SAP Minutes No. 2010-07 

A Set of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding: 

Re-evaluation of the Human Health Effects 

 of Atrazine: Review of Non-Cancer Effects and 

Drinking Water 

Monitoring Frequency

September 14 – 17, 2010 

FIFRA Scientific Advisory Panel Meeting 

Held at the 

Environmental Protection Agency Conference Center 

Arlington, VA 

NOTICE 

These meeting minutes have been written as part of the activities of the Federal 

Insecticide, Fungicide, and Rodenticide Act (FIFRA), Scientific Advisory Panel (SAP). 

The meeting minutes represent the views and recommendations of the FIFRA SAP, not 

the United States Environmental Protection Agency (Agency). The content of the 

meeting minutes does not represent information approved or disseminated by the Agency. 

The meeting minutes have not been reviewed for approval by the Agency and, hence, the 

contents of these meeting minutes do not necessarily represent the views and policies of 

the Agency, nor of other agencies in the Executive Branch of the Federal Government, 

nor does mention of trade names or commercial products constitute a recommendation 

for use. 

 The FIFRA SAP is a Federal advisory committee operating in accordance with 

the Federal Advisory Committee Act and established under the provisions of FIFRA as 

amended by the Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides 

advice, information, and recommendations to the Agency Administrator on pesticides and 

pesticide-related issues regarding the impact of regulatory actions on health and the 

environment. The Panel serves as the primary scientific peer review mechanism of the 

Environmental Protection Agency, Office of Pesticide Programs (OPP), and is structured 

to provide balanced expert assessment of pesticide and pesticide-related matters facing 

the Agency. FQPA Science Review Board members serve the FIFRA SAP on an ad hoc 

basis to assist in reviews conducted by the FIFRA SAP. Further information about 

FIFRA SAP reports and activities can be obtained from its website at 

http://www.epa.gov/scipoly/sap/ or the OPP Docket at (703) 305-5805. Interested 

persons are invited to contact Joseph E. Bailey, SAP Designated Federal Official, via email at bailey.joseph@epa.gov. 

 In preparing these meeting minutes, the Panel carefully considered all information 

provided and presented by EPA, as well as information presented by public commenters. 

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TABLE OF CONTENTS 

PARTICIPANTS ...................................................................................................................3 

INTRODUCTION .................................................................................................................6 

PUBLIC COMMENTS..........................................................................................................7 

SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS...........................8 

PANEL DELIBERATIONS AND RESPONSE TO CHARGE............................................17 

REFERENCES ......................................................................................................................76

ii 

2 
Federal Insecticide Fungicide and Rodenticide Act 

Scientific Advisory Panel Meeting 

September 14 - 17, 2010 

PARTICIPANTS 

FIFRA SAP Chair 

Steven G. Heeringa, Ph.D., Research Scientist & Director for Statistical Design, 

University of Michigan, Institute for Social Research, Ann Arbor, MI 

Designated Federal Official 

Joseph E. Bailey, FIFRA Scientific Advisory Panel, Office of Science Coordination and 

Policy, EPA 

FIFRA Scientific Advisory Panel Members 

John Bucher, Ph.D., DABT, Associate Director, National Toxicology Program, 

National Institute of Environmental Health Sciences, Research Triangle Park, NC 

Janice Chambers, Ph.D., DABT, Fellow ATS, Director, Center for Environmental 

Health Sciences, College of Veterinary Medicine, Mississippi State University, 

Mississippi State, MS 

Gerald A. LeBlanc, Ph.D., Professor and Department Head, Department of 

Environmental & Molecular Toxicology, North Carolina State University, Raleigh, NC 

Cary N. Pope, Ph.D., Professor, Head & Sitlington Chair of Toxicology, Department of 

Physiological Sciences, Oklahoma State University College of Veterinary Medicine, 

Stillwater, OK 

Kenneth Portier, Ph.D., Program Director, Statistics, American Cancer Society, 

National Home Office, Atlanta, GA 

Daniel Schlenk, Ph.D., Professor of Aquatic Ecotoxicology and Environmental 

Toxicology, Department of Environmental Sciences, University of California, Riverside, 

CA 

FIFRA Science Advisory Board Members 

Susan F. Akana, Ph.D., Associate Professional Researcher, Department of Physiology 

University of California San Francisco, San Francisco, CA 

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John C. Bailar, III, M.D., Ph.D., Scholar in Residence, The National Academies, 

Professor Emeritus, University of Chicago, Washington, DC 

Richard H. Coupe, Ph.D., Supervisory Hydrologist/Associate Water Science Director, 

U.S. Geological Survey, Pearl, MS 

Kenneth Barry Delclos, Ph.D., Pharmacologist, FDA, National Center for Toxicological 

Research, Jefferson, AR 

Penelope A. Fenner-Crisp, Ph.D., DABT, Consultant, North Garden, VA 

Ellen B. Gold, Ph.D., Professor and Chair, Department of Public Health Sciences, 

School of Medicine, University of California - Davis, Davis, CA 

Richard Greenwood, Ph.D., Professor of Environmental Science, University of 

Portsmouth, Portsmouth, UNITED KINGDOM 

Shelley A. Harris, Ph.D., Scientist, Prevention and Cancer Control, Cancer Care 

Ontario, Associate Professor, Dalla Lana School of Public Health, University of Toronto, 

Toronto, Ontario, CANADA 

Nelson D. Horseman, Ph.D., Professor, Department of Molecular and Cellular 

Physiology, Department of Medicine (Endocrinology & Metabolism), Program in 

Systems Biology and Physiology, University of Cincinnati, Cincinnati, OH, 

Kannan Krishnan, Ph.D., Professor, Department of Occupational and Environmental 

Health, University of Montreal, Montreal, CANADA 

Herbert K. H. Lee, Ph.D., Professor, Department of Applied Mathematics and Statistics, 

Associate Dean for Graduate Studies and Research, Baskin School of Engineering, 

University of California - Santa Cruz, Santa Cruz, CA 

Sandra J. Legan, Ph.D., Professor, Department of Physiology and Biophysics, 

University of Kentucky, Lexington, KY 

James L. McManaman, Ph.D., Professor, Department of Obstetrics and Gynecology, 

Division Chief, Basic Reproductive Sciences, University of Colorado – Denver, 

Aurora, CO 

M.E. Bette Meek, Ph.D., Associate Director, Chemical Risk Assessment, McLaughlin 

Centre for Population Health, University of Ottawa, Ottawa, Ontario, CANADA 

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Moiz M. Mumtaz, Ph.D., Science Advisor/Senior Toxicologist, Division of Toxicology 

and Environmental Medicine, Agency for Toxic Substances & Disease Registry, Centers 

for Disease Control & Prevention, Atlanta, GA 

Katherine Roby, Ph.D., Director, Reproductive Endocrinology Laboratory, Center for 

Advanced Reproductive Medicine, University of Kansas Medical Center, Kansas City, 

KS 

Wesley W. Stone, M.S., Hydrologist, U.S. Geological Survey, Department of Interior, 

Indianapolis, IN

5 
INTRODUCTION 

 The Federal Insecticide, Fungicide and Rodenticide Act Scientific Advisory Panel 

(FIFRA SAP) has completed its review of the Reevaluation of the Human Health Effects 

of Atrazine: Review of Non-cancer Effects and Drinking Water Monitoring Frequency. 

Advance notice of the meeting was published in the Federal Register on June 11, 2010. 

The review was conducted in an open panel meeting held in Arlington, VA, on 

September 14 - 17, 2010. Dr. Steven Heeringa chaired the meeting. Joseph E. Bailey 

served as the Designated Federal Official. 

 EPA is undertaking a re-evaluation of the human health effects of atrazine. The 

re-evaluation has involved three SAP meetings in 2010. The first was held in February 

2010 at which the Agency presented its preliminary reviews of several epidemiologic 

studies of the relation of atrazine to birth outcomes and described a plan to evaluate 

atrazine epidemiology data from the Agricultural Health Study. The second SAP 

meeting, held in April 2010, focused on 1) a preliminary review of experimental 

toxicology studies from laboratory mammals and in vitro studies and recent 

advancements in understanding atrazine’s mode of action along with 2) statistical 

approaches for evaluating monitoring frequency in community water systems (CWS). 

The third SAP in September 2010 focused on the non-cancer effects of atrazine. The 

Agency included studies available up through July 15, 2010. At this meeting OPP 

integrated data from in vitro and in vivo experimental toxicology studies along with 

preliminary review of non-cancer epidemiologic studies in a draft weight of the evidence 

(WOE) analysis. The Agency will use feedback received from the SAP at the September 

2010 meeting as it completes the non-cancer WOE analysis integrating experimental 

toxicology and epidemiology studies with statistical analysis for determining whether or 

not adjustments are necessary in the sampling frequency of CWS water monitoring. 

 Opening remarks at the meeting were provided by Steven Bradbury, Ph.D., 

Director, Office of Pesticide Programs and Tina Levine, Ph.D., Director, Health Effects 

Division, Office of Pesticide Programs. Agency presentations were given by Anna Lowit, 

Ph.D., Carol Christensen, Ph.D. and Chester Rodriguez, Ph.D., Health Effects Division; 

Nelson Thurman, M.S., and Mary Frankenberry, Environmental Fate and Effects 

Division, all of the Office of Pesticide Programs. In addition, a presentation was given by 

Suzanne Fenton, Ph.D., of the National Institute of Environmental Health Sciences, 

National Institutes of Health. 

6 
PUBLIC COMMENTS 

Oral Statements were presented as follows: 

Janis McFarland, Ph.D., Charles Breckenridge, Ph.D., Russell C. Hovey, Ph.D. 

(University of California - Davis), Robert Handa, Ph.D. (The University of Arizona), 

James Simpkins, Ph.D. (University of North Texas), David Kim, Ph.D., Tony M. Plant, 

Ph.D. (University of Pittsburgh) and Paul Hendley, Ph.D. all on behalf of Syngenta Crop 

Protection 

Tyrone B. Hayes, Ph.D. (University of California - Berkeley) on behalf of himself and 

other scientists 

Rod Snyder on behalf of the National Corn Growers Association 

Jere White, James C. Lamb, Ph.D., Gary Marshall, John Hall and 

David C. Bridges, Ph.D. (Abraham Baldwin Agricultural College) all on behalf of the 

Triazine Network 

Scott Slaughter on behalf of the Center for Regulatory Effectiveness 

Tyler Wegmeyer on behalf of the American Farm Bureau Federation 

Rebeckah Freeman Adcock on behalf of Croplife America 

Written Statements were provided by: 

Jean Public 

Dan Campbell on behalf of Syngenta Crop Protection 

Scott Slaughter on behalf of the Center for Regulatory Effectiveness 

Tyrone B. Hayes on behalf of himself and other scientists 

Michael Leggett, Ph.D., on behalf of CropLife America 

7 
Summary of Panel Discussion and Recommendations 

Charge Issue 1.1: Non-Cancer Epidemiology Reviews - EPA's critique of the atrazine 

epidemiology literature related to non-cancer outcomes is thoughtful and comprehensive; 

however, the Panel did not agree with EPA's conclusion that the database for non-cancer 

outcomes is strong. Of 29 candidate papers, 19 met the Agency's criteria for evaluation. 

