Charge Questions to the 

# FIFRA Scientific AdvisoryPanel2 

# Charge Question 1.0In 2003, the human health risk assessment was based on a mode ofaction in which exposure to atrazine leads to a reduced release ofgonadotropin releasing hormone(GnRH) from the hypothalamusthereby lessening the afternoon pituitary luteinizing hormone (LH)surge in female Sprague Dawley rats. As a result, the estrus cyclelengthens. This, in turn, leads toincreased estrogen levels and anincreased incidence of mammary tumors in female Sprague Dawleyrats. EPA determined that atrazine’s cancer mode of action (i.e.,premature reproductive aging) in the Sprague-Dawley rat is not likelyto be operative in humans (a conclusion consistent with the SAPrecommendation in 2000). The Agencywill be evaluating the cancerclassification further as the experimental toxicology data areintegrated with the epidemiologyliterature; new experimentaltoxicology studies (Section 3.6 ofthe draft issue paper) do notsuggest a change to the Agency’s previous conclusion that atrazine is“not likely to be carcinogenic to humans.”3 

# Charge Question 1.0 (2)Although the cancer mode of action may not beoperative in humans, it is not unreasonable to assumethat atrazine might cause adverse effects onhypothalamic-pituitary function in humans. Thus, thesame endocrine perturbations that induce tumors in ratsmay play a role in at least some reproductive/developmental effects (not associated with reproductiveaging) that may be relevant to humans. Accordingly, theAgency identified disruption of estrous cyclicity anddelays in puberty onset (males and females) occurring asa consequence of disruptions to the hypothalamicpituitary-gonadal (HPG) axis as the critical endpoints ofconcern (Sections 2.0 & 3.2 of the draft issue paper).4 

# Charge Question 1.0 (3)The Agency continues to believe that this disruption of theHPG axis is critical to affecting estrous cyclicity and delayedpuberty onset. However, as discussed in Questions 1.2-1.6below (See Figure 3 of the draft issue paper), new informationhas been published that indicates a more expandedunderstanding of how atrazine may perturb the pathwayleading to reproductive effects. These new data indicate thatatrazine disrupts the hypothalamic-pituitary-adrenal (HPA)axis altering the central nervous system’s control of thepituitary and adrenal which, in turn, disrupts the HPG axis.Furthermore, a hypothesis for a direct effect of atrazine onthe adrenal cortex and the gonads which may impactsteroidogenesis directly is alsosupported by the available data.5 

# Charge Question 1.1With the caveat that the review of theepidemiological literature is still on-going,please commenton the Agency’s preliminaryconclusion that new experimental data from invitro and in vivo laboratory animal studies donot support a change in the conclusions fromthe 2003 risk assessment that atrazine isunlikely to be a human carcinogen.6 

# Charge Question 1.2Based on an evaluation of the studies examining the mode of action ofatrazine on neuroendocrine function(Section 3.2 of the draft issue paper),the Agency has preliminarily concluded that atrazine affects both the HPGaxis and the HPA axis. With respectto the temporal concordance, recentstudies show that atrazine induces a rapid (within minutes) increase inACTH and adrenal cortical hormones (corticosterone and progesterone) inboth male and female rats (Fraites et al., 2009; Laws et al., 2009; Pruettet al., 2009), while changes in the HPG axis, such as the suppression ofthe LH surge, may take up to 2-3 days (Cooper et al., 2009). Thissequence of HPA alterations followedby HPG changes indicates that thesuppression of the surge may be, in part, mediated by activation of theHPA axis (i.e., a corticosterone suppression of the GnRH and LH release).In addition, with respect to dose-response concordance, atrazine-inducedincreases in ACTH, corticosterone and adrenal progesterone are seenfollowing a single dose of atrazine (50 mg/kg). In contrast, the HPG isnot altered following a single dose up to 200 mg/kg. However, whentreated for multiple days, the dose necessary to alter the HPA axis is lowerthan or equal to the one needed to affect the HPG axis.7 

