SCIENTIFIC ISSUES CONCERNING HEALTHEFFECTS OF CHLORPYRIFOS:REVIEW OF CHLORPYRIFOS BIOMONITORINGRESEARCH AND INTERPRETIVE APPROACHESAARON NIMAN, LT USPHSINDUSTRIAL HYGIENISTCHESTER RODRIGUEZ, PHDTOXICOLOGISTUSEPA, OFFICE OF PESTICIDE PROGRAMSApril 10, 2012FIFRA SAPOutlineChlorpyrifos Biomarker OverviewTCPy Biomonitoring Study ComparisonBiomonitoring and AChE InhibitionRisk-Based Biomonitoring InterpretationChallengesApproaches2Chlorpyrifos Biomonitoring:Overview3Chlorpyrifos biomarkers of exposure have been reviewed in the literature(Barr & Angerer, 2006; Eaton et al., 2008)Most common chlorpyrifos biomarkers of exposure include urinarymetabolite TCPy and the non-specific DAP metabolites DEP and DETPAlthough less common, chlorpyrifos has also been monitored directly inblood and other biologic media (e.g., Columbia Cohort)Biomarker/AnalyteBiologic MatrixPopulation Group3,5,6-trichloro-2-pyridinol(TCPy)UrineAllMeconiumPregnant Females, NeonatesDiethylphosphate (DEP)UrineAllDiethylthiophosphate (DETP)UrineAllChlorpyrifosBloodAllCord BloodPregnant Females, NeonatesCommon Chlorpyrifos Biomarkers of ExposureChlorpyrifos Biomonitoring:Urinary Biomarkers (DEP/DETP, TCPy)4SpecificityDEP and DETP are non-specific biomarkers of multiple of OP pesticidesTCPy is only metabolite of chlorpyrifos, chlorpyrifos-methyl, trichlopyrChlorpyrifos and other OP pesticides have been shown to formDEP, DETP,and TCPy in environmental media and foodMay overestimate chlorpyrifos exposure if assumed that urinary biomarkerlevels only attributable to chlorpyrifosVariability & Pharmacokinetic FactorsHigh variability observed in research suggests that spot-urine samplescannot accurately quantify pesticide exposure (Barr & Angerer, 2006)However, spot samples may be sufficient for assigning exposure categoriesin epidemiology studiesChlorpyrifos Biomonitoring:Blood Biomarkers5Measurement of chlorpyrifos in blood provides more directmeasure of internal chlorpyrifos doseBlood measurement is far less common in the literature thanurinary TCPy & DEP/DETPRequires more sensitive analytical methodMay place greater burden on study participantsBlood concentrations represent more narrow period of exposurerelative to urinary biomarkersTCPy Biomonitoring Study Comparison6Summarized TCPy biomarker levels in the general U.S.population and targeted study groupsIntended to provide broad comparison of study resultsKey Differences:Timing relative to residential phase-outStudy populations (age, location, setting)Study design and research objectivesSelected studies intended to represent major monitoring studiesNHANESORD observational studiesDow AgroSciences observational Studies (High-Low)Peer-Reviewed literature (2008 SAP; Eaton et al., 2008)Children’s Health CohortsTCPy Biomonitoring Study Summary7ReferenceStudy PopulationType ofStudy PopulationCollection PeriodHill et al., 1995General US Population20-59 year olds1988-1992NHANESGeneral US Population6-11 year olds, 20-59 yearolds1999-2000, 2001-2002Egeghy et al. 2011ORD - NHEXES, AZ5-12 year olds1995-1997ORD - MNCPES3-13 year olds1997ORD - CTEPP, NC2-5 year olds2000-2001ORD - CTEPP, OH2-5 year olds2001ORD - JAX4-6 year olds2001ORD - CPPAES2-4 year olds1999-2000Lu et al., 2008CPES-WA3-11 year olds2003--2004Lu et al., 2006WA Children3-11 year olds2003Alexander et al., 2006Farm families4-18 year olds, Adults2000-2001Fenske et al., 2002Farm Children≤6 years olds1995Curwin et al., 