andprovidethelistofunderlyingchemicalstructures.Thesestructuralalertsprovideacoarse-grainfilterforthepreliminaryscreeningofpotentialinvivomutagens. LISTOFABBREVIATIONSEPAEnvironmentalProtectionAgencyEUEuropeanUnionFDAFoodandDrugAdministrationHOMOHighestOccupiedMolecularOrbitalISSIstitutoSuperiorediSanita’JRCJointResearchCentreLUMOLowestUnccupiedMolecularOrbitalOECDOrganisationforEconomicCooperationandDevelopment(Q)SAR(Quantitative)Structure-ActivityRelationshipREACHRegistrationEvaluationandAuthorisationofCHemicalsROCReceiverOperatingCurveSAStructuralAlertSA_BBBenigni-Bossastructuralalertsformutagnicity/carcinogenicityinToxtreeSA_MicStructuralalertsrefersfortheinvivomicronucleusassayinToxtreeSA_ProtStructuralalertsforproteinbindingintheOECDQSARToolbox CONTENTS1.Introduction....................................................................................................62.Structuralalerts..............................................................................................83.Developmentofstructuralalertsfortheinvivomicronucleusassay..............104.Finalconsiderations......................................................................................205.References....................................................................................................21Appendix1..............................................................................................................23 1.IntroductionMutagenicitytestingisanimportantpartoftheregulatoryhazardassessmentofchemicals.Itisundertakenfortwomainreasons:a)todetectchemicalsthatmightcausegeneticdamageingermcells,andthusincreasetheburdenofheritable(genetic)diseaseinthehumanpopulation;andb)todetectchemicalsthatmightbecarcinogenic(basedontheassumptionthatmutagenesis,forexampleinsomaticcells,isakeyeventintheprocessofcarcinogenesis).Sincenomethodisablealonetodetectallpossiblegenotoxicevents,awidearrayoftestsystemshasbeendevelopedandacceptedinternationallyinregulatoryschemes.Mostoften,thesemethodsareusedwithina2-tieredintegratedtestingapproach:Tier1includesinvivoassays,andTier2includesinvivoassays.Asamatteroffact,mutagenicitytestingwasthefirsttoxicityendpointforwhichinvivoassayswereacceptedforregulatorytesting,some25yearsago.Thelatterusuallycomprisebacterialmutagenicityandcytogeneticstests,althoughgenemutationtestinginculturedmammaliancellsissometimesalsoundertaken.Tier2ofthetestingstrategyinvolvestheuseofshort-terminvivostudies(usuallyabone-marrowcytogeneticsassay)toassesswhetheranypotentialforgenotoxicitydetectedattheTier1invivostageisactuallyexpressedinthewholeanimal.Thus,negativeresultsinvivoareusuallyconsideredsufficienttoindicatelackofmutagenicity,whereasapositiveresultisnotconsideredsufficienttoindicatethatthechemicalrepresentsamutagenichazard(i.e.itcouldbeafalsepositive).Theabove1approachtogenotoxicitytestinghasbeenadoptedthroughouttheEU,andhasbeenrecommendedinternationallyaspartofthestrategyforpredictingandquantifyingmutagenicandcarcinogenichazard(Ashbyetal.,1996;Combesetal.,2007;KirklandandSpeit,2008;Lilienblumetal.,2008).1http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_r7a_en.pdf?vers=20_08_086 