Charge Question

FIFRA Scientific A

Panel
Charge Question 1.0

In 2003, the human health risk assessment was basaction in which exposure to atrazine leads to a redugonadotropin releasing hormone (GnRH) from the hthereby lessening the afternoon pituitary luteinizing surge in female Sprague Dawley rats. As a result, tlengthens. This, in turn, leads to increased estrogeincreased incidence of mammary tumors in female Srats. EPA determined that atrazine’s cancer mode opremature reproductive aging) in the Sprague-Dawto be operative in humans (a conclusion consistent recommendation in 2000). The Agency will be evalclassification further as the experimental toxicology integrated with the epidemiology literature; new extoxicology studies (Section 3.6 of the draft issue pasuggest a change to the Agency’s previous conclusi“not likely to be carcinogenic to humans.”
Charge Question 1.0 (

Although the cancer mode of action may noperative in humans, it is not unreasonablthat atrazine might cause adverse effects ohypothalamic-pituitary function in humanssame endocrine perturbations that induce may play a role in at least some reproductdevelopmental effects (not associated with aging) that may be relevant to humans. AAgency identified disruption of estrous cycdelays in puberty onset (males and femalea consequence of disruptions to the hypotpituitary-gonadal (HPG) axis as the critical concern (Sections 2.0 & 3.2 of the draft iss
Charge Question 1.0 (

The Agency continues to believe that this disHPG axis is critical to affecting estrous cyclicipuberty onset. However, as discussed in Qubelow (See Figure 3 of the draft issue paper)has been published that indicates a more expunderstanding of how atrazine may perturb tleading to reproductive effects. These new atrazine disrupts the hypothalamic-pituitary-aaxis altering the central nervous system’s conpituitary and adrenal which, in turn, disrupts Furthermore, a hypothesis for a direct effect the adrenal cortex and the gonads which masteroidogenesis directly is also supported by 
Charge Question 1.1

With the caveat that the review of tepidemiological literature is still on-gplease comment on the Agency’s pr

conclusion that new experimental davitro and in vivo laboratory animal snot support a change in the conclusthe 2003 risk assessment that atrazunlikely to be a human carcinogen.
Charge Question 1.2

Based on an evaluation of the studies examining the matrazine on neuroendocrine function (Section 3.2 of ththe Agency has preliminarily concluded that atrazine aaxis and the HPA axis. With respect to the temporal cstudies show that atrazine induces a rapid (within minACTH and adrenal cortical hormones (corticosterone aboth male and female rats (Fraites et al., 2009; Laws eet al., 2009), while changes in the HPG axis, such as tthe LH surge, may take up to 2-3 days (Cooper et al., sequence of HPA alterations followed by HPG changes suppression of the surge may be, in part, mediated by HPA axis (i.e., a corticosterone suppression of the GnRIn addition, with respect to dose-response concordancincreases in ACTH, corticosterone and adrenal progestfollowing a single dose of atrazine (50 mg/kg). In conot altered following a single dose up to 200 mg/kg. Htreated for multiple days, the dose necessary to alter tthan or equal to the one needed to affect the HPG axis
Charge Question 1.2 (

z Please comment on the Agency’s preliminary 

the mode of action involving atrazine’s alteratHPA and HPG axes. Does the document adeqclearly describe the hypothesis in the context pathway” (i.e. cellular response pathways thasufficiently perturbed, are expected to result effects)? Does the document clearly describe test the proposed hypothesis? To what extenavailable data establish key events in the proaction hypothesis? What are the strengths athe data available on this hypothesis. 

z Please include in your comments a discussion 

interpretations of the data linking the initial pHPA axis to changes in the HPG axis. 
Charge Question 1.2 (

Please comment on the evidence tha

perturbations in the HPA axis may leaimpairment in reproductive function adevelopmental consequences. Which is/are viewed as critical in leading to consequences? Are there data on othsubstances that would inform this qu
Charge Question 1.3

z The Agency has preliminarily conclude

directly targets cells within the HPA a3.2 & 3.3.2.3 of the draft issue paper) increased activity of the adrenal axis dnonspecific stress. Evidence for this cbased on the following observations:

 Several studies report increased adrenoc

hormone (ACTH) and corticosterone levefollowing a single exposure to atrazine anmetabolites (deisopropyl-atrazine, DIA; dDEA) but not diamino-s-chlorotriazine (D2009; Fraites et al., 2009; Pruett et al., 2

continued on the next page......
Charge Question 1.3 (

 A single intravenous administration of DE

et al., 2009) also causes an immediate inand adrenal hormone release, indicating tchlorotriazine-induced gastrointestinal disthe hormonal response.

