non-animal alternatives for research and testing...

THISWORKSHOPPAPERISAWORKINPROGRESS.DONOTCITEORQUOTETHISDRAFTPAPERWITHOUTEXPRESSWRITTENPERMISSIONOFTHEAUTHORS.

Locke&Goldberg Draftof11.01.161

Non-AnimalAlternativesforResearchandTestingundertheAnimalWelfareAct:AnAssessmentandVisionfortheFuture

PaulA.Locke,MPH,JD,DrPH1AlanM.Goldberg,PhD2

1. Introduction

In1966,theUSCongressfirstpassedlegislation,whichthenPresidentLyndonJohnsonsignedintolaw,designedtoaddresstheuseofsomeanimals(largelydogsandcats)inresearch,testing,exhibitionandtransportation.ThelawbecameknownastheLaboratoryAnimalResearchAct,whichwasshortenedtotheAnimalWelfareAct(AWA)insubsequentamendments.(Lee2016;CommitteeonLegalIssuesPertainingtoAnimals2003;Cohen2006).Inthepasthalfcentury,theAWAhasbeenamendedseveraltimes,andhasbeenextensivelyanalyzedinboththescientificandlegalliterature.ThisarticleexaminestherelationshipoftheAWAtothedevelopmentofnon-animalalternativesandtheiruseinscientificresearch,andaddresseswhethertheAWAshouldbeamendedtoenhancethedevelopment,useandfutureofnon-animalalternatives.Part1ofthisarticleconsistsofthisbriefintroduction.InPart2,thearticlecoverstheevolutionoftheAWA,areviewofthebackgroundofregulatinganimaluseinresearch,andthedevelopmentoftheAWAoverthepasthalfcentury,includinganexaminationofitslegislativehistory,commentariesandcriticisms,withafocusonthe“3Rs”(reduction,refinementandreplacement–discussedherein)andnon-animalalternatives.Part3setsoutsixscientificscenariostoserveasexamplesofthedevelopmentofthescienceunderlyingalternatives,andextractsfromthesescenariosfourprinciplesthatareimportanttokeepinmindwhenevaluatingpotentialchangesintheAWA.Part4positsaseriesofquestionsregardingamendingtheAWA,withthegoalofstimulatingdiscussionaboutwhethertheAWAshouldorcouldbeamendedtoactasacatalystforthedevelopmentanduseofalternatives.

1Dr.LockeisanAssociateProfessorattheJohnsHopkinsBloombergSchoolofPublicHealth,wherehedirectsaprogramontoxicologypolicythatisfocusedonthedevelopment,useandfutureofinvitroalternativesinthebiomedicalsciences.HeholdsanMPH(environmentalhealthsciences)fromYaleUniversitySchoolofMedicine;aJDfromVanderbiltUniversitySchoolofLaw;andaDrPH(environmentalhealthsciences)fromtheJohnsHopkinsBloombergSchoolofPublicHealth.2Dr.GoldbergisaProfessorattheJohnsHopkinsBloombergSchoolofPublicHealthandfoundingdirector(emeritus)oftheJohnsHopkinsCenterforAlternativestoAnimalTesting,aninternationallyrecognizedscientificcenterthatstudies,developsandadvancestheuseofhumanescienceandthe“3Rs”andalternativesinbiomedicalresearch,drugdevelopment,cosmeticsandtoxicitytesting.Hehasbeenactiveinanimalwelfareissuesforbothresearchanimalsaswellasfoodanimals.HeholdsaPhD(pharmacology)fromtheUniversityofMinnesotaandaBA(pharmacology)fromBrooklynCollegeofPharmacology.

2. TheEvolutionoftheAWAanditspolicytowardsanimalsinresearchA. Firststepstowardtoday’sAWA

Theissuesassociatedwithregulatingtheuseofanimalsinresearchandsciencehavebeenoflong-standingconcernintheUnitedStates.Effortsbeganinthelate19thcenturytocontrolanimalexperimentationthroughlegislation.Forexample,in1880abillaimedatregulatinganimalexperimentationintheDistrictofColumbia(withprovisionsroughlysimilartothe1876BritishCrueltytoAnimalsAct)wasintroducedbeforeCongress.Thislegislationwaseasilydefeatedbecausescientistsopposedit.(AnimalWelfareInstitute,1990[p.67].)Earlystatelegislationdidnotaddress,oractivelyexcluded,animalsusedinscience.Betweenthe1820sand1900,nearlyeverystateandterritoryintheUSpassedanti-crueltylegislation.Fourteenoftheselawsexplicitlyexemptedanimalsinresearch,andeveninstateswithoutexemptions,therewerenoknownprosecutionsforanimalcrueltyagainstanimalresearchersbefore1958.Sincethen,onlytwocaseshavebeenbroughtagainstlaboratoriesand/orresearchersbasedonstateanimalcrueltylaws.(AnimalWelfareInstitute,1958;Taub,1983.)ThepracticalimpactoftheTaublitigationhasbeentomakeitmuchmoredifficulttousestateanti-crueltylawsaslegaltoolstopursueresearchandtestingfacilitiesandresearchers.(SeeReppy,W.Jr.DoStateAnti-CrueltyLawsApplytoAnimalsUsedinScientificResearch?Availableathttp://scholarship.law.duke.edu/cgi/viewcontent.cgi?article=2470&context=faculty_scholarship(lastaccessed25October2016).)Atthefederallevel,concernaboutanimalwelfareissueswassubstantialbythemiddleofthetwentiethcenturyandledtoseverallegislativeeffortstorequirethehumanetreatmentoflaboratoryanimals.Inthe1960s,theUSCongressreceivedmoremailaboutanimalcareissuesthanaboutcivilrightsandthewarinVietnamcombined.(Kregeretal.1996,[p.vii].)Nevertheless,itwasdifficulttoadvancefederalanimalwelfarelegislation.Thefirstanimalwelfarebillwasintroducedin1960,anditwouldhaverequiredhumanetreatmentofanimalsusedinresearchbyUSfederalgrantrecipients.(See106Cong.Rec.,Part9,pp.1192–93(1960);S.3570,86thCong.,2dSess.(1960);AnimalWelfareInstitute,1990[p.72])Inthewordsofoneofitssponsors,SenatorWilliamProxmire:

“Clearly,therearetwosidestotheanimalexperimentationquestion.Ontheonehand,suchexperimentshavemadepossiblegreatscientificandmedicalachievements.Ontheotherhand,somethoughtlessorcarelessexperimentershaveinflictedunnecessarypainandsufferingonlaboratoryanimalsWhatisneededisanapproachtothisquestionwhichpreservesthenecessaryandusefulaspectsofanimalexperimentswhilepreventingtheabuses.”(106Cong.Rec.11963).

In1965,legislativeeffortswerevigorouslyrenewedafteraDalmatiandognamedPepperwasillegallytakenfromherowners’homebyadogdealer.ThedealersupposedlytookPeppertoahospitalinNewYorkCity,butPepperwasneverfound.ThereisspeculationthatPepperdiedontheoperatingtableandherbodywasincineratedatthehospital,butitwasneverproven

conclusively.(AnimalWelfareInstitute,1990[p.74]).ThisincidentledtotheintroductionofHR9743byCongressmanJosephResnickinthesummerof1965.Thisbillwasamended,andlaterreplacedbyasimilarbillintheHouse(HR13881),andacompanionbillintheSenate.Afteraseriesofhearings,amendments,andthepublicationofaconferencereport,thehousebill(withamendments)waspassedbyboththeHouseandSenate,andsignedintolawbyPresidentLyndonJohnsoninlatesummer1966.(Engber2009;CQAlmanac,“’Dognapping’billenactedafterheavylobbying(1966),availableathttps://library.cqpress.com/cqalmanac/document.php?id=cqal66-1301593(lastaccessed20October2016)).Asoriginallyenacted,thepurposeofthislawwas:• toprotecttheownersofdogsandcatsfromtheftofsuchpets;• topreventtheuseorsaleofstolendogsorcatsforpurposesofresearchor

experimentation;and• toestablishhumanestandardsforthetreatmentofdogs,cats,andcertainotheranimals

(monkeys,guineapigs,hamsters,andrabbits)byanimaldealersandmedicalresearchfacilities.Seehttps://www.animallaw.info/administrative/us-senate-report-1966-animal-welfare-act(lastaccessed20October2016)reprintingportionsofS.Rep.1281(1966).

The1966legislation,definedasthe“LaboratoryAnimalWelfareAct”initsregulations(see§1.1(a)ofthe1967rules)didnotaddressalternatives.ItdidauthorizetheUSDepartmentofAgriculture(USDA)topromulgateregulationsforhumanestandards,whichwerepublishedinfinalformon24February1967.(See32Fed.Reg.3270-3282(24February1967))availableathttp://archive.org/stream/federalregister32aunit#page/n1422/mode/1up(lastaccessed20October2016).Theseregulationscoverlicensingandregistrationofdealersandfacilitiesandrecording-keeping.Theyalsosetoutcertainstandardsforfeeding,watering,sanitationandveterinarycare.Thestandardsareaimedatprovidingadequatehusbandry,butarenotdirectlyrelatedtoeliminationorminimizationofpain,stressordistress.ItisnoteworthythattheUSDAwasassignedtheresponsibilitytoimplementtheAWA.AlmostallotherfederalhealthresearchrelatedlegislationsisunderthejurisdictionoftheDepartmentofHealthandHumanServices(HHS).TheLaboratoryAnimalWelfareActwasamendedin1970andrenamedthe“AnimalWelfareAct.”(SeePL91-579,approved24December1970.)Itwasalsoamendedin1976.Whiletheseamendmentscontainedimportantchanges,thechangesdidnotdirectlyaddressalternativesinresearch.The1985amendmentstotheAnimalWelfareAct(AWA),however,includedmanysignificantchangesrelatingtolaboratoryanimalresearchandtangentiallyaddressedalternatives.

