ILSI Health andEnvironmental SciencesInstituteEPA Difficult to TestSubstances WorkshopAlternative methods for EcotoxicityTesting11 September 2014Michelle EmbryHESIAgenda for this talk•Context•The ‘big picture’•Fish Embryo Test (FET)•Overview of several other new methodsunder development•Eco-TTC (non-test method)•ConclusionsContext for Alternatives DevelopmentRegulatory driversCosmetics DirectiveAnimal Protection DirectiveREAChTox21 (regulatory and scientific)OECD Fish Testing FrameworkScientific driversMechanistic ecotoxicologyAOPsEndocrine disruptionModeling mammalian toxicitySocietal driversAnimal welfare pressure (3R’s approach)Increased fish use as an alt to higher vertebratesReflected in regulatory and scientific worldsRegulatoryNeedsScientificNeedsSocietalNeedsNeed for Balance•Science has pushed the development of thesemethods:•A call to use new, more approaches in lieu ofolder, less informative test procedures (e.g.,AOPs)•A call to optimize resource use•Societal pressures have led industry to developalternative methods:•A call to reduce animal use•Regulatory acceptance / implementation is key•Global commerce underscores the need forharmonization (e.g., OECD MAD)•Issues arise when there are legal differences;what happens when methods are notaccepted in certain jurisdictions? (e.g., issueswith Cosmetics Directive, REACh, etc.)RegulatoryScientificSocietalWhy Alternatives Work is ImportantFrom**: van der Jagt , Munn, Tørsløv & de Bruijn. 2004.**Alternative approaches can reduce the use of test animals under REACH.0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00%Two generation reprotoxDevelopm. tox screeningLong-term fishDevelopm. tox studyCarcinogenicitySkin sensitisationShort term repeated doseFurther mutagenicityShort term fishBioAccumulation (fish)Long-term repeated toxSubchronic toxAcute oral toxAcute inhalation toxIn vivo eye irritationAcute dermal toxIn vivo skin irritationLong-term birdEstimated animal need in REACH for required endpoints(% total test animals needed)Additional potential rolesfor fish testingMissing: EDC testingMovement Towards IATA•IATA: Integrated Approaches toTesting and Assessment•OECD Fish Testing FW Projectan example•General push towardsconsideration of pathwaybased approaches, integrationof new technologies asnecessary and whenappropriate**http://www.oecd.org/officialdocuments/publicdisplay documentpdf/?c** **ote=ENV/JM/MONO(2012)16&doclanguage=en**Adverse Outcome Pathways (AOPs)ChemicalPropertiesReceptor/LigandInteractionDNA BindingProtein OxidationGeneActivationProteinProductionAlteredSignalingProteinDepletionAlteredPhysiologyDisruptedHomeostasisAltered TissueDevelopmentor FunctionLethalityImpairedDevelopmentImpairedReproductionCancerToxicantMacroMolecularInteractionsCellularResponsesOrganResponsesOrganismResponsesStructureRecruitmentExtinctionPopulationResponsesFish Life Stages and TestingOECD 236 TGFET(Fish Embryo test)OECD 203 TG4 dZF 2d 2dCompliant with Dir.2010/63/EU definition of animalsusedfor scientific purposesEgg(unfertilisedOocyte)EmbryoHatchingFertilisationSperm entrythrough themicropyleEMBRYONIC PERIODLARVA ADULTFree swimmingand feeding larvaMetamorphosis (differentiation of thedorsal and caudal fins)SecondarysexualcharacteristicsScaled bodyENDOGENEOUS FEEDING(autotrophic)EXOGENEOUS FEEDING(heterotrophic)resorption of yolk sacJUVENILEEleutheroembryo“ Sac fry ”Yolk sacFemaleMaleOECD 210 TGZF 32dEndpoints : lethality, growth, malformationsEndpoint : lethalityEndpoint : lethalityFish Embryo Test (FET)Alternative method to the 96h acute fish toxicity test (OECD TG 203)2005:•German Federal Environment Agency (UBA) submitted the draft test guideline“Fish embryo toxicity (FET) test” to the OECD TestGuideline Program•Used in Germany as a replacement for 48h fish effluent testing2006:•OECD established the ad hoc Expert Group on the FET.