SUMMARY & NEXT STEPSANNA LOWIT, PH. D.SENIOR SCIENTISTUSEPA, OFFICE OF PESTICIDE PROGRAMSApril 10, 2012FIFRA SAPOverview2Follow text in Section 6.0 of the 2012 SAPissue paperSummarize key points from previouspresentationsDiscuss weight of the evidence conceptKey science questionsQualitative & quantitative considerationsHighlight aspects of the charge questionsNRC ReportsNRC ReportsToxicity Testing in the 21st Century: A Vision and aStrategy (2007):Adverse outcome pathwaysIntegration across multiple lines of evidenceDisruption of homeostasis leading to adverse outcomesScience & Decisions (2009):Improved technical analysis (including mode of action)Evaluation of alternative interpretations/approachesBetter align risk assessment & risk managementUse of improved problem formulation3Stepwise, Transparent Science Review42008, FIFRA SAP: Preliminary review ofchlorpyrifos human health effects withfocus on infants, children & pregnantwomen2010, FIFRA SAP: Draft Framework forIncorporating Epidemiologic and HumanIncidence Data in Human Health RiskAssessmentStepwise, Transparent Science Review52011, Preliminary risk assessment on chlorpyrifos:Focus on AChE/ChE inhibition as the most sensitive doseresponse data for deriving points of departurePlan to conduct a full weight of evidence (WOE) evaluationintegrating the epidemiology studies with the experimentaltoxicology studies for the neurodevelopmental outcomes2012, FIFRA SAP: New data & more in-depth analysissince 2008Charge questions on strengths & uncertainties of differentlines of evidenceUncertainty analysis: qualitative & quantitativeNext step: Agency will consider input from SAP inintegrating data & developing the WOE2007NRCToxicityTestinginthe21stCenturyCompoundsAssessBiologicalPerturbationAffectedPathwayMeasures ofdose in vitroDose ResponseAnalysis forPerturbationsof ToxicityPathwaysCalibratingin vitro and humandosimetryHuman ExposureDataPopulation BasedStudiesExposureGuideline



# Mode of ActionChemicalCharacterizationDose ResponseAssessmentHazard CharacterizationRisk CharacterizationExposure AssessmentNRC, 2007. Fig 3-7 Risk Assessment ComponentsMetabolite(s)ChlorpyrifosAssessBiologicalPerturbationMeasures ofdose in vitroDose ResponseAnalysis forPerturbationsof ToxicityPathwaysCalibratingin vitro and humandosimetryUrinary biomonitoring ofTCPy in variouspopulationsChildren’s CohortsExposurePathways:Food, water,worker,air volatility,spray drift,cross-placental



# Mode of ActionChemicalCharacterizationDose ResponseAssessmentHazard CharacterizationRisk CharacterizationExposure AssessmentNRC, 2007. Fig 3-7 Risk Assessment ComponentsOxon, TCPyAffectedPathway•AChE inhibition•Plausible hypotheses forneurodevelopmental effectsWeight of Evidence (WoE)Process for characterizing the extent to which theavailable data support a hypothesis that an agentcauses a particular effectInforms Dose-Response AssessmentInvolves a number of stepsStarting with assembling the relevant dataEvaluating that data for quality and relevanceFollowed by an integration of the different lines ofevidence to support conclusionsWeight of Evidence (WoE)Transparency is importantexplains the kinds of data availablehow the data were selected and evaluatedhow the different lines of evidence fit together indrawing conclusionssignificant issues, strengths, and limitations of the dataand the uncertainties that deserve serious considerationare presentedmajor points of interpretation highlightedCharacterization of a range of probabilities andput in perspective (confidence/uncertainty)Outline of Presentations10Introduction (Anna Lowit)Adverse Outcome Pathways: Data for Chlorpyrifosat Varying Levels of Biological Organization(William Mundy)Chlorpyrifos Effects on the Developing Brain:Animal