Atrazine: Updates to the DoseResponse Assessment withResponseAssessmentwithImplications for WaterResponseAssessmentwithImplications for WaterMonitoring FrequencyP t ti t S i Ad i P lPresentationtoScienceAdvisoryPanelJuly 26-29, 2011Ch R d i Ph DChesterRodriguez,Ph.D.Health Effects DivisionUSEPA Office of Pesticide ProgramsOutline•Re-cap of September 2010 SAP meeting: internal measureof exposure•Analysis of additional animal pharmacokinetic studies withradiolabeled atrazine•Human pharmacokinetic study with atrazine•Pharmacokinetic modeling approach•Onecompartment linear model-compartmentlinearmodel•Model evaluation•Understanding of pharmacokinetics, internal dosimetry, andUnderstandingofpharmacokinetics,internaldosimetry,andLH attenuation•Estimating the human intake dose rate through AUC analysis2of water chemographs•Apply methodologies to case studiesHuman Health Risk Assessment:AtrazineHuman Health Risk Assessment:Atrzinea e•Previous risk assessment to support the RED• Based on a NOAEL/LOAEL approach• Critical study: 6 month dietarystudyyy• Key event: Luteinizing Hormone (LH) Attenuation•September 2010 SAP meeting• More sophisticated approaches introduced• Benchmark dose modeling• Internal measures of exposure3Internal Dose for LH AttenuationAtrazineExternal DosePharmacokineticsBody IntakeInternal Dosimetry(Atrazine & Metabolites)“The dose at the target site, theinternal dose, is the ultimatedeterminant of risk.”National Research Council,1994.Mode of Action Key Event(LH Attenuation)NationalResearchCouncil,1994.(LHAttenuation)Observed Toxicity4(e.g., ovarian cyclicity disruption,developmental delays)Internal Measure of Exposure for LHAttenuation in the RatAttenuationintheRatAttenuation•Why an “Internal Measure of Exposure”?•Atrazine is short-lived in the bodydueprimarilyto metabolismp y• Metabolites also active in attenuating LH•Area under the plasma concentration-time curve (AUC) asit l fnternalmeasure ofexposureSingle dose effect

l)Cooper et al. 200025Repeated daily dosing over 4 daysCooper et al. 2010

Serum LH (ng/m510152025

u tinizing Hormone Serum (ng/ml)0 1 3 61 3 6time (hr)0 50 100 200 300Atrazine Dose (mg/kg bw)1 3 6 1 3 6 1 3 6Vehicle1.563.126.2512.52505Atrazine (mg/kg-d)

Le u5• Duration is critical parameter for LH attenuation in ratsAUC = Levels x Duration(“how much x for how Long”)•AUC selected was for plasma triazines based onradiolabeled atrazine studies( g)radiolabeledatrazinestudies• Lack of detailed pharmacokinetic information for atrazineand its metabolitesR di l b l d t i t di i t il bl•Radiolabeledatrazine studiesin rats available• Radiolabel studies generally achieve a high degree ofmass balanceCl***HN NNHCl***HN NNHClNNCH3CH3HCH2CH3CHNNNCH3CH3HCH2CH3CH6Metabolically Stable Radiolabeled Triazine RingRationale for Study SelectionThede 1987 Radiolabeled Atrazine Pharmacokinetic Study:• Selected to learn about the temporal relationship betweenatrazine exposure and LH attenuation in rats• Similar to the 4-day LH attenuation study:• Used intact young female ratsInvolved repeated once daily dosing with atrazine for≥4•Involvedrepeatedoncedailydosingwithatrazinefordays• Covered a wide range of atrazine doses (1- 100 mg/kg bw)• Repeated plasma measurements including elimination phase7Plasma Profile from Thede 198781 mg/kg bw ATRA3 mg/kg bw ATRA7 mg/kg bw ATRA10 mg/kg bw ATRA

e s (mg/L)46

P lasma Triazin e02448729612014416819221624026428802time (hrs)