2005FarmersAdults2001, Spring-SummerCurwin et al., 2007Farm FamiliesChildren and Adults2001, Spring-SummerBerkowitz et al., 2003Mt. SinaiPregnant Female1998-2001Whyatt et al., 2009ColumbiaPregnant Female2001-2004Castorina et al., 2010CHAMACOSPregnant Female1999-2000Eskenazi et al., 2007CHAMACOSPregnant Female2000-200380.11101001000 NHANES 1999-2000 (n=481) NHANES 2001-2002 (n=573) NHEXAS-AZ 1995-97, 5-12 years (n= 21) MNCPES 1997, 3-13 years (n = 263) CTEPP-NC 2000-2001, 2-5 years (n=129) CTEPP-OH 2001, 2-5 years (n=123) JAX 2001, 4-6 years (n = 9) CPPAES 1999-2000, 2-4 (n=81) CPES-WA, Summer 2003-2004 (n= 21) CPES-WA, Fall 2003-2004 (n= 21) CPES-WA, Winter 2003-2004 (n= 21) CPES-WA, Spring 2003-2004 (n= 21) Conventional Diet, Pre, 2003 (n=87) Organic Diet, 2003 (n=116) Conventional Diet, Post, 2003 (n=155) Child 2001, farm (n=65) Child 2001, nonfarm (n=51 Farm families—pre-appl 2000-2001 (n=50) Farm families—post-appl 2000-2001 (n=50)CDC, 2009 (6-11years)Egeghy et al, 2011 Lu et al., 2008 (3-11 years) Lu et al.,2006 (3-11 years) Curwin et al, 2007 Alexander et al.,2006 (4-18 years)

TCPy Urinary Concentration (μg/L)ReferenceRangeGMGMTCPy Biomonitoring Study Summary: Child Study Populations9TCPy Biomonitoring Study Summary: Adult Study Populations.0.11101001000 NHANES III, 1988-1992 (n= 993) NHANES 1999-2000 (n=832) NHANES 2001-2002 (n=1,113) Residential Low- Crack & Crevice/Spot (n=8) Residential High- Turf Treatment (n=10) Worker Low- Re-Entry (n=10) Worker High- Mix/Load/Apply/Clean (n=15) Nonfarmers, chlorpyrifos not sprayed (n= 45) Farmers, chlorpyrifos not sprayed (n=45) Farmers, chlorpyrifos self-sprayed (n=2) Father, nonfarm (n=23) Father, farm (n=24) Mother, nonfarm (n=24) Mother, farm (n=24) Farm families—Applicators pre-appl. (n=34) Farm families, Applicators post-appl (n=34 Farm families, Spouses pre-appl Farm families, Spouses post-appl (n=34) Mother–child pair cohort 1998-2001 (n=365) Prenatal 1 (n=95) Prenatal 2 (n=83) Prenatal 3 (n=54) Prenatal 4 (n=21) Postnatal (n=73) 1st Pregnancy (n=442) 2nd Pregnancy (419) Prenatal 1 (n=538) Prenatal 2 (n=481) NHANES Subsample 1999-2002 (n=224)Hill et al, 1995;CDC, 2009Dow AgroSciences Curwin et al, 2005 Curwinet al, 2007 Alexander et al., 2006 Berkowitzet al., 2003Whyatt et al, 2009 Eskenazi etal, 2007Castorina et al,2010

TCPy Urinary Concentration (μg/L)ReferenceRangeMaternalCohortsGMGMGMGMGMGMGM* NHANES data represents all survey participants aged 20-59 years old.*100.1110100 NHANES III, 1988-1992 (n= 993) NHANES 1999-2000 (n=832) NHANES 2001-2002 (n=1,113) NHANES Subsample 1999-2002 (n=224) Prenatal 1 (n=538) Prenatal 2 (n=481) Mother–child pair cohort 1998-2001 (n=365) Prenatal 1 (n=95) Prenatal 2 (n=83) Prenatal 3 (n=54) Prenatal 4 (n=21) Postnatal (n=73) 1st Pregnancy (n=442) 2nd Pregnancy (419)Hill et al, 1995; CDC, 2009 Castorina et al, 2010 Berkowitzet al., 2003Whyatt et al, 2009 Eskenazi et al, 2007

