CHLORPYRIFOS EFFECTS ON THEDEVELOPING BRAIN:ANIMAL STUDIESGINGER MOSER, PHD, DABT, FELLOW ATSOFFICE OF RESEARCH AND DEVELOPMENTUS EPAApril 10, 2012FIFRA SAPBackgroundSAP in 2008 review of animal studiesconcluded:“independent of exact time frame of gestational orpostnatal exposure, only doses of 1 mg/kg orgreater demonstrate significant effects onbehavior”Two areas of concernInconsistent behavioral changesLack of dose-response and data on ChE inhibitionRecommended review of critical events at time ofexposure and targeted endpoints2Focus of Current DevelopmentalNeurotoxicity Review2012 review aims to assess neurobehavioraloutcomes in terms of:Domains of neurological functionCritical exposure periodsEvidence of brain ChE inhibition during criticalperiodsTargeted review of publications since 2008New informationIntegration with existing database3Existing Literature426 papers* from 11 laboratoriesInclude 9 since 2008, plus 2 new laboratoriesFocus on 20 papers from 9 laboratories25 actual studiesFocus review on papers with:Dosing any time up to weaningAssessments after weaning to reflect longer-term impactsLower doses, avoid toxic levels of ChE inhibitionRodents* As of March 29Existing Literature5Variability in these 25 studies include differences in:Exposure paradigms10 gestational studies with 7 dosing periods13 postnatal studies with 5 dosing periods2 perinatal studies with different dosing periodsRoutes of exposure and vehicles4 routes of administrationsubcutaneous, oral, dermal, implant4 vehiclesDMSO, peanut oil, corn oil, ethanolDose selections1 (10 studies), 2, 3, or 5 dosesSpeciesRat studies≈mouse studiesAgency Guideline Interpretations1998 US EPA Guidelines for Neurotoxicity RiskAssessment conclude:Developmental neurotoxicity effects in animal studiesindicate the potential for altered neurobehavioraldevelopment in humansSpecific types of developmental effects seen inexperimental animal studies may not be the same asthose seen in humans6Cognition Domain: OutcomesSpatial learning and memory: radial arm maze, Morriswater maze, foraging in radial arm mazeAltered function, mostly showing deficit and few improvement8 of 9 studies, gestational and early postnatal exposuresEffects at 1 mg/kg/d and above, maybe at 0.3 mg/kg/dLowest NOEL reported at 0.2 mg/kg/d using water mazeAlternation learning in T-mazeNo effect5 of 5 studies, gestational and postnatal exposuresClassical conditioning using passive avoidanceNo effect2 of 2 postnatal studies7Cognition Domain: ConsiderationsMost of these tests validated and accepted (exception,foraging)Morris water mazeReplications within one laboratory (2 studies)Radial arm mazeAcross laboratories, different procedures and equipmentGender differences in effect and controls not consistentActivity level or motor differences can confound performanceNo activity differences in 5 of6 studies (where reported) showingcognitive changesDose-response seen in 3 of 5 studies with 2 or more dosesEffect sizes sometimes smallDifferences sometimes transient, sometimes persistent acrosstraining8Replication of Cognitive Effects:Morris Water Maze9Billauer-Haimovitch et al., 2009; Turgeman et al., 2011Multiple studies in mice within the same laboratory, GD9-18 exposureSignificantly slower time to findplatform at 1 and 3 mg/kg/dNot 5 or 10 mg/kg/dNo changes in swim speedReplicated results insubsequent studies using3 mg/kg/dNo changes in swim speedReplication of Cognitive Effects:Radial Arm Maze10Significant across training for both sexesAldridge et al., 2005Reference memory errors in rats within the same laboratory1 mg/kg/d, PND1-4Increased errors in males, decreased in femalesMalesFemalesMales significant first trial onlyFemales significant across trainingLevin et al., 2001Anxiety and Emotion Domain:OutcomesAnxiety: elevated plus maze, light-dark boxAltered behaviors7 of 8 studies, gestational and postnatal exposuresEffects at 1 mg/kg/d and aboveEmotion: novelty and chocolate preferenceNo effect with novelty exploration in 2 postnatal studiesChange in chocolate preference in 1 postnatal studyEmotion and despair: forced swim testNo effect in 1 gestational study11Anxiety and Emotion Domain:ConsiderationsMost of these tests validated and accepted in literatureSpecific endpoints not consistently measured or alteredacross studiesDifferent procedures across studies and within same laboratoryMouse: of 6 studies, 3 showed increased and 3 decreasedanxietyRat: of 2 studies, 1 showed decreased anxietyActivity level