Radiolabeled Pstart of dosingpseudosteady state dosing stopped•Repeated dosing with atrazine leads to plasma accumulationthof triazines to a plateau/pseudosteady state level by the 4thday Plateau/pseudosteady state:• Plasma levels staywithin a specific range as longas8y p ggdosing continues at the same level and frequencyPseudo-Steady State ConceptSteady State Concept•Hypothetical:repeated dosing for 3 different duration leadingto the samepseudosteady state plasma levelstothesamesteadystateplasmalevels600Hypothetical400Pseudosteady stateElimination Phase(dosing stopped)

L evels020040060080002004 days14 days6 monthstime (hr)

Plasma L• As long as dosing continues at the same level and frequency,plasma levels will remain within the same rangetime(hr)9plasmalevelswillremainwithinthesamerange.Critical LH Attenuation Studies ofDifferent Durations• The bioaccumulative profile of plasma triazines is consistentwith the similar LOAELs for LH attenuation across differentDifferentDurationsdurations of atrazine exposureStudy Duration LOAEL (mg/kg bw/dayatrazine)Cooperet al20104 days3 12..Morseth 1996 6 months 3.65• Proposed a daily steady state AUCfor internal doseresponse analysis10Linear Pharmacokinetic Behavior:Thede 1987Study• Other features noted in the behavior of plasma triazinesThede1987StudyLinear Pharmacokinetic Behavior15001 mg/kg bw/day AtrazineAUC for plasma triazinesscales directly with atrazine doseLinear elimination kinetics750100012501500y= 13.0x - 8.57R2= 0.994

udo -daily y state AUC m g/L-h)-2.0-1.8-1.6

m a triazines)0204060801000250500ATRA dose(mg/kg/day)

pse stead y ( m240252264276288300-2.4-2.2time (hrs)

Ln (plas m11( g g y)Linear Elimination KineticsChange in plasma concentrationElimination rate constantRate of eliminationΔCpΔt= kel * CpdCpdt==Integrated form: LnCp = LnCp – kel*t0Rate of elimination:•Linear expression with kel as slopeLinearexpressionwithkelasslope• Proceeds at a rate that is independent of plasma levels• Rate determined solely by kel (a constant)C t t l h lflif (i tl 2/k l)di ti12•Constantplasmahalf-life(i.e.,t1/2=ln2/kel)- predictivefor extrapolation across dose levelsPlasma Elimination PhaseAnalysis: Thede 1987 Study81 mg/kg bw ATRA3 mg/kg bw ATRA7 mg/kg bw ATRA10 mg/kg bw ATRA

n es (mg/L)EliminationAnalysis:Thede1987StudyAnalysis:246

e d Plasma Triazi nPhase0244872961201441681922162402642880start of dosingpseudosteady statedosing stoppedtime (hrs)

Radiolabel eTh d 1987startofdosingpseudosteadystatedosingstoppedAtrazine Dose Elimination rate constantPlasma half-lifeThede1987(mg/kg bw/day) (kel, hr-1) (hrs)1 0.010 72.6 (3.0 days)3 0.015 47.2 (2.0 days)137 0.013 52.9 (2.2 days)10 0.014 50.3 (2.1 days)Outline•Re-cap of September 2010 SAP meeting: internal measureof exposure•Analysis of additional animal pharmacokinetic studies withradiolabeled atrazine•Human pharmacokinetic study with atrazine•Pharmacokinetic modeling approach•Onecompartment linear model-compartmentlinearmodel•Model evaluation•Understanding of pharmacokinetics, internal dosimetry, andUnderstandingofpharmacokinetics,internaldosimetry,andLH attenuation•Estimating the human intake dose rate through AUC14analysis of water chemographs•Apply methodologies to case studiesAddressing Limitations of theThede 1987StudyLimitations of the Thede 1987 Pharmacokinetic Study:•Only 2 animals per dose groupThede1987StudyOnly2animalsperdosegroup• Elimination phase estimated with only 3 data points from asingle animalPlasma elimination analysis from other rat studieswith radiolabeled atrazine:P lt l1993 l t lti f•aueal.1993: plasma measurements resultingfromsingle oral gavage doses of 1or 100 mg/kg bw radiolabeledatrazine administered to groups of 3 male rats• Simoneaux 1985: plasma measurements at various dayspost-dosing resulting from oral gavage doses of 0.4 or 4.0mg/kg bw radiolabeled atrazine given to 36 male rats (3 per15mg/kgbwradiolabeledatrazinegivento36malerats(3pertime point) for 7 daysAdditional Rat Studies with RadiolabeledAtrazineSingle dose of 1 mg/kg bw14C- atrazine

