CHARGE QUESTIONSCHARGEQUESTIONSCharge Question 11. One approach for evaluating the performance of monitoring sampling designs is to simulatedifferent sampling frequency strategies using robust (daily or near-daily sampling during thehighuse/runoffperiod)monitoringdataAsnotedinpreviousSAPs(April2010andSeptemberuse/runoffperiod)monitoring.previousSeptember2010), Heidelberg University’s National Center for Water Quality Research (NCWQR)monitoring of selected watersheds in Ohio and the Atrazine Ecological Exposure MonitoringProgram (AEEMP) monitoring of headwater streams in the corn belt provide the most robustdatasetsforatrazineUsingexamplesfromthesedatasetstheUSEPApresentedamatrixdatasetsatrazine.examplesdatasets,presentedapproach for deriving a bias factor for different sampling frequencies (e.g., 4, 7, 14, 28 dayintervals) for use in estimating concentrations for different exposure durations of concern (e.g.,4, 14, 28, 90 day durations).a) Given that the factors are likely to vary based on watershed size and water body type,comment on the level of detail we would need to develop (e.g., flowing watervs.reservoir; smallvs.mediumvs. large watershed area). How many datasets would weneed to analyze to provide a reasonable representation of a bias factorforeachcategory?b)Pleasecommentontheadvantagesanddisadvantagesofderivingbiasfactorscommentadvantagesdisadvantagesderivingfactorsbased on analyses of individual sites and years compared to taking percentilesof averages across sites/years.2Chargeuestion 2Q2. The September 2010 FIFRA SAP on atrazine recommended combining a regressionmodelsuchasWARPwitheitheradeterministicmodelsuchasPRZMorageostatisticaldeterministicgeostatisticalapproach in order to estimate a time series of atrazine concentrations from less frequentmonitoring. In developing methods based on the SAP’s recommendations, the USEPAwas able to derive reasonable estimations of the time series for sampling intervals of 7-daysorshorterusingconditionalsimulationsofvariogrammodelswithoutincorporatingshorterconditionalsimulationsvariogramwithoutincorporatingadditional models. Although the simulations underestimated the 1-day maximums, theyappeared to provide reasonable bounds for rolling average concentrations as short as 4days.a) Please comment on the Agency’s method of estimating time series usingconditional simulations of variograms for monitoring data sets such as the AMPCWS monitoring that have 7-day sampling frequencies.b) Based on the USEPA’s analysis using WARP with longer duration samplingintervals (Appendix D.1), what advantages does the SAP see of including WARPmodelingin this approach(i.e.,betterestimation of the dailymaximum value)?3Charge Question 3ChargeQuestion33. Given that most monitoring data for pesticides are based on lessfrequentsamplingintervals(egbiweeklymonthly)theUSEPAalsofrequentsamplingintervals..,-,monthly),explored additional approaches that combined variogram models withcovariate approaches – in particular, correlation with flow within a narrowwindow–orwithWARP.Wealsoconsideredmethodsoffillingintimeconsideredmethodsfillingseries concentrations using a deterministic model such as PRZM or amass balance model, although such approaches would be moreresource/data intensive.a) Please comment on these additional modeling approaches forinterpretingsparse(lessfrequentlysampled)monitoringdatasets.interpretingfrequentlysampled)monitoringdatasets4Charge Question 44. In characterizing overall uncertainty inexposure estimates from monitoring data, theUSEPAmust also considerhow manyyears of monitoringare necessarytoprovide areasonable bound on the year-to-year variability or, alternatively, develop methods ofplacing the existing monitoring data in context of likely exposures that may occur over atime period of interest (for instance, 15 year cycles for registration review).a) Please comment on the sufficiency of existing atrazine/triazine monitoring dataavailable to the Agency – in particular the Atrazine Monitoring Program (AMP)coupled with the earlier Voluntary Monitoring Program (VMP), which conceivablyspanfrom1993tothepresentforsomeCWSforuseincharacterizingthelikelypresent–characterizingrange in year-to-year variability in atrazine/TCT concentrations.b) Please