Studies (Virginia Moser)Review of Children’s Health Epidemiology CohortStudies (Carol Christensen)Review of Chlorpyrifos Biomonitoring Researchand Interpretive Approaches (Aaron Niman)Summary & Next Steps (Anna Lowit)Summary112011 Preliminary Risk AssessmentAChE/ChE inhibition remains the most sensitive doseresponse data for deriving points of departureConsistent with 2008 SAP recommendationsRoute & lifestage specificRobust BMD analysisAcute, oral: BMDL100.36 mg/kg for RBC AChE inhibition inPND11 ratsChronic, oral: BMDL100.03 mg/kg/day for RBC AChEinhibition from pregnant ratsSummary12Compared to 2008, 2012 analysis incorporatesnew data available and/or provides more in-depthanalysisKey scientific questionsStrength/limitations of evidence that suggestchlorpyrifos exposure results in long-termneurodevelopmental effects from early life exposureStrength/limitations of evidence that adverse effectsmay be attributed to doses lower than those which elicit10% inhibition of AChE, i.e., the dose levels used forregulatory decision makingSummary-Qualitative lines of evidence: Long-termneurodevelopmental effects from early life exposure13Strength/limitations of evidence that suggestchlorpyrifos exposure results in long-termneurodevelopmental effects from early lifeexposure-Mechanistic studiesIn vivoexperimental toxicology studiesEpidemiologySummary - Qualitative lines of evidence: Long-termneurodevelopmental effects from early life exposure14Charge Question 2.1Mechanistic studiesAt this time, a mode of action/adverse outcomepathway has not been established forneurodevelopmental outcomesGrowing body of literature does demonstrate thatchlorpyrifos and/or its oxon are biologically activeon processes that affect the developing brainSummary -Qualitative lines of evidence: Long-termneurodevelopmental effects from early life exposure15Charge Question 3.1In vivoexperimental toxicology studiesMultiple studies and laboratories reporting changes inaspects of neurobehavioral function in adult rodentsfollowing developmental exposureEffects seen long after chemical exposure and any ChEinhibition, if present, had recoveredBehavioral alterations show some consistency in termsof broad domains of neurological functionMost consistent effects on cognition, anxiety, social behaviorSummary - Qualitative lines of evidence: Long-termneurodevelopmental effects from early life exposure16Charge Question 4.1 & 4.2Epidemiology studiesCurrent database strengthens conclusion from 2008“chlorpyrifos likely plays a role” in the neurodevelopmentaloutcomes specifically those reported by Columbia UniversityStrength of observed associations high in some cases, but not allNon-differential exposure measurement error could reduce magnitudeSome evidence of exposure-response in studies, when availableConsistency in Brazelton (abnormal reflexes), ADHD-like behaviorproblems, Bayley (particularly MDI), and IQSignificant given differing exposure measuresAlternative explanations investigated by authorsSummary17From a qualitative perspective---Agencypreliminarily concludes:Lines of evidence together support a conclusion thatexposure to chlorpyrifos results in adverseneurodevelopmental outcomes in humans, at leastunder some (still unclear) conditionsMechanistic studiesIn vivoexperimental toxicology studiesEpidemiologySummary18Key science questionStrength/limitations of evidence that adverseeffects may be attributed to doses lower than thosewhich elicit 10% inhibition of AChE, i.e., the doselevels previously used for regulatory decisionmakingQuantitative considerationsMechanistic studiesIn vivoexperimental toxicology studiesEpidemiologySummary - Quantitative Lines of Evidence: Potential forEffects < Doses Eliciting 10% AChE Inhibition19Charge