They were spread over many different health outcomes and had many important 

limitations as noted by EPA and the Panel's detailed analysis. The Panel had the 

following comments regarding the methodology used to evaluate the papers and 

recommendations: 1) the criteria for including studies should be described in greater 

detail; 2) papers that were excluded from full evaluation should be discussed in an 

appendix with the rationale for their exclusion; 3) two of the 29 candidate papers were 

excluded primarily because they did not independently measure atrazine or triazines, but 

this was not well justified by EPA; 4) the 46 exposure assessment studies identified in the 

literature search should be reviewed and summarized in any future cancer risk evaluation, 

and it was suggested that the review and summary be provided to the SAP for 

consideration; 5) a system of scoring study quality should be developed and each study 

should be independently scored by at least two reviewers; 6) ecologic studies should 

rarely be used for evaluating weight of evidence and are useful only for purposes of 

hypothesis generation; and 7) additional studies other than those reviewed have assessed 

some of the outcomes better and have stored biologic samples and thus have the potential 

to examine the relation of atrazine to non-cancer outcomes more effectively, and the EPA 

should pursue measurement of atrazine and analyses of the existing outcomes in these 

studies. In general, the Panel believed the 19 non-cancer outcome studies reviewed were 

suggestive of possible adverse health outcomes, but had limitations that need to be 

addressed more fully. 

Charge Issue 1.2a & b: Non-Cancer Epidemiology Database & Risk Assessment - 

Overall, the Panel agreed with EPA's conclusions that the current non-cancer 

epidemiologic database provides some useful information for hazard identification but is 

too limited to provide sufficient information for credible dose-response assessment or 

risk characterization. To integrate epidemiology and experimental toxicology 

information, the following are needed: 1) a strong experimental toxicology database; 2) 

understanding of toxicological mode(s) of action; and 3) a strong epidemiology database. 

For atrazine, a robust experimental toxicology database exists and understanding its 

modes of action is increasing. However, with regard to human epidemiologic studies, 

studies are needed that are specifically designed to evaluate relevant outcomes and that 

are based on defined mode(s) of action. The 19 epidemiology studies evaluated presented 

very little information collected in humans that support the outcomes observed in 

animals; therefore, the Panel concluded none of the non-cancer epidemiology studies 

were useful other than for problem formulation. The Panel agreed with EPA's conclusion 

that the experimental toxicology data and not the epidemiologic data are best suited for 

dose-response and risk assessment at the present time, but that the epidemiologic and 

toxicologic databases, in combination, can be used to inform hazard identification and 

characterization. Contrary to the Agency's opinion stated in the Issue Paper, the Panel 

8 
believed that epidemiologic findings could be used for endpoint selection if the studies 

are well-designed and experimental data are not available. 

Charge Issue 2.1: Mammary Gland Development Studies (Rayner et al., 2004, 2005 

and Coder, 2010) - The Panel pointed out important experimental design and 

methodology differences between the Rayner and Coder studies related to the following 

aspects: 1) composition of the diet consumed by the dams; 2) light-dark photoperiod 

employed; 3) inclusion/absence of pair-fed controls; 4) methods used for mammary gland 

development analysis; and 5) differences in maternal stress levels. The Panel concluded 

that these methodological and procedural differences confounded the understanding of 

the toxicological significance of exposure to atrazine on certain aspects of female 

reproductive system development. It was the Panel’s opinion that unless/until these 

differences are resolved, an understanding of the effects of atrazine on mammary gland 

development will be hampered. Consequently, the Panel believed that use of the existing 

data on rat mammary gland development to assess the potential human risk of atrazine is 

not warranted at the present time. 

Charge Issue 2.2 & 2.3: Enoch (2007) Mammary Gland Study and Use of Rayner, 

Coder and Enoch Studies in Hazard Assessment - The Panel agreed with EPA’s 

conclusion that the Enoch study is not suitable for use in quantitative risk assessment. 

However, the study does raise some interesting points and dilemmas. Certainly, the very 

low doses at which effects on mammary gland development were reported could be of 

importance in assessing the risks of atrazine exposure. However, since these effects 

occurred at doses so much lower than observed in other studies, replication of the study is 

needed. The observations of this study have not been corroborated using other mixtures 

of parent atrazine and metabolites reflective of temporal and spatial occurrence or for 

atrazine parent at equivalent doses. Atrazine itself will, once absorbed, yield a mixture of 

free and conjugated metabolites (but little or no hydroxyatrazine) and the proportion of 

metabolites would likely change temporally. Any replication would need to evaluate the 

same endpoints measured in this study and, perhaps, other physiological endpoints. The 

Panel concurred with the Agency’s conclusion that the few available mammary gland 

studies do not constitute a sufficient body of evidence to be used in a quantitative risk 

assessment (i.e., for establishing a Point of Departure (PoD)). However, the Panel was 

clear in stating that this conclusion should not be construed to mean that the mammary 

gland is not a legitimate target for further analysis and concluded that the suggestive 

evidence in these studies argues strongly for development of useful and standardized 

methods for assessing mammary gland functional outcomes. 

Charge Issue 3.1a & b: Thede (1987) Pharmacokinetic (PK) Data and PK PseudoSteady State - The Panel believed it would be prudent for the Agency to work with the 

current PBPK model described by the registrant and make appropriate modifications. The 

Panel recommended that the Agency be more proactive in this area, and be more rigorous 

in addressing the uncertainties of extrapolation from rodent studies to low dose regimens 

in humans. However, in the absence of a functional PBPK model and appropriate data, 

the Panel strongly supported the work of the Agency in pursuing a dose-response analysis 

based on an internal dose metric, as an alternative to the administered dose metric used in 

9 
the 2003/2006 risk assessment. The Panel agreed with the Agency that, on the basis of 

the currently available data, plasma appears to be a reasonable biological compartment 

that is reflective of tissue dose, and that use of area under the plasma concentration time 

curve (AUC) provides an appropriate measure of internal exposure. It is the best way of 

approaching the problem of relating the exposure of rats to atrazine to the magnitude of 

resulting biological responses, and extrapolating to the prediction of human exposure 

levels. In the absence of good data on the plasma concentrations of parent compound and 

individual metabolites at various elapsed times after dosing, the use of total chlorotriazine 

based on total 14C-compounds is a reasonable first step (particularly in the absence of 

information on the pharmacodynamic activity of the parent compound and individual 

metabolites). However, the Panel believed that the Thede (1987) data, had limitations, 

and should be interpreted with caution. 

 In the opinion of the Panel, the approach taken by the Agency is correct, but 

requires better data than those currently available. The pharmacokinetic study in progress 

as described in the registrant’s presentation at the meeting and in Breckenridge, et al. 

(2010) will provide data of the required quality since the use of frequent sampling and 

mass spectrometric detection of the analytes is providing tight definitions of the PK 

profiles of the individual compounds (parent compound and analytes). This type of study 

lends itself to analysis of relationships between the AUCs of the different compounds and 

biological responses. This information combined with good estimates of plasma partition 

coefficients should allow the development of physiologically based pharmacokinetic 

models that can be used to extrapolate from rodents to other species, including humans. 

This would provide a sound basis for the setting of benchmark doses, and PoDs, 

providing that it is developed for the rat strain in which the PoD or dose-response is 

characterized. 

 Pseudo steady state implies that the absorption and distribution processes are 

matched by the elimination processes. The interpretation of the levels presented in 

Figure 5.5 of the Issue Paper is misleading since all plasma samples were taken at 24 h 

after the previous dose. The points defining the profiles in Figure 5.5 are the minima in 

the sawtooth profile, and not the average levels. Perhaps if the samples had been taken at 

12 h after dosing, the profile would have been different. If the samples had been taken at 

random times after dosing, there would not have been such a smooth plateau. The real 

profile will not have such small amplitude fluctuations as illustrated in Figure 5.4 of the 

Issue Paper (see Figure 6 in Breckenridge, et al. (2010)). This means that the AUC is 

underestimated. The bias will be approximately constant across the plateau area, and so 

it will not change the goodness of fit of AUC versus administered dose, and could give a 

false sense of security. 

Charge Issue 3.1c: PK Pseudo-steady State and Attenuation of LH Surge - The 

Panel was asked to comment on the Agency's analysis and preliminary conclusions 

related to Figure 5.8 in the Issue Paper which shows a plot of LH attenuation data versus 

administered atrazine across a range of exposure durations. The Panel consensus was that 

Figure 5.8 is an imaginative distillation of data from two different rat strains across 15 

years of data collection from four laboratories. The Figure serves as a visual meta10 
analysis and assists in the understanding of the atrazine dose-related attenuation of the 

LH surge. It is remarkable that the overall response is similar given the relatively great 

diversity in the study paradigms: different rat strains and different dosing regimens. The 

model presented in Figure 5.8 represents a good, initial approach to data integration and 

has great utility for exploring concepts and formulating approaches to test hypotheses 

further. The PK data indicated that a pseudo-steady state was achieved over the four days 

of dosing in the Thede experiments. However, it is important that interpretation of these 

data not use the idea that a pseudo-steady state is necessary. The data do not support this 

interpretation because no other dosing pattern was used. Overall, the Panel agreed that 

several aspects of Figure 5.8 need refinement in terms of the axis scaling, winnowing of 

LH data, inclusion of the higher doses to incorporate the full range of data and the 

separate evaluation of data from two rat strains. All these aspects can be corrected and 

may yield a more accurate and sensitive model. 

Charge Issue 3.2a: Benchmark Dose Analysis - The Panel agreed with the Agency's 

conclusion that, based on the available data, a benchmark dose (BMD) modeled from 

data on suppression of the LH surge appears to be protective for other endpoints, since 

this phenomenon occurs at doses lower than for the wide range of effects identified in a 

rather extensive toxicological database. While attenuation of the LH surge over one 

estrous cycle is a sensitive biological response, a causal association between the modest, 

yet statistically significant, observed changes in LH surge peak and adverse fertility 

measures has not been made. The relatively small attenuation of the LH surge may well 

be a harbinger of a possible future adverse event, but at present this remains to be 

demonstrated and other possible endpoints should continue to be investigated. 

 The biological significance and degree of adversity of the effect used to derive the 

benchmark value remains to be addressed robustly and needs to be considered carefully 

in relation to other endpoints on continued exposure, based on the totality of data, 

including that from more traditional studies. It may be helpful, then, to develop a variety 

of potential points of departure for relevant endpoints with a view to characterizing their 

degree of protection (in relation to biological significance of the observed effect) and 

associated uncertainty. This would serve as a critical step for the interpretation of the 

derived BMD, not just to provide understanding of the biological significance of 

observed effects, but also as a tool to compare and contrast the uncertainties associated 

with various options. Comparative uncertainty analysis would include consideration of 

appropriate interspecies and intraspecies adjustments, in the context of their associated 

degree of uncertainty. 

 The existing data on the dose response of the attenuation of the LH surge 

associated with exposure to atrazine (in rats) are sufficient to permit robust analysis for 

the benchmark dose. These include significant data in several strains exposed to a range 

of doses delivered either by oral gavage or in the diet. The Agency modeled four relevant 

candidate data sets, representing the four studies summarized in Figure 5.8 of the Issue 

Paper and selected the “new” NHEERL 4-day data (Cooper 2010, unpublished) as the 

most appropriate for deriving a PoD. Selection of this study as the basis for BMD 

modeling is appropriate, not only because of the range of doses used, and the lower 

11 
variability of the data, but also owing to the inclusion of data on animals exhibiting a 

statistically defined LH surge. 