B. TheImprovedStandardsforLaboratoryAnimalsActof1985(ISLAA)andlateramendmentstotheAWA

The1985amendmentstotheAWA,passedbyCongressastheISLAA,werethemostsignificantadditiontothestatutefromtheperspectiveofanimalsusedinresearchsettingsandlaboratories.(Lee,2016;Dukes1986.)Unlikeearlieramendmentsandtheoriginallegislation,theISLAAauthorizedUSDAtopromulgaterulesthataffectedanimalsduringexperimentation.Amongotherthings,theISLAArequired:

• Minimizingpainanddistressduringexperimentation(unlesstheexperimentrequiredotherwise)byrequiringtheuseofanalgesicsandanesthetics;

• EstablishinganInstitutionalAnimalCareandUseCommittee(IACUC)atfacilitiessubjecttotheAWA;

• Traininganimalcarepersonnel,includingprincipalinvestigators;• EstablishinganinformalserviceattheNationalAgriculturalLibrarythatwouldserveasa

resourcetoreduceunintendedduplicationofexperiments,helpreplaceandreduceanimaluse,andassistinminimizingpainanddistress;and

• Requiringthatprincipalinvestigatorsconsideralternativestoanyprocedureslikelytoproducepainanddistress.

Inaddition,theUSDAmustinspecteachfacilityatleastoneperyear.(Dukes,1986;Lee,2016.)TheILSAAalsore-affirmedaprinciplethatwasincorporatedintotheAWAinits1970amendments–theUSDAwasprohibitedfromregulatingresearch.Theresearchscientiststillheld“thekeystothelaboratorydoor.”(Dukes,1986[p.522].Morespecifically,theILSAAstatesthattheUSDAcannotregulatethedesign,outline,orguidelinesofactualresearchortheconductofactualresearchbeyondtherequirementsinthelawforreducingand/oreliminatingpainanddistress.(Cohen,2006[p.13].Thestatutorylanguageisfoundat7USC§§2143(a)(3)(E)and(a)(6)(A).)TheAWAwasamendedagainin1990and2002.In1990,additionalprovisionswereaddedtoprotectcatsanddogsbyprovidingaholdingperiodbeforetheiruseinresearch.The2002amendmentsexpandedtheAWAprohibitiononanimalfighting,andchangedtheAWAdefinitionof“animal”byexcludingfromthedefinitionrats,miceandbirdsbredforresearchpurposes.3(Cohen,2006).4TheAWAwasalsoamendedin2007,2008and2014.Theseamendmentsstrengthenedanimalfightingprohibitionsanddogresalerequirements.The2014amendmentsaddeda“de

3Thereviseddefinitionof“animal”isfoundat7USC§2132(g).Section2132(g)(1)excludes“birds,ratsofthegenusRattus,andmiceofthegenusMusbredforuseinresearch.”4Becausethisarticleisfocusedonalternativesitdoesnotdiscussthisamendment,oritsimpact,ingreatdetail.(Seesection4,partiv,infra.)Obviously,the2002changeinthedefinitionofanimalisverysignificantbecauseitexcludesfromtheAWAapproximately90to95%ofallanimalsusedinresearch.Itsimpacthasbeenexaminedatgreatlengthinboththelegalandscientificliterature.

minimis”provisionthatallowstheUSDAtoexemptcertainsmallexhibitorsanddealersfromlicensureandregistration.(Cowan,2016.)

C. Non-animalalternativesandtheAWA

i. Whatisan“alternative?”

Theunderstandingandevolutionoftheterm“alternative”asappliedtoanimalsusedinlaboratorieshasarichandcomplexhistory.(TannenbaumandTaylor,2015.)Asaresult,thereisnouniversallyagreedupondefinitionforthistermand,especiallyinthelegalliterature,ithasnotbeenusedconsistently.Thissectionofthearticletraceshowthistermhastransmutedovertimeandoffersadefinitionforitthatisconsistentwithitscontemporaryuse.Thedefinitionof“alternatives”todayisoftenunderstoodtoincludethe“3Rs”–reduction,replacementandrefinement–conceptsfirstproposedbytheBritishscientificteamofWMSRussellandRLBurch.(RussellandBurch,1959.)The3Rsoriginatedaswaystodiminishorremove“inhumanity”inexperimentation.Intheirtreatise“ThePrinciplesofHumaneExperimentalTechnique,”RussellandBurchintroducedthesethreeideas.Astheyexplained:“Replacementmeansthesubstitutionforconsciouslivinghigheranimalsofinsentientmaterial.Reductionmeansreductioninthenumbersofanimalsusedtoobtaininformationofgivenamountandprecision.Refinementmeansanydecreaseintheincidenceorseverityofinhumaneproceduresappliedtothoseanimalswhichstillhavetobeused.”(RussellandBurch,1959[p.66])(Emphasisadded.)Notsurprisingly,the3Rsconceptshaveevolvedoverthepasthalfcentury.TheUSGuidefortheCareandUseofLaboratoryAnimals(theGuide),publishedbytheNationalAcademyofSciencesNationalResearchCouncil(NAS2011)5,andadoptedbytheUSNationalInstitutesofHealthandAAALACInternational,offerthefollowingdefinitionsofthe3Rs:“Overtheyears,theThreeRshavebecomeaninternationallyacceptedapproachforresearcherstoapplywhendecidingtouseanimalsinresearchandindesigninghumaneanimalresearchstudies.Replacementreferstomethodsthatavoidusinganimals.Thetermincludesabsolutereplacements(i.e.,replacinganimalswithinanimatesystemssuchascomputerprograms)aswellasrelativereplacements(i.e.,replacinganimalssuchasvertebrateswithanimalsthatareloweronthephylogeneticscale).Refinementreferstomodificationsofhusbandryorexperimentalprocedurestoenhanceanimalwell-beingandminimizeoreliminatepainanddistress.Whileinstitutionsand

5Oneoftheauthorsofthispaper(PAL)wasamemberoftheNationalAcademyofSciencescommitteethatwrotethelatestversionoftheGuidefortheCareandUseofLaboratoryAnimals.(NAS,2011p.v.)

investigatorsshouldtakeallreasonablemeasurestoeliminatepainanddistressthroughrefinement,IACUCsshouldunderstandthatwithsometypesofstudiestheremaybeeitherunforeseenorintendedexperimentaloutcomesthatproducepain.Theseoutcomesmayormaynotbeeliminatedbasedonthegoalsofthestudy.Reductioninvolvesstrategiesforobtainingcomparablelevelsofinformationfromtheuseoffeweranimalsorformaximizingtheinformationobtainedfromagivennumberofanimals(withoutincreasingpainordistress)sothatinthelongrunfeweranimalsareneededtoacquirethesamescientificinformation.Thisapproachreliesonananalysisofexperimentaldesign,applicationsofnewertechnologies,theuseofappropriatestatisticalmethods,andcontrolofenvironmentallyrelatedvariabilityinanimalhousingandstudyareas.”(NAS2011[p.__],)Theterm“alternatives”doesnotappearinRussellandBurch’sbook.Itcameintouselater,andhasbeenusedtorefertothe3Rscollectively,aswellasonlyoneofthe3Rs--replacement.Thisuseoftheterminbothofthesewayshascreatedconfusion.(TannenbaumandTaylor,2015.)6Inthisarticle,weadoptasthedefinitionof“alternatives”thedefinitionof“replacement”fromtheGuide.Inotherwords,analternativeinvolves(1)avoidingtheuseofanimalsinexperimentswheneverpossible(e.g.,usingcellculturesystemsorcomputationaltechniques),and/or(2)usinganimalsthatareloweronthephylogeneticscaleinsteadofanimalsthatarehigheronthephylogeneticscale.(e.g.,usingaworm(c.elegans)insteadofamouse;usingazebrafishinsteadofaguineapig).Weusetheterm“non-animalalternatives”torefertothosemethodsandtechniquesthatdonotdirectlyemploylivingcreatures.Inotherpublications,theterms“invitro”and“insilico”aredefinedasexamplesofnon-animalalternatives,andweagreewiththatcharacterization.(Lee,2016.)Webelievethatthedevelopmentanduseofalternativesresultsinstrongerscientificresearchandthecreationofbetterdatafordecision-makingbecausetheprocessbywhichalternativesareconceived,developed,testedandappliedtoscientificquestionstendstobemoreforwardlookingandcanavoidmanyofthescientificshort-comingsoftraditional,animalbasedmethods.

ii. AnalysisofalternativesinthekeystatutoryprovisionsoftheAWArelatingtolaboratoryanimaluseandresearchfacilitiesthatuseanimals

AreviewoftheAWA’sprovisionsapplicabletolaboratoryandresearchfacilitiesrevealsthattheActcontainsveryfewreferencestoalternativesandnospecificreferencestothe3Rs.AmoredetailedanalysisofkeyAWAlanguageissetoutintheparagraphsthatfollow.