differences can confound measurementNo differences in 5 of 6 studies,when reported, with anxiety changesDose-response in only 1 of 4 studies with 2 or more dosesEffect sizes sometimes small12Replication of Anxiety Effects:Elevated Plus Maze & Light-Dark Box13Increased anxiety in mice using two differenttests; Female offspring, exposed GD15-LD14Braquenier et al., 2010Light-Dark BoxElevated Plus MazeDecreased anxiety in mice using elevated plusmaze; Offspring exposed PND11-14Ricceri et al., 2006Social/Maternal Behavior Domain:Outcomes and ConsiderationsSame-sex social interactionsAltered behaviors6 of 10 studies, gestational and postnatal exposuresIncreased social interactions or agonistic responses (males only)Maternal behaviors4 of 5 studies, gestational and postnatal exposuresDecreased reactions to intruder, increased or decreased behaviorstowards pupsAll studies from same laboratory, in miceEndpoints not standardized, even within same laboratorySometimes just one or few significant changes out of manymeasured behaviorsTests of social behavior have not been widely used indevelopmental neurotoxicity studies14Motor Activity Domain:Outcomes and ConsiderationsSpontaneous, exploratory activity, including locomotion andrearing: figure-8 chambers, open fieldsAltered activity levels8 of 14 studies, gestational and postnatalRat: increased (1), decreased (2), or no effect (2) postnatalexposures; no effect with gestational or perinatal exposuresMouse: increased (3), gestational and postnatal exposuresHabituation over test session, different from activity levelsAltered in 3 of 6 studies where analyzedDifferent statistical approaches across laboratoriesMotor activity is accepted and widely used as an indicatorof general behavioral changes15Neuromotor Domain:Outcomes and ConsiderationsAuditory startle responseNo effects in 2 studies in rats, gestational or perinatalexposuresNeuromuscular performanceAltered in 1 gestational study in ratsVery limited literature16Measurements of ChE Inhibition17ExposureperiodSpeciesDosingTime of measurementGestationalRatRatGD6-20 po1GD17-20 sc2No inhibition 4 hr after dose to dam on GD20(also at 0.3 mg/kg/d)No inhibition 24 hr after dose to dam on GD20EarlypostnatalRatMouseSingle dose sc3PND1-4 sc4Inhibition 2 and 4 hr after dose on PND1Inhibition 1 hr, not 4 hr, after dose on PND4LatepostnatalRatMouseSingle dose sc3PND11-14 sc4,5Inhibition 2 and 4 hr after dose on PND11No inhibition 1, 4, or 24 hr after dose on PND14ChE inhibition in pup or fetal brain at 1 mg/kg, given once or repeatedly;This dose produced effects in almost all domains across studiesNo information about:Fetal ChE inhibition at shorter times after dosingor on other daysNo-effect dose for PND1 to 41Mattsson et al., 20002Qiao et al., 20023Dam et al., 20004Ricceri et al., 20035Ricceri et al., 2006Sources of Variability in Outcomes18Experimental designProcedural and equipment differences, within and across laboratoriesInsufficient evaluation of dose-responseSpecies, strains, routes of exposure, vehiclesAges of exposure and subsequent testingOutcomesControl values and treatment effects not always exactly reproducedBrain ChE inhibition in fetus/pupGestational exposures maybe not, postnatal yesInterpretationsStatistical analyses and acceptance criteria not applied consistentlySubtle effects vs false positivesPotential for false negatives by conservative acceptance criteria orunderpowered studiesPublication bias against studies of negative dataSummary19Multiple studies and laboratories reporting changes inaspects of neurobehavioral function in adult rodentsfollowing developmental exposureEffects seen long after chemical exposure and any ChE inhibition,if present, had recoveredBehavioral alterations show some consistency in termsof broad domains of neurological functionMost consistent effects on cognition, anxiety, social behaviorDifferences in direction and magnitude of change, genderspecificityDifferences in procedures, exposures, etc. impact comparabilityacross studiesSummary20No clear evidence of critical period of exposure orspecies differencesNew publications support previous analyses,strengthen conclusion of 2008 SAPNeurobehavioral effects at doses≥1 mg/kg/dInsufficient dose-response data for effects and ChE inhibitionNot enough data to definitely conclude relativesensitivity between developmental outcomes and ChEinhibitionNeed robust dose-response and time-course data withsystematic replications of exposure periodsQuestions?21