# Charge Question 1.2 (2)zPlease commenton the Agency’s preliminary hypothesis forthe mode of action involving atrazine’s alteration of both theHPA and HPG axes. Does the document adequately andclearly describe the hypothesis in the context of a “toxicitypathway” (i.e. cellular response pathways that, whensufficiently perturbed, are expected to result in adverse healtheffects)? Does the document clearly describe the data used totest the proposed hypothesis? To what extent do theavailable data establish key events in the proposed mode ofaction hypothesis? What arethe strengths and limitations ofthe data available on this hypothesis.zPlease includein your comments a discussion of the Agency’sinterpretations of the data linking the initial perturbations inHPA axis to changes in the HPG axis.8 

# Charge Question 1.2 (3)Please commenton the evidence that the initialperturbations in the HPA axis may lead toimpairment in reproductive function and/ordevelopmental consequences. Which event(s)is/are viewed as critical in leading to healthconsequences? Are there data on othersubstances that would inform this question?9 

# Charge Question 1.3zThe Agency has preliminarily concluded that atrazinedirectly targets cells within the HPA axis (Sections3.2 & 3.3.2.3 of the draft issue paper) and that theincreased activity of the adrenal axis does not reflectnonspecific stress. Evidence for this conclusion isbased on the following observations:Several studies report increased adrenocorticotropichormone (ACTH) and corticosterone levels immediatelyfollowing a single exposure to atrazine and its twometabolites (deisopropyl-atrazine, DIA; deethyl-atrazine,DEA) but not diamino-s-chlorotriazine (DACT; Laws et al.,2009; Fraites et al., 2009; Pruett et al., 2009). 

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# Charge Question 1.3 (2)A single intravenous administration of DEA in vivo (Fraiteset al., 2009) also causes an immediate increase in ACTHand adrenal hormone release, indicating that achlorotriazine-induced gastrointestinal distress is not drivingthe hormonal response.The adrenal corticosterone response to continued oralexposure to atrazine did not habituate in rats (Fraites et al.,2009; Laws et al., 2009) or mice (Pruett et al., 2009).Although, atrazine induced a rapid increase in ACTH,prolactin secretion was not affected in the same animals(Laws et al., 2009). This is unusual in that an increase inboth ACTH and prolactin are typically observed in responseto a number of physiological and psychological stressors.11 

# Charge Question 1.3 (3)Please commenton the extent to which theavailable evidence supports the preliminaryconclusion that atrazine and its intermediatemetabolites (DIA and DEA) induce changes inthe HPA axis as a result of a direct action onthe HPA tissue and such changes are not dueto a generalized or non-specific stressresponse. Are there data on other substancesthat would support this conclusion?12 

# Charge Question 1.4Based on a review of in vitro studies evaluating the effects ofatrazine on estrogen production, the Agency has preliminarilyconcluded that atrazine does not have a direct effect on thecatalytic activity of aromatase. However, with continuedexposure (> 24 hrs) atrazine can cause increased estrone andestradiol production in the H295R and JEG-3 cell lines(Sanderson et al., 2000; Laville 2006; Higley et al., 2010).These changes in estrogen production have been associatedwith increased cAMP and CYP19 mRNA (Sanderson et al., 2000,2001) that are part of a complex mode of action through whichatrazine up-regulates the gene expression of aromatase andpossibly other enzymes within the steroidogenic pathway(Section 3.3.2.3 & Figure 3, Arrow 7 of the draft issue paper). 

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# Charge Question 1.4 (2)The hypothesis that atrazine alters multiple steroids issupported by increased testosterone concentrations in H295Rcells following exposure to atrazine (Higley et al., 2010), and invitro studies demonstrating that atrazine affects a number ofsignal transduction pathways and/or transcription factors in avariety of cell lines (Suzawa and Ingraham, 2008; Albanito et al.,2008). Up- regulation of the expression of a group of majorsteroidogenic genes has been observed in JEG-3 cells (Suzawaand Ingraham, 2008). In vivo, there is little evidence thatatrazine alters aromatase per se (Modic, 2004) and theevidence associating atrazine exposures to increases in serumestrogens appears to be related to a general increase ingonadal and adrenal progesterone and androstanedione (Modic2004; Laws et al., 2009).14 