6AsTannenbaumandTaylorpointout,ina1995speechWMSRussell,agreeingwithoneoftheauthorsofthispaper(AMG),pointedoutthattheuseoftheterm“alternatives”torefertoallthreeRswasbothunfortunateandconfusing.(TannenbaumandTaylor,2015[p.123])

TheAWAbeginswithastatementofpolicy.TheCongressionalfindingsdoechosomewhattheconceptsofreplacementandreduction,althoughneitherofthesetermsisused.Forexample,7USC§2131(2)statesthat“methodsoftestingthatdonotuseanimalsarebeingandcontinuetobedeveloped…andfurtheropportunitiesexistforthedevelopmentofthesemethodsoftesting,”and§2131(3)statesthat“measureswhicheliminateorminimizetheunnecessaryduplicationofexperimentsonanimalscanresultinmoreproductiveuseofFederalfunds.”Theplainlanguagemeaningofthesestatementsseemstobeacknowledgementsthatnon-animaltestsareevolvingandthatduplicationisapooruseofFederalfunds.Theseareonlytangentiallyrelatedtotheconceptsofreplacementandreduction,however,andarenotwrittenasgoalsorobjectivesofthestatute.ThedefinitionssetforthintheAWA7USC§2132donotexplicitlyorimplicitlyidentifyalternativesorthe3Rs.Theomissionofthe3RsfromthesesectionsissignificantbecauseiftheAWAwerea3Rslaw,itwouldbeexpectedthatinthesesectionsreplacement,reductionandrefinementwouldatleastbereferencedandperhapsendorsedand/ordefined.Theterm“alternatives”andlanguagethatresonateswithoneofthe3Rsconceptsiscontainedin7USC§2143,whichisthecentralprovisionoftheAWAapplicabletoresearchfacilities.Section2143(a)(3)requiresthattheUSDApromulgateminimumrequirementsforcare,treatmentandpracticesthatensurethatpainanddistressareminimizedthroughadequateveterinarycareandpainrelievingdrugs.Italsorequiresthattheprincipalinvestigatorofanexperimentconsideralternativestoanyprocedurethatwouldproducepainordistress.ForproceduresthatwouldcausepaintheAWArequiresthataveterinarianisconsulted,thatpainkillingdrugsareused(unlessitisscientificallynecessarytowithholdthem,andtheyshouldbewithheldonlyforaslongasscientificallynecessary);thatpre-andpost-operativecaremeetsveterinarystandards;andthatnoanimalisusedinmorethanonemajoroperation(unlessitisscientificallynecessaryorinotherspecialcircumstancessetoutbyUSDA).Theseprovisionscapturemanyoftheideasthatunderlietheconceptofrefinement,especiallyasthattermisdefinedbytheGuide(seeabove).Section2143(a)(7)hasrequirementsforresearchfacilityreporting.FacilitiesmustreporttotheUSDAatleastannually,providinginformationaboutanypainfulprocedures,orthoselikelytocausedistress,andassuretheUSDAthattheprincipalinvestigatorconsideredalternativestothosepainfulprocedures.Anydeviationsfromthisstandardmustbeexplained.Insection2143(d),theAWArequiresthateachresearchfacilityshalltrainitspersonnelinresearchandtestingmethodsthatminimizeoreliminateanimaluse,andlimitpainanddistress.ThistrainingistiedtotheuseoftheNationalAgriculturalLibrary,whichisdiscussedinsection2143(e).

iii. DoestheAWAembracealternativesand/orthe3Rs?BasedonthekeyprovisionsintheAWAthataddressanimalexperimentationandresearchfacilities,itdoesnotappearthattheAWAdirectlyembracesthe3Rsoralternativesinany

comprehensiveorproactiveway.7NeitherthestatementsofCongressionalintentnorthedefinitionsspecificallymentionreplacement,reductionandrefinement.Someoftherequirementsin7USC§2143espouseportionsofthe3Rsconcepts.Perhapsthestrongestevidenceforassumptionofoneofthe3Rs–refinement–iscontainedinthissection,whichdoesmentiontheneedtominimizeoreliminatepainand/ordistressinlaboratoryanimals,consistentwithscientificgoals.TraininginresearchmethodsthatminimizeoreliminatetheuseofanimalsisalsorequiredbytheAWA.ThistrainingislinkedtotheestablishmentanduseoftheNationalAgriculturalLibrary,whichmustprovideinformationonimprovedmethodsoflaboratoryanimaluse,especiallypaineliminationandmanagementandreductionandreplacement.Finally,intheeventthatanexperimentislikelytoproducepainanddistress,assurancesmustbeprovidedthattheprincipalinvestigatorconsideredalternatives.8ThisconclusionthattheAWAdoesnotfullyembraceoradoptthe3RsisatoddswithatleastonestatementintheAWA’slegislativehistory,publicstatementsbytheregulatedcommunityandlanguageinseverallawreviewarticles.Forexample,legislativehistoryassociatedwiththe1985amendmentstotheAWA(theISLAA)statesthattheamendmentswereintendedto“reflecttheimportanceofthe3Rs.”(CongressionalRecord,1991[pE1296].)Theregulatedcommunityhasacknowledgedthatthe3RsaredeeplyingrainedintheAWAandlaboratoryanimalpractice.AccordingtothegroupSpeakingofResearch,anadvocacyorganizationthatprovidesinformationabouttheimportanceofanimalresearchinmedicalandveterinaryscience:“The3RsareimplicitintheAWAandanyscientistplanningtouseanimals(exceptrats,mice,andbirds,whicharenotincludedintheAWA)intheirresearchmustfirstdemonstratewhythereisnoalternative;andthatthenumberofanimalsused,andanysufferingcaused,willbekepttoaminimum.”Seehttps://speakingofresearch.com/facts/animal-welfare-the-3rs/(lastaccessed6October2016).OnelawreviewarticleacceptsthattheAWAisa3Rslaw:“In1985,thegrowingpoweroftheanimalrightsmovement,thedocumentationofegregiousabusesofanimalsatseveralresearchinstitutions,andthegeneralacceptanceoftheThreeR'sbyadvocatesandresearcherspromptedCongresstoincorporatetheThreeR'sintotheAWA.”(Ibrahim,2006[p.206]).Thisbeliefisalsoadoptedinalaterlawreviewarticle,citingassupportthis2006paper:

7WhileadetailedreviewoftheAWAregulationsisbeyondthescopeofthisarticle,webelievethattheregulationsareconsistentwiththelegislationintheirapproachtothe3Rs.8Interestingly,implementationofthisrequirementseemstobelimitedtoaliteraturesearchforalternativeproceduresverylateinthegame.WehavereachedthisconclusionbasedonareviewofpubliclyavailableIACUCformsfromvariousuniversitiesandotherentities,whichshowthatthealternativesanalysisisonlyconductedaftertheexperimentaldesignhasbeenestablished–and,perhapsmoreimportantly,afterfundinghasbeenawarded.Forexample,seehttp://web.jhu.edu/animalcare/forms.html(“Newprotocol/Thirdyearrenewalform”)lastaccessed29October2016.

“[I]SLAAincorporatedintoitslanguageapolicyknownatthe“ThreeRs”totrytoincreasehumanenessoflaboratorytesting.”(Lee,2016[p.200.])OuranalysissupportstheconclusionthattheassertionthattheAWAisa3Rslawisnotcorrectandshowsthat,atbest,theAWAonlypartiallyandhalf-heartedlycapturesaportionofthe3Rsconcepts.WeconcludethattheAWAappearstobeatmosta“1R”law,largelyalignedwiththerefinementprincipleofreducingoreliminatingpainanddistresswhenscientificallyfeasible.93. Sixscenariosdemonstratingthegrowthof,andevolutionin,non-animalalternatives

andfourprinciplesthatemergefromthem.

A. Sixscenariosthatdemonstratehowalternativesevolve.BeforeembarkingonananalysisofwhethertheAWAshouldbeamendedtoincorporatethe3Rsmorefully(especiallythereplacementR),and(ifso)howitshouldbeamended,webelieveitisimportanttoattempttoillustrateandcharacterizethescopeofscientificactivitiesthatareimpactedbytheAWA,andinparticularpointoutsimilaritiesanddifferencesalongthecontinuumofscientificactivitiesthatareoftenlumpedtogetheras“laboratoryanimalscience.”Asexplainedingreaterdetailbelow,wethinkitisusefultoparsethesescientificactivitiesintofourdistinctbutoverlappingdomains,becauseeachofthesedomainsutilizelaboratoryanimalscience,andthedataitproduces,differently.Tomorefullydescribethesefourdomains,wefirstofferaseriesofsixscenariosinvolvingscientificproblemsandalternativestoanimalsandissuesacrossthecontinuumofresearch.Wethenextractanddiscussfourprinciplesthatarisefromthesescenarios.TheseprinciplesanddomainswillbeusefulinanalyzingthefuturedirectionoftheAWA.

ii. “Therabbitdied”Inthe1950’sand1960’s,ifyouheardtheterm“therabbitdied”itreferredtosomeonebeingpregnant.Considerthepopular1960’ssituationcomedy,theDickVanDykeshow.In1962,theshowrananepisodeinwhichMr.VanDyke’stelevisionwife,playedbyMaryTylerMoore,announcesherpregnancywiththatphrase.(seehttp://www.imdb.com/title/tt0559862/andhttp://dictionnaire.sensagent.leparisien.fr/Rabbit%20test/en-en/#cite_note-2(lastaccessed21October2016)).Thistest,developedinthe1920’s,usedurinefromawomanthoughttobepregnant.Afterpurification,theurinewasinjectedintoarabbitormouse,andafter24hours,theanimalwaskilledtoseeifithadovulated(bloodyovaries).Ifyes,thenpregnancywas

9WhilewearguethattheAWAisnota3Rsbasedlaw,webelievethatalmostallanimalcareanduseprogramsatinstitutionsthatuseanimalsforresearcharebasedonthe3Rs.Theseprogramsarefoundedupongenerallyacceptedpractices,suchastheGuide,which(alongwithtwootherstandardsdocuments)formthebasisforaccreditationbyAAALACInternational.(Seehttp://www.aaalac.org/about/guidelines.cfm(lastaccessed29October2016)).

presumed.(Engelfried,HawkinsonandGalandey,1945).Thephrase“therabbitdied”was1960svernacularforapositivetest.