Charge Issue 3.2b: Benchmark Response for LH Attenuation - A solid understanding 

of a benchmark response is needed for BMD analysis. In this case, attenuation of the LH 

surge is a sensitive measure of atrazine exposure. It directly relates to mode of action, 

being one of the key precursor events to the ultimate endpoint(s) of toxicity, and it 

exhibits dose response characteristics. Thus, it is a suitable benchmark response. Even so, 

inherent difficulties exist. These include the wide range in LH surge amplitude normally 

observed in both rodents and women, and the variability in the exact time of day the 

actual peak in LH surge occurs. There are ample data in the literature to demonstrate that 

complete (or nearly complete, i.e. ~ 80%+) inhibition of the LH surge, does directly 

correlate with adverse fertility outcomes, including altered cyclicity and inhibition of 

ovulation. Outside of these very dramatic changes in LH surge (at or near complete 

abrogation), data correlating more modest, yet statistically significant, changes in LH 

surge characteristics to key fertility events are lacking. These data are not only lacking 

from the series of studies presented in the Issue Paper, but importantly, there are not 

sufficient data in the broad body of literature (rodent or human) related to reproduction 

and fertility to directly link small or moderate changes in LH surge characteristics with 

fertility outcomes. This does not negate the possibility that they do exist. 

 Based on the Panel's current understanding of effects related to atrazine exposure 

together with its level of understanding of long-term effects of modest changes in LH 

surge and fertility, they concluded that the available data are insufficient to directly relate 

a specific percent change in either amplitude or AUC of the LH surge with fertility 

outcomes. At the present time, use of data on attenuation of the LH surge is a suitable 

benchmark response and directly relates to the present understanding of mode of action. 

However, additional endpoints, including but not limited to growth hormone activity, 

should not be overlooked. Furthermore, consideration of additional endpoints and/or 

additional modes of action may allow for integration of both cancer and non-cancer 

health effects of atrazine. 

Charge Issue 4.1: EPA's Framework for Designing Monitoring Study - Regarding 

EPA's recommended framework for designing a drinking water monitoring study, the 

Panel noted that if the true purpose of a monitoring program is to generate valid “human 

exposure estimates,” then sampling the finished water of a community water system 

(CWS) is more appropriate than sampling its raw input water. EPA requested the Panel to 

specifically comment on four elements of its recommended framework for designing a 

monitoring study. The Panel agreed that targeting the most vulnerable areas is 

appropriate; however, caution was urged in how areas are defined as “vulnerable.” 

Frequency of sampling and selection of criteria for inclusion in the monitoring program 

should be carefully considered to avoid underestimating true atrazine concentrations. 

The Panel noted that monitoring programs need flexibility to accommodate year to year 

variability in agricultural practices. The Panel agreed with EPA that it is appropriate to 

sample intensively during periods expected to have high occurrence of exceedances. To 

help predict occurrence, some Panelists suggested use of an equation that considers 

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atrazine application timing and amounts along with hydrology of the site. Further, the 

Panel indicated that reservoirs warranted separate consideration when designing a 

monitoring program. Basing sampling frequency on the toxicological exposure duration 

of concern, as proposed by EPA, would be appropriate if there was agreement on what 

constitutes toxicological exposures of concern. Some Panel members expressed the 

opinion that there was not strong evidence of adverse health impacts from atrazine 

exposure at levels currently observed in surface waters. On the other hand, the fact that 

atrazine is so widely used and found in many environmental compartments raises the 

importance of further monitoring. The Panel agreed that it is important to continue to 

monitor atrazine to better understand exposure levels and to ensure that exposure levels 

continue to decline. Finally, the Panel agreed with EPA that autosamplers are appropriate 

to use to collect data for exposure periods of interest when quality assurance and quality 

control programs are in place to ensure reliable information. 

Charge Issue 4.2a: Use of Chemograph Shapes to Match CWS with Intensively 

Monitored Datasets - Given the likelihood of missing short-duration concentration 

peaks and the variability of chemograph shapes over time at a site, using the chemograph 

shape as the mechanism to link CWS sites and the high sampling frequency sites, may be 

overly difficult and may provide a false sense of confidence in terms of assessing the 

sampling frequency adequacy for a site. Functional Data Analysis (Ramsey and 

Silverman, 2002; 2005) was suggested as one approach to grouping sites based on 

chemograph shapes; however, care must be taken in accounting for missing peak 

concentrations in this analysis. As an alternative approach, the Panel suggested matching 

high frequency sampling sites to community water supply sites based on water body and 

watershed characteristics. 

Charge Issue 4.2b: Heidelberg and AEEMP Datasets for Analysis of Flowing Water 

and PRZM/EXAMS for Lakes and Reservoirs - When developing methodology, it is 

important to have true daily data, as both the Heidelberg and AEEMP datasets do. The 

Heidelberg dataset is of high quality and reflects frequent sampling, but it does not 

represent geographical diversity. Therefore, generalization from this dataset to the data 

from other watersheds would need to be done with care to avoid less than accurate 

predictions. The Perry Lake dataset would be more relevant to reservoirs. 

PRZM/EXAMS modeling is also useful, providing calibrated curves with simulated daily 

values; however, it should be noted that these models provide approximations of true 

values and that appropriate caution should be used in drawing conclusions from these 

data. PRZM/EXAMS is a very detailed model, not the easiest to use in practice, and 

needs to be tailored to each individual watershed. There was some sentiment among the 

Panel members that a more practical use of resources could be the collection of more 

varied, intensely-sampled datasets (particularly for reservoirs), rather than attempting to 

fit complex, labor-intensive, site-specific models to less frequently-sampled datasets. 

Data from intensely sampled sites would be expected to give much more accurate results 

than a linear or stair-step interpolation of less frequently sampled data (such as weekly 

data). The Panel noted that there is substantial literature on the use of computer models to 

augment physical data, and when properly calibrated (i.e., adjusted to match the available 

sparser real data), the approximations can be very good. 

13 
Charge Issue 4.3: Regression-based Modeling Combined with Random Function 

Modeling - Combining a deterministic model, like SEAWAVE-Q or PRZM/EXAMS, 

with a regression-based model, like WARP, appears to be the most promising approach 

to deal with sparse data in flowing water (the scenario for which these models were 

designed). This approach makes use of information about the pathways and aggregate 

data that are not incorporated by purely statistical methods such as kriging or neural 

networks. Combining a model such as WARP with a purely statistical method will give 

better interpolation than simple methods such as linear interpolation, but it would fail to 

make use of information about the expected shape of concentration curves that could be 

predicted by a deterministic model. However, there is a trade-off in the difficulty of 

implementing these methods, and it may be easier to implement a combination of WARP 

and a relatively simple statistical approach such as kriging, compared with combining 

WARP with a deterministic model. Such a consideration might have ramifications in the 

field. As EPA and the registrant continue to explore sampling plans for monitoring, some 

thought should be given to actually using the simulation models and CWS 

characterizations as part of the monitoring process. In particular, it is feasible that models 

will eventually be accurate enough to provide predictions of atrazine concentrations in 

source waters to a CWS for the coming crop season. Instead of requiring a CWS to 

collect and analyze water samples in their output stream (drinking water) at some predefined frequency (e.g., daily or weekly in the case of some sites), it should be possible to 

use the models to facilitate targeting sampling to periods of time most likely to 

experience an exceedance. The Panel suggested a sampling plan that closely follows 

sampling protocols typically used for obtaining information on rare events. It would 

control the sampling effort and, at the same time, focus sampling on periods with the 

highest likelihood of actually seeing measurable concentrations. 

Charge Issue 5: Potential Sensitivity of Infants and Children - Consideration of the 

value of the FQPA safety factor is seemingly best predicated on transparent and 

systematic consideration of the most important qualitative and quantitative uncertainties 

associated with both exposure and effect, relevant to susceptible life stages, in a context 

consistent with that for other pesticides. While considerable information related to water 

monitoring is available, dietary intake of atrazine in young children or adolescents 

remains somewhat uncertain and at the present time, it remains unclear what the relevant 

duration of dosing for either adults or developing organisms may be. There is a general 

consensus from the results of the animal studies that atrazine can influence the 

gonadotropin-releasing hormone (GnRH)-mediated LH surge following oral exposures 

leading to reproductive and/or developmental toxicity. One of the most important 

uncertainties from available toxicokinetic studies is the magnitude of the internal dose in 

the adults and the fetuses/pups, but newer studies seem to be pointing in the right 

direction to characterize these values. An extension of the data on internal dose is needed 

for developing a PBPK model that will better estimate the internal concentrations in 

people. This activity seems to be underway and so a PBPK model should be 

forthcoming. None of the experimental studies done thus far indicate that the prenatally, 

lactationally, or peripubertally-exposed animal exhibits higher sensitivity to 

developmental disruption than the effects in adults related to changes in LH surge. Thus, 

14 
it remains unclear whether existing data coupled with as yet unfinished studies will 

answer sufficiently the questions about the toxicity profile for all important life stages 

(gestational, lactational and peri-pubertal) and whether there are any life stage-related 

differences in sensitivity. One of the biggest uncertainties at the present time is the 

validity of the very low dose effects observed on mammary gland development in the 

Enoch et al. (2007) paper which suggests that alterations in development occur following 

repeated exposures to a mixture of atrazine and metabolites/degradates. It should be 

stressed that earlier studies also reported mammary gland effects with early in life 

atrazine exposure (Rayner et al., 2005), but with markedly higher exposures. The study 

reported by Enoch et al. (2007) is the only study evaluating mixtures of atrazine and its 

degradation products. While the mechanism for the non-cancer mammary gland effects 

following atrazine/degradate exposures is unknown, findings from studies to date, along 

with the apparently substantial accumulation of atrazine and/or its metabolites in the 

mammary gland, provide some concern for higher sensitivity in developing organisms. 

The (re)-consideration of the FQPA 10X Safety Factor should start with documentation 

and conclusions regarding the adequacy of the data describing atrazine’s toxicity profile 

in adults in order to make intelligent conclusions regarding comparative sensitivities. 

Once that is completed, the data for earlier life stages can be presented and more clearly 

interpreted. 

Charge Issue 6: Implications of MOA and Toxicity Profile on Water Monitoring - 

With regard to the water sampling frequency, several Panel members indicated that they 

believed the sampling frequency currently being conducted by the registrant was 

adequate. No one raised strong objections or could offer a rationale for an alternative 

sampling frequency based on the collected information discussed during the SAP 

meeting. There are some assumptions and extrapolations that contribute to the proposed 

critical window of human exposure but given the collective uncertainties that these 

assumptions introduce, the imprecision in the Agency’s proposed sampling frequency 

seems justified. This may be about as precise an estimate as can be obtained when 

starting with the experimental animal data and the exposure requirements for LH surge 

suppression as opposed to using outcomes that are more unequivocally adverse. One 

question that clearly needs further consideration is whether there is a critical exposure 

(such as a minimum AUC at the target site) that leads to a given level of suppression of 

the LH surge. 

 The Panel offered the following approach for setting the boundaries on exposures 

of concern for human health effects. The Agency has appropriately concluded that the 

limited epidemiological human evidence is insufficient to establish causality and does not 

provide sufficient quantitative exposure information to use in a risk assessment. 