comment on the Agency’s suggestion for using a PRZM hybrid model,calibrated on the current years of monitoring, to provide estimates fora widertimeframe by modeling additional years using weather data that span a 30- to 50-yearperiod.c) What other possible approaches can the SAP recommend for capturing year-toyear variability?5Chargeuestion5uestionuestionuestionQ5. In 2000, the SAP agreed with the Agency’s proposal for atrazine’sneuroendocrine MOA, and they further concluded that it is unlikely that the mode ofactionbywhichatrazineinducesmammarytumorsinadultfemaleSpragueDawleyatrazineinducesmammarySpraguerats could be operational in humans. The SAP further concluded that it is notunreasonable to expect, however, that atrazine might cause adverse effects onhypothalamic-pituitary-gonadal (HPG) function in humans if exposures weresufficiently high and that perturbation of the HPG axis was relevant fordevelopmental and reproductive effects (FIFRA SAP, 2000). In the 2003 IRED, theAgency identified perturbations of the neuroendocrine system (particularly LHregulation)leadingtoreproductivetoxicityasthemostbiologicallyplausibleandregulation)leadingreproductivetoxicitybiologicallyplausiblesensitive effects attributable to atrazine exposure. The adverse outcomesoccurring as a consequence of disruptions to the HPG axis in rats includedisruption of estrous cyclicity and delays in puberty onset (males and females).Additi lff ttdi tlli k dtLHdi tiithd dn additionaleec–norecyneosrupon–seecreasedsuckling-induced prolactin release in milk early in life (perinatally), which leads toincreased incidence of prostatitis in young adult rats. All of these effects can belinkedtoand/oroccurathigherdosesthantheatrazine-inducedchangesinLHatrazineinducedchangessecretion.6ChargeQuestion 5(Contd.)uestion 5uestion 5uestion 5Q ( )Research conducted since the 2003 IRED continues to point to LH surge attenuationas the most sensitive effect in the atrazine database. Consequently, the Agency willtithiLHtiti ld itfHPGconnueo use cangesnsecreon as a senneenponorperturbations and the basis of the atrazine risk assessment. The September 2010SAP “ agreed with the Agency's conclusion that, based on the available data, abenhmrkdose(BMDmodelfromdatonsupprinof theLHsurappearc a)edaess ogesto be protective for other endpoints, since this phenomenon occurs at doses lowerthan for the wide range of effects identified in a rather extensive toxicologicaldatabase.”a) Currently available data show that in the rat a brief exposure (as brief as 4days) to low levels of atrazine can elicit decreases in LH. Please commentonthebiologicalplausibilityofthesebriefchangesleadingtoanadversebiologicalplausibilitychangesleadingadverseoutcome taking into account typical variability and how long and how much aLH surge reduction is needed to cause the observed adverse effects (i.e.,disruptions in cyclicity, delayed puberty, and prostatitis).7Chargeuestion 6Q6. During the September 2010 SAP, the Panel raised the issue of singlevs.multipleatrazine exposure effects on the LH surge. In theirreport to the Agencthe SAPycommented:“Data are clear in identifying that a greater-than-one pulse of exposure to atrazine isnecessary forattenuation of the LH surge. Forexample, single high doses (over100mg/kg) administered on the morning of proestrus did not alter characteristics of theLH surge occurring later the same day. Additional data clearly demonstrate a oncedailydosefor4daysandbeginningonestruscaninducesignificantinhibitionofthebeginningsignificantinhibitionLH surge peak. In this instance, a dose response is observed. However, what is notclear is if less than 4, but greater than 1 days’ exposure is sufficient to alter the LHsurge. Further complicating the matter, it is not clear if a 4-day exposure, beginningdiff tdfthlillltihithLHi iltthon aerenay oe cyce, wresun cangesnesurge smaroosewhen dosing begins on the morning of proestrus. Understanding of the relationshipbetween duration of exposure and phase of the cycle will be key in translating rodentdata to humans forrisk assessmentpuroses.”p8ChargeQuestion 6(Contd.)uestion 6uestion 6uestion 6Q ( )In response to the Panel’s comments, EPA scientists in the Office