•Following expert meetings, OECD decided to performa validation studycoordinated by EURL ECVAM and steered by a validation management group(VMG)2009 – 2011:•The validation study aimed to assess the transferability, intra- and interlaboratoryreproducibility of the zebrafish embryo toxicity test (ZFET) as well as expand thedomain of chemicals tested2013•Validation study reviewed and endorsed by ECVAM inaddition to the relationshipto acute fish toxicity, approved at EU level for implementation•FET became OECD TG 236Fish Embryo Test (FET)Parallel to the line of 1:1correspondenceWide range of potencies, largedatabase**(Currently ~300 chemicals with different MOA and physico** **chemical properties).**In practical terms:•0.1 mg/L FET = 0.07 mg/Lfish•1 mg/L FET = 0.7 mg/L fish•10 mg/L FET = 6.2 mg/L fish**Belanger et al. 2013. Environ. Toxicol. Chem. 32: 1 768-83** Fish Embryo Test (FET): Summary•Correlation between FET and acute fish test is better than acutefish tests between species•Little difference between zebrafish and fathead minnow withchemicals tested thus-far•Already being submitted for registrations / dossiers•Use of OECD TG 236 data has not translated to acceptance in allregulatory jurisdictions•Potential applications beyond acute testing...Fish Embryo Test (FET) – chronic toxicity•Work initiated by HESI to explore application /optimization of the FET to predict chronic toxicity•Focus on OECD 210 TG (Fish Early Life Stage Test);mostfrequently used bioassay to predict chronic toxicity•Ongoing work to discover, characterize, and annotate FELSAOPs and prioritize their development (CEFIC LRI, others)•Ultimate goal is to identify alternative test methods topredict chronic toxicity in fish**Villeneuve et al. 2014. Environ. Toxicol. Chem. 33: 158-169** Fish Embryo Test (FET) – other applicationsEDC testing•Development of alternatives for in vivo EDC tests –emerging need•In-line with current pathway-based efforts (AOPs)•Review paper on existing alternative methodologies(for fish andamphibians)•HESI committee is currently developing criteria forselection of referencechemicals to use in validating / developing new methodologies.**Scholz et al. 2013. Crit Rev in Toxicol 43: 45-72**Effluent testing•Effluent testing is the single largest source of use of aquatic vertebratesglobally for a regulatory purpose and is a subjectseparate from chemicalregulation•FET has been used in Germany for effluent testing since 2005•How might alternative approached be utilized for effluent testing? IATA,FET, etc.Bioaccumulation•Increase in regulatory needs for bioaccumulation assessment,e.g. UNEP Stockholm Convention on POP chemicals (2004);Canadian Domestic Substance List (2006); EU REACH (2006)•Several strategies* for bioaccumulation testing (orwaiving) havebeen published taking into account:•Existing information•Non-testing methods•Testing methods*Examples:ECETOC report (de Wolf et al, 2007)HESI in vitro workshop (Weisbrod et al, 2009)REACH guidance (ECHA 2008, 2012)OSIRIS project (Lombardo et al 2014)Bioaccumulation: in vitro metabolismLoss viaegestion(kE)Loss viagills(k2)Loss viametabolism(kMET)Uptake viagills(k1)Uptake viafoodAccumulation of a chemical in anorganism is the result of absorption,distribution, metabolism, and excretion(ADME).•BCF models based on log KOWare used toscreen chemicals for potentialbioaccumulative properties: Do notaccount for “M” (i.e., biotransformation)!•Some QSAR models predict metabolismrates based on chemical structure (incl. inBCF/BAFTM programme of US EPA Episuite)•Only waterborne uptake•BCF = k1/ (k2+ kE+ kMET)In vitro methods?In vitro Metabolism Ring-Trial•Further evaluate in vitro methods (trout S9 and hepatocytes) used to assesschemical biotransformation in fish in order to support OECD TG development.