However, what if one assumes that the reported human health outcomes are, in fact, due 

to current levels of exposure to atrazine? The patterns of atrazine concentrations in water 

could then be used to provide reasonable estimates of the extent and duration of human 

consumption of atrazine following agricultural applications for pre-emergent weed 

control. Simple models could be used to estimate human exposures corresponding to a 

range of times of exposure to the elevated concentrations observed in the field, given the 

expected maximum water consumption. Where spikes in water concentration are short 

15 
lived and concentrations of atrazine change over a short time scale, then steady state 

blood levels will not be achieved. In this instance, one might consider what would be 

average levels of exposure, relate this to the equivalent internal exposure in the rat, and 

use the promised PBPK model to extrapolate to humans. This may represent a reasonable 

alternative approach to getting at levels of atrazine in drinking water that may represent 

risks to human health. These risks could be compared, certainly on an order of magnitude 

scale against those calculated from the animal data, and may provide a lower bound 

conservative floor from which to work, and provide a different perspective on the water 

sampling frequency problem. This would put the Agency in a much better position if, in 

fact, the Agricultural Health Study or other epidemiology studies provide further support 

for human health effects as the results continue to accumulate. 

 The other consideration when faced with the uncertainty over a critical exposure 

period of from a few days to 4 weeks is whether basing sampling frequency on human 

health effects is, in fact, the best course of action. It may be more useful to consider a 

strategy that attempts to capture the pattern of atrazine concentrations in the source water 

of each CWS based on the characteristics of that particular water system, as opposed to a 

one-size-fits-all approach based on the series of health-based considerations put forth by 

the Agency. Given the collective limitations of the health outcome-based approach, this 

would seem prudent, and would again put the Agency in a better position to take further 

action should the results of ongoing or future epidemiology studies prove more 

convincing. In the meantime, since water is the primary source of environmental 

exposures to atrazine and its metabolites/degradates, there is value in doing a better job of 

establishing the relationship between the measured concentrations at the community 

level, and the resulting absorbed dose in humans. 

16 
PANEL DELIBERATIONS AND RESPONSE TO CHARGE 

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

FIFRA Scientific Advisory Panel (SAP) were scheduled in 2010. The first two meetings 

were held in February and April. The purpose of the September meeting was to solicit 

feedback from the SAP on the status and overall scientific direction of the Agency’s reevaluation on several topics including our preliminary conclusions regarding the noncancer epidemiology literature on atrazine, as well as the human health risk implications 

of the experimental toxicology data. The Agency also sought the Panel’s feedback on the 

proposed updates to the dose-response assessment including the use of an internal dose 

metric and EPA’s benchmark dose analysis. With respect to drinking water exposure, the 

Agency sought the Panel’s feedback on a general framework for designing monitoring 

studies along with basic approaches for analyzing sampling strategies and other methods 

for estimating exposures from less frequent existing monitoring data. Finally, the SAP 

was asked to consider and comment on the implications of atrazine’s toxicity profile and 

its MOA on the development of a water monitoring strategy. 

Question 1.0: Non-cancer Epidemiology 

Section 3.0 and Appendix B of the draft Issue Paper provide the Agency reviews and 

synthesis of the non-cancer epidemiology studies available for atrazine. These include 

studies on a variety of topics, notably female and male reproductive outcomes and birth 

outcomes in addition to other topics. Section 4.0 integrates the findings of the 

epidemiology and experimental toxicology studies. 

Question 1.1 

Please comment on the sufficiency of the Agency’s non-cancer epidemiology reviews with 

respect to identifying the major strengths and limitations of each study. 

Panel Response 

 The Panel congratulates EPA on its inclusion and evaluation of the epidemiologic 

literature as a component of the risk assessment process. The EPA critique of the 

literature on non-cancer outcomes in relation to potential atrazine exposure has been 

thoughtful and comprehensive. While it is easy to criticize human studies because, 

generally, one cannot control exposures and behaviors as well as in laboratory animal 

studies, human data do have strengths that are important for risk assessment. Many of 

the problems in measuring exposures and outcomes, especially non-cancer outcomes, can 

be mitigated with robust study designs. 

 

 Some of the general comments and recommendations which follow also were 

made in the February 2010 Scientific Advisory Panel review but do not appear to have 

been heeded in the present draft of the Issue Paper. We encourage EPA to incorporate the 

earlier advice regarding evaluation of epidemiologic studies. 

17 
 The Panel disagreed with the Agency’s conclusion that the database for noncancer outcomes is strong (i.e., it started with 29 candidate papers and reduced the 

number to 19 papers; 38 additional papers were cancer-related). Only 19 papers met the 

Agency's criteria for evaluation and these covered many different health outcomes of 

interest and had many important limitations, as were noted both by EPA and in the 

Panel’s own detailed analysis presented below. The cancer study database will be larger 

and more relevant for men. Women are generally under-represented in studies of 

occupational exposures. 

 The Panel had a number of comments and recommendations regarding the 

methodology used for evaluating these papers. First, the criteria for including studies 

should be described in greater detail. Second, EPA should include an appendix with a 

full citation and abstract for each paper excluded from full evaluation and a summary 

describing why the paper was excluded so that EPA can demonstrate the completeness of 

its review and justify the exclusions. Third, of the 29 papers identified (page 324 of the 

Issue Paper), two were excluded primarily because they did not independently measure 

atrazine or triazines. This is not well justified in the text. Other studies that were 

included, either ecologic or observational in design, did not report any semi-quantitative 

measures of atrazine or triazine exposure. For example, season of birth or distance to 

corn or soybean crops may be a surrogate or proxy of exposure, but these are not direct 

measures of atrazine or triazine exposures. Therefore, neither of these surrogates is likely 

to be correlated closely with actual exposures or absorbed doses in humans. Fourth, of the 

studies identified in the literature search, 46 identified as exposure assessment studies 

were excluded from further evaluation. For the purposes of the epidemiologic review of 

reproductive and other effects, these are not needed, but in the future (especially for the 

evaluation of cancer risks), these should be reviewed and summarized by EPA exposure 

assessors and epidemiologists and it was suggested that the review and summary be 

provided to the SAP for consideration. When the epidemiologic database is sufficiently 

strong to support dose-response assessment, such studies can be used to connect the 

exposure assessment data with the questionnaire and biomarker data, given knowledge of 

the toxicokinetics of the herbicide. 

 Fifth, it may be efficient to develop a scoring system for the quality of studies 

(i.e., how well do they meet the inclusion criteria) to show how each paper addresses the 

key factors identified by the EPA and the February 2010 SAP Panel (statistical power, 

sample size, generalizability, accurate and precise exposure and outcome assessment 

methods, sophistication of statistical analysis, control for confounding, minimized 

participation and selection biases, etc.). These have been developed for other types of 

epidemiologic analyses (e.g., meta analysis, Cochrane reviews) and could be applied 

easily by EPA. Also, in future reviews of other pesticides and for larger epidemiologic 

databases, EPA may want to have two reviewers independently examine and score each 

paper (perhaps, initially on quality and then on potential for use in dose-response 

assessment). 

 Lastly, it seems that the bar for review of the ecologic studies in the present Issue 

Paper was set rather low. Ecologic studies depend on aggregate data for exposure and 

18 
outcome, and the Panel believed that this study type should be considered in a lower class 

separate from cohort and case-control studies of individual exposures and outcomes, that 

they should rarely be used for evaluating weight of evidence, and are useful only for 

purposes of hypothesis generation. If some reasonable measure or proxy of exposure 

(e.g., surface, groundwater or finished drinking water contamination levels) for the group 

level data is available, the studies that include such exposure measures should be given 

more weight than those that have no measures or proxies (e.g., season of birth). 

 In general, EPA has done a good job of improving an Issue Paper that was already 

strong. Four problems are typically common to all major public health issues involving 

quantitative data. They are the following: 1) the data are vast; 2) the data are highly 

complex (in disciplines, problems addressed, methods, settings, etc.); 3) the data are 

generally of poor quality, and 4) the data may just not be what assessors would like to 

have (e.g., high-dose lifetime animal ingestion studies are conducted when low-dose 

intermittent human exposures by inhalation or consumption of drinking water would be 

of more interest). These problems also apply with some force to the data relevant to 

atrazine and it is apparent that EPA has largely attempted to consider and address these 

problems in evaluating the literature. 

 The Panel’s evaluation of the relevant epidemiology literature and of the 

Agency’s review of these studies follows. 

Female Reproductive System

(1) Farr et al. (2004), (pages 30 and 335-6 of the Issue Paper): The EPA 

critique was largely appropriate in highlighting the strengths and limitations of the 

Farr et al. (2004) analytic approach to evaluating the relationship of atrazine 

exposure to menstrual cycle characteristics in the Agricultural Health Study 

(AHS). EPA was correct in noting that lifetime exposure is assessed while 

menstrual cycle outcomes are captured either within the past 12 months or as 

average cycle characteristics, which might result in non-differential 

misclassification of either the relevant exposure or the outcome, and thus would 

tend to attenuate any true association, if one exists. This approach also does not 

address the temporal relationship of exposures to outcomes. The Issue Paper 

might also note that Farr et al. (2004) relied on recall of exposures and menstrual 

cycle outcomes, and, although it was a cohort study, it was analyzed with odds 

ratios (OR), which might enhance estimates of risk somewhat. Some important 

aspects of this study were not described in the Issue Paper. First, the participation 

rate of women included in this analysis, based on data from the AHS, was only 

57%, which raises the potential for participation bias (e.g., hypothetically, women 

with menstrual abnormalities or concerns about pesticide exposure might have 

been more likely to participate, making the data less representative and potentially 

overestimating the relationship of atrazine to long menstrual cycles and missed 

periods, the strongest associations found for atrazine). Secondly, at issue is the 

representativeness of the study population; the AHS is 97% white and fairly well 

educated so that the generalizability of the findings from this study is somewhat 

19 
limited, and any effect modifiers, such as race/ethnicity or socioeconomic status, 

cannot be examined effectively. This point was included in the February 2010 

SAP comments, but not in the September EPA draft Issue Paper. 

(2) Farr et al. (2006), (pages 30-31 and 332-3 of the Issue Paper): The EPA 

critique appropriately described the strengths and limitations of the second paper 

by Farr et al. (2006)on the AHS. This paper examined the relationship of 

exposure to age at menopause, an indicator of subsequent disease risk and 

longevity (i.e., later age at menopause is associated with reduced risks to, and 

mortality from, a number of chronic diseases and, therefore, with increased 

longevity, although it is associated also with an increased risk of breast cancer). 

However, the ninth line on page 31 is incorrect; a hazard ratio of 0.79 for atrazine 

exposure does not constitute a “greater” association than that for use of 

hormonally active or ovotoxic pesticides, which was 0.77, because the closer the 

hazard ratio is to 1.0 (regardless of whether the hazard ratio is less than or greater 

than 1.0), the smaller the association. The EPA critique is correct in pointing out 

that a non-standard question was used to assess menopause and peri-menopause, 

that pesticide exposure was assessed, rather than, specifically, atrazine exposure, 

and that timing of exposure relative to the outcome could not readily be addressed 

in this study. The Issue Paper should mention that the initial participation rate in 

this portion of the AHS was relatively low (59.5%), thus again creating the 

potential for participation bias, albeit probably not great for this outcome. Also, 

as before, the generalizability of the results from the AHS is somewhat limited 

due to the lack of heterogeneity in race/ethnicity, socioeconomic characteristics 

and lifestyle of the study sample. 

(3) Saldana et al. (2007), (pages 31 and 354-5 of the Issue Paper): The EPA 

critique is appropriate in the analysis of the AHS by Saldana et al. (2007) on the 

relation of pesticide exposure to gestational diabetes. EPA might add to the next 

version of the Issue Paper the point that while the relationship of atrazine 

exposure to gestational diabetes is depicted only graphically, the adjusted odds 

ratio is in excess of 1.0, and the 95% confidence limits do not appear to include 

1.0, which differs from the indication on page 354 that the association was not 

statistically significant. Limitations of the generalizability of the results of the 

AHS due to inclusion of a primarily white and relatively homogeneous study 

sample, as noted above, apply to this paper as well. 