AccordingtoanassessmentcarriedoutbytheformerEuropeanChemicalsBureau(ECB),theinvivomutagenicitystudies,shortlyfollowedbycarcinogenicity,areposinghighdemandfortest-relatedrecourses(Pedersenetal.,2003;VanderJagtetal.,2004).Amongthose,themicronucleustestinrodentsisthemostwidelyused,asfollowuptopositiveinvivomutagenicityresults.Arecentsurveyofthe(Q)SARmodelsformutagenicityandcarcinogenicity(performedjointlybyISSandtheJRC)hasindicatedthatno(Q)SARmodelsforinvivomicronucleusareavailableinthepublicdomain(Benignietal.,2007):therefore,thedevelopmentandextensiveuseofestimationtechniquessuchas(Q)SARs,read-acrossandgroupingofchemicals,mighthaveahugesavingpotentialforthisendpoint.Inthisreport,wedescribe:a)thecollectionofdataonchemicalstestedwiththeinvivomicronucleusassay;b)preliminaryanalysesofthedata;c)theidentificationofStructuralAlerts(SA)propertothistoxicologicalendpoint.First,somebackgroundinformationontheconceptofSAisprovided.7 2.StructuralalertsTheSAsforatoxicologicalendpointaremolecularfunctionalgroupsorsubstructuresknowntobelinkedtothattypeoftoxicity.TheSAsareacoarse-grainedapproachtotheuseofStructure-ActivityRelationships(SAR)tounderstandthetoxicitymechanismsandtopredictthetoxicactivityofchemicals.Becauseoftheirnature,theSAshavetheroleofpointingtochemicalspotentiallytoxic,whereasnoconclusionsorindicationsaboutnontoxicchemicalsarepossible(exceptbyexclusion)(BenigniandBossa,2006;BenigniandBossa,2008).AsetofchemicalscharacterizedbythesameSAconstituteafamily(class)ofcompoundsthatsharethesamemechanismofaction.ThereactivityofaSAcanbemodulatedorabolishedbytheremainingpartofthemoleculeinwhichtheSAisembedded.Atacoarse-grainlevel,suchmodulatingeffectscanberepresentedbyothermolecularsubstructures(e.g.,bulkygroupsorthotoanaromaticaminegroup)thatareknowntohaveaninfluenceonthereactivityoftheSA.Usually,theknowledgeonthemodulatingsubstructuresisquitelimitedformostoftheSAs,thusitprovideslimitedhelpindecidingwhichchemicalsinaclasswillactuallybetoxicandviceversa.ApowerfulgeneralizationoftheStructure-ActivityRelationshipsisprovidedbytheQuantitativeStructure-ActivityRelationship(QSAR)analysis,whichproducesamathematicalmodelthatlinksthebiologicalactivitytoalimitednumberofphysicalchemicalorothermolecularproperties(descriptors)withgeneralrelevance.Sincemostofthedescriptorshavecontinuousvalues,theQSARsprovidefine-tunedmodelsofthebiologicalactivity,andcangiveaccountofsubtledifferences.GeneralintroductionsonQSARaregivenelsewhere(HanschandLeo,1995,Hanschetal.,2002).ThustheSAsarenotadiscriminantmodelonthesamegroundoftheQSARmodels:thelatterproduceestimatesforbothpositiveandnegativechemicals,aswellasforthegradationoftoxicpotency.ThemainroleoftheSAsisthatofpreliminary,orlarge-scalescreenings.Theyareexcellenttoolsforcoarse-graincharacterizationofchemicals,including:descriptionofsetsofchemicals,preliminaryhazardcharacterization,categoryformationandprioritysetting(enrichment).Sincefine-tunedQSARsdonotexistformanytypesofchemicals,themodelsbasedonSAsholdaspecialplaceinpredictivetoxicology.The8 knowledgeontheactionmechanismsasexemplifiedbytheSAsisroutinelyusedinSARassessmentintheregulatorycontext(see,forexample,themechanistically-basedreasoningaspresentedinWooetal.(2002).Inaddition,theSAsareatthebasis2ofpopularcommercial(e.g.,DEREK,byLhasaLtd.)andnon-commercialsoftware3systems(e.g.,Oncologic,byUSEnvironmentalProtectionAgency[EPA]).Recently,asfollow-upofthecollaborationbetweenISSandJRC,arulebaseformutagensandcarcinogenshasbeendesignedandimplementedinthesoftwareToxtree1.51.Itusesastructure-basedapproachconsistingofanewcompilationofSAsforcarcinogenicityandmutagenicity.ItalsooffersthreemechanisticallybasedQSARsforcongenericclasses(aromaticaminesandaldehydes)(Benignietal.,42008a).Toxtree1.51isfreelyavailablefromtheJRCwebsite.2http://www.lhasalimited.org/3http://www.epa.gov/oppt/newchems/tools/oncologic.htm4http://ecb.jrc.ec.europa.eu/qsar/qsar-tools/index.php?c=TOXTREE9 