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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF 

PREVENTION, PESTICIDES AND 

TOXIC SUBSTANCES 

MEMORANDUM

DATE: April 5, 2010

SUBJECT: Charge questions for the FIFRA Scientific Advisory Panel meeting 

on “Re-Evaluation of Human Health Effects of Atrazine: Review of 

Experimental Animal and In Vitro Studies and Drinking Water 

Monitoring Frequency” to be held April 26-29, 2010

FROM: Tina Levine, Ph.D. 

Director

Health Effects Division (7509P)

TO: Joseph Bailey

Designated Federal Official

FIFRA Scientific Advisory Panel

Office of Science Coordination and Policy (7201M)

On March 30, 2010, EPA transmitted its draft issue paper entitled “ReEvaluation of Human Health Effects of Atrazine: Review of Experimental Animal 

and In vitro Studies and Drinking Water Monitoring Frequency” to the FIFRA 

Scientific Advisory Panel (SAP) in preparation for the April 26-29, 2010 meeting. 

This memo contains the charge questions for the April SAP meeting.
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SAP CHARGE

As part of the re-evaluation of the health effects of atrazine, three meetings of 

the FIFRA Scientific Advisory Panel (SAP) are scheduled in 2010. The first of 

these was held February 2-4, 2010. In preparation for the April meeting, the 

Agency developed a draft issue paper focused on 1) a review of the recent 

scientific literature on the mode of action and toxicological profile of atrazine and 

2) approaches to determine appropriate drinking water monitoring frequencies. 

The purpose of the April meeting is to solicit feedback from the SAP on the status

and overall scientific direction of the Agency’s re-evaluation in these two areas

including preliminary conclusions about some aspects of the experimental 

toxicology data. The Agency will use the Panel’s feedback to inform the weight 

of the evidence (WOE) analysis that will be developed in the coming months and 

reviewed at the September 2010 SAP. The experimental toxicology portion of 

the draft April issue paper (Sections 3 & 4, Appendices A & B) represents the 

state of the science up to January 30, 2010. Since that time, new data have 

become available and more will become available in the spring and summer of 

2010 that will provide further characterization of some key areas, such as 

describing the causal linkage between key events in the mode of action, the

dose-response concordance of these key events, and the relationship between 

the precursor events with functional outcomes. These new studies will be 

incorporated into the September SAP.

Question 1.0: In Vivo & In Vitro Experimental Toxicology in Mammals

In 2003, the human health risk assessment was based on a mode of action in 

which exposure to atrazine leads to a reduced release of gonadotropin releasing 

hormone (GnRH) from the hypothalamus thereby lessening the afternoon 

pituitary luteinizing hormone (LH) surge in female Sprague Dawley rats. As a 

result, the estrus cycle lengthens. This, in turn, leads to increased estrogen 

levels and an increased incidence of mammary tumors in female Sprague 

Dawley rats. EPA determined that atrazine’s cancer mode of action (i.e., 

premature reproductive aging) in the Sprague-Dawley rat is not likely to be 

operative in humans (a conclusion consistent with the SAP recommendation in 

2000). The Agency will be evaluating the cancer classification further as the 

experimental toxicology data are integrated with the epidemiology literature; new 

experimental toxicology studies (Section 3.6 of the draft issue paper) do not 

suggest a change to the Agency’s previous conclusion that atrazine is “not likely 

to be carcinogenic to humans.” 

Although the cancer mode of action may not be operative in humans, it is not 

unreasonable to assume that atrazine might cause adverse effects on 

hypothalamic-pituitary function in humans. Thus, the same endocrine 

perturbations that induce tumors in rats may play a role in at least some 

reproductive/developmental effects (not associated with reproductive aging) that 

may be relevant to humans. Accordingly, the Agency identified disruption of 
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estrous cyclicity and delays in puberty onset (males and females) occurring as a 

consequence of disruptions to the hypothalamic-pituitary-gonadal (HPG) axis as 

the critical endpoints of concern (Sections 2.0 & 3.2 of the draft issue paper). 