Thepregnancyhadtobepost6weekstohaveadequatelevelsofthehormone(hCG,HumanChorionicGonadotropin),andthetesttook24to48hoursfrominjectionoftheurinetothekillingoftheanimal.(Engelfried,HawkinsonandGalandey,1945).Inthe1950’sthistestcostabout$25(1950dollars)andwasconsideredtobehighlyaccurate.10Bytheendofthe20thcentury,theanimaltestforpregnancytestinghadbeenabandoned.Itwasreplacedbyarapidhomepregnancytestthatwasquick,reliable,inexpensive,andnolongerrequiredalivinganimal.Intheprivacyofone’shome,awomancantestherurinewithindaysofamissedperiod(orpossiblyearlier)bypurchasingapregnancytestkitatthelocalpharmacyandwithinminuteshaveagoodideaifsheispregnant.Thecostoftheinvitrotestisusuallylessthan$10(2015dollars).Insummary,thehometestingkitisquicker,easier,lessexpensive,morereliable,anddoesnotrequiretheuseofananimal.ThisispossiblythefirstinvitrotesttoreceiveFDAregulatoryclearanceandiscurrentlythegoldstandardforpregnancytesting.Seehttp://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfIVD/Results.cfm(lastaccessed29October2016).

iii. ThecreationoftheAmestest

Inthe1970’sBruceAmesandhisgrouppublishedaclassicpaperonmutagenesis.11(Ames1973.)Ames’articledescribedatestthatprovidesaquick,non-animalmethodforidentifyingmutagens.TheAmestestusesbacteriatotestwhetheragivenchemicalcancausemutationsinthebacteria’sDNA.Thesebacteriaarefirstmodifiedsothattheyrequirehistidine(anaminoacid)forgrowth,butcannotproduceit.TheAmestestassessesthecapabilityofasyntheticchemicalthatisintroducedtothebacteriafromaforeignsource(axenobiotic)tomutatethecellssothattheycangrowonahistidine-freemedium.Apositivetestindicatesthatthechemicalismutagenicand,inaddition,suggeststhatitmaybecarcinogenic.Asaresult,theAmestestwas(andstillis)usedtoscreenforpossiblecarcinogens,althoughitspredictiveabilityisrelativelylow,inthe50%range.TheAmestestisausefultechniqueforscreeningbecauseifacompoundisfoundtobeamutagenusingtheAmestest,furthertestingcanbeusedtoevaluateitscarcinogenicity.Thetestbecamewidelyusedindrugdevelopmentandtoxicitytesting,andtothisday,isstillconsideredabasicandnecessaryscreeningtestforproductdevelopmentandsafety,andrequired(byregulation)forregistrationundertheFederalInsecticide,Fungicide,andRodenticideAct(FIFRA).(Farmer,2006).

10Statementsaboutthehistoricalcostsandaccuracyofthistestearebasedonpersonalknowledgeofoneoftheauthors(AMG),whoworkedasapharmacistatthestartofhiscareer.11Mutagenesisisaprocessinwhichgeneticinformationischanged,resultinginamutation.

TheAmestestwasoneofthefirstandmostwidelyusedinvitroassays.Itwasnotdevelopedtoreplaceananimaltest;itwascreatedtoprovidemorerapid,accessibleandbetterscientificinformationfordecision-making.However,italsobecamethestimulusforthedevelopmentofotherinvitrotestsandhasprovidedarationalefortheuseofinvitro,non-animal,assaysintoxicologyandsafetytesting.

iv. TheThalidomidetragedy–overrelianceonanimalmodels

Formuchresearch,especiallydiscoveryorbasicresearch,animalmodels,includingrodentmodels,mightnotbeagoodpredictorofhumanresponsestochemicalsanddrugs.Rodentmodelsarewidelyusedandhaveturnedouttobemodelsofimportanceinmanyinstances.Thereareclearexamplesofnon-humananimalstudiesthathavecontributedgreatlytoimprovinghumanandanimalhealthandqualityoflife.(NAS,1991.)Unfortunately,therearealsonumerousexampleswherenon-humananimalstudieshaveproducedmisleadingscience,resultinginharm.Thalidomide(usedasadrugtopreventmorningsickness)isbutoneexample.(See,http://www.thalidomide.ca/recognition-of-thalidomide-defects/(lastaccessed28October2016).)Thewrongspecies--dog,rat,amongothers--wereusedtopredictthehumanresponseandthesestudiesindicatedthatThalidomidewassafe.WenowknowthatthehumanmetabolismofThalidomideisdifferentfromanimalmetabolism.(Kim,2011.)IfThalidomideistakenduringacriticalphaseofhumanfetaldevelopmentitcancausesignificantdamageinhumans(Brent,1988.)

v. Advancingcell,tissueandorganculturesandorgansonachip

Cellortissuecultureisthegrowthofagroupofcells,ortissues,independentfromalivingcreature.Cellortissueculturesaregenerallygrowninanutrientrichmedia(oftencalledbrothoragar).Cellandtissuecultureshavemanyusesinscientificresearch,includingstudyinghowpotentiallytoxic(ortherapeutic)chemicalsinteractwithcellularortissuemachinery.Mostsimplecellandtissueculturesareonedimensionalortwodimensional,andarethereforelimitedinreplicatingthecomplexreactionsofinvivoorgansandlivingcreatures.Morerecently,threedimensionalculturesarebeingcreated.These3Dsystemshavegreaterabilitiestomimicinvivoconditionsandcouldbeinsightfulforstudyingcomplexdiseasessuchascancer.(Bielecka,2016.)Thefirstattemptsatcellculturewerecarriedoutintheearly1800’s,andtissueculturehaditsstartatJohnsHopkinsin1907,underthedirectionofRossHarrison.(Seehttp://www.frame.org.uk/1907-harrison-grows-from-nerve-cells-by-hanging-drop-technique/(lastaccessed28October2016)).Significantadvancesincell,tissueandorganculturesoccurredoverthefirst50to60yearsofthetwentiethcentury,andmajoradvancescontinuetoday.Muchoftheworkwasindefininghowbesttogrowcellsandtissueinvitro.Oneofthefirstattemptstodevelopaninvitromechanisticbasedmammaliantestwasundertakenbyoneoftheauthorsofthispaper.(Goldberg,1980.)1212AnabbreviatedhistoryofcellandtissueculturecanbefoundinAppendixCofZurlo,1994.

Three-dimensional(3D)organcultureshaveamorerecenthistoryandarebecomingmorecommoninallaspectsofmedicalresearch,diseasestudiesandtoxicology(Alepee,2014.)Thesetechnologiesareadvancingrapidlyandhavethepotentialtoraiseinvitrosciencetoanewlevelofphysiologicalrelevance.AttheWyssInstituteatHarvard,3Dorgansarebeingcombinedwithmicrochipsandscaffoldingthatallowsbetterapproximationofthephysicalfactorsthataffectorganfunction.(Seehttps://wyss.harvard.edu/technologies/?taxonomy=focus_area&term=38(lastaccessed30October2016).)Usingpulmonary(lung)tissue,thescaffoldallowsbothliquidperfusionoftheorgananduniquelyproducesstretchingofthetissuemimickingrespiration.Studiesconductedusingthesetechnologiesallowformoresophisticatedresearchthatcanaddresscomplexquestionsandresultinmorerelevantdata.Theseorgansonachipareseenasthenextstepinadvancinginvitrotoxicologicalresearch.(Bahinski,2016.)

vi. Improvingtoxicitytestingandconfrontingthetoxicsinformationgap

Untilitsamendmentthispastsummer,theToxicSubstancesControlAct(TSCA),enactedandsignedintolawin1976,wasthepremierUSlawthatregulateschemicalsincommerce.(SeePublicLaw94-469,94thCongress,90Stat.2003(11October1976).13Inthe40yearssinceTSCAwaspassed,ithascomeundersubstantialcriticism.(See,e.g.,http://www.sciencemag.org/news/2015/03/congress-takes-another-crack-reforming-chemical-testing-system(lastaccessed28October2016)).OneofthemajorcriticismsofthislawwasthatitwasnoteffectiveinproducingthekindoftoxicologicalknowledgethatEPA,citizensandthebusinesscommunityneededtomakedecisionsaboutthehazardsofchemicals.(EnvironmentalDefenseFund,1997.)Ithasbeenestimatedthatoftheapproximately85,000chemicalsincommerce,wehavesolidtoxicologicalinformationonfewerthan1000.Seehttp://thehill.com/blogs/congress-blog/energy-environment/246196-toxic-ignorance-and-the-challenge-for-congress(lastaccessed28October2016)).In2007,inresponsetoarequestfromtheEnvironmentalProtectionAgency(EPA)andinparttoaddressthistoxicignorancegap,theNASpublishedaseminalreport“ToxicityTestinginthe21stCentury:AVisionandaStrategy”(TT21C).Thereportisadetailedcritiqueofthecurrentanimalbasedtoxicitytestingparadigmandaplanforitsevolution.Itrecommendsthatthecurrenttestingparadigmshouldbechangedinawaythatallowsforthedevelopmentofmuchimproved,predictive,humancelllinebasedscience.