m )Single dose of 100 mg/kg bw14C-atrazine

m )•Paulet al.1993 StudyAtrazine0.40.6Paul et al. 1993

e d Equivalents (pp m10152025Paul et al. 1993

e led equivalents (pp m048961441922402883360.00.2time (hr)

Plasma radiolabel e0244872961201441681922162402642883123360510time (hr)

Plasma radiolab eSingle dose of 1 mg/kg bw14C-atrazine-20Slope = - 0.013±0.002R2=0 74

valents) (ppm)Single dose of 100 mg/kg bw14C-atrazine4Slope = - 0.014±0.001R2= 0 94

ents) (ppm)-8-6-42Paul et al. 1993R0.74

a sma radiolabeled equi4896144192240288336384-202R=0.94Paul et al. 1993time (hr)

m a radiolabeled equival160 48 96 144 192 240 288 336time (hr)

ln (Pl a-4

ln (Plas mAdditional Rat Studies with RadiolabeledAtrazineRepeated dosing with 0 4 mg/kg bw14C AtrazineRepeated dosing with 4 0 mg/kg bw14C AtrazineAtrazineSimoneaux 1985Repeateddosingwith0.4mg/kgbw14C-Atrazine0.060.080.10

e quivalents (ppm)Repeateddosingwith4.0mg/kgbw14C-Atrazine1.01.5

e quivalents (ppm)0246810121416180.000.020.04Simoneaux 1985time (days)

Plasma radiolabeled e0246810121416180.00.5Simoneaux 1985time (days)

Plasma radiolabeled etime(days)time(days)Elimination Phase Analysis: 0.4 mg/kg bw/day14C-atrazine1

s ) (ppm)Elimination Phase Analysis: 4.0 mg/kg bw/day14C-atrazine1

t s) (ppm)1012141618-4-3-2-101Simoneaux 1985time (days)

d iolabeled equivalent s10121416183-2-10Simoneaux 1985time (days)