The Agency continues to believe that this disruption of the HPG axis is critical to 

affecting estrous cyclicity and delayed puberty onset. However, as discussed in 

Questions 1.2-1.6 (See Figure 3 of the draft issue paper), new information has 

been published that indicates a more expanded understanding of how atrazine 

may perturb the pathway leading to reproductive effects. These new data 

indicate that atrazine disrupts the hypothalamic-pituitary-adrenal (HPA) axis 

altering the central nervous system’s control of the pituitary and adrenal which, in 

turn, disrupts the HPG axis. Furthermore, a hypothesis for a direct effect of 

atrazine on the adrenal cortex and the gonads which may impact steroidogenesis 

directly is also supported by the available data. 

Question 1.1

With the caveat that the review of the epidemiological literature is still on-going, 

please comment on the Agency’s preliminary conclusion that new experimental 

data from in vitro and in vivo laboratory animal studies do not support a change 

in the conclusions from the 2003 risk assessment that atrazine is unlikely to be a 

human carcinogen.

Question 1.2

Based on an evaluation of the studies examining the mode of action of atrazine 

on neuroendocrine function (Section 3.2 of the draft issue paper), the Agency 

has preliminarily concluded that atrazine affects both the HPG axis and the HPA 

axis. With respect to the temporal concordance, recent studies show that

atrazine induces a rapid (within minutes) increase in ACTH and adrenal cortical 

hormones (corticosterone and progesterone) in both male and female rats 

(Fraites et al., 2009; Laws et al., 2009; Pruett et al., 2009), while changes in the 

HPG axis, such as the suppression of the LH surge, may take up to 2-3 days

(Cooper et al., 2009). This sequence of HPA alterations followed by HPG

changes indicates that the suppression of the surge may be, in part, mediated by 

activation of the HPA axis (i.e., a corticosterone suppression of the GnRH and

LH release). In addition, with respect to dose-response concordance, atrazineinduced increases in ACTH, corticosterone and adrenal progesterone are seen 

following a single dose of atrazine (50 mg/kg). In contrast, the HPG is not 

altered following a single dose up to 200 mg/kg. However, when treated for 

multiple days, the dose necessary to alter the HPA axis is lower than or equal to 

the one needed to affect the HPG axis. 

Please comment on the Agency’s preliminary hypothesis for the mode of action

involving atrazine’s alteration of both the HPA and HPG axes. Does the 

document adequately and clearly describe the hypothesis in the context of a 
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“toxicity pathway” (i.e. cellular response pathways that, when sufficiently 

perturbed, are expected to result in adverse health effects)? Does the document 

clearly describe the data used to test the proposed hypothesis? To what extent 

do the available data establish key events in the proposed mode of action

hypothesis? What are the strengths and limitations of the data available on this 

hypothesis? 

Please include in your comments a discussion of the Agency’s interpretations of 

the data linking the initial perturbations in HPA axis to changes in the HPG axis. 

Please comment on the evidence that the initial perturbations in the HPA axis

may lead to impairment in reproductive function and/or developmental 

consequences. Which event(s) is/are viewed as critical in leading to health 

consequences? Are there data on other substances that would inform this 

question?

Question 1.3

The Agency has preliminarily concluded that atrazine directly targets cells within 

the HPA axis (Sections 3.2 & 3.3.2.3 of the draft issue paper) and that the 

increased activity of the adrenal axis does not reflect nonspecific stress. 

Evidence for this conclusion is based on the following observations. 

a. Several studies report increased adrenocorticotropic hormone 

(ACTH) and corticosterone levels immediately following a single 

exposure to atrazine and its two metabolites (deisopropylatrazine, DIA; deethyl-atrazine, DEA) but not diamino-schlorotriazine (DACT; Laws et al., 2009; Fraites et al., 2009; 

Pruett et al., 2009). 

b. A single intravenous administration of DEA in vivo (Fraites et 

al., 2009) also causes an immediate increase in ACTH and 

adrenal hormone release, indicating that a chlorotriazineinduced gastrointestinal distress is not driving the hormonal 

response.

c. The adrenal corticosterone response to continued oral 

exposure to atrazine did not habituate in rats (Fraites et al., 

2009; Laws et al., 2009) or mice (Pruett et al., 2009). 

d. 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 in both ACTH and 

prolactin are typically observed in response to a number of 

physiological and psychological stressors.

Please comment on the extent to which the available evidence supports the 

preliminary conclusion that atrazine and its intermediate metabolites (DIA and 
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DEA) induce changes in the HPA axis as a result of a direct action on the HPA 

tissue and such changes are not due to a generalized or non-specific stress 

response. Are there data on other substances that would support this 

conclusion?