ofResearchandDevelopmenthaveundertakenaseriesofexperimentstoResearchDevelopmentundertakenexperimentstry to elucidate the nexus between phase of the cycle and duration ofexposure. This research is in the early stages. Initial results suggestthat a single high dose of atrazine (100 mg/kg bw) can affect the LHsurge. However, the effect seen was an increase in LH rather than thedecrease observed after 4 days of exposure.a) Please comment on the potential relevance of one day exposureto elicit an adverse outcome(s) and the significance of an increasevs.a decrease in LH.9Chargeuestion 7Q7. An increased incidence of prostatitis has been observed in the offspringof rats exposed to atrazine from PND 1 to 4. This effect was not linked toan LH alteration, but ratherthe atrazine related suppression ofsucklinginduced prolactin release in the lactating dams. Prolactin plays a crucialrole in the neonatal brain for normal TIDA neuron development. In thed ltff ithi idTIDAl tiifl t dbl tdauosprng,emparedreguaons reecey eevaeprolactin levels (hyperprolactinemia). It is this elevated level of circulatingprolactin in the adult male rats that has been linked to an increasedincidenceofprostatitisItisunknownwhentheTIDAneuronsdevelopinincidenceprostatitis.unknownneuronsdevelopthe human fetus or whether this development is dependent on thematernal prolactin concentrations.a) Given the biological processes involved in the atrazine-mediatedprostatitis in rats, please comment on the human relevance of thesefindingsinratsfortheoverallhazardcharacterizationforatrazinefindingsoverallcharacterizationatrazine.10Charge Question 88. When evaluating the data on mammary gland development, the September 2010 SAPreport concluded that the “use of the existing data on rat mammary gland development toassessthepotentialhumanriskofatrazineisnotwarrantedatthepresenttime”Regardingpotentialatrazinewarrantedpresent.Regardingthe methodological differences between the Rayneret al.,and the Coder studies, the Panelcommented that “it is surprising that they did not employ both qualitative and quantitativescoring measures of mammary gland development, which would have provided a definitiveinterstudycomparison”InresponsetothiscommenttheAgencyhasconductedasetof-comparison.responsecomment,conductedexperiments investigating the potential impact ofin uteroatrazine exposure on mammarygland development (MGD) in Sprague Dawley rats using both the subjective scoringmethodology described by Rayneret al.and a computer-based quantitative methodology(morphometricanalysis)Usingeitherquantitativeorsubjectivemeasurestoevaluatemorphometricanalysis).quantitativesubjectivemeasuresevaluatemammary gland development, no differences in MGD were found between control oratrazine-exposed rats indicating that gestational atrazine exposure had no demonstrableeffect on normal mammary gland development.a) Please comment on the Agency’s findings in addressing the issues raised by the SAPduring the September 2010 meeting. Please comment on whether this study (alongwith the negative studies by Coder) adds to the weight of evidence that it is unlikelythat atrazine impacts mammary gland development.11Chargeuestion 9Q9. In 2003, an FQPA Safety Factor was retained in part to address the lackof data evaluating the potential for differences in sensitivity across lifestages. Since then, multiple studies evaluating atrazine’s potential impactfollowing exposure during the prenatal, perinatal, prepubertal, and adulttflifhbd tdtddthd tNfsages oeaveeen conuceo aresseseaa gaps.one othe new studies has identified an enhanced sensitivity in the young and theLH surge attenuation observed in females of reproductive age continues tobethemostsensitiveendpoint.a) Please comment on the weight of the evidence analysis conductedbthAdtht tthi htht i tiltdtyegency ane exeno wce unceranes reaeothe potential for differential sensitivity of the young are addressedwith the additional data.12Chargeuestion 10Q10. Section 3.0 and Appendices B-3 through B-5 of the draft Issue Paperprovide the Agency’s reviews and synthesis of the atrazine cancerepidemiology studies. These studies examine a variety of cancer endpoints,notably reproductive and endocrine system tumors including prostate,b tidth idtfthl h h t i tireas, ovaran