TCPy Urinary Concentration (μg/L)Reference RangeMaternalCohorts*TCPy Biomonitoring Study Summary: NHANES and Children’s Health Cohort Results* NHANES data represents all survey participants aged 20-59 years old.TCPy Comparison Results: Findings11TCPy levels of mothers in the CHAMACOS, Columbia, andMount Sinai cohorts varied in comparison with TCPy levelsobserved in NHANESMedian TCPy levels higher in CHAMACOS and Mount Sinai Cohorts(Eskanazi et al., 2007; Castorina et al., 2010)Median TCPy levels lower in Columbia Cohort (Whyatt et al., 2009)However, in contrast to the other cohorts, data were collected, in part,during a time period of rapid decline in use of chlorpyrifosTCPy Biomonitoring and AChE Inhibition12AChE Monitoring OverviewRed Blood Cell (RBC) AChE and Plasma BuChE most commonbiomarkers of ChE activityRBCAChE inhibition considered an early marker of potentialchlorpyrifos toxicityPlasmaBuChE has been shown to be inhibited at lower levelsof exposure thanRBCAChEAs a result,PlasmaBuChE may be a more sensitive biomarkerfor monitoring potential physiological change.TCPy Biomonitoring and AChE Inhibition13Previous Agency FindingsAgency previously evaluated relationship between TCPy andRBC/Plasma AChE activity at 2008 SAPRBC/Plasma AChE inhibition observed in some studyparticipants of registrant studiesHowever, large variability in response to the administereddose of chlorpyrifosAs a result, difficult to draw definitive conclusions about therelationship between urinary TCPy levels and RBC/PlasmaAChE inhibition.TCPy Biomonitoring and AChE Inhibition14Recent Study Findings (Post-2008 SAP)Two observations studies have been published that furtherevaluate the relationship between TCPy on a creatinineadjusted basis and RBC/Plasma AChE inhibitionGarabrant et al. (2009)Evaluated group of exposed workers at a chlorpyrifosmanufacturing facilities and a reference group of chemicalmanufacturing workersFarahat et al. (2011)Evaluated cotton field workers in Egypt followingchlorpyrifos application to cotton fieldsComparisonofCreatinine‐AdjustedUrinaryTCPylevels(μg/gcreatinine)reportedbyGarabrant etal.(2009)andFarahatetal.(2011)withtheGeneralU.S.Population.NR– NotreportedTCPy Biomonitoring and AChE Inhibition15PopulationGroupTimePeriodnTCPy(μg/gcreatinine)AverageMedian(95%CI)95thPercentile(95%CI)NHANES(CDC,2009)GeneralPopulation1999‐20001,994NR1.5(1.2‐1.7)8.4(6.3‐11.6)2001‐20022,508NR1.9(1.6‐2.2)9.2(6.9‐12.3)Garabrant etal,2009ExposureGroupFall199953NR37.5432.6Spring200053NR40.7436.3Turnaround50NR59.27,403.8Fall200052NR51.5987.2ReferenceGroupFall199960NR3.213.9Spring200057NR4.811.2Turnaround57NR6.613.0Fall200058NR6.315.5Farahatetal,2011Applicators1‐to2‐weekExposurePeriod146,437NRNRTechnicians12184NRNREngineers12157NRNRGarabrant et al. (2009) Results:TCPyÅÆAChE RelationshipRBC AChEPlasma BuChE16Farahat et al. (2011) Results:TCPyÅÆAChE relationship17RBC AChETCPy Biomonitoring and AChE Inhibition18Garabrant et al. (2009)No association observed between TCPy andRBCAChEStatistically significant association observed between TCPy andPlasmaBuChE levels above a cutpoint of 110 μg TCPy/g creatinineFarahat et al. (2011)Identified an inflection point representing a significant decrease inRBCAChE activity at TCPy levels greater than 3,148μg TCPy/g creatinineReported that both studies observed similar inflection point of inhibition ofPlasmaBuChE activityGarabrant et al. (2009) 110 μg TCPy/g creatinineFarahat et al. (2011) 114 μg TCPy/g creatinineRisk-Based Biomonitoring Interpretation19NAS 2006 Biomarker Interpretation FrameworkQualitativeSemi-QuantitativeQuantitativeBiomonitoring Interpretation:ChallengesSpecificityDirect exposure to metabolites in the environment (TCPy, DEP/DETP)Longitudinal VariabilityDifficult to relate single biomarker measurementHighly variable depending on sampling time/method, pharmacokineticbehavior, biomarker specificity, exposure routes, etc.Toxicological RelevanceNot