Question 1.4

Based on a review of in vitro studies evaluating the effects of atrazine on 

estrogen production, the Agency has preliminarily concluded that atrazine does 

not have a direct effect on the catalytic activity of aromatase. However, with 

continued exposure (> 24 hrs) atrazine can cause increased estrone and 

estradiol 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 associated with increased cAMP and CYP19 mRNA (Sanderson et al., 

2000, 2001) that are part of a complex mode of action through which atrazine upregulates the gene expression of aromatase and possibly other enzymes within 

the steroidogenic pathway (Section 3.3.2.3 & Figure 3, Arrow 7of the draft issue 

paper). The hypothesis that atrazine alters multiple steroids is supported by 

increased testosterone concentrations in H295R cells following exposure to 

atrazine (Higley et al., 2010), and in vitro studies demonstrating that atrazine 

affects a number of signal transduction pathways and/or transcription factors in a 

variety of cell lines (Suzawa and Ingraham, 2008; Albanito et al., 2008). Upregulation of the expression of a group of major steroidogenic genes has been 

observed in JEG-3 cells (Suzawa and Ingraham, 2008). In vivo, there is little 

evidence that atrazine alters aromatase per se (Modic, 2004) and the evidence 

associating atrazine exposures to increases in serum estrogens appears to be 

related to a general increase in gonadal and adrenal progesterone and 

androstanedione (Modic 2004; Laws et al., 2009). 

Please comment on the extent that the available data do or do not support the 

Agency’s preliminary conclusion that atrazine has a general stimulatory effect on 

steroidogenesis, as opposed to a direct effect on aromatase. 

Question 1.5

A series of studies evaluating the effect of either peripubertal or gestational 

exposure of atrazine in the male rat indicate that atrazine decreases testosterone 

concentrations and may lower androgen dependent tissue weights but that it has 

little effect on basal LH secretion in the male. This response generally occurs at 

doses of 50 mg/kg and above. In contrast, atrazine in vitro has been found to 

consistently increase steroid hormone production. Thus, there is an apparent 

discrepancy between the in vivo and in vitro data. However, our understanding 

of the interplay between the adrenal and the gonadal axes may provide an 

explanation. At the doses used in the in vivo studies, atrazine induces an 

activation of the adrenal axis (increased ACTH and corticosterone) and the 

increase in corticosterone does not habituate (i.e., it does not lessen with 
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repeated dosing). There are a number of studies in the literature demonstrating 

that prolonged corticosterone stimulation will impair testosterone production by a 

direct action on steroidogenesis in the Leydig cells. Therefore, the in vivo effects

on testosterone synthesis are likely the result of an adrenal hormone-mediated

down regulation of HPG axis (through corticotropin releasing hormone [CRH] 

modulation of GnRH pulsatility) or directly on the testis (through a glucocorticoid 

receptor mediated change in steroidogenesis). 

Please comment on the degree to which the proposed mode of action (Figure 3 

of the draft issue paper) provides a biologically plausible explanation for the 

decrease in serum and testicular testosterone identified in the in vivo studies. 

Please comment on the extent that the available data do or do not support this 

hypothesis. 

Question 1.6

Based on a review of the neurotoxicity studies (Section 3.4 of the draft issue 

paper), the Agency has preliminarily concluded that several recent studies 

provided further support for the concern that dopaminergic neurotransmission 

may be affected by atrazine, a concern that was raised by studies prior to 2003. 

However, the Agency has also concluded that several aspects of these studies of 

the dopaminergic neuronal pathways, in particular the changes noted in 

stereological evaluations and the observed changes in behavior, should be 

considered as preliminary findings. This conclusion is based on the notable 

limitations identified in the data including: (1) lack of clear dose-response 

relationships, (2) lack of inclusion of suitable positive controls to confirm the 

competency and reliability of the procedures utilized in examining dopaminergic 

systems in the brain, (3) limited data to corroborate stereological findings, (4) 

limited or no supporting histological and behavioral assessments, and (5) no 

consideration of the potential role of the HPA axis (e.g. alterations in 

corticosterone). EPA has further determined that two non-dopaminergic 

neurotoxicity studies--one on brain somatostatinergic systems and the other on 

neurobehavior in mice -- also have significant limitations (e.g., lack of details on 

source and purity of atrazine, and age and body weights of the mice; poor quality 

of amino cupric silver staining photomicrographs; limited presentation of reversetranscriptase polymerase chain results; lack of data to corroborate conclusion of 

neuronal degeneration; reference citations did not support statements made in 

the text; no explanation of biological plausibility 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). Please comment on these preliminary

conclusions regarding the neurotoxicity findings. 