 The adrenal corticosterone response to co

exposure to atrazine did not habituate in 2009; Laws et al., 2009) or mice (Pruett e

 Although, atrazine induced a rapid increa

prolactin secretion was not affected in the (Laws et al., 2009). This is unusual in thaboth ACTH and prolactin are typically obsto a number of physiological and psycholo
Charge Question 1.3 (

Please comment on the extent to w

available evidence supports the preconclusion that atrazine and its intemetabolites (DIA and DEA) induce cthe HPA axis as a result of a direct the HPA tissue and such changes ato a generalized or non-specific streresponse. Are there data on other that would support this conclusion?
Charge Question 1.4

Based on a review of in vitro studies evaluatinatrazine on estrogen production, the Agency hconcluded that atrazine does not have a direccatalytic activity of aromatase. However, with exposure (> 24 hrs) atrazine can cause increaestradiol production in the H295R and JEG-3 c(Sanderson et al., 2000; Laville 2006; Higley eThese changes in estrogen production have bwith increased cAMP and CYP19 mRNA (Sand2001) that are part of a complex mode of actiatrazine up-regulates the gene expression of apossibly other enzymes within the steroidogen(Section 3.3.2.3 & Figure 3, Arrow 7 of the drthis paragraph is continued on the next page...
Charge Question 1.4 (

The hypothesis that atrazine alters multiple stsupported by increased testosterone concentcells following exposure to atrazine (Higley et vitro studies demonstrating that atrazine affecsignal transduction pathways and/or transcripvariety of cell lines (Suzawa and Ingraham, 202008). Up- regulation of the expression of a gsteroidogenic genes has been observed in JEGand Ingraham, 2008). In vivo, there is little eatrazine alters aromatase per se (Modic, 2004evidence associating atrazine exposures to incestrogens appears to be related to a general igonadal and adrenal progesterone and andros2004; Laws et al., 2009). 
Charge Question 1.4 (

Please comment on the extent th

available data do or do not suppAgency’s preliminary conclusion atrazine has a general stimulatoron steroidogenesis, as opposed direct effect on aromatase. 
Charge Question 1.5

A series of studies evaluating the effect of peripubertal or gestational exposure of atramale rat indicate that atrazine decreases teconcentrations and may lower androgen deweights but that it has little effect on basal the male. This response generally occurs amg/kg and above. In contrast, atrazine in found to consistently increase steroid hormproduction. Thus, there is an apparent disbetween the in vivo and in vitro data. Howunderstanding of the interplay between the the gonadal axes may provide an explanati

this paragraph is continued on the next pag
Charge Question 1.5 (

At the doses used in the in vivo studies, atraactivation of the adrenal axis (increased ACTcorticosterone) and the increase in corticostehabituate (i.e., it does not lessen with repeatThere are a number of studies in the literatudemonstrating that prolonged corticosterone impair testosterone production by a direct acsteroidogenesis in the Leydig cells. Thereforeeffects on testosterone synthesis are likely thadrenal hormone-mediated down regulation (through corticotropin releasing hormone [CRof GnRH pulsatility) or directly on the testis (glucocorticoid receptor mediated change in s
Charge Question 1.5 (

Please comment on the degree to 

proposed mode of action (Figure 3 draft issue paper) provides a bioloplausible explanation for the decreserum and testicular testosterone in the in vivo studies. Please comthe extent that the available data not support this hypothesis. 
Charge Question 1.6