a diolabeled equivalen t17-6-5slope = -0.236±0.031 day-1= -0.010±0.001 hr-1

Ln (Plasma ra d-4-3slope = -0.258±0.019 day-1= -0.011±0.008 hr-1

Ln (Plasma r aConsistent Plasma Elimination RateConstants Across StudiesConstantsAcrossStudies• A very consistent elimination rate constant acrossdifferent studies and oral doses of radiolabeled atrazineAtrazine Dose(mg/kg bw/day)Elimination rateconstant (kel, hr-1)Plasma half-life(hrs)Study0.4 0.010 72.6 (3.0 days) Simoneaux 19851 0.013 53.3 (2.2 days) Paulet al.19931 0.010 72.6(3.0 days)Thede 1987y)3 0.015 47.2 (2.0 days) Thede 19874 0.011 63.0 (2.6 days) Simoneaux 198570 01352 9 (2 2days)Thede 1987.52.9(2.2Thede198710 0.014 50.3 (2.1 days) Thede 1987100 0.014 50.3 (2.1 days) Paulet al.199318Pharmacokinetic Studies with RadiolabeledAtrazine in NonAtrazine in Non-Human Primates (Monkeys)Human Primates (Monkeys)• Huiet al.2011: rhesus monkeys dosed via oral gavage with1,10or 100 mgof radiolabeled atrazine, g• Plasma was sampled for radioactivity as various time pointspost-dosingMost important observations•AUC scaled linearly with atrazine dose• Linear elimination kinetics14C-Atrazine(mg)AUC0→∞((mg/L)*hr)kel(hr-1)Plasma Half-life(hr)11.61±0.350.04±0.0320±81013.42±3 580.06±0 0316±919±3.58±0.03100151±240.03±0.0024±1Huiet al.2011Outline•Re-cap of September 2010 SAP meeting: internal measureof exposure•Analysis of additional animal pharmacokinetic studies withradiolabeled atrazine•Human pharmacokinetic study with atrazine•Pharmacokinetic modeling approach•Onecompartment linear model-compartmentlinearmodel•Model evaluation•Understanding of pharmacokinetics, internal dosimetry, andUnderstandingofpharmacokinetics,internaldosimetry,andLH attenuation•Estimating the human intake dose rate through AUC analysis20of water chemographs•Apply methodologies to case studiesHuman Pharmacokinetic Study•Human information very limited• Davidson 1985 report:• 6 human volunteers received a single oral dose of atrazine(0.1 mg/kg bw)•Whole blood from a single subject analyzed for atrazine and itsWholebloodfromasinglesubjectanalyzedforatrazineanditschlorotriazine metabolites DEA, DIA, and DACT• Urine from all subjects analyzed for atrazine, DEA, DIA, andDACTResults:• Only DIA and DACT detected in whole bloodO l DEA DIA d DACT d t t d i i•OnlyDEA,DIA, andDACTdetectedin urine• Chlorotriazines detected exhibited linear elimination kineticswhen analyzed in blood (DIA & DACT) and urine (DEA, DIA, & DACT)21• Lack of mass balance: DEA, DIA, and DACT accounted foronly 14.5% of the administered atrazine doseSummary of Human PharmacokineticStudyHuman Pharmacokinetic ParametersWhole BloodChlorotriazine tmax(hr)Linear EliminationRate Constant (kel, hr-1)Elimination HalfLife (hr)AtrazineND ND NDDEAND ND NDDIA≤20.252.8DACT ~ 5 0.039 17.8UrineAt iN/ANDNDrazneDEAN/A0.29 2.4±1.0DIAN/A0.30 2.3±0.522DACTN/A0.060 11.5±0.4Davidson 1985ND = not detectedN/A = not applicableSummary of PharmacokineticInformation•Use of radiolabeled plasma equivalents includes atrazined ll f it t b litandallofits metabolites• AUC as internal measure of exposure accounts forduration–important for LH attenuation• Plasma triazines (atrazine and its metabolites) accumulateupon repeated dosing with atrazine to reach al t / d t d t t l l b th 4thd fd iplateau/pseudo steady statelevelbythe4thday ofdosing;LH attenuation also observed by 4thday of once dailydosing• Proposed internal dose metric: daily steady state AUC forplasma triazinesLi b h i f l t i i i t t f t23•Linearbehavior ofplasmatriazines consistentfeatureacross doses and speciesNew Pharmacokinetic Information• Anin vivopharmacokinetic study in rats recently submitted bythe Registrant•A new PBPK modeling effort based on this new dataset recentlyAnewPBPKmodelingeffortbasedonthisnewdatasetrecentlysubmitted by