anyroumors, cancers oelymphohematopoieticsystem including non-Hodgkin lymphoma (NHL), and other cancer typesincluding glioma, pediatric cancers and colon cancer. Section 3.3 integratesthefindingsoftheepidemiologyandexperimentaltoxicologystudiesfindingsepidemiologyexperimentaltoxicologystudies.a) Please comment on the sufficiency of the Agency’s cancerid i liithttid tif ithjt thepemoogy revews wrespecoidentifyinge maorsrengsand limitations of each study, and overall synthesis of results bycancer types.13Chargeuestion 11Q11. There is no compelling evidence in the available experimental toxicologydatabasethatatrazinemaybecarcinogenicinhumansThisdatabasewasrevieweddatabaseatrazinecarcinogenic.databasereviewedby the SAP in April 2010. Briefly, the results of both guideline and non-guidelineinvivoas well asin vitrostudies do not suggest atrazine has mutagenic orcarcinogenic properties. With regard to mammarytumors, several internal andexternal peer reviews have concluded that the mode of action through whichatrazine influences mammary tumor development is not operational in humans.Further, EPA’s current review of the atrazine cancer epidemiology database did notidentifyevidenceacrossanyofthecancerspecificdatabasesevaluatedastotheidentifyevidence-specificdatabasesevaluatedcarcinogenic potential of atrazine that EPA finds sufficiently convincing to change itsconclusions. Therefore, in view of the evidence in the experimental toxicology andepidemiologic databases, EPA concludes atrazine is not likely to be carcinogenic inthe human population. The observational data lend furthersupport forthe humanrelevance of the laboratory rodent tumor findings,i.e., the databases lack evidenceof an association between atrazine and cancer in the human population.14Charge Question 11 (Contd.)a) Please comment on the extent to which the scientific informationsupports the integrative analysis contained in Section 3.3 withrespect to the similarities, differences of the experimental toxicologydid i l ifi diPlti ifi tanepidemiologicnngs.ease commenon any significantuncertainties in the epidemiologic findings.b) Please comment on whether the epidemiology literature publishedsince the last SAP review including the AHS findings is sufficient tojustify changing the Agency’s conclusions that atrazine is not likelyto be carcinogenic to humans.15Chargeuestion 12Q12. The proposed refined dose response assessment for atrazine will be based on internalfAtthSt b2010SAPtithAt dmeasures oexposure.eepemermeeng,egency preseneestimates of area under the concentration-timecurve (AUC) for plasma triazines based on arat [14C]-atrazine study (i.e., Thede 1987). The Panel concluded that “...on the basis of thecurrently available data, plasma appears to be a reasonable biological compartment that isfl tiftiddth tfdthlt titireecve ossueose, anause oarea unere pasma concenraonme curve(AUC) provides an appropriate measure of internal exposure.” The Panel also noted that “theuse of total chlorotriazine based on total14C-compounds is a reasonable first step(particularly in the absence of information on the pharmacodynamic activity of the parentddi di id lt b lit )HthP lb li dth tthTh d(1987)compounanindividualmetabolites).owever,eaneeeveaeeedata, had limitations, and should be interpreted with caution.” In response to the commentsby the Panel, the Agency has evaluated additional pharmacokinetic studies involving differentspecies including humans and two additional rat14C-atrazine studies that support the plasmalti tbt i dfthTh d1987t dB dthit tlicearance esmaes oaneromeeesuy.aseone conssennearpharmacokinetic behavior of plasma triazines resulting from orally administered doses ofatrazine across the different studies, the Agency proposes to use an interim pharmacokineticmodeling approach based on a one-compartment linear model to inform internal dosimetryth ttd tld ibthh ki tiflti iitfaseemso aequaelyescree pharmacokinetics opasmaraznesn rasromorally administered atrazine.16Charge Question 12 (Contd.)a) Please comment on the strengths and limitations associated with this simplifiedpharmacokineticmodelingapproachforhumanextrapolationpharmacokineticmodelingapproachextrapolation.b) Compare and contrast the strengths and weaknesses