Based on a review of the neurotoxicity studies (Sectioissue paper), the Agency has preliminarily concluded studies provided further support for the concern that neurotransmission may be affected by atrazine, a conraised by studies prior to 2003. However, the Agency that several aspects of these studies of the dopaminepathways, in particular the changes noted in stereologand the observed changes in behavior, should be conpreliminary findings. This conclusion is based on the identified in the data including: (1) lack of clear doserelationships, (2) lack of inclusion of suitable positive the competency and reliability of the procedures utilizdopaminergic systems in the brain, (3) limited data to stereological findings, (4) limited or no supporting hisbehavioral assessments, and (5) no consideration of tthe HPA axis (e.g. alterations in corticosterone). this paragraph is continued on the next page
Charge Question 1.6 (

EPA has further determined that two non-dopamin

neurotoxicity studies--one on brain somatostatinerthe other on neurobehavior in mice -- also have silimitations (e.g., lack of details on source and puri

age and body weights of the mice; poor quality of silver staining photomicrographs; limited presentatranscriptase polymerase chain results; lack of dat

conclusion of neuronal degeneration; reference citsupport statements made in the text; no explanatiplausibility of alterations in somatostatinergic receinappropriate statistical evaluation of data (litter vdiscrepancies in number of pups examined for behand lack of objective and validated behavioral test

comment on these preliminary conclusions regardi

neurotoxicity findings. 
Charge Question 1.6 (

The Agency has preliminarily concluded that studies indicate that the neurotoxicity endpoare not more sensitive than those evaluated neuroendocrine function following atrazine eattenuation of LH surge and estrous cycle dfemale Sprague-Dawley rats which form the current chronic RfD). In addition, the Agencthat there is no association between atrazine development of Parkinson’s Disease on the bspecificity of effects on brain dopaminergic shistological and behavioral features characteParkinson’s Disease, and results of epidemioPlease comment on this preliminary conclus
Charge Question 1.7

Based on a review of two studies of the poteatrazine on the prostate (Section 3.3.2.2 of tpaper), the Agency has concluded that the reRayner et al. (2007) study of pregnant rats tgestation support previous observations (Stothat atrazine treatment to the rat dam either early postnatally can increase prostate weighincrease in inflammatory infiltrate) in the maThis effect on the offspring was shown to be suppression of prolactin in the atrazine-expolactation, and is consistent with the mode of atrazine on neuroendocrine function. 

this paragraph is continued on the next page
Charge Question 1.7 (

The Agency also preliminarily concluded thresearch is needed to provide more convinthat atrazine-mediated suppression of prosthe probasin/SV40T antigen transgenic rat dependent prostate cancer rodent model) be due to caloric restriction rather than endas postulated by Kandori et al. (2005). Pleon these preliminary conclusions regarding prostate findings.

Please comment on the extent to which the 

support the proposed mode of action for prthe appropriateness of the rodent model in human health. 
Charge Question 1.8 

Based on data obtained in studies to assesatrazine on the immune system following dand adult exposure, as well as in vitro mecstudies, EPA concluded that atrazine has thaffect the immune system. However, the Aconcluded that underlying mechanisms of amediated immunotoxicity and its relevance adverse health effects in humans are still nunderstood. It is well established that prodendocrine system modulate immune functiothis paragraph is continued on the next pag
Charge Question 1.8 (

However, adult rodent studies have not dewhether immune system effects are caused effects of atrazine and/or its metabolites or are the result of modulated endocrine hormproduction, which in turn affects immune cpublished studies indicate that exposure to immune system development may result in immune function in offspring, although no were drawn on the potential adversity of thbecause immunosuppression was observed and immunoenhancement was observed in 
Charge Question 1.8 (

Please comment on potential explanations 

disparate findings reported by Rooney et aal. Many immunotoxicologists consider immunomodulation, i.e., suppression or enimmune function, as a potentially adverse ahomeostasis, because both have been assodisease states. Thus, while the results RooRowe et al. may appear to be contradictorydata indicate unintended immunomodulatioapproximately the same dose in two specie