 In summary, these studies evaluating the associations of atrazine with female 

reproductive outcomes (longer menstrual cycles, more missed periods, later age at 

menopause, and gestational diabetes) are suggestive, but inconclusive because of the lack 

of representativeness of the study sample, the potential for participation bias, nonconcordance of the periods of exposure with those of the outcomes, and use of nonstandard questions for some outcomes. Also, the use of odds ratios may overestimate 

relative risks and, thus, the magnitude of association. Some of these issues regarding the 

limitations of the AHS were raised in the February 2010 SAP’s report on the use of 

epidemiologic studies in risk assessment, but appear not to have been incorporated in the 

20 
critiques included in this Issue Paper. If EPA disagrees with the Panel’s comments, the 

next version of the Issue Paper should cite the reasons. 

 Furthermore, while the AHS included a study sample with likely substantial 

pesticide exposure, thus, representing a potentially high risk population, it was not 

designed to address questions related to reproductive outcomes. Other epidemiologic 

studies have been better designed to assess menstrual cycle characteristics and age at 

menopause, have collected biologic samples that could be assessed for atrazine exposure, 

and/or have better representativeness and diversity in their study samples than the AHS. 

Examples of such studies include the Study of Women’s Health Across the Nation 

(SWAN) (Gold et al., 2001; Sowers et al., 2000) and perhaps the Midlife Women’s 

Health Study (Bromberger et al., 1997) or the Australian longitudinal study of midlife 

women (Burger et al., 1995), the Semiconductor Women’s Health Study (Gold et al., 

1995), and/or the Sacramento Community Health Study (Gold et al., 2010). Evaluating 

data and samples from these other studies would make the findings more generalizable, 

and would permit examination of potentially important modifications of the effects by 

race/ethnicity, lifestyle or health-related factors, which are important in geneenvironment interactions and potential epigenetic effects. Additionally, the concern 

expressed in the EPA critique regarding adequate control of physical activity is 

overstated for age at menopause as few, if any, studies have shown this to be an 

important determinant of age at menopause; key factors for this outcome are smoking, 

educational level and use of oral contraceptives. Finally, in several reports, atrazine was 

studied along with other pesticides. Results for atrazine then are presented in comparison 

with the results for all pesticides. This is incorrect; the result of such an analysis is to 

reduce the apparent strength of evidence for some effect. The comparison should be 

between atrazine and all other pesticides in the study or to no pesticide exposure. It may 

be that the original authors often or always reported the wrong comparisons in a way that 

cannot be corrected, but EPA should at least note the error. 

Male Reproductive System

(1) Swan et al. (2003), (pages 32-3 and 362-4 of the Issue Paper): The EPA 

critique of the strengths and limitations of the Swan et al. (2003) cross-sectional 

case-control study is appropriate. Like the AHS, the study sample, derived from 

the multicenter Study of Future Families, was almost entirely white, once again 

limiting the generalizability of the findings and the ability to examine interactions. 

In addition, the participation rate was low (27%), and the sample size was small; 

however, the fact that the study was hypothesis-driven and biomarkers of 

exposure and outcomes were used were important strengths. A small correction 

should be made in the second line of the critique on page 362 to indicate that this 

study was of non-persistent pesticides (rather than persistent, as currently 

indicated on this page). The study authors found that high atrazine levels were 

associated with poor semen concentration, morphology and motility. 

 This single study examining the relationship of atrazine exposure to reduced 

sperm quality was suggestive with the important strengths of being hypothesis-driven and 

21 
using biomarkers of exposures and outcomes, but its limitations need to be addressed in 

additional studies. 

Fetal and Perinatal Outcomes 

(1) Arbuckle et al. (2001), (pages 33 and 327-8 of the Issue Paper): The EPA 

critique of Arbuckle et al. (2001) is mostly appropriate with the exception of the 

exposure assessment issue described below. The Issue Paper should note that the 

Ontario Farm Family study used a historical cohort design. Data were analyzed 

with odds ratios, which may overestimate relative risk, without adjusting for 

confounders. Some analyses appeared to use post-conception atrazine or other 

herbicide exposure or later preconception exposure as the referent category, 

presumably to try to address potential recall bias, but the number of women who 

reported spontaneous abortions and were exposed in this latter category was 

small. This publication also was unclear about how multiple pregnancies from 

the same woman, which represent lack of statistical independence, were handled 

statistically. (Tests for non-independent outcomes exist, but were not used here.) 

The Issue Paper also should emphasize that the associations of preconception 

atrazine exposure with early spontaneous abortions were modest in magnitude and 

not statistically significant (ORs ranged from 1.3-1.7, depending on the referent 

used, all with 95% confidence intervals including 1.0). In addition, the last 

paragraph of the Agency’s review of this paper should refer to “odds” not “risks” 

because odds ratios and not relative risks were determined. The EPA critique of 

this and the Savitz et al. (1997) paper seems to discount results because they used 

farm level exposures rather than direct personal exposure measurements (page 

328) so that farm level exposures of male and female applicators cannot be 

distinguished. This approach, however, might represent a much better effort at 

historical pesticide exposure reconstruction than the self-reported measures used 

in other observational studies (e.g., the AHS ever/never classification). The Issue 

Paper correctly notes that women who were aged 35 years or older and reported 

preconception exposure to triazines had a nearly three-fold, statistically 

significantly, increased odds of spontaneous abortion (OR 2.7, 95% CI 1.1, 6.9). 

Women aged 35 years or older who reported preconception use of both triazines 

and thiocarbamates had an OR=7.5 for spontaneous abortion, all of which 

suggests that older maternal age may modify the overall risk of preconception 

triazine use by increasing the odds of spontaneous abortion. 

(2) Savitz et al. (1997), (pages 34, 39and 358-9 of the Issue Paper): The EPA 

critique of the historical study by Savitz et al. (1997) is appropriate. In addition 

to not being able to link farm activities with the application of crop herbicides and 

the use of specific herbicides (as noted in the critique), the small sample size of 

women exposed to atrazine who had some of the outcomes (e.g., preterm and 

small-for-gestational-age deliveries) and the modest participation rate (64%) also 

are limitations, although they probably resulted in less likelihood of detecting 

differences as statistically significant. Paternal use of atrazine on crops had an 

22 
adjusted odds ratio for miscarriage of 1.5 (95% CI 0.9-2.4), for preterm delivery 

of 2.4 (95% CI 0.8-7.0) and for small-for-gestational-age of 0.5 (95% CI 0.2-1.3). 

 In summary, these two studies of the relationship of atrazine exposure to fetal and 

perinatal outcomes were suggestive, with largely positive but non-significant associations 

that were modest in magnitude. They also possessed a number of limitations, including 

use of odds ratios instead of relative risks and small sample sizes for some outcomes, 

resulting in wide confidence intervals, lack of adequate statistical power, lack of clarity in 

the handling of lack of independence of multiple pregnancies from the same woman, lack 

of data on specific herbicide exposure, and potential for participation bias. These deficits 

should be addressed in additional studies. 

Birth Defects

(1) Mattix et al. (2007), (pages 34-5 and 344-5 of the Issue Paper): The EPA 

critique of the paper by Mattix et al. (2007) correctly notes its major limitation: 

the ecologic study design which does not permit assessment of actual maternal 

exposure to atrazine in the drinking water. Thus, the correlation between atrazine 

concentrations in surface water and month of conception of fetuses with 

abdominal wall defects does not mean that the individual mothers who delivered 

such infants actually consumed water with elevated atrazine. 

(2) Hornemann et al. (2009) (pages 35 and 340 of the Issue Paper): The EPA 

critique appropriately notes the limitations of this ecologic study by Hornemann 

et al. (2009). These authors examined seasonality of omphalocele prevalence rate 

during a time when atrazine was banned from use in agriculture in Germany so 

that they had little likelihood of observing differences in prevalence rates by 

season. Also, the ecologic design would not permit assessment of actual 

individual maternal prenatal atrazine exposure in relation to occurrence of 

omphalocele. 

(3) Winchester et al. (2009) (pages 35-7 and 370-1 of the Issue Paper): The 

EPA critique appropriately indicates that the major limitation of the Winchester et 

al. (2009) study is its ecologic design, which does not permit assessment of the 

individual maternal exposures to atrazine or other contaminants consumed in 

drinking water in relation to occurrence of birth defects. The critique also 

appropriately notes that the effect sizes are small. 

(4) Ochoa-Acuna and Carbajo (2009a) (pages 36 and 352 of the Issue Paper): 

The EPA critique correctly identifies the major limitation of the Ochoa-Acuna 

(2009) study as being the use of residence near corn fields as a surrogate of 

potential chemical exposure, so that actual individual maternal exposures were 

not measured. In addition, the sample sizes were small for several types of birth 

defects, and it was likely that chance could have resulted in at least some of the 

significant findings due to the multiple statistical tests that were performed. Also, 

while the authors indicated that atrazine is used largely on corn fields, rather than 

23 
soybean fields, other chemicals such as fertilizer nutrients also are preferentially 

applied to corn fields. The study authors did not indicate whether other pesticides 

also were used on corn fields, which might also be a potential explanation for the 

observed findings. 

(5): Waller et al. (2010) (pages 36-7 and 368-9 of the Issue Paper): The EPA 

critique of the population-based case-control study of gastroschisis reported by 

Waller et al. (2010) is quite appropriate in noting the strengths of the case-control 

approach, including individual information on exposures and outcome, compared 

with that of an ecologic design, and the large sample size studied, while also 

noting the limitations, including: a) that the exposure was largely ecologic (i.e., 

distances of residence to “high” atrazine surface water monitoring site, rather than 

actual maternal tap water exposure); b) the lack of analysis of surface water 

atrazine concentration by maternal age; c) the possibility of misclassification of 

exposure due to monitoring of sites that were not necessarily the source of 

drinking water and the possibility of mothers relocating residence between early 

pregnancy and delivery; and d) the lack of clarity of selection of confounders to 

control in multiple logistic regression models. In addition to the variables already 

listed in the Issue Paper, marital status, race and income also should be noted as 

differing significantly between cases and controls but not controlled in the 

multivariable analyses. The study authors concluded that the occurrence of 

gastroschisis was significantly inversely related to distance of the maternal 

residence from the closest “high” atrazine monitoring site. 

 In summary, three of the five studies of birth defects used the sub-optimal 

ecologic design so that atrazine exposures and outcomes were not assessed on an 

individual level. This is a major limitation. The other two studies are also considered 

only suggestive because one did not examine individual maternal exposures to atrazine 

and the other used a largely ecologic measure, rather than an individual measure of 

atrazine exposure. Additional limitations included: multiple statistical testing that might 

have resulted in statistical significance due to chance alone because the p values were not 

adjusted for multiple testing; the analyses did not account for other exposures that might 

explain the observed effect (Ochoa-Acuna); and confounding variables were not 

adequately described and adjusted for in analyses (Waller). 

Adverse Birth Outcomes

(1) Ochoa-Acuna et al. (2009b) (pages 38 and 349-50 of the Issue Paper): The 

EPA critique of the population-based, historical cohort study by Ochoa-Acuna et 

al. (2009) on the relationship of atrazine concentrations in drinking water to the 

prevalence of pre-term and small-for-gestational-age infants appropriately noted 

the strengths of using a more refined exposure measure of atrazine based on 

monitoring data and adjustment for multiple individual-level confounding 

variables. The Issue Paper, however, also should note the strength of the large 

sample size, as well as the limitation that individual maternal tap water 

consumption was not obtained, which might have resulted in some 

24 
misclassification of exposure. No association was found between atrazine 

concentrations and preterm births, but a significant positive association (a 20 % 

increase) was found with small-for-gestational-age. 