The Agency has preliminarily concluded that the available studies indicate that 

the neurotoxicity endpoints examined are not more sensitive than those 

evaluated for neuroendocrine function following atrazine exposure (e.g., 
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attenuation of LH surge and estrous cycle disruptions in female Sprague-Dawley 

rats which form the basis for the current chronic RfD). In addition, the Agency 

has concluded that there is no association between atrazine exposure and 

development of Parkinson’s Disease on the basis of non-specificity of effects on 

brain dopaminergic systems, lack of histological and behavioral features 

characteristic of Parkinson’s Disease, and results of epidemiological studies. 

Please comment on this preliminary conclusion.

Question 1.7

Based on a review of two studies of the potential effects of atrazine on the 

prostate (Section 3.3.2.2 of the draft issue paper), the Agency has concluded that 

the results of the Rayner et al. (2007) study of pregnant rats treated during 

gestation support previous observations (Stoker et al., 1999) that atrazine 

treatment to the rat dam either perinatally or early postnatally can increase 

prostate weights (due to an increase in inflammatory infiltrate) in the male 

offspring. This effect on the offspring was shown to be due to a suppression of 

prolactin in the atrazine-exposed dams during lactation, and is consistent with the 

mode of action of atrazine on neuroendocrine function. The Agency also 

preliminarily concluded that further research is needed to provide more 

convincing evidence that atrazine-mediated suppression of prostate cancer in the 

probasin/SV40T antigen transgenic rat (androgen-dependent prostate cancer 

rodent model) may possibly be due to caloric restriction rather than endocrinerelated as postulated by Kandori et al. (2005). Please comment on these

preliminary conclusions regarding the recent prostate findings.

Please comment on the extent to which the available data support the proposed 

mode of action for prostatitis and the appropriateness of the rodent model in the 

context of human health.

Question 1.8

Based on data obtained in studies to assess the effects of atrazine on the 

immune system following developmental and adult exposure, as well as in vitro

mechanistic studies, EPA concluded that atrazine has the potential to affect the 

immune system (Section 3.5 of the draft issue paper). However, the Agency has 

also concluded that underlying mechanisms of atrazine-mediated immunotoxicity 

and its relevance to potential adverse health effects in humans are still not 

thoroughly understood. It is well established that products of the endocrine 

system modulate immune function. However, adult rodent studies have not 

determined whether immune system effects are caused by direct effects of 

atrazine and/or its metabolites or whether they are the result of modulated 

endocrine hormone production, which in turn affects immune cells. Two 

published studies indicate that exposure to atrazine during immune system 

development may result in altered immune function in offspring, although no 

conclusions were drawn on the potential adversity of the effects, because 
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immunosuppression was observed in one study and immunoenhancement was 

observed in the other. Please comment on potential explanations for the 

disparate findings reported by Rooney et al. and Rowe et al. Many 

immunotoxicologists consider immunomodulation, i.e., suppression or 

enhancement of immune function, as a potentially adverse alteration of 

homeostasis, because both have been associated with disease states. Thus, 

while the results Rooney et al. and Rowe et al. may appear to be contradictory, 

together the data indicate unintended immunomodulation at approximately the 

same dose in two species. Please comment on characterizing the effects of 

gestational atrazine exposure as immunomodulation, when describing the 

immunotoxicological outcome of developmental immunotoxicity studies.

Appendix B of the draft issue paper describes experiments conducted by EPA 

scientists on the potential developmental immunotoxicity of atrazine. The results 

of the experiments provide conflicting results. Additional studies did not provide 

a suitable explanation of the differences. The Agency believes both sets of data 

are of high quality. However, in the context of hazard assessment, such 

differences are difficult to interpret. Please comment on the information 

contained in Appendix B and provide suggestions for interpreting such data as 

part of the atrazine re-evaluation.