13TSCAwassubstantiallyamendedinJune2016,inparttotryandaddressthistoxicsinformationgap.SeeFrankR.LautenbergChemicalSafetyforthe21stCenturyAct,PublicLaw114-182,130Stat.448.22June2016.ThischangeinthelawdoesnotimpacttherecommendationsofTT21C.

TTT21Creachedfourmajorconclusions:

• Animalstudiesaretimeconsumingandexpensive;• Theyarenotalwayspredictiveofhumanresponse;• Inthefuture,weshouldencouragetheuseofhumancells,tissuesandorgansfor

regulatorytoxicitytesting;and• Systemsbiologyandpathwaysoftoxicitywillprovidebetterscienceforregulatory

decision-making.Sincethereport’srelease,theUSfederalgovernmenthasspentaconsiderableamountoftimeandefforttomakeitsvisionandstrategyareality.(http://www.inderscience.com/info/ingeneral/forthcoming.php?jcode=ijram.(lastaccessed28October2016.))14Therearenowpublishedapproachestoinvitrotoxicitytestingforallorgansofthebody.Someareveryadvanced(e.g.,skinandliver)whileotherareinlatestagesofdevelopment(e.g.,thenervoussystem).Alloftheseareavailableinacademicandmostindustrialtoxicologylaboratories.Thestateofdevelopmentisalsoverysophisticatedinthatassayscanbepurchasedcommercially,asdo-it-yourselftestingkits.Therearenowmanycommercialcontractresearchlaboratories,andinmanycasestheselaboratoriesaredevotedtoinvitrostudiesonly.(Seehttp://www.iivs.org/(lastaccessed29October2016).)TheTT21Creport,andthenewinvitroandinsilicotechnologies,arerapidlychangingthepracticeofregulatorytoxicology.Ifoneattendsameetingofprofessionaltoxicologiststoday,80-85%ofthepaperspresentedinvolvetheuseofinvitromethodologies.Inthe1970,approximately95%ofthepapersatsuchmeetingswerebasedonanimalstudies.Thischangehasbeendramatic.15

B. Fourprinciplesthatcanbeextractedfromthegrowthandevolutionofnon-animalalternatives

i. Therearefourseparate,butoverlapping,domainsofsciencethatuse

laboratoryanimals.Generally,tofacilitatethediscussionaboutalternatives,wefinditusefultosub-dividetheuniverseofanimaluseinscienceamongfourseparate,butoverlappingdomains.Thescientificinformationrequiredineachcanvarysignificantly.Thesedomainsare(1)discoveryorbasicresearch(e.g.,developingnewknowledgeandtacklingverycomplex,oftencuttingedgeproblems,suchasacureofcancer),(2)pharmaceuticalanddrugdevelopment(e.g.,theinvestigationofnewmoleculesfortheirpotentialasmedicines,questforbetter,moreeffectivemedicinestotreatchronicconditions),(3)screening,toxicityandsafetytestingforchemicalsusedincommerce(e.g.,TT21C,TSCAandregulatorytoxicology)and(4)safetyandtoxicitytestingforcosmeticsandtheircomponents.

14ThislinkconnectstoaspecialissueoftheInternationalJournalofRiskAssessmentandManagementthatisdevotedtotheTT21Creportanditsimplementation.15Personalobservation,AMG.

Discoveryorbasicsciencemeansexperimentationandscientificexplorationthatseekstofindnewknowledgeabouthowtheworldworks.Morespecifically,inthefieldsofpublichealthandmedicinediscoveryresearchisworkthatstudiescomplexproblemsaboutwhichmoreneedstobeknownsothatacauseofadiseaseorcondition,and/oritscure,canbediscerned.Discoveryresearchisoftencomparedtoappliedresearch,whichistheutilizationofscienceandscientificmethodstodesignsolutions,orsetexposurelevelsoransweralegalorpolicyquestion.Basicordiscoveryresearchisalsodescribedashighrisk,highpay-offresearchthatseeksfarsightedsolutions.IntheUnitedStates,theNationalInstitutesofHealthareoneofthemajorfundersofdiscoveryorbasicresearch.Academicsettings(largelycollegesanduniversities)maintainsophisticatedlaboratorieswherediscoveryresearchisconducted.Ingeneral,basicordiscoveryresearchdoesnotseektoaddress“realworld”problems;itisfocusedoncreatinginformationtofillknowledgegaps.Scienceassociatedwithpharmaceuticalanddrugdevelopmentincludessomediscoveryresearch,butalsoinvolvesscienceaimedatpracticalproblems.Forexample,indevelopingapotentialdrugmoleculeitisimportanttodeterminethebiologicalmechanismbywhichthemoleculeworks–thatis,howitinteractswithalivingsystem–whichisabasicresearchproblem.Itisalsoimportanttoevaluatehowapotentialdrugmoleculecanbedeliveredtothesystemofinterest(i.e.,lung,heart)andhowmuchofitshouldbeintroduced(i.e.,dosage),andhowitshouldbeintroduced(i.e.,routeofadministration).Thesescientificquestionscallforappliedresearch,orresearchthatisaimedatsolvingrealworldproblems.Screening,toxicityandsafetytestingforchemicalsincommercearethebackboneofregulatorytoxicology.Regulatorytoxicologyistheassessmentandevaluationofinformation,usuallybyfederalagencies,todeterminewhetherexposuretoacompoundissafeforhumans,andthelevelsatwhichsuchexposureissafe.IntheUnitedStates,theEPAisthemajorpractitionerofregulatorytoxicology,althoughotheragencies,suchastheUSDA,canplayarole.Regulatorytoxicologyemploystestsforscreeningcompoundstomakeabasicdeterminationabouttheirsafety.Thesetestsrangefromverysimpletechniques,suchastheAmestest,tomoresophisticatedprotocolsinvolvinganimals.AspracticedintheUnitedStatestodayregulatorytoxicologyconsumesmanyanimals,althoughevolutiontowardnon-animalmethodsistakingplace.Safetyandtoxicitytestingforcosmeticsiscloselyakintoregulatorytoxicologyforchemicalsusedincommerce.Italsoemploysaseriesoftestsandtechniquesthatseektoestablishwhethercosmeticsandtheircomponentsaresafeforhumanuse,andthedosageatwhichtheyaresafe.Cosmeticsandtheircomponentsarealmostalwaystestedforskinandeyereactions,andsomeoftheoldestsafetytestingprotocols,suchastheDraizetest,weredevelopedforcosmetics.(Wildhelmus,2001.)Inmanynationstherearelawsinplacepreventingtheuseofanimalsforcosmeticstesting.Forexample,theEuropeanUnionCosmeticsRegulationpreventsthemarketingofanycosmeticsproductifthefinishedproduct,oranycomponent,weretestedonanimalsaftertheeffectivedatesintheRegulation.(SeeRegulation1223/2009,foundat

http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:02009R1223-20150416&from=EN(lastaccessed23October2016).)16

i. Non-animalmethodsandalternativeswillbemorerapidlyandsuccessfullydevelopedifthebiologicalmechanism(s)leadingtotheconditionofinterestarewellunderstoodandcanbereplicatedinnon-livingsystems.

Whenthebiologicalmechanism(s)leadingtotheconditionofinterestarewellcharacterized,itiseasiertoreplicatetheminnon-livingsystems.Thisisoneoftheimportantprinciplestokeepinmindwhenassessingtheeaseatwhichitispossibletomovefromananimaltonon-animalmodel.Thetrajectoryoftheimprovementinpregnancytestingdemonstratesarelativelysmoothtransitionfromananimaltonon-animaltest.Fromitsfirstincarnationasananimalmodelinthe1920s,pregnancytestingevolvedrelativelyrapidly.Theinvitrohomepregnancytestoftodaywasfirstdevelopedin1968(patentUS3579306)andfoundinwidespreadusebythelate1970’s(https://history.nih.gov/exhibits/thinblueline/timeline.html).Theso-calledrabbittestisbasedonthebiologicalfactthataveryspecificsetofmolecules(called,collectively,humanchorionicgonadotropin,orhCG)aresecretedinhigherlevelsthannormalveryearlyinpregnancy.(Cole,2012).ThesehigherthanusualamountsofhCGcanbecollectedinurineandpurified,andtestedtoshowbiologicalevidencethatawomanislikelytobeexpecting.WhiletheinformationavailableabouthCGismuchgreatertodaythanitwaswhentherabbittestwasfirstproposed,thebasicfactsabouthCGwereknownandunderstoodinthe1920s.(Seehttps://history.nih.gov/exhibits/thinblueline/timeline.html(lastaccessed29October2016).)Armedwiththisknowledge,twoearlytwentiethcenturyscientistswereabletodeveloptherabbittest.(Ruediger,1936.)17Thegreatertheunderstandingofthebiologicalmechanismsleadingtotheconditionofinterest,whetheritispregnancyorahealthconditionsuchasasthma,diabetesorcancer,themorelikelyitistobeabletoeffectivelydesignexperimentsandcarryoutscientificexplorationinnon-livingsystems.Acorollarytothispropositionisthatbiologicalmechanismsthataremorecomplex,andonlypartiallyunderstood,areusuallynotcandidatesfornon-animalmodels.Thispropositionanditscorollaryarepartiallythereasonswhyitisfeasibletoeliminatetheuseofanimaltestingformanycosmeticsproducts,whichareoftenconcernedwithskinandeyeirritationorcorrosion(forwhichthereisconsiderablescientificknowledge).Bythesametoken,pursuitofdiscoveryresearch(thestudyandassessmentofchronic,verycomplexconditions,suchasasthma,diabetesandcancer),arenotwellenoughunderstoodtodaytodevelopreplacementsforanimalmodels.Forstudyingtheseconditions,thedefault16AbillthatissimilartothisEURegulation,calledtheHumaneCosmeticsAct,hasbeenintroducedbeforetheUSHouseofRepresentatives.Oneoftheauthorsofthispaper(PAL)haspubliclyendorsedthisbill.Seehttps://www.scientificamerican.com/article/beauty-and-the-beasts-the-u-s-should-ban-testing-cosmetics-on-animals/(lastaccessed30October2016)andhttps://issuu.com/aavs/docs/aavs_av-magazine_2016_cruelty-free-(p.7)(lastaccessed30October2016).17TheoriginaltestwasdevelopedbyAscheimandZondek.TheirpaperiswritteninGermanandiscitedintheRuedigerarticle.Thetestfirstusedonlyrabbits,butwaslaterextendedtomiceandrats.