•As of April 2014, the HESI ring trial is part of the official OECD Test GuidelinesProgramme Workplan [U.S. and European Commission areleads]•Aims:• Compare in vitro assay performance across participating laboratories• Compare predicted BCF values using the in vitro data to in vivomeasured BCF values• Provide recommendations for future use of these methods inbioaccumulation assessments• Provide data that can be used to validate modeled bioaccumulationpredictions• Provide information required to develop an OECD TGfor these methodsBioaccumulation: in vitro metabolismREACH endpoint specific guidance – Bioaccumulation (ECHA, 2012)“In vitro methods have the potential to provide important data onbioaccumulation assessments...These methods may become an importantpart of future test strategies, but their applicability is currently limited dueto thelack of standardised protocols, limited validationbased on smalldata sets. Further evaluation work is necessary before they can berecommended for use within an ITS.”Gill Cell Line Ring TrialRationale:•Gill epithelia is the primary target and uptake site of water contaminants•Damage to gill epithelia may reflect impaired functioning of the organismand eventually death•Cells tolerate exposure to simple buffersallows direct analysis ofenvironmental samples•Ongoing round-robin test of the RTgill-W1 cell lineassay to predict fishacute toxicity – led by Eawag (7 labs and 6 test chemicals)•Follow-up to the CEFIC-LRI CellSens ProjectNon-testing Approaches: eco-TTC Development**Threshold of Toxicological Concern (TTC)**•An approach for establishing an exposure level forchemicals, below which no appreciable risk tohuman health and/or the environment is expected•TTC proposes that a**de minimis**value for toxicitycan be identified for many chemicals, includingthose of unknown toxicity•TTC was originally applied to assess chemicals infood:•Flavorings•Food contact materials•Pharmaceutical impuritiesEnvironmental TTC Approaches (eco-TTC)•De Wolf et al. 2005. ET&C 24:479•Compiled aquatic toxicity data from:•ECETOC EAT database•USEPA fathead minnow database•EU Existing substances database•Utrecht guppy database•Binned by Mode of Action (MoA)•PNECs derived using EU procedures /Application Factors•Eco-TTC of 0.1 μg/L for Verhaar MoA 1-3**See also: Gross et al. (2010); Williams et al. (20 11) for additional eco-TTC like approaches** Benefits of an eco-TTC•Maximizes resource use (animals, time, $$)•Allows screening-level assessment forchemicals with little or no toxicity data•Groups without a QSAR (or no hope for one)polymers? UVCBs*?•New chemistries•Difficult to test substances (?)•Particularly useful for screening assessmentwhen production is low•Supports read-across***Unknown or variable composition, complex reaction p roducts, biological materials** HESI eco-TTC Project•Led by the HESI Animal Alternatives in EnvironmentalRisk Assessment Committee•Scoping meeting held Feb 2014; multi-sectorparticipation•General strategy to explore this approach in a step-wise manner developed•Initial analysis is ongoing, utilizing readily-availabledatabases (D. deZwart, RIVM) and industry datawhen made available•Strong interest from multiple sectors / groups•Exploring additional data sources and partnersOverall Conclusions•Several new, alternative eco test methods availableor indevelopment•Integration into an IATA approach is necessary•Stakeholder involvement early and often is key toapplication and adoption of methods•Must work together to overcome barriers to developmentand use of alternative methods**See: Braunbeck et al., “Bottlenecks in the developm ent and** **implementation of alternative experimental test met hods in** **ecotoxicology” ALTEX, in press.**Questions?membry@hesiglobal.org