(2) Villanueva et al. (2005) (pages 38-40 and 365-6 of the Issue Paper): The 

EPA critique of the Villanueva cross-sectional study appropriately noted its 

strength in using measurements of atrazine in drinking water and the major 

limitation of uncertainty in using these measurements to estimate individual 

maternal atrazine exposures as well as the large number of measurements below 

the limit of detection and the resulting narrow range of exposure levels, which 

reduced the ability to detect significant differences in exposure groups. The 

study’s sample size was moderate for the three outcomes of interest: low birth 

weight, preterm delivery and small-for-gestational-age. Indeed, no significant 

associations were detected between atrazine concentrations in drinking water and 

these three pregnancy outcomes. The Issue Paper also should note that the 

findings are suggestive of elevated odds of low birth weight (which was not 

statistically significant) and small-for-gestational-age in pregnancies whose third 

trimester overlapped high atrazine use periods (50% increase) and, similarly, for 

preterm deliveries whose first trimester overlapped these periods (not statistically 

significant). Importantly, it also should be noted that the analyses in this paper 

were not adjusted for variables that are often influential in these outcomes, such 

as maternal smoking, educational level and parity. 

(3) Munger et al. (1997) (pages 38-9 and 347-8 of the Issue Paper): The EPA 

critique of the ecologic study by Munger et al. (1997) is quite appropriate in 

noting that the correlation of drinking water levels of atrazine and other triazine 

herbicides with low birth weight, prematurity, and intrauterine growth retardation 

is useful only for generating hypotheses to be studied in methodologically 

stronger epidemiologic approaches because time periods for the exposure levels 

and birth outcomes were not completely comparable. The analyses assume that 

the relative ranking of the communities with regard to drinking water 

contaminants remained the same throughout the study period. Again, the study 

population was largely white (thus limiting the generalizability of results). The 

Issue Paper should note that this study did not examine individual maternal 

drinking water exposures and controlled only for some of the important 

confounding variables (e.g., smoking and maternal educational level) on a 

community rather than individual level, thus potentially misclassifying or 

inadequately controlling for confounders on an individual level. 

(4) Dabrowski et al. (2003) (pages 38-9 and 330 of the Issue Paper): The EPA 

critique of the population-based, case-control study by Dabrowski et al. (2003) of 

the relationship of pesticide exposure during pregnancy to birth weight and 

pregnancy duration appropriately notes that the strengths of the study included its 

population-based comparison with a control group similar to the cases, 

ascertainment of birth weight from medical records, paternal confirmation of 

pesticide exposure reported by mothers and adjustment for multiple confounding 

25 
variables. The critique also highlights the major limitation that the analysis is not 

specific for atrazine exposure, and the sample size was too small to examine 

specific pesticides. The results indicated a significant reduction in birth weight 

and pregnancy duration with pesticide exposure in the first or second trimester 

after adjustment for important confounders. 

(5) Sathyanarayana et al. (2010) (pages 39 and 356 of the Issue paper): The 

Sathyanarayana et al. (2010) study analyzed the AHS for the association of 

maternal pesticide exposure to birth weight appropriately. EPA noted that the 

study did not specifically address atrazine exposure. Additionally, the number of 

low birth weight or preterm infants was too small to assess individual pesticide 

exposures or to detect meaningful differences while adjusting for multiple 

confounding factors. Indeed, the study found no significant effect of maternal 

pesticide exposure, even direct agricultural exposure, on birth weight. A strength 

of the study was the control for multiple confounding variables in the analyses. 

As noted previously, the AHS is comprised of a largely white population sample, 

which limits the generalizability of the findings and the ability to examine any 

modification of effects by race/ethnicity, socioeconomic status or lifestyle or 

health factors. 

 In summary, these five studies of adverse pregnancy outcomes ranged from 

ecologic to population-based case-control and historical cohort designs, the latter having 

the strengths of better individual assessment of exposure to atrazine. Strengths included 

some large sample sizes and adjustments for multiple confounding variables to assess the 

independent effect of atrazine exposure, although individual maternal water consumption 

was not assessed to improve estimates of individual exposures. The findings suggest a 

relation of atrazine exposure to small-for-gestational-age, but two of the non-ecologic 

study designs focusing on birth weight were not specific for atrazine. Small-forgestational-age has many causes, and more than one cause (if any) may be affected by a 

pesticide. Lack of agreement among studies may be a result of multiple mechanisms. 

For example, atrazine may have a direct effect on the fetus at some time or in some 

settings and work through the mother’s endocrine system in others. 

 Respiratory Effects

(1) Hoppin et al. (2002) (pages 40 and 338-9 of the Issue Paper): The EPA 

critique appropriately noted that this paper reporting on a cross-sectional 

association of wheeze with pesticide use in the AHS showed a modest but 

significant association of atrazine with wheeze (odds ratio =1.2, 95% CI 1.07, 

1.34) with a significant dose-response trend observed with days of use, although 

this was not an a priori exposure hypothesis. As noted in the critique, this study 

did have a large sample size and was able to control for multiple confounding 

factors, especially exposure to corn and grain dust. The Issue Paper also should 

note that this study had the same limitations noted previously for the AHS, 

particularly the potential for participation bias and the lack of diversity in the 

study sample which reduces generalizability and the ability to examine 

26 
modification of the effects by race/ethnicity, socioeconomic status and lifestyle or 

health factors. As noted in the Issue Paper, a subsequent paper with overlapping 

authorship examined the same study sample and found that stratifying on two 

different conditions (allergic and non-allergic asthma) and controlling for 

additional potential confounding factors resulted in no significant relationship of 

atrazine exposure to these outcomes (Hoppin et al., 2009). 

(2) Kossman et al. (1997) (pages 40-1 and 342-3 of the Issue Paper): The EPA 

critique correctly identifies the limitations of the cross-sectional study by 

Kossman et al. (1997), including the inability to evaluate the specific relationship 

of atrazine exposure to impaired respiratory function and the lack of control of 

confounding factors (particularly importantly, smoking). In addition, the Issue 

Paper should note that the sample size was small and would not have provided 

adequate statistical power to detect meaningful associations if specific chemicals 

were evaluated and if the associations were controlled for confounders. 

(3) Kluchinski et al. (2001) (pages 41 and 341 of the Issue Paper): The EPA 

critique correctly identified the limitations of this cross-sectional study to include 

the inability to evaluate the specific relationship of atrazine exposure to immune 

parameters that may be biomarkers of impaired respiratory function and the lack 

of control of confounding factors (particularly smoking). In addition, the Issue 

Paper should note that the sample size was small and would not have provided 

adequate statistical power to detect meaningful associations if specific chemicals 

were evaluated and if the associations were controlled for confounders. 

 In summary, these three cross-sectional studies of the relation of pesticides to 

respiratory impairment had significant limitations, including sample sizes inadequate to 

provide sufficient statistical power to detect meaningful effects associated with specific 

chemicals while controlling for confounding variables; any observed associations were 

modest in magnitude. 

 It appears that relatively little still is known about human fertility related to 

atrazine exposure. This reality should be noted in the Issue Paper. If additional 

epidemiologic studies are undertaken, they should be well-designed with sample sizes 

sufficient to identify meaningful effects while controlling for confounding factors and 

permitting examination of effect modification by important genetic/demographic/ 

lifestyle/health factors, adjustment for multiple comparisons, standard and precise 

assessment of individuals’ actual exposure and outcomes and more representative and 

diverse study samples. New research should focus on relevant health outcomes in 

individuals who have relevant, real-life exposures (e.g., in occupational settings, such as 

farm or manufacturing workers or individuals with likely or known drinking water 

exposures), perhaps in appropriate agricultural areas. Such studies will need to be 

supported by laboratory data bearing on important public health issues so that significant 

effects or observations of no significant association(s) in humans can be appropriately 

interpreted. 

 

27 
 One Panelist expressed concern about the handling of studies with substantial 

numbers of analytical chemistry results below the limit of detection (LOD). A lot of 

useful information can be derived from such findings, even when they account for most 

of the observations, and especially when interest is focused primarily on the highest 

exposures, as is the case in many of the studies presented in the Issue Paper. At the very 

least, one can do a rough test by first assuming that all of the “below LOD” values are, in 

fact, zero, then assuming that all are at the upper end of the no-detect range. Regardless 

of whether these two analyses are in rough agreement on the most important conclusions, 

the Issue Paper should say so. More sophisticated ways are available to approach the 

LOD issue. 

Question 1.2 

At this time, atrazine’s non-cancer epidemiologic database is not robust enough for 

inclusion in quantitative risk assessment and does not support the ability to determine 

causal associations. There are several limitations present in the current database, and 

the quality of atrazine exposure assessment is paramount among the limitations. In 

Section 4.0 of the draft Issue Paper, the Agency describes the qualitative similarities and 

differences between the epidemiologic findings and the experimental toxicology database. 

In short, the observational studies – particularly those related to reproductive effects in 

adult females, as well as small for gestational age in newborns – lend further support for 

the human relevance of the laboratory animal findings. 

a. Please comment on the scientific information that does and does not support the 

Agency’s conclusions (as described in Section 3.0) with respect to the characterization of 

quality, and limitations of the non-cancer epidemiologic database and its utility in hazard 

characterization, dose response analysis, and quantitative risk assessment. 

Panel Response 

 EPA states (Section 4, pg 58) that the epidemiology and toxicology databases can 

be used together to inform hazard identification, dose-response assessment and hazard 

characterization, but for purposes of quantitative risk assessment (i.e., risk 

characterization), the experimental toxicology data (particularly, in vivo data from the rat) 

will be used for endpoint selection and dose-response assessment. 

 EPA has conducted a thorough study-by-study review of the non-cancer 

epidemiologic papers; the SAP critique of the Agency review is presented in the response 

to Question 1.1. Overall, the SAP agrees with the Agency’s conclusions that the current 

non-cancer epidemiologic database (n=19 studies) provides some useful information for 

hazard identification but is too limited to provide sufficient information for credible dose 

response assessment or risk characterization. As the Agency states, exposure 

characterization is the primary limitation; it would be impossible to reconstruct individual 

level exposures using the available observational data. Furthermore, many of the 19 

studies are ecologic in design and should be given little weight for risk assessment 

purposes. 

28 
 Some general comments on the broader issues regarding interpretation of the 

integrated data were presented and deserve more explicit acknowledgement and 

discussion in the EPA draft. These extend across a whole set of studies and are not 

limited to the interpretation of single studies. First, a general failing in most studies of 

toxic effects is the lack of sufficient attention to matters of statistical power. “Lack of 

evidence showing an effect” is simply not the same as “evidence showing a lack of 

effect.” The EPA draft would be stronger if it discussed the likelihood that critical 

(apparently) negative results might be reversed by better and stronger designs and, 

especially, larger studies (i.e. with greater statistical power), whether in humans or 

animals and whether findings, even when non-significant, show consistency across 

studies with different designs performed in different populations. 

 

 Similarly, the “multiple comparison problem” is not sufficiently appreciated. 