assumptioninscientificresearchisthatanimalmodelsarenecessaryandappropriate.Livingnon-humansystemsareconsideredtobeaccuratebutimperfectreplicationsofhumansystemswhenseekingtoexpandknowledge.Incontrast,forthecaseoftoxicityorsafetytesting,screeningtestscanoftenbethebestfirststep.Forexample,thenewTSCAlegislationrequiresprioritization(seePublicLaw114-82,114Cong.,2dSess.,130Stat.448,22June2016),andalternativescantodayplayanimportantroleinthisprocess.Prioritizationtests(suchastheAmestest)usuallyevaluateasimplemechanismorreactionthatwillprovideevidenceforwhetheracompoundshouldbeeither(1)furtherevaluatedor(2)placedonalistofcompoundsthatdonotrequireadditionalevaluation.(Sunita,2010.)

ii. Thereisasocietalneedanddesireandincreasingsocietalpressurefortoxicityandsafetytestingthatdoesnotuseanimals.

Consumersbegantodemandfasterandmoreaccuratetestinginthemid-twentiethcentury,andthatcontributedtothedevelopmentofafaster,cheaperandpredictivenon-animaltestforpregnancy.Aswehavealsopointedoutabove,societaldemandtofillthetoxicsinformationgaphascontributedtoapushtouseinvitrotoxicologytoimproveregulatorytoxicologyandsafetytesting.BothTT21CandthenewTSCAlegislationareevidenceofsocietalresponsetothispressure.Inaddition,cosmeticscompanieshavemovedawayfromusinganimalsforsafetytestinginpartbecauseofconsumerdemandandpressurefrominterestgroups.(Basketter,2010.)Fordiscoveryorbasicresearch,societalpressurestomoveawayfromanimalmodelsexist,buttoalesserextent.IntheUnitedStates,attitudestowardanimalsinresearchareclosetoevenlysplit.(Seehttp://www.pewinternet.org/2015/07/01/chapter-7-opinion-about-the-use-of-animals-in-research/(lastaccessed30October2016).Asmentionedabove,animalmodelsarethede-factowaythatbiomedicalresearchfordiscoveryisdone.Animalmodelsarealsorequiredbyregulationfordevelopingnewdrugs,butalternativesarealsousedinthisprocess,andwithgreaterfrequency.Ourscenarios,especiallythediscussionsoftheTT21Creportandtheprogressoftheinhomepregnancytest,demonstratethatUSconsumerswanttousefeweranimalsinthedevelopmentofconsumerproductsandchemicalstesting.Inordertomeetthisgoal,itwillbenecessarytogetabetterunderstandingofhumanbiologicalmechanisms.

iii. Non-animalbasedtechnologiescanemergebecausetheyrepresentscientificadvancesandarerecognizedasthebestscience.

TheAmestestwascreatedbecauseDr.Amesandhisteamwerelookingforabetterwaytotestformutagens,notbecausetheysoughttodevelopareplacementforananimaltest.Webelievethatmoreandmoreinvitrotests–humanescience–representthebestapproachestoadvancingknowledge.

Insomecases,lawssuchastheEuropeanCosmeticRegulation(Regulation1223/2009,foundathttp://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:02009R1223-20150416&from=EN(lastaccessed23October2016))canadvancetheuseofinvitromethods.18Betterscience,however,isalsoapowerfulandsignificantdrivingforce.Cell,tissueandorganculturesandorgansonachipallowtheuseofhumancellsforbiomedicalscience,thestudyofdiseaseandfortoxicologicalandsafetyassessment.Thereareseveralotherdriversfordevelopinginvitromethods.AsexemplifiedbypregnancytestingandtheconclusionsoftheTT21Creport,invitromethodscanbequicker,lessexpensiveand,whenusinghumancellsmorepredictive.Animaltoxicologystudiesarenotcapableofdevelopingthedatainareasonableamountoftimeoratareasonablecost.Inmanycases,alargeshortcomingofanimalstudiesisalackofpredictability.AtameetingatJohnsHopkinsanumberofyearsagofocusingonthedogasamodelforbiomedicalsciences,manypresentationsindicatedthatthedogisthebestmodelforpreclinicaltestingofdrugsforhumanuse.Asthemeetingdiscussiondevelopeditbecameclearthatthedog,althoughthe“best”modelwasnottrulypredictiveforhumans.Thedatathatwassharedisthatapproximately95%ofdrugsthatweresuccessfulintheanimal(dog)preclinicaltrialsfailedinhumans.TheFDAinthelast15orsoyearsrequiresthatmetabolismstudiesofdrugsbedoneusinginvitrohumanlivercultures.Thefailurerateofdrugssotestedfellto35%.(AlbertLi,PersonalcommunicationwithAMG)AsThomasKuhnsaidwhendescribingscientificrevolutions,“Eachofthemnecessitatedthecommunity’srejectionofonetime-honoredscientifictheoryinfavorofanotherincompatiblewithit.Eachproducedaconsequentshiftintheproblemsavailableforscientificscrutinyandinthestandardsbywhichtheprofessiondeterminedwhatshouldcountasanadmissibleproblemorasalegitimateproblem-solution.”(Kuhn,1970[p.6].)Itishardtopredictwhenthescientificcommunitywillturnthiscorner,butthereareindicationsthatalternativesaregaininggroundinallfourdomains.

4. ShouldtheAWAbeamendedtoembracealternativesinlaboratoryscience?

i. ShouldtheAWAbeamendedtofullyadoptthe3Rs?Asathresholdquestion,westartbyexaminingwhethertheAWAshouldbeamendedtoadoptthe3Rsinfull.Morespecifically,shouldtheAWAbebasedonallthree3Rs,andinparticularbemoreofaforceinencouragingalternatives?Whileitiscorrecttosaythatalmostalllaboratoryanimalwelfareprogramsarebasedonthe3Rs,andthatthe3Rsverymuchinformthepractical18AbillcalledtheHumaneCosmeticsAct(HCA)wasintroducedintheUSHouseofRepresentativesin2014thatissimilarinintenttotheEUCosmeticsRegulation.Oneoftheauthorsofthisarticle(PAL)hasco-writtentwoarticlessupportingtheHCA.Seehttps://www.scientificamerican.com/article/beauty-and-the-beasts-the-u-s-should-ban-testing-cosmetics-on-animals/(lastaccessed22October2016andhttps://issuu.com/aavs/docs/aavs_av-magazine_2016_cruelty-free-(page7)(lastaccessed22October2016).

decisionsinlaboratoryanimalwelfareandprograms,ascurrentlywritten,aswepointedoutearlier,theAWAisatbesta1R(refinement)law.IftheAWAwereamendedtoincorporatethe3Rs,itwouldbenefitlaboratoryanimalresearchinseveralways.First,itwouldmaketheAWAconsistentwithmodern,humane,internationallyacceptedlaboratoryanimalprinciples.ItcouldbearguedthatformostUSlaboratories,thisamendmentwouldhavelittleoperationaleffect.TheselaboratoriesarelikelycertifiedbyAAALACInternational,and/orregulatedbyotherorganizationsthatrequire3Rscompliance.Asonerecentlawreviewarticlepointsout,astrongercommitmenttothe3Rscouldincreasetherecognitionanduseofvalidatedalternativemethods.(Lee,2016.)Second,itmightacttoraisethestandardsofthoseanimalresearchlaboratoriesthatarenotapplyingthe3Rs,eitherbecausetheyareverysmall,ortheyarenotrequiredbytheirfundingagenciestocomplywiththe3Rs.Third,itcouldbeseenasawaytoencouragegoodscientificpractice.The3Rsareaffirmativedutiesthatresearchersundertakebecausehumanetreatmentofanimalsisuniversallyacknowledgedtoimprovescience.(NAS,2011.)Incontrast,thefullincorporationofthe3RsintotheAWAmightbeseenasunnecessaryorilladvised.TheAWAcontainsaclearstatementthatregulationismeanttostopatthelaboratorydoor,andanAWAamendmentmakingtheActafull3Rslawcouldbeinterpretedasanassaultonthisprinciple,anopeningofthelaboratorytoregulatoryagencies.Whenproperlyapplied,the3Rsdoseekaffirmativelytoprobeaboutresearchquestiondesign,choiceofspecies,number(s)ofanimalsused,andotherfeaturesoftheexperimentalprotocol.Furthermore,itcouldbearguedthatfullincorporationofthe3Rswouldraisecoststocarryourresearchatinstitutions.(Thulin,2014;Haywood,2008.)Makingdiscoveryresearchmoreexpensivebyaddingadministrativeburdensmightbeseenasawasteofgovernmentalresources,becausesomuchdiscoveryworkisfundedbytheNationalInstitutesofHealth.