Question 2.2Mechanistic studiesMultiple biologically plausible hypotheses are beingpursued by researchers; no one pathway has sufficientdata to be considered more credible than the others.Cannot establish key events, causal linkages or dose-responselinkagesDose metric uncertain (peak exposures vs. AUC)There is some evidence of possible effects which aresimilarly sensitive or more sensitive than AChE inhibition.This remains an area of uncertaintyQuantitative Lines of Evidence: Potential forEffects < Doses Eliciting 10% AChE Inhibition20Charge Question 3.2In vivoexperimental toxicology studiesMany studies use only one, maybe two doses, and in severalcases the lower but not higher dose level produced significantneurodevelopmental effectsMost studies use of 1 mg/kg/day or higherDose that produces some amount of brain ChE inhibition when givendirectly to the pups post-natallyLimited or no dose response data on inhibition of fetal brain ChEactivity below 1 mg/kg when given to the dams gestationallyOne study (Braquenier et al., 2010) using lower doses,administered to the dam on GD15-LD14, reported a NOEL of0.2 mg/kg/dSimilar to the acute, oral BMDL100.36 mg/kgAbout 10X higher than chronic, oral BMDL100.03 mg/kg/dayQuantitative Lines of Evidence: Potential forEffects < Doses Eliciting 10% AChE Inhibition21Effects seen6.17 pg/gupper-tertileof >LOD(Rauh et al.2006)YearNMeanCPF(pg/g)1999875.0(4.1‐6.2)2000831.9(1.4‐2.4)2001330.5(0.3‐0.6)1998‐20023144.0(sd 6.1)Range:ND‐632001‐200465<LOD2.5(95th)Whyatt et al. 2003, 2004, 2009Summary - Quantitative Lines of Evidence: Potential forEffects < Doses Eliciting 10% AChE Inhibition22Evidence suggesting that pg/g levels of chlorpyrifosunlikely to elicit AChE inhibitionNolan, 1984: Single dose, deliberate dosing study in adult males0.5 mg/kg in single oral doseNo RBC AChE inhibition observedPlasma ChE was inhibited 83-89% relative to pre-dose activities followingthe oral dosePeak blood levels of 0.01-0.03 ug/mL5 mg/kg, single dermal doseNo RBC AChE or plasma ChE inhibition observedMostly non-detects, maximum of 0.01 ug/mLComparison to epidemiology studiesneeds to be interpreted with care:Males vs. Pregnant femalesSingle Dose vs. Repeating exposureControlled, clinical setting vs. “Real world”Oral route vs. Multiple routes/pathwaysFigure 40, Dow PBPK-CARES SAP Issue Paper, 2010230.11101001000100001000001000000100000001000000000.01 0.1 1 10

Peak Blood Concentration (pg/g)Dose mg/kgRelationships between Single Oral Dose and Peak Blood Concentrations ofChlorpyrifos and Chlorpyrifos Oxon in Adults and InfantsAdult ChlorpyrifosAdult Chlorpyrifos oxonChild ChlorpyrifosChild Chlorpyrifos oxon50 per. Mov. Avg. (Adult Chlorpyrifos)50 per. Mov. Avg. (Adult Chlorpyrifosoxon)Quantitative Lines of Evidence: Potential forEffects < Doses Eliciting 10% AChE Inhibition24Exposure range cord blood 3-6 pg/gRemaining Uncertainties:Fetal exposure during critical window(s) ofdevelopmentIncluding blood levels or the AChE inhibition at the timeof chlorpyrifos applicationContribution of other ChE-inhibiting pesticides toOP exposureDiazinon (ρ=0.63), propoxur (ρ=0.21)Whyatt et al. 2009Summary - Quantitative Lines of Evidence: Potential forEffects < Doses Eliciting 10% AChE Inhibition25Charge questions 5.3 & 5.4Given the uncertainties, area of whereadditional analyses may provide helpfulcharacterization of biomonitoring dataSummary26Charge Question 6.0Assessing and presenting the range ofplausible responses at given doses, andthe effect of the overall uncertainty andvariability around that range.Ensure scientific judgements are transparentIdentify areas of uncertaintyPractical steps towards further refinement ofexisting information with existing informationSAP QuestionsQuestions?28