# Charge Question 1.4 (3)Please commenton the extent that theavailable data do or do not support theAgency’s preliminary conclusion thatatrazine has a general stimulatory effecton steroidogenesis, as opposed to adirect effect on aromatase.15 

# Charge Question 1.5A series of studies evaluating the effect of eitherperipubertal or gestational exposure of atrazine in themale rat indicate that atrazine decreases testosteroneconcentrations and may lower androgen dependent tissueweights but that it has little effect on basal LH secretion inthe male. This response generally occurs at doses of 50mg/kg and above. In contrast, atrazine in vitro has beenfound to consistently increase steroid hormoneproduction. Thus, there is an apparent discrepancybetween the in vivo and in vitro data. However, ourunderstanding of the interplay between the adrenal andthe gonadal axes may provide an explanation. 

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# Charge Question 1.5 (2)At the doses used in the in vivo studies, atrazine induces anactivation of the adrenal axis (increased ACTH andcorticosterone) and the increase in corticosterone does nothabituate (i.e., it does not lessen with repeated dosing).There are a number of studies in the literaturedemonstrating that prolonged corticosterone stimulation willimpair testosterone production by a direct action onsteroidogenesis in the Leydig cells. Therefore, the in vivoeffects on testosterone synthesis are likely the result of anadrenal hormone-mediated down regulation of HPG axis(through corticotropin releasing hormone [CRH] modulationof GnRH pulsatility) or directly on the testis (through aglucocorticoid receptor mediated change in steroidogenesis).17 

# Charge Question 1.5 (3)Please commenton the degree to which theproposed mode of action (Figure 3 of thedraft issue paper) provides a biologicallyplausible explanation for the decrease inserum and testicular testosterone identifiedin the in vivo studies. Please comment onthe extent that the available data do or donot support this hypothesis.18 

# Charge Question 1.6Based on a review of the neurotoxicity studies (Section 3.4 of the draftissue paper), the Agency has preliminarily concluded that several recentstudies provided further supportfor the concern that dopaminergicneurotransmission may be affected by atrazine, a concern that wasraised by studies prior to 2003. However, the Agency has also concludedthat several aspects of these studies of the dopaminergic neuronalpathways, in particular the changesnoted in stereological evaluationsand the observed changes in behavior, should be considered aspreliminary findings. This conclusionis based on the notable limitationsidentified in the data including:(1) lack of clear dose-responserelationships, (2) lack of inclusion ofsuitable positive controls to confirmthe competency and reliability of the procedures utilized in examiningdopaminergic systems in the brain, (3) limited data to corroboratestereological findings, (4) limited orno supporting histological andbehavioral assessments, and (5) no consideration of the potential role ofthe HPA axis (e.g. alterations in corticosterone). 

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# Charge Question 1.6 (2)EPA has further determined that two non-dopaminergicneurotoxicity studies--one on brain somatostatinergic systems andthe other on neurobehavior in mice -- also have significantlimitations (e.g., lack of details onsource and purity of atrazine, andage and body weights of the mice; poor quality of amino cupricsilver staining photomicrographs; limited presentation of reversetranscriptase polymerase chain results; lack of data to corroborateconclusion of neuronal degeneration; reference citations did notsupport statements made in the text; no explanation of biologicalplausibility of alterations in somatostatinergic receptor subtypes;inappropriate statistical evaluation of data (litter vs. pup);discrepancies in number of pups examined for behavioral endpoints,and lack of objective and validated behavioral tests). Pleasecommenton these preliminary conclusions regarding theneurotoxicity findings.20 