This problem is widely recognized as it applies to single studies; but it applies equally to 

any collection of papers or other sets of independent results. Many studies have 

examined the health effects of atrazine, each with multiple endpoints accompanied by 

many analyses of their data. At the traditional default 5% level for statistical significance 

in scientific analyses, an average of one in 20 tests, in which no effect truly exists, will be 

labeled “statistically significant.” This 5% approach (or 95%, in the case of confidence 

bounds) has been very productive for many decades across all areas of science, as 

important results can be evaluated in a broad context of prior knowledge and, sometimes, 

by the collection of new data. The multiple comparisons problem is pervasive and hard 

to deal with in reviews that cover large numbers of reports. The question in such reviews 

is not whether some result is statistically significant, but whether the overall pattern of 

results labeled significant actually lies outside the expected range. Roughly speaking, if 

the literature reported 100 tests of data in which no true effect exists, one should expect 

about 5 to be “significant.” If say, 10 are significant, or if some of the reported p-values 

are quite small, an effect may be present. The SAP urges EPA to review the critical 

positive findings and to comment at greater length on the plausibility that such findings 

are a result of multiple comparisons. 

 One more point is that statistical confidence bounds and p-values measure the 

effects of random error, and random error only. Bias increases the uncertainty, so that 

calculated p-values are too small and confidence bounds are too narrow, and the effect 

estimate is generally not accurate. Bias can be made rather small in designed laboratory 

studies, but is a major threat to conclusions in other settings unless sufficient attention is 

given to minimizing selection and participation biases in the design and implementation 

of the study. 

 These points can be summarized to say that negative findings do not always mean 

no effect is present, and positive findings do not always mean that such an effect is truly 

operating. This is as true for laboratory studies as it is for epidemiology. Educated 

scientific judgment cannot be replaced by reliance on mechanical rules about what is a 

real effect. It would be helpful to have more explicit acknowledgment of points for 

which judgment (informed by statistical analysis) still has an important role. 

29 
 The Panel also presented some specific comments on the epidemiology portion of 

the Issue Paper. The Issue Paper indicates that no national birth defect registry exists in 

the US (page 37, line 18). In fact, CDC has operated a very broad-based registry for 

many years which could be used in geographic studies of atrazine. EPA has presented 

the data for gastroschisis and omphalocele separately; this is appropriate because while 

they are both abdominal wall defects (AWDs), they are entirely different conditions and 

almost certainly brought about by different mechanisms. Finally, while exposures may 

be of short duration, the effects might be expressed over much longer times (e.g., for 

outcomes such as cancer, age at menopause and menstrual disturbances and possible 

hormonal disturbances and even birth defects). Thus, an exclusive focus on exposures 

occurring based only on the first trimester of gestation is not recommended. 

b. Please comment on scientific information that does and does not support the 

integrative analysis and conclusions, contained in Section 4.0, with respect to the 

similarities, differences, and uncertainties of the experimental toxicology and 

epidemiologic findings. 

Panel Response 

 

 In the February 2010 SAP meeting on the development of a framework for 

incorporating epidemiologic data into the risk assessment process, two case studies were 

presented (diazinon incident data and atrazine epidemiologic studies), and a draft 

framework was developed and presented by the EPA. This was reviewed by the SAP at 

that time and recommendations for improvements to the framework were provided. The 

work presented by the EPA at the September 2010 atrazine meeting and summarized in 

Appendices B1 and B2 reflects consideration of the majority, but not all, of these 

comments/recommendations. 

 Well-designed observational studies in humans that test hypotheses which are 

developed, in part, based upon the adverse effects observed in animals and an 

understanding of the toxicological mode(s) of action are needed to be able to incorporate 

the epidemiologic data into the risk assessment process and integrate it with the 

toxicology database. The comments from the Panel members included in this report 

reflect their assessment of the literature and associated materials that were provided for 

the September 14-17 meeting and, to a lesser degree, the additional data/studies that were 

presented in the public comment session on September 15, 2010. 

 To discuss the integrative analysis of the epidemiology and toxicology literature, 

the Panel assumed that atrazine or a mixture of parent and/or its relevant metabolites, 

following repeated exposures, attenuates the LH surge in animals, and when this 

attenuation is significant, this can result in adverse reproductive effects in animals. 

Furthermore, the Panel assumed that the mode of action (MOA) is relevant to humans, 

although some evidence to the contrary was presented in the public comment session (re: 

hypothalamus GnRH). 

30 
 While some major uncertainties in the toxicology database (which includes 

hundreds of studies) were identified in the SAP meeting, atrazine exposure disrupts the 

regulation of pituitary luteinizing hormone secretion and can alter a number of 

reproductive functions in rats based on the experimental toxicology. Attenuation of LH 

may be a precursor to a number of adverse events (depending, in part, upon life stage of 

the exposed individual), and might lead to altered reproductive function. The available 

data did not allow a clear consensus to emerge on the necessary percent attenuation 

needed to yield adverse developmental and reproductive effects. The form of the dose, 

route of dosing, and frequency of administration all impact on the size of dose that will 

produce a given defined response. Multiple lower dose exposures over time (as seen in 

the Cooper et al., 2010, studies of 4 day exposures in rats) were more effective for LH 

surge attenuation than a single high dose (Cooper et al., 2000). Repeated atrazine 

exposures at doses as low as 3.65 mg/kg/day led to disruption of estrous cyclicity and 

early reproductive senescence. Greater than 80% attenuation of the LH surge in any 

given 4-day estrous cycle would be needed to observe deleterious effects in the 

reproductive system effects in rats. There was significant discussion of the effect of the 

route of administration on the pharmacokinetic behaviour, internal exposure and 

magnitude of the attenuation of the LH surge or other relevant precursor events. It was 

noted that the same applied dose had different effects when administered by feeding and 

oral gavage. A dose by the latter route that produced reproductive toxicity was without 

effect when administered in the food (according to data presented by the registrant in its 

public comments). This may reflect different pharmacokinetic profiles being produced by 

the different methods of administration. The importance of the form of the dose is well 

known and had been discussed at an earlier SAP (page 14 of the report of the SAP, April 

26 - 29, 2010). Work is ongoing to resolve these problems, but clearly there is a need for 

data that are relevant to human exposure via drinking water. Moreover, some of the 

toxicologists speculated that the rodent may not be a good model for the preovulatory LH 

surge in humans. All of these factors are important for the interpretation and design of 

both the animal and epidemiologic studies. Assuming a relevant MOA, it will be 

necessary to consider route and duration of exposure, as well as magnitude of 

exposure/dose, to assess the potential for risks of adverse reproductive outcomes in 

humans. 

 

 Furthermore, some humans may be exposed to atrazine and/or its metabolites over 

a significant portion of their lifespan; these exposures are not consistent within or 

between life stages and may be predominantly oral/dietary (unless the population is 

occupationally exposed, which may represent predominantly dermal exposure) and are 

also likely to occur in mixtures. In most animal studies, exposures are mainly to single 

compounds, generally at higher doses than those to which humans would be exposed. In 

addition, real-world human exposures may differ in route of administration than those 

used in the animal studies. Lastly, few mixtures of triazines alone or in combinations with 

other environmentally-detected substances are generally tested in animal experiments. 

 

 As EPA noted, in order to integrate the epidemiology and experimental 

toxicology information, a strong experimental toxicology database, an understanding of 

toxicological mode(s) of action, and a strong epidemiology database is needed. For 

31 
atrazine, a robust experimental toxicology database exists, including hundreds of studies, 

and understanding of its likely mode(s) of action is increasing. To complement this 

toxicology database, we need human epidemiologic studies which have been specifically 

designed to evaluate relevant outcomes and that are based on defined mode(s) of action. 

Significant improvements in the epidemiologic literature will be necessary for the 

integration of these results into quantitative risk assessment. 

 Since some of the reproductive effects or their precursor events (e.g., estrous 

cycle characteristics, long or missed cycles, and attenuation of the LH surge) observed in 

animals are acute in nature and occur after relatively short periods of exposure, 

prospective studies can be designed to evaluate these outcomes in humans. These can be 

conducted at relatively low cost (as compared with chronic outcomes such as cancer). To 

achieve exposures/doses that are measurable and relevant (meaning that exposures are 

repeated over time, not simply a single occurrence), these types of studies could be 

conducted in occupational cohorts, such as farm or manufacturing workers, or 

alternatively, in women living on farms who are likely to have higher exposures than the 

general population (whose primary source of exposure is via drinking water). Ideally, 

one would obtain information on current (external) exposures (via direct measurement, 

questionnaire, records, and/or diaries) and at the same time, collect information on 

absorbed dose over time (biological samples including urine analyzed for atrazine parent 

and active metabolites as well as inactive conjugates). This type of information would be 

far more useful for risk assessment than the measures extant in the epidemiologic atrazine 

literature to date. 

 Currently, the epidemiology database consists of 19 non-cancer studies which met 

EPA’s selection criteria for detailed review. EPA states that this is a larger database than 

is typically available, but these studies cover a broad variety of non-cancer outcomes 

(ranging from reproductive effects to wheeze) and are not of sufficient quality to include 

in a comprehensive risk assessment. These 19 epidemiology studies present very little 

information collected in humans that support the outcomes observed in animals; 

therefore, the Panel concluded none of these studies were useful for hazard, risk or doseresponse assessment and are likely useful only for problem formulation. Epidemiologic 

data relevant for female or male reproductive health may be obtained from existing 

cohort studies, which were designed for assessing these outcomes in relation to other 

exposures or factors and could perhaps be used in hypothesis-generation, but rarely 

should it be expected that the data will be of sufficient quality to be useful for a risk 

assessment of outcomes that differ from those of the primary hypotheses. For example, 

many of the primary outcomes of interest in the Agricultural Health Study (AHS) are 

cancers and, thus, the methods of exposure assessment were designed to be relevant for 

cancer outcomes (i.e., long latent periods, some consideration for frequency (seasonal) 

and duration of exposure (years) and a few select factors that may modify exposures, 

such as protective clothing worn and equipment used but not genetic, lifestyle or other 

health-related factors). Furthermore, the AHS was not designed to and thus did not 

measure well other outcomes (such as menstrual or reproductive outcomes) nor was its 

study population adequately diverse and large to be more generalizable and to examine 

effect modification adequately as noted in the previous sentence. 

32 
 

 The two studies published by Farr et al. in 2004 and 2006, which examined 

menstrual cycle characteristics and delayed menopause, made use of the AHS cohort but 

are severely limited by the availability of relevant exposure data. The SAP does not 

consider that dichotomous exposure categories, which are classified as “ever/never” and 

the corresponding results or odds ratios will be useful other than for hypothesis 

generation. They provide no information on the timing, frequency, duration or magnitude 

of dose – all of these measures are important for the integration of the epidemiologic data 

with the toxicology data for risk assessment. Although the AHS will provide very 

important information related to pesticide and other agricultural exposures over the years, 

particularly in relation to cancer and other chronic diseases, it was not designed to 

identify accurately these specific reproductive outcomes. The critique of the exposure 

assessment methods is provided knowing that the study was not designed to test 

hypotheses about pesticide or atrazine-specific exposure in relation to these reproductive 

outcomes and thus is not appropriate for assessing risk for these outcomes. 

 

 An example of an inappropriate “ever/never” classification of an exposure could 

be based on the following question: “Have you ever smoked one cigarette?” A large 

percentage of the population would answer “yes” to this question and would be classified 

as exposed. This measure would have little relevance for most health outcomes. The 

more relevant question for a cancer outcome would be “Have you smoked at least 10 

cigarettes a day for 10 or more years?” For short-term effects, the question “Have you 

smoked 10 or more cigarettes/day over the past month?” might be asked. Thus, the 

hypothesis or study question will dictate the type of questions being asked or the 

exposure measures used. Furthermore, the use of exposure categories such as “all 

pesticides” or “herbicides,” “insecticides,” “fungicides,” is not particularly useful. 