3.Developmentofstructuralalertsfortheinvivomicronucleusassay3.1DataThecompilationofSAsfortheinvivomicronucleusassayinrodentsprovidedhere,isbasedonboththeexistingknowledgeonthemechanismsoftoxicactionandastructuralanalysisofthechemicalstestedintheassay.Theinvivomicronucleusdatainthepublicdomainisquitelimited.AsearchoftheChemicalCarcinogenesisResearchInformationSystem(CCRIS)attheToxnet5websitewiththequery:“invivomicronucleus”pointsonlyto240chemicals.Forthiswork,theremarkablylargercommercialdatabasebyLeadscopeInc.,called6“FDASARGenetoxDatabase”wasused.Thisdatabasecontainsmorethan700chemicalstestedininvivomicronucleuswithrodents,andincludesdatafromboththepublicdomainandtheUSFoodandDrugAdministration(FDA)files.Alargemajorityofdatawerebasedontheanalysisofmicronucleiinbonemarrowcells;fordetailsonthetechnique,seeforexample,KrishnaandHayashi(2000).3.1PreliminaryanalysesSincethemainroleoftheinvivomicronucleusassayinregulatoryschemesisthatofconfirming(ordisproving)thepositiveinvitroresults,itisofinteresttocheckhowtheinvivomicronucleusresultsrelatetotherodentcarcinogenicitydataandtotheprimaryinvitropredictiontest,i.e.,theSalmonellatyphimurium(Ames)test.TablesIandIIdisplaytherelationshipsbetweentheinvivomicronucleusadthetworeferencetests.TheresultsforrodentcarcinogenicityandtheAmestestwere7retrievedfromthefreelyavailableISSCANv3adatabase,whichischaracterizedby:5http://toxnet.nlm.nih.gov/cgi-bin/sis/search6http://www.leadscope.com/product_info.php?products_id=777http://www.iss.it/ampp/dati/cont.php?id=233&lang=1&tipo=710 a)thehighqualityofbothchemicalandbiologicalinformation;b)theQSAR-readyformat(Benignietal.,2008b).Obviously,thetotalnumbersofchemicalsinthetwotablesarerelativeonlytothosechemicalstestedinbothsystems.TableI.ContingencytablecomparingtheresultsoftherodentcarcinogenicitytestwiththemicronucleustestCarcinogenicitytestMicronucleustestNegativePositiveTotalNegative301040Positive8657143Total11667183TableII:ContingencytablecomparingtheresultsoftheSalmonellatyphimuriumassaywiththemicronucleustestSalmonellaassayMicronucleustestNegativePositiveTotalNegative7436110Positive413475Total11570185TableIshowsthatistheinvivomicronucleusassayispoorlysensitivetotherodentcarcinogens:about60%oftherodentcarcinogensarenotdetectedbythemicronucleus.ThepoorsensitivityofthemicronucleusassaytopotentialgenotoxinsisalsoapparentfromTableII.ItshouldbeemphasizedthatthepresentresultsobtainedwiththelargeLeadscopemicronucleusdatabaseareinagreementwithpreviousanalysesbasedonsmallerdatasetsinthepublicdomain(Benigni,1995).11 Inasecondroundofanalyses,theextenttowhichthemicronucleusdataarerelatedtowellestablishedindicatorsofDNAandproteinbindingwaschecked.Thisinviewoftheplethoraofthereportedmechanismsofmicronucleusinduction.Asamatteroffact,micronucleiaremarkersofbothaneugenic(changeinthechromosomesnumber,usuallybyloss)andclastogenic(chromosomebreakage)effects.Itisgenerallyassumedthatsucheffectsaregeneratedthrougharangeofdifferentpathways.Evidence(mainlygatheredfrominvitrostudies)indicatesthatmicronucleicanbeinducede.g.,bytypicalDNA-attackingagents(e.g.,alkylatingagentslikemethylmethanesulfonate),bymitoticspindlepoisons(e.g.,colcemide,vincristine),orbyinhibitorsofcytokinesis(e.g.,cytochalasinB).Thelattereffectsareprobablyduetointerferencewithproteins.Otherchemicalsarethoughttobeclastogenicthroughaspecificdisturbanceofcytokinesisduetolipophilicity(Dornetal.,2007).TherelativeinfluenceofDNAandproteinbindingonmicronucleusgenerationwascheckedbyrecordingthedistributionofstructuralalertsforthetwoeffectsintheLeadscopeinvivomicronucleusdatabase.AsprobesforDNAbinding,weusedthestructuralalertsforcarcinogenicity/mutagenicityimplementedinToxtree1.51.Asamatteroffact,thelargemajorityofthesealertsrefertogenotoxiccarcinogenicity,whichisassumedtobecausedthroughdirectinteractionwithDNA(BenigniandBossa,2008).Asprobesforproteinbinding,weusedthealertsimplementedinthe8OrganisationforEconomicCooperationandDevelopment(OECD)QSARToolbox.Thesealertsweremainlydevelopedfromthemechanisticknowledgeonskinsensitization,andmodelthecovalentbindingtoproteins.TheresultsoftheaboveanalysisisdisplayedinFigure1asaROCgraph.Itappearsthatthestructuralalertsforcarcinogenicity/mutagenicitycorrelatetosomeextentwiththeinductionofmicronuclei,whereasthoseforproteincovalentbindingshownocorrelation(inthegraph,theyareonthediagonallinewhichrepresentsrandomresults).8http://www.oecd.org/document/23/0,3343,en_2649_34379_33957015_1_1_1_1,00.html12 Figure1.ReceiverOperatingCurveshowingtheconcordanceoftwosetsofstructuralalertswiththeresultsoftheinvivomicronucleusassay(SA_BBreferstotheBenigni-BossaalertsinToxtree;SA_ProtreferstothealertsforproteinbindingintheOECDQSARToolbox)3.3StructuralAlertsforinvivomicronucleusassaySincetheaboveanalysespointedtogenotoxiceffectsasanimportantdeterminantofmicronucleiinduction,wedevelopedthelistofStructuralalertsforinvivomicronucleususingthecarcinogenicity/mutagenicityalertsinToxtreeasacore,andthensearchingforadditionalsubstructuresspecifictothemicronucleus-positivechemicals.FromtheToxtreealertsforcarcinogenicity/mutagenicity,weexcludedfouralertsspecificfornon-genotoxicmechanismsofcarcinogenicity.UsinglineardiscriminantanalysisasananalyticaltoolandROCplotsasagraphicaltool,aseriesofadditionalsubstructureswereadded/removedto/fromtheToxtreealertsinordertoincreasesensitivityandspecificity.Intheseexploratoryanalyses,we13 screenedtheverylargecollectionofsubstructuralpatternsandfunctionalgroups(morethan27,000)containedinthesoftwareLeadscopeEnteprise2.4.15-6.Wealsore-checkedtheToolboxproteinbindingalertsforindividualsubstructuresrelatedwithmicronucleusinduction.TheresultistheoptimizedlistofalertsinAppendix1.TogetherwiththeToxtreealerts,itcontainsfiveadditionalsubstructuresidentifiedinthecourseofthisresearch.Forthesakeofclarity,thecodesofthealertsinToxtreearemaintained,whereasthefiveadditionalalertshavenewcodes.Figure2displaystheagreementbetweenthealertsforinvivomicronucleus,andtheexperimentalresultsforthisendpoint.Outof547negatives,thespecificityoftheSAsis0.57.Thesensitivityis0.65outof182positives.Theoverallaccuracyis0.59.Foracomparison,theROCgraphshowsthenewlydevelopedalertsformicronucleustogetherwiththoseforDNAandproteinbinding.ItappearsthattheperformanceofthefinallistofalertsisconsiderablyhigherthanthatoftheDNAbindingandProteinbindingalerts.TableIIIgivesthetruepositiveratefortheindividualalerts.14 Figure2ReceiverOperatingCurveshowingtheconcordanceofstructuralalertsfortheinvivomicronucleusassaywiththeexperiemtnalresultsforthisassay(SA_MicreferstotheinvivomicronucleusalertsinToxtree)15 TableIII:CharacterisationofStructuralAlerts.No.ofTrueNo.SubstancesSTRUCTURALALERTpositivePositivesfiredsubstances(%)SA_1:acylhalides00SA_2:alkyl(C<5)orbenzylesterofsulphonic4375orphosphonicacidSA_3:N-methylolderivatives100SA_4:monohaloalkene33100SA_5:SorNmustard44100SA_6:propiolactonesorpropiosultones00SA_7:epoxidesandaziridines201260SA_8:aliphatichalogens35926SA_9:alkylnitrite11100SA_10:a,bunsaturatedcarbonyls581628SA_11:simplealdehyde9222SA_12:quinones9444SA_13:hydrazine600SA_14:aliphaticazoandazoxy00SA_15:isocyanateandisothiocyanategroups200SA_16:alkylcarbamateandthiocarbamate9222SA_18:PolycyclicAromaticHydrocarbons11100SA_19:heterocyclicPolycyclicAromatic700HydrocarbonsSA_21:alkylandarylN-nitrosogroups6583SA_22:azideandtriazenegroups22100SA_23:aliphaticN-nitrogroup215016 