# Charge Question 1.6 (3)The Agency has preliminarily concluded that the availablestudies indicate that the neurotoxicity endpoints examinedare not more sensitive than those evaluated forneuroendocrine function following atrazine exposure (e.g.,attenuation of LH surge and estrous cycle disruptions infemale Sprague-Dawley rats which form the basis for thecurrent chronic RfD). In addition, the Agency has concludedthat there is no association between atrazine exposure anddevelopment of Parkinson’s Disease on the basis of nonspecificity of effects on braindopaminergic systems, lack ofhistological and behavioral features characteristic ofParkinson’s Disease, and results of epidemiological studies.Please commenton this preliminary conclusion.21 

# Charge Question 1.7Based on a review of two studies of the potential effects ofatrazine on the prostate (Section 3.3.2.2 of the draft issuepaper), the Agency has concluded that the results of theRayner et al. (2007) study of pregnant rats treated duringgestation support previous observations (Stoker et al., 1999)that atrazine treatment to the rat dam either perinatally orearly postnatally can increase prostate weights (due to anincrease in inflammatory infiltrate) in the male offspring.This effect on the offspring was shown to be due to asuppression of prolactin in the atrazine-exposed dams duringlactation, and is consistent with the mode of action ofatrazine on neuroendocrine function. 

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# Charge Question 1.7 (2)The Agency also preliminarily concluded that furtherresearch is needed to provide more convincing evidencethat atrazine-mediated suppression of prostate cancer inthe probasin/SV40T antigen transgenic rat (androgendependent prostate cancer rodent model) may possiblybe due to caloric restriction rather than endocrine-relatedas postulated by Kandori et al. (2005). Please commenton these preliminary conclusions regarding the recentprostate findings.Please commenton the extent to which the available datasupport the proposed mode of action for prostatitis andthe appropriateness of the rodent model in the context ofhuman health.23 

# Charge Question 1.8Based on data obtained in studies to assess the effects ofatrazine on the immune system following developmentaland adult exposure, as well as in vitro mechanisticstudies, EPA concluded that atrazine has the potential toaffect the immune system. However, the Agency has alsoconcluded that underlying mechanisms of atrazinemediated immunotoxicity and its relevance to potentialadverse health effects in humans are still not thoroughlyunderstood. It is well established that products of theendocrine system modulate immune function. 

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# Charge Question 1.8 (2)However, adult rodent studies have not determinedwhether immune system effects are caused by directeffects of atrazine and/or its metabolites or whether theyare the result of modulated endocrine hormoneproduction, which in turn affects immune cells. Twopublished studies indicate that exposure to atrazine duringimmune system development may result in alteredimmune function in offspring, although no conclusionswere drawn on the potential adversity of the effects,because immunosuppression was observed in one studyand immunoenhancement was observed in the other.25 

# Charge Question 1.8 (3)Please commenton potential explanations for thedisparate findings reported by Rooney et al. and Rowe etal. Many immunotoxicologists considerimmunomodulation, i.e., suppression or enhancement ofimmune function, as a potentially adverse alteration ofhomeostasis, because both have been associated withdisease states. Thus, while the results Rooney et al. andRowe et al. may appear to be contradictory, together thedata indicate unintended immunomodulation atapproximately the same dose in two species.Please commenton characterizing the effects ofgestational atrazine exposure as immunomodulation,when describing the immunotoxicological outcome ofdevelopmental immunotoxicity studies.26 

# Charge Question 1.8 (4)Appendix B of the draft issue paper describes experimentsconducted by EPA scientists on the potentialdevelopmental immunotoxicity of atrazine. The results ofthe experiments provide conflicting results. Additionalstudies did not provide a suitable explanation of thedifferences. The Agency believes both sets of data are ofhigh quality. However, in the context of hazardassessment, such differences are difficult to interpret.Please commenton the information contained in AppendixB and provide suggestions for interpreting such data aspart of the atrazine re-evaluation.27 

# Charge Question 1.8 (5)The available data do not indicate that atrazineinduced immunotoxicity is a more sensitiveendpoint than the atrazine-induced effects onneuroendocrine function,(e.g., attenuation of LHsurge and estrous cycle disruptions which formthe basis for the current chronic RfD) in femaleSprague-Dawley rats. Please commenton thedegree to which these preliminary conclusions aresupported by the available data28 