the Registrant in collaboration with the HamnerInstitutes•The Agency has notcompleted a thorough review andevaluation of a new PBPK modeling effort24Outline•Re-cap of September 2010 SAP meeting: internal measureof exposure•Analysis of additional animal pharmacokinetic studies withradiolabeled atrazine•Human pharmacokinetic study with atrazine•Pharmacokinetic modeling approach•Onecompartment linear model-compartmentlinearmodel•Model evaluation•Understanding of pharmacokinetics, internal dosimetry, andUnderstandingofpharmacokinetics,internaldosimetry,andLH attenuation•Estimating the human intake dose rate through AUC25analysis of water chemographs•Apply methodologies to case studiesPharmacokinetic Modeling• Estimate of internal dose metric for LH attenuation in rats•Extrapolate of internal dose for LH attenuation to differentExtrapolateofinternaldoseforLHattenuationtodifferentspecies (humans), life-stages, exposure conditions, etc.• Relate the human ingested dose through drinking water toh l l l th t b d t th t lhuman plasmalevelsthatcanbe comparedtothe ratplasmapoint of departure level for LH attenuationIdeal Approach: A Physiologically Based PharmacokineticModelIn the absence of a PBPK model,the Agency is consideringother options for informing pharmacokinetics, internal dose, andwater monitoring26watermonitoringProposed Interim PharmacokineticModeling ApproachModelingApproachA one compartment linear modelPlasma Triazines(Cp, mg/L)kel(hr‐1)Triazines eliminatedOral dose ofAtrazine(mg)Fast/completeabsorptionVdss(L)•Linear behavior: consistent feature of plasma triazines acrossdifferent studies doses and speciesdifferentstudies,doses,andspecies• AUC scales directly with atrazine dose• Elimination is linear (constant plasma elimination half-life)C i t t ith i ff t b Ti h lk t l 1990•Consistentwithprevious effortsbyTimchalketal.1990,McMullin et al. 2007 and Hui et al. 2011 in modelingchlorotriazine equivalents27• Modeling overallbehavior of allplasma triazines resulting fromatrazine exposureProposed Interim PharmacokineticModeling ApproachModelingApproachA one-compartment linear modelPlasma Triazines(Cp, mg/L)kel(hr‐1)Triazines eliminatedOral dose ofAtrazine(mg)Fast/completeabsorptionVdss(L)• Only two parameters are needed• volume of distribution (Vd)•kel• Vd and kel at steady state (Vdss)• Vdss and kel estimated independently from single dosel dt i P lt l199328plasmadatainPauletal.1993Evaluation of OneEvaluation of One-CompartmentCompartmentLinear ModelPlasma Triazines(Cp, mg/L)kel(hr‐1)Triazines eliminatedOral dose ofAtrazineFast/completeabsorptionV(L)LinearModel(mg)Vdss(L)Vdss= 6.55 L/kg bwkel = 0.013 hr-1Analytical solution to a onecompartment linear model:Analyticalsolutiontoaone-compartmentlinearmodel:Doserate(mg/kgbw/day)Vdss (L/kgbw)*kel (hr‐1)*24hr/dayCpss (mg/L)=Expression relates daily dose rate of atrazine to steady state plasma levels (Cpss)Can the model be used to predict plasma triazine levels reported inthe Thede 1987 study?animal 1 animal 2Atrazine dose rate (mg/kg/day) Cpss (mg/L) Cpss (mg/L)1 0.56 0.623 1.88 2.16Model Predicted0.491.47297 3.84 3.4510 5.11 4.8350 22.80 25.61100 53.10 56.843.434.8924.4748.93Outline•Re-cap of September 2010 SAP meeting: internal measureof exposure•Analysis of additional animal pharmacokinetic studies withradiolabeled atrazine•Human pharmacokinetic study with atrazine•Pharmacokinetic modeling approach•Onecompartment linear model-compartmentlinearmodel•Model evaluation•Understanding of pharmacokinetics, internal dosimetry, andUnderstandingofpharmacokinetics,internaldosimetry,andLH attenuation•Estimating the human intake dose rate through AUC30analysis of water chemographs•Summary and next stepsIntegrating Internal Dosimetry and LHAttenuation in RatsAttenuationinRatsAtrazineO ld( /d )