of using total radioactivity for apharmacokinetic analysis (as presented in the Agency’s issue paper) as opposed tofusing available pharmacokinetic dataorthe parent and the chloro-s-triazinemetabolites that have similar toxicological properties to the parent?c)Aspointed out in theAgencyissuepaper,we are still reviewinga PBPK modelyp ,submitted by Syngenta. As we complete our review of the Syngenta model, pleasecomment on key aspects that EPA should be considering concerning a PBPK modelincluding model credibility (e.g., structure, parameter values, documentation), modelreliability(e.g.,how well does the model simulate the dose metrics relevant to themode of action), and model applicability (e.g., does the model have essential featuresfor intended application).d)Pleasecommentontheextenttowhichtheonecompartmentlinearmodeloftotalcomment-compartmentplasma radioactivity derived from14C labeled atrazine may account for interspeciesdifferences in pharmacokinetics.17Charge Question 1313. Any risk assessment of atrazine ideally should account for the temporal relationship betweenexposure and toxicological endpoint(i.e.,LH attenuation). TheAgencyis relyingon rat studiesyginvolving constant dosing frequency and dose level to elucidate this relationship. The time-to-effectin rats appears to be based on the build-up of plasma triazines topseudosteady state (i.e.,plasmatriazine levels stay within a constant range). The Agency is using this information to estimate theallometricallyscaled equivalent human time-topseudosteadystateplasma triazine levels.However, the Agency is cognizant that human exposure is different from controlled rat studies inthat both the dose level and the frequency of exposure through drinking water are variable. Thus,the Agency has identified several possible durations of human exposure, which collectively bracketthe exposure intervals of concern forLH attenuation in humans:• 28 days: a duration predicted by allometrically scaling the rat plasma elimination kinetics. In both ratsand humans, the time estimated to reachpseudosteady state plasma triazines coincides with therespective length of their ovarian cycles(i.e., 4 and 28 days, respectively).y)• 14 days: Proposed to serve as a midpoint between the other two durations for better characterization ofwater monitoring frequencies.• 4 days: the duration of exposure needed to reach pseudo steady state in rats and corresponding to thefollicular phase in the human menstrual cycle.18ChargeQuestion 13(Contd.)uestion 13uestion 13uestion 13Q ( )a)Please comment on the rationale used bythe Agencforselectingtheseyexposure duration options. Please discuss the rationale for other alternativedurations of concern, if any.b) Please comment on which exposure duration in humans most closelycorresponds to the exposure duration found to cause adverse effects in rats.c) Please comment on the approach used by the Agency (i.e.,the onecompartment linear model) to relate atrazine levels from the waterchemographs to predict corresponding human plasma triazine levels for theproposeddurationsofconcern.Inparticular,pleasecommentontheproposeddurationsconcernparticular,commentAgency’s proposed approach to use water AUC estimates to calculate atime-weighted daily average of atrazine exposure for a given duration ofconcern. Please suggest alternative approaches as appropriate.19Charge Question 1414. For the case study, the Agency used the 95thand 5thpercentile ofconditionalsimulationsofdailyconcentrationsfromvariogrammodelsconditionalsimulationsconcentrationsvariogrambased on 7-day sampling intervals to predict human plasma AUC fortriazines. The estimations of daily concentrations from weekly samplingwereusedtocalculatetherollingaverageconcentrationsforthepotentialcalculaterollingaverageconcentrationspotentialcritical window of exposure from 4 to 28 days. The simulation modelsunderestimated a single day peak concentration but appear to providereasonable approximations ofrollingaverage concentrations withdurations as short as 4 days as well as the length of time of the potentialexceedance.a) Please comment on the use of a 95thpercentile of the conditionalsimulations for providing an upper bound on rolling averageconcentrations in the case study.20