ii. ShouldtheAWAbeamendedtobean“alternativesfirst”law?ItisalsoimportanttoexplorewhethertheAWAshouldbeamendedtofavorthealternativesR.Inotherwords,iftheAWAwereamendedsothatitdidincorporateallofthe3Rs,shouldtheAWAalsobeamendedsothatitactuallyrequiredprincipalinvestigatorstoaffirmativelyseekoutexisting,and/ordevelopnew,experimentalprotocolsthatusednon-animalmethodsbeforeanylaboratoryanimalresearchtookplace?IftheAWAwereamendedtomakeitanalternatives-firstlaw,theuseofanimalsinsciencewouldalmostcertainlydecrease,andanimaladvocatesmightseethatasanadvantagetosuchanamendment.Anotherpossibleadvantagemightbethatanalternatives-firstprovisionwouldsparkinnovation,andacceleratethedevelopmentandutilizationofnon-animaltestmethods.Suchaprovisionwouldfallmostheavilyonscientistspursuingbasicordiscoveryresearch,becauseinthisdomainthedefaultpositionisthecreationanduseofananimalmodeltostudycomplexdiseasesandconditions.Whileanimalmodelsarefarfromperfect,and(astheThalidomidescenariodemonstrates)canmissimportanteffects,intheopinionofmostbasicresearchersthebenefitsofanimalmodelsoutweightheirburdens.

Forthoselaboratoriesinvolvedinsafetyand/ortoxicitytesting,suchachangewouldraisesome,butfewerbarriers.19Thesescientistsarelikelyusingalternativesalreadybecausechemicalsandcosmeticstestinginvolvessimplerendpointsandmoreinvitrotestsarealreadyavailableforuse.ThisprovisionwouldalsobeconsistentwiththeHumaneCosmeticsAct,recentlyintroducedintheUSHouseofRepresentatives,whichwouldbananimaltestingonfinishedcosmeticsproductsandallcomponents.(Moran,2014.)

AmendingtheAWAinthiswaycouldbringitintoconflictwithotherfederallaws,suchastheFederalFood,DrugandCosmeticsAct(FFDCA),whichrequirescertainanimalteststobeperformedbeforetheFDAwillconsiderapotentialdrugmoleculeforhumanclinicaltrials.Seehttp://www.fda.gov/AboutFDA/Transparency/Basics/ucm194932.htm(lastaccessed30October2016;DiSPirito,2015)andtheFederalInsecticide,FungicideandRodenticideAct(FIFRA)(EPA,2002.)However,suchconflictscouldbeaddressedbystatutorydraftingthatexcludedtheseregulatorytestingrequirements.Moresignificantly,analternatives-firstamendmentwouldlikelyincreasecostsassociatedwithresearchanddelayon-goingorlongstandingworkcenteredaroundanimalmodels.

iii. ProposedamendmentsinlightoftheISLAA’sCongressionalfindingsTheISLAAsetoutfourCongressionalfindings:“(1)theuseofanimalsisinstrumentalincertainresearchandeducationforadvancingknowledgeofcuresandtreatmentfordiseasesandinjurieswhichafflictbothhumansandanimals;(2)methodsoftestingthatdonotuseanimalsarebeingandcontinuetobedevelopedwhicharefaster,lessexpensive,andmoreaccuratethantraditionalanimalexperimentsforsomepurposesandfurtheropportunitiesexistforthedevelopmentofthesemethodsoftesting;(3)measureswhicheliminateorminimizetheunnecessaryduplicationofexperimentsonanimalscanresultinmoreproductiveuseofFederalfunds;and(4)measureswhichhelpmeetthepublicconcernforlaboratoryanimalcareandtreatmentareimportantinassuringthatresearchwillcontinuetoprogress.”(Emphasisadded.)(PL99-198,subtitleF(§§ 1751–1759),§ 1751,Dec.23,1985,99Stat.1645)(foundat99Stat.1645(lastaccessed30October2016).)Theadoptionofamendmentstofullyincorporatethe3RsintotheAWAwould,onbalance,beconsistentwithISLAA’sCongressionalfindings.Ifappropriatelyimplementedafull3Rsstrategywouldrecognizethatcertainresearch(especiallybasic/discoveryresearch)stillrequiresanimal

19Oneoftheco-authorsofthispaper(PAL)advocatedforanalternatives-firstapproachtoTSCAtoxicitytesting.(Locke,2011)

models(finding1).Atthesametime,agreateremphasisonreductionandreplacementwouldservefinding2,especiallyinthedevelopmentofmethodsfortoxicityandsafetytesting.Minimizingduplicationwouldbepartiallyaccomplishedbycloseradherencetoreduction,althoughreductioneffortsaredirectlylargelyatkeepingthenumberofanimalsinanyoneexperimenttoaminimumwhilemaintainingthescientificvalueoftheresearch.Finally,publicconcernforlaboratoryanimalswouldbesupportedbecausethe3Rs,andareplacement-firststrategy,couldreducethenumberofanimalsthatareusedoverall.Animaladvocateshavepromotedbothcitizensuitprovisions,andstandingforanimals,asamendmentstotheAWA(orotherfederallaws)tobetterpoliceAWAenforcementandlaboratoryanimalwelfare,andasameasuretohelpaddresspublicconcernforlaboratoryanimaltreatment(finding4).(Frasch,2016(inpress);Swanson,2002.)Thesesuggestionshavebeencounteredbypointingoutthatanimalresearchisalreadyheavilyscrutinized,andthecostsassociatedwithanimalresearchoversightrepresentasignificantburden.(Thulin,2014;Haywood,2008.)Thisdebateislikelytocontinue.

iv. OtherpossibleAWAamendmentsInadditiontoexplicitlyincorporatingthe3RsintotheAWAaspartofitspolicystatements,andmakingtheAWAareplacementfirstlaw,thereareotheramendmentsthatcouldstrengthentheAWA’scommitmenttothefourCongressionalfindingssetoutintheILSAA.Forexample,theAWAcouldrequirethatfacilities,throughtheirIACUCs,keepa“3Rsscorecard.”Thisscorecardcouldtrack,intermsofanimaluse,thenumberofanimalstowhichrefinementwasapplied;thenumberofanimalsnotusedbecausereductionmeasureswereputintoplace;andthenumberofanimalsreplacedbynon-animalalternatives.Inaddition,theAWAcouldrequirethatthesereportsaremadeavailabletomembersofthepublic.TheIACUCprovisionsoftheAWAcouldalsobechanged.TheISLAAamendmentstotheAWAestablishedtheseinternalcommitteesatfacilitiestooverseeanimaluseinresearch.TheAWArequirestheappointmentofanindividualtotheIACUCwhocanrepresentsocietalinterests.Thisprovisioncouldbemademoreeffectiveinbringingthepublic’svoiceintoresearchfacilitiesandlaboratoryanimalwelfaredecision-making.Inasystemofself-regulationandoversightsuchastheoneestablishedpursuanttotheAWA,itisimperativethatthispublicrepresentativebeeffectiveandinformed.Trainingisanotherareawhereimprovementscouldbemade.Asitiscurrentlywrittenandimplemented,thetrainingrequiredundertheAWAislefttoeachfacilityorinstitution.Perhapstrainingshouldbestandardized,andtheAWAshouldbeamendedtoestablishanationaleducationalbodywhosemissionistotrainlaboratoryanimalresearchers.Eachresearcherandlaboratoryworkerwouldberequiredtotakeastandardizedcurriculum,andthatcurriculumcouldbefocusedonthefourCongressionalfindingsintheISLAA.Finally,itisimportanttoconsideranamendmenttotheAWAthatwouldchangethedefinitionof“animal”sothatitdoesnotexcluderats,miceandbirdsbredforresearchpurposes.Forthe

protectionsandbenefitstolaboratoryanimalwelfareandscientificresearchtobefullyrealized,itwouldseemobviousthatthebulkofanimalsusedinresearchshouldnotbeexcludedbythisdefinitionfromtheAWA.ChangingthedefinitionofanimalinthiswaywouldmaketheAWAmoreconsistentwithotherfederalrequirementsthatgovernlaboratoryanimalresearch(Seehttp://grants.nih.gov/grants/olaw/references/phspolicylabanimals.pdf(lastaccessed30October2016),settingouttherequirementsforanimalresearchforrecipientsoffundsfromthePublicHealthService,includingadefinitionof“animal”thatdoesnotexcluderats,miceandbirdsbredforresearchpurposes;seealsotheGuide,whichdefinesananimalas“asanyvertebrateanimal(i.e.,traditionallaboratoryanimals,agriculturalanimals,wildlife,andaquaticspecies)producedfororusedinresearch,testing,orteaching.")