# I NOne-compartment linearmodel expression ford lOralgavagedose(mg/day)100%absorption



# N PUTAbsorbedDose(mg/day)(Dose rate)ratadult rats:AUC =Plasmatriazines (Cp,mg/L)Bodyvolumeofdistribution(Vd,L)clearanceEliminationkel (hr‐1)OUTPUTContinuedexposureVdssrat* kelratAUC=OUTPUTContinuedexposureSteadystate(Vdss,L)bioaccumulationSteadystateplasmatriazines (Cpss,mg/L)DailysteadystateAUC((mg/L)*hr)DurationLHattenuation31Relating Animal to Human SituationsAtrazineAnimal StudiesAtrazineHuman SituationOrally administered atconstant dose level &frequencyDrinking water withvariable dose level &and frequencyPharmacokineticsPharmacokineticsandfrequencyPharmacokineticsInternal Dosimetry(Atrazine & Metabolites)PharmacokineticsInternal Dosimetry(Atrazine & Metabolites)LH attenuation32LHattenuationHuman Internal Dosimetry ApproachWaterConsumption(L/day)Chlorotriazinesinwater(μg/L)One-compartment linearmodel with allometricallyscaled parameters for adultAbsorbeddose(mg)100%absorption



# INPUT(Dose rate)humanscaledparametersforadulthumans:AUC=clearanceEliminationkel (hr‐1)ContinuedexposureBodyvolumeofdistribution(Vd,L)*Plasmatriazine concentration(Cp,mg/L)Vdsshuman* kelhumanAllometricallyscaledAUCOUTPUTBioaccumulationEquivalenttimetosteadystate(Vdss,L)yfrom rat valuesAveragedailyplasmaconcentration(Cpave,mg/L))durationHumanaveragedailyAUC((mg/L)*hr)LHattenuation33HumanaveragedailyAUC((mg/L)hr)Outline•Re-cap of September 2010 SAP meeting: internal measureof exposure•Analysis of additional animal pharmacokinetic studies withradiolabeled atrazine•Human pharmacokinetic study with atrazine•Pharmacokinetic modeling approach•Onecompartment linear model-compartmentlinearmodel•Model evaluation•Understanding of pharmacokinetics, internal dosimetry, andUnderstandingofpharmacokinetics,internaldosimetry,andLH attenuation•Estimating the human intake dose rate through AUC34analysis of water chemographs•Apply methodologies to case studiesEstimating the Human Dose Rate•Variable dose rate and frequencyHypothetical WaterChemographHypotheticalWaterChemograph304050

e Levels01020

Triazin eCriticaldurationTime•At the April 2010 SAP meeting, the Panel recommended ...”the use ofan integrated (or daily average) internal measure along with a relatedanintegrated(ordailyaverage)internalmeasurealongwitharelateddrinking water exposure monitoring approach based on the area underthe concentration-time curve (AUC)...”•Integrated (or daily average) internal measure=plasma AUC35Integrated(ordailyaverage)internalmeasureplasmaAUC• Proposed approach for water exposure = water chemograph AUC(AUCwater)Estimating the Human Dose Rate

e Levels LevelsA BHypothetical Water Chemograph304050

L evelstime

Chlorotriazin e ChlorotriazinetimeCriticalduration0102030Time

Triazine LCritical durationdivided into trapezoidsas dictated by the samplingfe erequencyArea of trapezoid=average levels*durationAUCwaterfor given duration = Area sum of all trapezoidsAreaoftrapezoidaveragelevelsduration[atrazine]ave(mg/L)=AUCwater((mg/L)*hr)forgivenduration#days*24hrs(4)36Expression 4 gives a time-weighted average level of atrazine in water whichalong with water consumption informationresults in a human dose rate estimateEstimating Human Equivalent PlasmaAUCs From Water ChemographsHypothetical Water Chemograph304050

L evelsAUCsFromWaterChemographsChemographsCriticalduration0102030Time

Triazine L[atrazine]ave(ug/L) = AUCwater((mg/L)*hr) for given duration# days * 24 hrs[triazine water levels]ave(μg/L) * water consumption (L/day)kel (hr-1) * Vdss (L)=Human averagedaily plasma AUCAllometrically scaled from rat values37Summary•AUC: reasonable internal dose metric since duration isimportant for LH attenuationimportantforLHattenuation•Accumulation of plasma triazines topseudosteady stateconsistent with temporalityof LH attenuationy•Linear pharmacokinetics of plasma triazines acrossdifferent doses, studies, and species (including humans)support the use of a one-compartment linear model•Extrapolation to adult humans possible with allometricallyscaled parameters (Vd and kel)scaledparameters(Vdandkel)•Human dose rate estimated through AUC analysis of waterchemographs38•Apply methodology to case studies