The available data do not indicate that atrazine-induced immunotoxicity is a more 

sensitive endpoint than the atrazine-induced effects on neuroendocrine function, 

(e.g., attenuation of LH surge and estrous cycle disruptions which form the basis 

for the current chronic RfD) in female Sprague-Dawley rats. Please comment on 

the degree to which these preliminary conclusions are supported by the available 

data

Question 1.9

After the April 2010 SAP, the Agency will evaluate the weight of evidence (WOE) 

for atrazine by integrating the experimental toxicology data with the 

epidemiological studies. As part of this work, the Agency will consider the 

available data on hormonal changes and functional outcomes that may be used 

as endpoints for deriving PoDs across different durations of exposure and for 

different populations (including potentially sensitive ones). Important studies 

evaluating the dose-response relationships for hormones involved in the HPA 

axis are still on-going. These studies are expected to provide high quality data 

that can be used to characterize the entire dose response curve. With the caveat

that these data are not available now, given the current understanding of 

atrazine’s mode of action, please comment on what would be appropriate 

endpoints to consider for use in deriving points of departure in a possible future 

risk assessment.

Given that the duration of the toxicological concern is a key factor in evaluating 

the frequency of drinking water monitoring, please include in your response a 
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consideration of the magnitude and duration of changes in key events in the 

toxicity pathway that are sufficient perturb normal function and comprise human 

health. In defining the exposure window of interest, please comment on the 

toxicokinetic/dynamic considerations with respect to atrazine's effects on the 

HPA/HPG axis. 

Question 2.0: Approaches to Evaluating Water Sampling Strategies & 

Frequency of Monitoring

In conjunction with the toxicological review presented in the issue paper, the 

Agency has also discussed methods for re-evaluating the sampling frequency 

that is necessary for determining, with confidence, concentrations of the pesticide 

in water that sources drinking water. These have included different methods for 

estimating pesticide concentrations between known sampling events and

examining the performance of different sampling strategies for averaging periods

of different durations. The Agency seeks feedback from the Panel with regard to 

how the uncertainty and variability in both the monitoring data and in the toxicity 

data (i.e., point of departure) can be integrated to characterize and to interpret 

the potential significance of atrazine concentrations in drinking water. 

Question 2.1

 

Given the nature of the temporal patterns of pesticide occurrence in surface 

waters described in Section 5.2, including serial correlations from day to day, 

periodicity in elevated concentrations within seasons and from year to year, 

below quantitation data, and uncertainty in the shape of the pesticide 

distributions in surface waters--what statistical approaches should the Agency 

consider in determining confidence bounds on exposure estimates from 

monitoring data? Please comment on how the approach may vary depending on 

the duration of concern.

Question 2.2

The first two simulation methods presented in Section 5.5 are applicable to the 

specific data sets they describe, although some generalities regarding shape 

patterns appear to exist. Given this information, please comment on the 

strengths and weaknesses of the approaches and on the practical merits of 

pursuing them or some other numerical approach with a larger set of higher 

concentration systems. Please comment on how the methods for determining 

confidence bounds might apply given these considerations. 

Question 2.3

As described in Section 5.4.2, the Agency is considering the use of a confidence 

interval or prediction interval approach to characterize the uncertainty of 

exposure estimates derived from monitoring data of varying sampling 
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frequencies. Please comment on the strengths and weaknesses of either placing 

confidence bounds on the rolling average estimates and comparing the upper 

limit from monitoring against the level of concern (LOC) or, conversely, placing 

confidence bounds on the LOC. 

Question 2.4

Please comment on the relative merits of the various modeling approaches the 

Agency described in Section 5.4.1 and 5.6 for interpolating pesticide 

concentrations between sampling points and, in particular, on the strengths and 

weaknesses of these methods as the frequency of samples decreases. 

Considering the health endpoint(s) being considered for atrazine, particularly 

data for the HPA axis, and the exposure time frame needed to induce the health 

effect(s) which is shorter than that used in the 2003 risk assessment, please 

comment on the advantages and disadvantages of each model for evaluating the 

likely occurrence and exposure via drinking water of short, moderate, and long 

duration concentrations. Please comment on the Agency’s proposed approach 

for evaluating these methods, as described in Section 5.7.1. To what extent 

should the Agency consider other factors, such as the shape of the chemograph 

(Section 5.5.3), weather patterns, stream flow, and/or pesticide use patterns in 

evaluating the modeling approaches?