SA_24:a,bunsaturatedaliphaticalkoxygroup11100SA_25:aromaticnitrosogroup00SA_26:aromaticringN-oxide00SA_27:nitro-aromatic17212SA_28:primaryaromaticamine,hydroxyl501938amineanditsderivedestersSA_28bis:aromaticmono-anddialkylamine5240SA_28tris:aromaticN-acylamine200SA_29:aromaticdiazo8450SA_30:coumarinsandFurocoumarins300SA_32:1,3-dialkoxy-benzene6583SA_33:1-phenoxy-benzene5480SA_34:hacceptor-path3-hacceptor1635534SA_35:cxolane21943SA_36:carbodiimides221003.4FurtheranalysesonthealertsformicronucleusAstrikingevidenceinTableIIIistherelativelylowpercentageoftruepositivesidentifiedbymanySAs.Inotherwords,oftenthetoxicpotentialofthealertsisnottranslatedintoactualtoxicityintheexperimentalsystem.Foracomparison,theTruePositiveRateofthevariousalertsformutagenicity/carcinogenicityinToxtreeisremarkablyhigher,rangingfrom70to100%(BenigniandBossa,2008).TheaboveresultcontributestobetterunderstandtheevidenceinTablesIandII,whereitappearsthatthemicronucleusassayhasmanymorenegativesthanthecarcinogenicitybioassayandtheSalmonellamutagenicitytest.TableIIIindicatesthatthelowsensitivityofthemicronucleusassayislargelyduetothefactthatoften,17 chemicalfunctionalitiesandsubstructureswhicharesupposedtobereactivedonotexerttheirpotentialreactivityinthisexperimentalsystem.Theissueofthelowsensitivityofthemicronucleusassayhasbeenrecognizedbyscientistsinvolvedinresearchaimedatimprovingtheavailableshort-termmutagenicityassays;asamatteroffact,validationoffurther,moresensitiveinvivoassays(e.g.,invivoCometassay)ispresentlyinprogress(KirklandandSpeit,2008).Inthecontextofthisresearch,weinvestigatedifageneraleffectofbioavailabilityonthelimitedsensitivityofmicronucleuswasapparent.Tothisaim,weconsideredtwochemicaldescriptorswellknownastobelinkedtobioavailability:logP(hydrophobicity)andMolarRefractivity(MR)(HanschandLeo,1995).Thetwo9descriptorswerecalculatedwiththeC-QSARsoftware(Daylight,Inc.)forallthechemicalsinthemicronucleusdatabase.Forthetwoparameters,TableIVreportstherangesofvaluesforpositiveandnegativemicronucleusresults.TableIV:RangesofC-logPandC-MRinchemicalsassayedwiththemicronucleustestC-logPC-MR(x10-1)MicronucleusNegatives-18.64–20.430.10–33.73MicronucleusPositives-9.58–15.230.15–32.91ItappearsthatthemicronucleuspositivescoveramorelimitedrangeoflogPvaluesthanthemicronucleusnegatives;however,theconsiderationofexclusionvaluesforlogPincombinationwiththeSAsdidnotimprovetheoverallperformance(resultsnotshown).WhereasnogeneraleffectoflogP(orMR)wasfound,analysesontheindividualchemicalclassesshowedthatlogPcut-offscanbeidentifiedfortheclassesofNitroaromatics(NegativesatlogP>0.0),AromaticDiazo(NegativesatlogP<3.7),9http://www.daylight.com/about/index.html18 andOxolanes(NegativesatlogP>1.5).Theconsiderationofthesecut-offsincreasesthespecificityoftheSAsfrom0.57to0.60.Theaboveresultsuggestsapossiblestrategytounderstandandmodelingthemanynegativeresultsobservedwiththemicronucleus.Sincethebonemarrow(maintargetofthetest)isanorganeasilyaccessiblebythebloodstream,itcanbehypothesizedthatthelackofeffectshownbyseveralchemicalswithSAs(hencepotentiallyreactive)isduetothemanypossibletargetsforreactionencounteredintheinvivosituation;thisdiminishestheprobabilityforthechemicalsofreaching,andinteractingwiththemoleculartarget(s)ofthemicronucleustest.Forexample,highlyreactivechemicalswillprobablyreactwithanytargetencounteredintheirway(e.g.,proteins,water)beforereachingthebonemarrow.ThusitcanbeenvisagedthatQSARsforindividualchemicalclassesshouldbedeveloped,andthattheyshouldconsiderparameterslinkedtochemicalreactivity(suchasHOMOandLUMOenergies).ItcanbehypothesizedthatthemodelsderivedfromtheseQSARswillcontributetomodulatetheindividualSAs.19 