Please comment on characterizing the effec

gestational atrazine exposure as immunomwhen describing the immunotoxicological odevelopmental immunotoxicity studies.
Charge Question 1.8 (

Appendix B of the draft issue paper describconducted by EPA scientists on the potentiadevelopmental immunotoxicity of atrazine. the experiments provide conflicting results. studies did not provide a suitable explanatidifferences. The Agency believes both sets high quality. However, in the context of haassessment, such differences are difficult to Please comment on the information contain

B and provide suggestions for interpreting part of the atrazine re-evaluation.
Charge Question 1.8 (

The available data do not indicate thinduced immunotoxicity is a more seendpoint than the atrazine-induced eneuroendocrine function, (e.g., attensurge and estrous cycle disruptions wthe basis for the current chronic RfD) Sprague-Dawley rats. Please comme

degree to which these preliminary cosupported by the available data
Charge Question 1.9

After the April 2010 SAP, the Agency will evaluatevidence (WOE) for atrazine by integrating the etoxicology data with the epidemiological studies. work, the Agency will consider the available data changes and functional outcomes that may be usfor deriving PoDs across different durations of exdifferent populations (including potentially sensitImportant studies evaluating the dose-response hormones involved in the HPA axis are still on-goare expected to provide high quality data that cacharacterize the entire dose response curve. Witthese data are not available now, given the curreof atrazine’s mode of action, please comment on 

appropriate endpoints to consider for use in derivdeparture in a possible future risk assessment.

this paragraph is continued on the next page......
Charge Question 1.9 (

Given that the duration of the toxicological key factor in evaluating the frequency of drmonitoring, please include in your response 

consideration of the magnitude and duratiokey events in the toxicity pathway that are perturb normal function and comprise humdefining the exposure window of interest, pon the toxicokinetic/dynamic considerations to atrazine's effects on the HPA/HPG axis. 
Charge Question 2.0

In conjunction with the toxicological review pissue paper, the Agency has also discussed mevaluating the sampling frequency that is necdetermining, with confidence, concentrations in water that sources drinking water. These hdifferent methods for estimating pesticide conbetween known sampling events and examiniperformance of different sampling strategies fperiods of different durations. The Agency sefrom the Panel with regard to how the uncertvariability in both the monitoring data and in (i.e., point of departure) can be integrated to and to interpret the potential significance of aconcentrations in drinking water. 
Charge Question 2.1

Given the nature of the temporal patterns ooccurrence in surface waters described in Sincluding serial correlations from day to daelevated concentrations within seasons and year, below quantitation data, and uncertashape of the pesticide distributions in surfawhat statistical approaches should the Agedetermining confidence bounds on exposurfrom monitoring data? Please comment on approach may vary depending on the dura
Charge Question 2.2

The first two simulation methods preSection 5.5 are applicable to the specthey describe, although some generaregarding shape patterns appear to ethis information, please comment on 

strengths and weaknesses of the appon the practical merits of pursuing thother numerical approach with a larghigher concentration systems. Pleason how the methods for determining bounds might apply given these cons
Charge Question 2.3

As described in Section 5.4.2, the Agency ithe use of a confidence interval or predictioapproach to characterize the uncertainty of estimates derived from monitoring data of sampling frequencies. Please comment on 

and weaknesses of either placing confidencthe rolling average estimates and comparinlimit from monitoring against the level of coor, conversely, placing confidence bounds o
Charge Question 2.4

Please comment on the relative merits of the 

approaches the Agency described in Section 5interpolating pesticide concentrations betweeand, in particular, on the strengths and weakmethods as the frequency of samples decreas

Considering the health endpoint(s) being conatrazine, particularly data for the HPA axis, atime frame needed to induce the health effecshorter than that used in the 2003 risk assesscomment on the advantages and disadvantag

for evaluating the likely occurrence and expowater of short, moderate, and long duration c
Charge Question 2.4

Please comment on the Agency’s proposed a

evaluating these methods, as described in Swhat extent should the Agency consider othas the shape of the chemograph (Section 5.patterns, stream flow, and/or pesticide use evaluating the modeling approaches?