directly comparable to regulatory screening valueNot always aligned with a toxicologically-relevant internal dose metricTCPy formation reflective of detoxification and can occur in absence ofchlorpyrifos-oxonTCPyÅÆAChE relationship unclear (at environmental exposure levels)20Biomonitoring Interpretation:Qualitative ApproachesEvaluate biomonitoring data without estimating chlorpyrifosexposureNon-ComparativeUrinary TCPy data for different time periods may be compared fortemporal trends in exposure (e.g., pre- versus post-mitigation)No link to chlorpyrifos, exposure routes, or health outcomesComparativeComparison to controlled human studiesPrimary comparison is to Nolan et al.,1984: measured urinary TCPy inhuman volunteers (males) following a single oral dose of 0.5 mg/kg bwchlorpyrifos21Biomonitoring Interpretation:Semi-Quantitative ApproachesEstimate biomarker levels likely associated with regulatoryexposure guidelinesBiomonitoring EquivalentsScreening level approach for analyzing biomonitoring dataUses pharmacokinetic information and forward dosimetry approachesto convert an exposure guideline value (e.g., cRfD) to a biomarkerlevel that can be compared to biomonitoring dataHays et al., 2007 and 2008ConsiderationsBiomarker Specificity: approach highlydependent on biomarker specificityDirect exposures to TCPy take placeToxicological Relevance: biomarker must bealigned with a toxicologically-relevantinternal dose metricTCPy is reflective of detoxification of chlorpyrifos and can form in the absence ofchlorpyrifos-oxon22Biomonitoring Interpretation:Semi-Quantitative ApproachesEstimate chlorpyrifos exposures from biomarker levelsSemi-Quantitative Reverse DosimetryUtilizes creatinine-normalized urinary levels of biomarker;lifestage-changes may be incorporatedCorrects for incomplete clearance of parent chemical as reflectedby biomarkerMage et al. 2004ConsiderationsEstimates represent net absorbeddoses without route-specificinformationBiomarker Specificity; assumes biomarker is derived from parentchemicalDirect exposures to TCPy take place23Quantitatively evaluate risk using exposure linkedphysiologically based pharmacokinetic (PBPK) modeling24Biomonitoring Interpretation:Quantitative ApproachesDowAgroSciences (2011).Source‐to‐OutcomeModelingPhysiologicallyBasedPharmacokinetic/Pharmacodynamic (PBPK/PD)ModelLinkedtoaDietaryExposureModel:Chlorpyrifos asaCaseStudy.FIFRAScientificAdvisoryPanel,Washington,DC,February15‐18,2011.Biomonitoring Interpretation:Quantitative Approaches25Chlorpyrifos PBPK ModelSeveral groups working on chlorpyrifos PBPK modelingTimchalk et al. (2002) PBPK ModelRefined since originally developed in 2002Evaluated at 2011 FIFRA SAPLimitationsBased on the “typical” adult human; does not incorporate life-stage relatedchanges (e.g., pregnancy or childhood)Can only simulate oral exposures to chlorpyrifos with particular emphasis ondietary exposuresSeveral efforts to expand the model to other life-stages have yet toproduce a model robust enough to inform human health risk assessment.26SummarySeveral approaches can be used to analyze the biomonitoringdata associated with chlorpyrifos exposureEach approach has limitations that need to be carefullyconsidered for any type of interpretationAgency soliciting recommendations from the SAP Panelregarding strengths and limitations of approaches, as well asalternative approaches not yet considered by the AgencyQuestions27