REFERENCES

1. Alépée,N,Bahinski,A,Daneshian,M,DeWever,B,Fritsche,EGoldberg,A,

Hansmann,J,Hartung,T,Haycock,J,Hogberg,HTHoelting,L,Kelm,JM,Kadereit,S,McVey,E,Landsiedel,R,Leist,M,Lübberstedt,M,Noor,F,Pellevoisin,C,Petersohn,DPfannenbecker,U,Reisinger,K,Ramirez,T,Rothen-Rutishauser,B,Schäfer-Korting,MZeilinger,KandZurich,M-GState-of-the-artof3Dcultures(organs-on-a-chip)insafetytestingandpathophysiology.ALEX31(4):441-77.2014

2. Ames,BN,Durston,WE;Yamasaki,EandLee,FD.CarcinogensareMutagens:A

SimpleTestSystemCombiningLiverHomogenatesforActivationandBacteriaforDetection.PNAS.70(8):2281–5.(1973).

3. AnimalWelfareAct,7USC§§2131–2159.4. AnimalWelfareInstitute.InformationReport:TheOverholtClinicCase.May-June

1958.5. AnimalWelfareInstitute.AnimalsandTheirLegalRights:ASurveyofAmerican

Lawsfrom1641to1990(4thEdition).Washington,DC:AWI(1990).6. Bahinski,A,Horland,R,Huh,D,Mumery,C,Tagle,DandMacGill,T.ThePromise

andPotentialof“Organs-on-Chips”asPreclinicalModels.AppliedInVitroToxicology1(4)235(2015).

7. Basketter,D,Clewell,H,Kimber,I,Rossi,A,Blaauboer,B,Burrier,R,Daneshian,M,

Eskes,C,Goldberg,A,Hasiwa,N,Hoffmann,S,Jaworska,J,Knudsen,T,Landsiedel,R,Leist,M,Locke,P,Maxwell,G,McKim,J,McVey,E,Ouédraogo,G,Patlewicz,G,Pelkonen,O,Roggen,E,Rovida,C,Ruhdel,I,Schwarz,M,Schepky,A,Schoeters,G,Skinner,N,Trentz,K,Turner,M,Vanparys,GP,Yager,J,Zurlo,JandHartung,T.ARoadmapfortheDevelopmentofAlternative(Non-Animal)MethodsforSystemicToxicityTesting.ALTEX29:1–91.January2012.

8. Bielecka,Z,Maliszewska-Olejniczak,K,Safir,IJ,Szczylik,C,andCzarnecka,AMThree-

dimensionalcellculturemodelutilizationincancerstemcellresearch.BiologicalReviews2016.

9. Brent,R.andHolmes,LB.Clinicalandbasicsciencelessonsfromthethalidomide

tragedy:whathavewelearnedaboutthecausesoflimbdefects?Teratology.38(3):241-51.Sept.1988.

10. Cohen,H.TheAnimalWelfareAct.2JournalofAnimalLaw13.2006.

11. Cole,LA.ThehCGassayorpregnancytest.ClinicalChemistryandLaboratoryMedicineJournal.50(4):617–630.2012.

12. CommitteeonLegalIssuesPertainingtoAnimals.ReportoftheCommitteeonLegal

IssuesPertainingtoAnimalsoftheAssociationoftheBaroftheCityofNewYorkRegardingitsRecommendationtoAmendtheAnimalWelfareAct.9AnimalLaw345(2003).

13. Cowan,T.TheAnimalWelfareAct:BackgroundandSelectedAnimalWelfare

Legislation.CongressionalResearchService,5January201614. DiSpirito,NP,Hall,RF,Hill,MJ.BringingYourPharmaceuticalDrugtoMarket.FDLI:

Washington,DC.2015.15. Dukes,E.TheImprovedStandardsforLaboratoryAnimalsAct:WillitEnsurethat

thePolicyoftheAnimalWelfareActBecomesaReality?31St.LouisLawJournal519(1986)

16. Engber,D.PepperGoestoWashington.Slate.3June2009.17. Engelfried,JJHawkinsonGandGalandey,JF.ThereliabilityoftheAschheim-Zondek

testinpregnancy;areportoftwothousandroutinetests.JournalofClinicalEndocrinologicalMetabolism5:419,1945.

18. EnvironmentalProtectionAgency,HealthEffectsTestGuidelines(OPPTS870.1000)

AcuteToxicityTesting—Background.December2002.19. EnvironmentalDefenseFund.ToxicIgnorance:TheContinuingAbsenceofBasic

HealthTestingforBestSellingChemicalsintheUnitedStates.199720. Farmer,PB,Walker,JM.TheMolecularBasisofCancer.KriegerPublishingCompany.

2006.21. Frasch,P.GapsinUSAnimalWelfareLawforLaboratoryAnimals.ILARJournal:

57(3):___[publicationinprogress]2016.22. Goldberg,AM,Brookes,NandBurt,DR.TheUseofSpinalCordCellCulturesinthe

StudyofNeurotoxicologicalAgents.Toxicology,17:233-235,1980.23. HaywoodJR.andGreeneM.Avoidinganoverzealousapproach:Aperspectiveon

regulatoryburden.ILAR49:4426-429.2008.24. Ibrahim,D.Reduce,Refine,Replace:TheFailureofthe3RsandtheFutureof

AnimalExperimentation.2006UniversityofChicagoLegalForum195.

25. Kim,JHandScialli,AR.Thalidomide:TheTragedyofBirthDefectsandtheEffective

TreatmentofDisease.ToxicologicalSciences122(1),1–6.2011.26. Kreger,MD,Jensen,DJB,Allen,T.AnimalWelfareAct1966-1996:Historical

PerspectiveandFutureDirections(SymposiumProceedings).Beltsville,MD.199827. Kuhn,TS.TheStructureofScientificRevolutions(2ndEdition,Enlarged).

InternationalEncyclopediaofUnifiedScience,TheUniversityofChicagoPress.1970.28. Lee,CG.TheAnimalWelfareActatFifty:ProblemsandPossibilitiesInAnimal

TestingRegulation.95NebraskaLawReview194.2006.29. Locke,PAandMyers,DB.Areplacement-firstapproachtotoxicitytestingis

necessarytosuccessfullyreauthorizeTSCA.ALTEX28(4),266-272.April2011.30. Moran,JandLocke,P.BeautyandtheBeasts:U.S.ShouldBanTestingCosmeticson

Animals.ScientificAmericanForum.28May2014.(availableathttps://www.scientificamerican.com/article/beauty-and-the-beasts-the-u-s-should-ban-testing-cosmetics-on-animals/(lastaccessed30October2016)).

31. NationalAcademyofSciences,NationalResearchCouncil.Science,Medicineand

Animals.Washington,DC.NationalAcademiesPress.1991.32. NationalAcademyofSciences,NationalResearchCouncil.GuidefortheCareand

UseofLaboratoryAnimals(8thEdition).Washington,DC.NationalAcademiesPress.2011.

33. NationalAcademyofSciences,NationalResearchCouncil.ToxicityTestinginthe

Twenty-firstCentury:AVisionandaStrategy.Washington,DC.NationalAcademiesPress,2007.

34. PL91-579.91stCongress,__Sess.,__Stat.__.24December1970.35. PL99-198,99thCong.,___Sess.,99Stat1354.23December1985.36. Ruediger,EH.TheAscheim-ZondekTestForPregnancy.CaliforniaandWestern

Medicine44(3):157-59.March1936.37. Russell,WMSandBurch,RL.ThePrinciplesofHumaneExperimentalTechnique.

SpecialEdition:1992(firstpublishedbyMethuen&Co.,London,1959)

38. SunitaJ.,Shukla,SJ,HuangR,Austin,CPandXia,M.TheFutureofToxicityTesting:AFocusonInVitroMethodsUsingaQuantitativeHighThroughputScreeningPlatform.DrugDiscoveryToday15(23-24):997–1007.December2010.

39. Swanson,K.CarteBlancheforCruelty:TheNon-EnforcementoftheAnimalWelfare

Act.35UniversityofMichiganJournalofLawReform937.Summer2002.40. Taubv.StateofMaryland.296Md.439(Md.1983).41. Tannenbaum,JandTaylor,B.RussellandBurch’s3RsThenandNow:TheNeedfor

ClarityinDefinitionandPurpose.JournaloftheAmericanAssociationofLaboratoryAnimalScience.54(2)120(2015).

42. Thulin,JD,Bradfield,Jf,Bergdall,VK,Conour,LA,Grady,AW,Hickman,DL,Norton,

JNandWall,JM.Thecostofself-imposedregulatoryburdeninanimalresearch.TheFASEBJournal28(8):3297–3300.August2014.

43. Wildhelmus,KR.TheDraizeEyeTest.SurveyofOpthamology45(6):493–515.

May-June2001.44. Zurlo,J,Rudacille,DandGoldberg,A.AnimalsandAlternativesinTesting:History,

ScienceandEthics.NewYork:MaryAnnLiebertPublishers,1994.

non-animal alternatives for research and testing...

Documents

petition to inclxde cephalopods as animals´ deserving of...

geosciences testing and research inc

bioscience research hiv testing

research on tensile testing of materials

research reports - educational testing service

electrical research and testing laboratory nuremberg

cavitation testing methods and previous research

patient self testing monitor name research

microbiology laboratory services guide · antimicrobial...

high voltage testing and research centre

research on internet recruiting and testing:

testing in scala by adform research

siemens.com/energy/transformers electrical research and...

research on testing & fp7 bastion

rubber research institute testing fee

hypothesis testing research methodology

research projects in software testing

preservation research and testing the science of...

being agile: ux research & usability testing€¦ · being...

research and testing