4.FinalconsiderationsStructuralalertspointtoclassesofchemicalswiththepotentialtocausetoxiceffects(here,invivomicronucleus).Sincethispotentialismodulatedineachmoleculebytherestofthestructure(e.g.,otherfunctionalgroups,electronicstructure,bulkygroups),notallchemicalsinaclassareequallytoxic.InthecaseoftheSAsidentifiedinthepresentstudyfortheinvivomicronucleustest,thepercentageofchemicalsthathaveSAsbutarenotactiveinthetestsystemisparticularlyhigh.Thisevidenceagreeswith,andrationalizesthenotionthatthistestsystemissensitivetogenotoxinstoalimitedextent,anddoesnotrespondtoalargenumberofrecognizedcarcinogensandmutagens.Forthisreason,apositiveinvivomicronucleusresultaddsastrongweighttoaninvivopositivemutagenicityresult,whereasanegativeinvivomicronucleusresulthasamuchlowerrelevance.Theavailabilityofawiderrangeofinvivomutagenicityassaysisapriorityforthepresentregulatorystrategies.Withintheaboveperspective,theSAsidentifiedinthisstudyprovideacoarse-grainfilterforapreliminaryscreeningofpotentiallyinvivomutagens.Inariskassessmentprocess,furtherinformation(e.g.,QSARsforindividualclasses,experiments)isnecessarytocompletethisinitialscreeningstep.20 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Appendix1STRUCTURALALERTDETAILSANDEXAMPLESSA_1:AcylhalidesOR=anyatom/group,exceptOH,SHR[Br,Cl,F,I]NorepresentativesSA_2:alkyl(C<5)orbenzylesterofsulphonicorphosphonicacidORR=AlkylwithC<5(potentiallysubstitutedbyhalogens),orbenzylO--R1=anyatom/groupexceptOH,SH,O,SR1SOOPORRR1OOH3COSCH3OName:EthylMethanesulfonateCAS:62-50-0InvivoMicronucleus(Rodent):PositiveReference:NTPOOSCH3H3COName:MethylMethanesulfonateCAS:66-27-3InvivoMicronucleus(Rodent):PositiveReference:CCRIS23 SA_3:N-methylolderivativesHOCH2R=anyatom/groupRNRNopositiverepresentativeSA_4:MonohaloalkeneR1[Br,Cl,F,I]R1,R2(orR3)=HorAlkylR3(orR2)=anyatom/groupexcepthalogensR2R3ClClName:1,3-dichloropropeneCAS:542-75-6InvivoMicronucleus(Rodent):PositiveReference:NTPH3CH3CClName:DimethylvinylChlorideCAS:513-37-1InvivoMicronucleus(Rodent):PositiveReference:NTPSA_5:SorNmustardRN[Br,Cl,F,I][Br,Cl,F,I]R=anyatom/grouporS[Br,Cl,F,I][Br,Cl,F,I]24 OClOHNClName:ChloroambucilCAS:305-03-3InvivoMicronucleus(Rodent):PositiveReference:NTPClNOHH2NOClName:MelphalanCAS:148-82-3InvivoMicronucleus(Rodent):PositiveReference:NTPSA_6PropiolactonesorpropiosultonesOOOSAnysubstancewiththedisplayedsubstructuresOOorNorepresentativesSA_7:EpoxidesandaziridinesRONR=anyatom/grouporO25 Name:EthyleneOxideCAS:75-21-8InvivoMicronucleus(Rodent):PositiveReference:CCRISNNNNNNName:TriethylenemelamineCAS:51-18-3InvivoMicronucleus(Rodent):PositiveReference:NTPSA_8:AliphatichalogensRR=anyatom/groupR[Br,Cl,I]HBrBrName:1,2-dibromoethaneCAS:106-93-4InvivoMicronucleus(Rodent):PositiveReference:NTPClCH3ClName:1,1-dichloroethaneCAS:75-34-3InvivoMicronucleus(Rodent):PositiveReference:CCRISSA_9:AlkylnitriteR=anyalkylgroup26 ONORCH3OOH3CNName:IsobutylNitriteCAS:542-56-3InvivoMicronucleus(Rodent):PositiveReference:NTPSA_10:a,bunsaturatedcarbonylsR1R1andR2=anyatom/group,exceptalkylchainswithC>5oraromaticrings.