# Charge Question 1.9After the April 2010 SAP, the Agency will evaluate the weight ofevidence (WOE) for atrazine by integrating the experimentaltoxicology data with the epidemiological studies. As part of thiswork, the Agency will considerthe available data on hormonalchanges and functional outcomesthat may be used as endpointsfor deriving PoDs across different durations of exposure and fordifferent populations (including potentially sensitive ones).Important studies evaluating thedose-response relationships forhormones involved in the HPA axisare still on-going. These studiesare expected to provide high quality data that can be used tocharacterize the entire dose response curve. With the caveat thatthese data are not available now,given the current understandingof atrazine’s mode of action, please commenton what would beappropriate endpoints to considerfor use in deriving points ofdeparture in a possible future risk assessment. 

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# Charge Question 1.9 (2)Given that the duration of the toxicological concern is akey factor in evaluating the frequency of drinking watermonitoring, please includein your response aconsideration of the magnitude and duration of changes inkey events in the toxicity pathway that are sufficientperturb normal function and comprise human health. Indefining the exposure window of interest, please commenton the toxicokinetic/dynamic considerations with respectto atrazine's effects on the HPA/HPG axis.30 

# Charge Question 2.0In conjunction with the toxicological review presented in theissue paper, the Agency has also discussed methods for reevaluating the sampling frequency that is necessary fordetermining, with confidence, concentrations of the pesticidein water that sources drinking water. These have includeddifferent methods for estimating pesticide concentrationsbetween known sampling events and examining theperformance of different sampling strategies for averagingperiods of different durations. The Agency seeks feedbackfrom the Panel with regard to how the uncertainty andvariability in both the monitoringdata and in the toxicity data(i.e., point of departure) can beintegrated to characterizeand to interpret the potential significance of atrazineconcentrations in drinking water.31 

# Charge Question 2.1Given the nature of the temporal patterns of pesticideoccurrence in surface waters described in Section 5.2,including serial correlations from day to day, periodicity inelevated concentrations within seasons and from year toyear, below quantitation data, and uncertainty in theshape of the pesticide distributions in surface waters--what statistical approaches should the Agency consider indetermining confidence bounds on exposure estimatesfrom monitoring data? Please commenton how theapproach may vary depending on the duration of concern.32 

# Charge Question 2.2The first two simulation methods presented inSection 5.5 are applicable to the specific data setsthey describe, although some generalitiesregarding shape patterns appear to exist. Giventhis information, please commenton thestrengths and weaknesses of the approaches andon the practical merits of pursuing them or someother numerical approach with a larger set ofhigher concentration systems. Please commenton how the methods for determining confidencebounds might apply given these considerations.33 

# Charge Question 2.3As described in Section 5.4.2, the Agency is consideringthe use of a confidence interval or prediction intervalapproach to characterize the uncertainty of exposureestimates derived from monitoring data of varyingsampling frequencies. Please commenton the strengthsand weaknesses of either placing confidence bounds onthe rolling average estimates and comparing the upperlimit from monitoring against the level of concern (LOC)or, conversely, placing confidence bounds on the LOC.34 

# Charge Question 2.4Please commenton the relative merits of the various modelingapproaches the Agency described in Section 5.4.1 and 5.6 forinterpolating pesticide concentrations between sampling pointsand, in particular, on the strengths and weaknesses of thesemethods as the frequency of samples decreases.Considering the health endpoint(s) being considered foratrazine, particularly data for the HPA axis, and the exposuretime frame needed to induce the health effect(s) which isshorter than that used in the 2003 risk assessment, pleasecommenton the advantages and disadvantages of each modelfor evaluating the likely occurrence and exposure via drinkingwater of short, moderate, and long duration concentrations.35 

# Charge Question 2.4Please commenton the Agency’s proposed approach forevaluating these methods, as described in Section 5.7.1. Towhat extent should the Agency consider other factors, suchas the shape of the chemograph (Section 5.5.3), weatherpatterns, stream flow, and/or pesticide use patterns inevaluating the modeling approaches?