-ROR=anyatom/group,exceptOH,O2ROCH3OHOName:MaltolCAS:118-71-8InvivoMicronucleus(Rodent):PositiveReference:CCRISNH2H2COName:AcrylamideCAS:79-06-1InvivoMicronucleus(Rodent):PositiveReference:CCRISSA_11:SimplealdehydeR=aliphaticoraromaticcarbona,bunsaturatedaldehydesareexcluded27 OHROOH3CName:PyruvaldehydeCAS:78-98-8InvivoMicronucleus(Rodent):PositiveReference:LeadscopeCH3OSName:3-(methylthio)propionaldehydeCAS:3268-49-3InvivoMicronucleus(Rodent):PositiveReference:LeadscopeSA_12:QuinonesOOAnysubstancewiththedisplayedsubstructuresOOorOOName:9,10-AnthraquinoneCAS:84-65-1InvivoMicronucleus(Rodent):PositiveReference:NTP28 H2NOOOHCH3H2NOHNNHH3COHName:MitomycinCCAS:50-07-7InvivoMicronucleus(Rodent):PositiveReference:CCRIS&NTPSA_13:HydrazineRRR=anyatom/groupNNRRNopositiverepresentativeSA_14:AliphaticazoandazoxyR2+-NNNNR1=AliphaticcarbonorhydrogenR1R1orR2orR2,R3=Anyatom/group-R4=AliphaticcarbonO+R4NNR3NorepresentativesSA_15:isocyanateandisothiocyanategroupsOSR=anyatom/groupRCRCNorNNopositiverepresentativeSA_16:alkylcarbamateandR=AliphaticcarbonorhydrogenthiocarbamateR1=Aliphaticcarbon29 [O,S]RR1N[O,S]ROH2NOCH3Name:UrethaneCAS:51-79-6InvivoMicronucleus(Rodent):PositiveReference:CCRIS&NTPSA_18:PolycyclicAromaticHydrocarbonsThreeormorefusedrings,notheteroaromaticCH3H3CName:7,12-Dimethylbenz(a)anthraceneCAS:57-97-6InvivoMicronucleus(Rodent):PositiveReference:CCRIS&NTPSA_19:HeterocyclicPolycyclicAromaticThreeormorefusedrings,heteroaromaticHydrocarbonsNopositiverepresentativeSA_21:alkylandarylN-nitrosogroupsR1R1=Aliphaticoraromaticcarbon,R2=Anyatom/groupNOR2NNH2ONNH3CO30 Name:N-methyl-N-nitrosoureaCAS:684-93-5InvivoMicronucleus(Rodent):PositiveReference:NTPH3CNNOCH3Name:N-nitrosodimethylamineCAS:62-75-9InvivoMicronucleus(Rodent):PositiveReference:CCRISSA_22:azideandtriazenegroupsR-NR=Anyatom/group+NNRNRNRorNNNNHName:DiazoaminobenzeneCAS:136-35-6InvivoMicronucleus(Rodent):PositiveReference:CCRIS&NTPOCH3HNONHOO-+NNNName:ZidovudineCAS:30516_87-131 InvivoMicronucleus(Rodent):PositiveReference:NTPSA_23:aliphaticN-nitrogroup-O+RNR=AliphaticcarbonorhydrogenNORONNNHO+H3CN-NHOName:N-methyl-N'-nitro-N-nitrosoguanidineCAS:70-25-7InvivoMicronucleus(Rodent):PositiveReference:CCRISSA_24:a,bunsaturatedaliphaticalkoxygroupHR1=AnyaliphaticCarbonR2=AliphaticoraromaticcarbonR1R2OHOH3COCH2Name:VinylAcetateCAS:108-05-4InvivoMicronucleus(Rodent):PositiveReference:NTPSA_25:aromaticnitrosogroupNAr=Anyaromatic/heteroaromaticringArONopositiverepresentative32 SA_26:aromaticringN-oxide-O+NAnyaromaticorheteroaromaticringNopositiverepresentativeAr=Anyaromatic/heteroaromaticringSA_27:Nitro-aromatic·Chemicalswithortho-disubstitution,orOwithanorthocarboxylicacidsubstituent+areexcluded.ArN·Chemicalswithasulfonicacidgroup(--OSO3H)onthesameringofthenitrogroupareexcluded.N-O+NNCH3OOHName:MetronidazoleCAS:443-48-1InvivoMicronucleus(Rodent):PositiveReference:CCRIS-OCH3+NONSNH2NNNName:CL64855CAS:19622-55-0InvivoMicronucleus(Rodent):PositiveReference:CCRISAr=Anyaromatic/heteroaromaticringSA_28:primaryaromaticamine,hydroxylR=Anyatom/groupamineanditsderivedesters·Chemicalswithortho-disubstitution,or33 OwithanorthocarboxylicacidsubstituentOROareexcluded.HHHN·Chemicalswithasulfonicacidgroup(-NRNHSO3H)onthesameringoftheaminoArorArorArgroupareexcluded.oraminegeneratinggroup:OCH2NNArorArNH2Name:AnilineCAS:62-53-3InvivoMicronucleus(Rodent):PositiveReference:CCRIS&NTPNH2Name:4-BiphenylamineCAS:92-67-1InvivoMicronucleus(Rodent):PositiveReference:NTPAr=Anyaromatic/heteroaromaticringR1=Hydrogen,methyl,ethylR2=Methyl,ethylSA_28bis:Aromaticmono-and·Chemicalswithortho-disubstitution,ordialkylaminewithanorthocarboxylicacidsubstituentR1R2Nareexcluded.·Chemicalswithasulfonicacidgroup(-ArSO3H)onthesameringofthenitrogroupareexcluded.34 CH3CH3NNH3CCH3Name:LeucomalachiteGreenCAS:129-73-7InvivoMicronucleus(Rodent):PositiveReference:NTPCH3NNNCH3Name:4-DimethylaminoazobenzeneCAS:60-11-7InvivoMicronucleus(Rodent):PositiveReference:CCRISAr=Anyaromatic/heteroaromaticringSA_28tris:aromaticN-acylamineR=Hydrogen,methylO·Chemicalswithortho-disubstitution,orwithanorthocarboxylicacidsubstituentRareexcluded.NR·Chemicalswithasulfonicacidgroup(-SO3H)onthesameringofthenitroArgroupareexcluded.NopositiverepresentativeSA_29:AromaticdiazoAr=Anyaromatic/heteroaromaticring·Chemicalswithasulfonicacidgroup(-NNSO3H)onbothringsthatcontainlinkedtothediazogroupareexcluded.ArArClNNClClCl35 Name:3,3’,4,4’-TetrachloroazobenzeneCAS:14047-09-7InvivoMicronucleus(Rodent):PositiveReference:NTPNCH3NNCH3Name:4-DimethylaminoazobenzeneCAS:60-11-7InvivoMicronucleus(Rodent):PositiveReference:CCRISSA_30:CoumarinsandFurocoumarinsAnysubstancewiththedisplayedsubstructureOONopositiverepresentativeSA_32:1,3-dialkoxy-benzeneROR=anyalkylgroupOR36 H3CH3CH3COOOOOH3CHNHOCH3Name:ColchicineCAS:64-86-8InvivoMicronucleus(Rodent):PositiveReference:CCRISH3CONNHHHOCHOOH3COOOH3COOCCH3Name:ReserpineCAS:50-55-5InvivoMicronucleus(Rodent):PositiveReference:NTPSA_33:1-phenoxy-benzeneAnysubstancewiththedisplayedsubstructure.37 OOFFFOClHCON3CH3Name:Lambda-cyhalothrynCAS:91465-08-6InvivoMicronucleus(Rodent):PositiveReference:CCRISSA_34:hacceptor-path3-hacceptorA=Anyatom,exceptHydrogenAH-bond-AccH-bond-Acc=AnyatomthatisapotentialH-bond-AccAHydrogenbondacceptorOOName:p-DioxaneCAS:123-91-1InvivoMicronucleus(Rodent):PositiveReference:CCRIS&NTP38 HOOHOOHName:3,4-DihydroxycinnamicacidCAS:331-39-5InvivoMicronucleus(Rodent):PositiveReference:NTPSA_35:OxolaneAnysubstancewiththedisplayedsubstructure.ONH2NNHONOOHOOHName:5-AzacytidineCAS:320-67-2InvivoMicronucleus(Rodent):PositiveReference:NTPOHHOONHONNH2ONName:RibavirinCAS:36791-04-5InvivoMicronucleus(Rodent):PositiveReference:NTPSA_36:CarbodiimidesR=anyalkylgroup39 RRNCNCH3H3CNCCCNCH3CH3Name:DiisopropylcarbodiimideCAS:693-13-0InvivoMicronucleus(Rodent):PositiveReference:NTPNCNName:DicyclohexylcarbodiimideCAS:538-75-0InvivoMicronucleus(Rodent):PositiveReference:NTP40 EuropeanCommissionEUR23844EN–JointResearchCentre–InstituteforHealthandConsumerProtectionTitle:DevelopmentofStructuralalertsfortheinvivomicronucleusassayinrodentsAuthor(s):BenigniR,BossaC,TcheremenskaiaOandWorthALuxembourg:OfficeforOfficialPublicationsoftheEuropeanCommunities2009–42pp.–21x29.7cmEUR–ScientificandTechnicalResearchseries–ISSN1018-5593AbstractInvivomutagenicityandcarcinogenicitystudiesareposingahighdemandfortest-relatedresources.Amongthesestudies,themicronucleustestinrodentsisthemostwidelyused,asfollowuptopositiveinvitromutagenicityresults.Arecentsurveyofthe(Q)SARmodelsformutagenicityandcarcinogenicityhasindicatedthatno(Q)SARmodelsforinvivomicronucleusareavailableinthepublicdomain.Therefore,thedevelopmentandextensiveuseofestimationtechniquessuchas(Q)SARs,read-acrossandgroupingofchemicals,promisestohaveahugeanimalsavingpotentialforthisendpoint.Inthisreport,wedescribetheidentificationofstructuralalertsfortheinvivomicronucleusassay,andprovidethelistofunderlyingchemicalstructures.Thesestructuralalertsprovideacoarse-grainfilterforthepreliminaryscreeningofpotentialinvivomutagens. ThemissionoftheJRCistoprovidecustomer-drivenscientificandtechnicalsupportfortheconception,development,implementationandmonitoringofEUpolicies.AsaserviceoftheEuropeanCommission,theJRCfunctionsasareferencecentreofscienceandtechnologyfortheUnion.Closetothepolicy-makingprocess,itservesthecommoninterestoftheMemberStates,whilebeingindependentofspecialinterests,whetherprivateornational.