Analyses based on individual chemicals, relevant mixtures, or chemical classes are far 

more relevant (e.g., triazines as a group) and categorization based on suspected mode of 

action (i.e., targets a specific gene or endocrine modulators, although this is very broad as 

well) at least have some biological basis for hypothesis generation. 

 Of the other studies that indicate some adverse effects in humans, EPA notes the 

Arbuckle et al. (2001, spontaneous abortion), Waller et al. (2010, AWDs) and Villanueva 

et al. (2005, preterm delivery, small-for-gestational-age, low birth weight) studies 

conducted hypothesis testing (Table 3.1, EPA Issue Paper), and two others had nonsignificant results. Unfortunately, inadequate exposure assessment was a major 

limitation in these studies. The other studies reviewed, including the ecological studies of 

group data, should be given little consideration in EPA's analysis. In addition, it should 

be very clear in the EPA reviews of individual studies that the important distinction be 

made between ecological studies and the “ecological approach” for exposure assessment. 

For example, assigning an area value (i.e., average or median concentration of atrazine in 

finished drinking water from a community water system) to an individual study 

participant is not an ecological study design. That would represent an observational 

study with individual assignment of exposure. As noted previously, ecologic studies 

should be considered only for hypothesis generation. 

 

33 
 The Panel agreed that the integration of study findings, interpretations and 

conclusions in Section 4 of the Issue Paper is largely correct. However, Section 4 does 

not adequately address the significant limitations of the AHS that were noted directly 

above and in the response to Question 1.1. Given these limitations and the likely 

different mechanisms whereby ovarian aging occurs in rodents compared to humans, it 

may be expected that the experimental toxicology and the limited epidemiologic findings 

would differ with regard to these effects. Furthermore, as stated previously, other human 

studies of menstrual cycle characteristics and/or age at menopause are likely to have been 

better designed to assess these outcomes with less misclassification than the AHS, and 

many of these other studies have stored biologic specimens that could be measured for 

atrazine, its metabolites and other environmental contaminants and would do a better job 

that the AHS of assessing any association of atrazine exposure with these outcomes. 

 While the limitations of the Swan et al. (2003) study have been noted above in the 

response to Question 1.1, and are important to consider in interpreting the findings, it is 

also important to note that the findings are congruent with some of the non-rodent 

findings that were presented at the meeting regarding adverse male reproductive effects. 

While the participation rate in the Swan et al. (2003) study was quite low at 27%, as the 

authors note, this is not an infrequent occurrence in human studies of semen 

characteristics and was unlikely to be related to the findings. Furthermore, while the 

numbers were small, resulting in wide confidence intervals, the effect estimate was large, 

and the confidence intervals did not include 1.0. It is true that further investigation is 

needed on the mode of action. 

 The human data on small-for-gestational-age, low birth weight and prematurity 

are suggestive and not inconsistent with some of the animal study findings. The Issue 

Paper appropriately points out the possibility of misclassification, particularly of 

exposure and/or possibly, of outcome in the human studies. It should be noted that on the 

fifth line on page 58 the first use of “atrazine” should be “pesticide.” It should also be 

noted at the end of this same paragraph that one would not necessarily expect an effect on 

birth weight to result from exposure only in the first trimester of pregnancy because an 

effect resulting from exposure in the third trimester, when fetal weight increase is 

generally greatest, is at least equally plausible. 

 An integrative analysis of the epidemiology and toxicology databases could be 

strengthened with additional information on the toxicokinetics and toxicodynamics in 

humans or an effective PBPK model, which is expected by 2011. The registrant 

presented information in the public comment session that humans primarily form the 

DEA metabolites, while rats primarily form the DIA metabolites. Similar formation of 

the DACT metabolites is expected. This information is helpful for dose reconstruction in 

humans based on biological monitoring studies. In addition, a thorough review of the 

human exposure data (including biological monitoring studies) in both occupational and 

environmental settings is absolutely necessary to be able to connect potential exposures 

and absorbed dose measures in humans with doses administered to animals. Such an 

analysis should be brought to the SAP for peer review. 

34 
The EPA conclusions in Section 4 of the Issue Paper indicate that “the non-cancer 

epidemiology database provides important information that adds to the human relevance 

of the animal findings, particularly related to female and male reproductive effects and 

small-for-gestational-age birth outcome.” The SAP suggests that only “limited 

information” is provided. Furthermore, the following statement appears in the EPA Issue 

Paper: “Among the key studies in the epidemiology database, studies in which the 

potential for other non-causal explanations is comparatively lower, evidence indicates a 

possible role for atrazine exposure (Farr et al., 2004; Farr et al., 2006; Swan et al., 2003; 

Ochoa-Acuna et al., 2009; and, Villanueva et al., 2005).” The SAP disagrees with this 

statement. Given the limitations of these studies as discussed in the Panel’s responses to 

Questions 1.1 and 1.2a, the potential for other non-causal explanations is still very high in 

these studies; they provide only limited evidence of human relevance. 

 The Panel agreed with the conclusion in the Issue Paper that the experimental 

toxicology data and not the epidemiologic data are best suited for dose-response and risk 

assessment at the present time, but that the epidemiologic and toxicologic databases, in 

combination, can be used to inform hazard identification and characterization. Contrary 

to the opinion stated in the Issue Paper, the Panel believed that the epidemiologic 

findings could be used for endpoint selection if the studies are well-designed (even if not 

achieving unattainable perfection) and experimental data are not available. 

 Thus, based on an understanding of the mode of action in animals anticipated to 

pertain to humans, additional research should be focused on selecting appropriate 

biomarkers of exposure and effect with support from laboratory studies and with special 

attention to exposure levels of relevance to apical events in human populations. 

Adequate sample size to achieve sufficient statistical power to detect meaningful effect 

sizes in the outcomes of interest will be a major consideration in low-dose research, as 

will adjustment for appropriate known confounding factors for some outcomes, such as 

maternal age and smoking for low birth weight. 

 The concerns about effects not yet identified in humans, but seen in laboratory 

findings are recognized but further efforts in epidemiology will produce additional 

equivocal findings unless studies are well-designed (i.e., have good exposure and 

outcome measurement in individual men and women with adequate sample sizes, 

adjustment for confounding factors, and minimal participation and selection biases) and 

evaluate health effects of doses that are relevant to real-life human exposures either in the 

occupational setting (e.g., farm workers) and real-life drinking water exposures (e.g., 

studying populations likely or known to consume atrazine-contaminated water, perhaps 

in agricultural settings). 

 In conclusion, it is not a simple task to develop a satisfactory framework to 

integrate the results of epidemiologic, clinical and laboratory studies on an issue, such as 

the health risks of atrazine. At the February 2010 meeting of the SAP, the registrant 

proposed a framework for a weight-of-evidence approach to the interpretation of findings 

that bear on human risks of toxic exposures. The proposed framework does not obligate 

the generation of new data but has the virtue of bringing out assumptions and judgments 

35 
that might otherwise be harder to identify. The background materials for this meeting 

included a paper based on the application of the registrant’s proposed framework entitled 

"Preliminary Review of Recently Identified Atrazine Ecologic and Retrospective Studies" 

by Breckenridge, Pastoor and Scialli (2010). While the Panel disagreed with a number of 

the critical judgments presented in Breckenridge et al. it would be useful to have a clearer 

articulation of differences of opinion (and, similarities) between these authors and EPA. 

 Finally, the Issue Paper is largely correct in its conclusions that 1) the animal data 

are likely to be relevant to humans, although different mechanisms of toxicity may also 

play a role in humans and 2) the human epidemiologic data suggest possible adverse 

reproductive effects. EPA is encouraged to collaborate with other federal agencies or 

other parties that have funded human studies of these types of outcomes, have biologic 

samples stored and potentially available for assessment of exposure and have done a 

good job of investigating the association of the exposure(s) of interest with these 

outcomes. 

Question 2.0: Review of Studies on Mammary Gland Development 

 At the April SAP, the Agency evaluated effects of atrazine on a number of apical 

endpoints including neuroendocrine, neurotoxicity, and immunotoxicity effects. At that 

time, the Agency deferred review of experimental toxicology studies on mammary gland 

development to the September SAP meeting. Development of mammary tissue occurs in 

defined stages linked to sexual maturation and reproduction including embryonic, preand peripubertal periods, as well as pregnancy and lactation. Given that atrazine affects 

the hormonal environment and reproductive system, it is not unreasonable to hypothesize 

that it may also affect mammary gland development. The database of mammary gland 

development studies includes four studies---three from same the research group (Rayner 

et al., 2004; Rayner et al., 2005; Enoch et al., 2007) in addition to one conducted by 

Coder (2010). It is important to note that the key events leading to reported delays in 

mammary gland development in the young following gestational exposure to atrazine are 

not known and none of these studies propose a mechanism/mode of action. 

 

Question 2.1 

 The studies from the two different research groups yielded different results 

regarding the impact of atrazine on mammary gland development. While the Rayner and 

Enoch studies report delays in mammary gland development, the Coder study does not. 

The basis of these different findings is unknown. However, one notable difference in the 

study design used by the two research groups is the scoring system. The Rayner et al. 

and Enoch et al. studies used a subjective scoring scale with a 1-4 scoring system (1 = 

poor development/structure; 4 = normal development/structure) with criteria which vary 

depending on the age of the pups. In contrast, Coder employed a quantitative 

morphometric analysis to evaluate mammary gland development. In addition, the Coder 

study also included the use of BrdU labeling to assess cell proliferation in the mammary 

tissue. 

36 
 Please comment on the quality, strengths, and limitations of the mammary gland 

development studies by Rayner et al. (2004, 2005) and Coder (2010) studies. Please 

discuss in your comments factors which could lead to different findings. Please comment 

on the Agency’s conclusions regarding these studies on atrazine. 

Panel Response 

 Each of the three studies investigated the effects of atrazine administration to 

pregnant dams on the body weights, mammary gland development and age of vaginal 

opening of their female offspring. All three studies used the Long Evans strain of rats 

and oral gavage as the route of administration. Each study appeared to be well-executed. 

However, the studies differed in several aspects of experimental design and methodology 

including: (1) composition of the diet consumed by the dams; (2) light-dark photoperiod 

employed; (3) inclusion/absence of pair-fed controls; (4) methods used for mammary 

gland development analysis; and (5) differences in maternal stress levels. Each of these 

aspects is significant when comparing the findings and conclusions of the studies, which 

differed in several ways. While each found that atrazine at higher doses (50 mg/kg and 

100 mg/kg) affected both dam and offspring body weights as well as mammary gland 

development in the offspring, the Rayner studies found effects of atrazine on the age at 

vaginal opening that were not detected in the Coder (2010) study. In addition, the Rayner 

studies differed from the Coder study in the nature of the effects that atrazine produced 

on postnatal mammary gland development. 

The Panel concluded that numerous significant methodological and procedural 

differences in the Rayner and Coder studies have confounded the understanding of the 

toxicological significance of atrazine exposure on certain aspects of female reproductive 

system development. It was the Panel’s opinion that unless/until these differences are 

resolved, understanding of the effects of atrazine on mammary gland development will be 

hampered. Consequently, use of the existing data on rat mammary gland development to 

assess the potential human risk of atrazine is not warranted at the present time. 

 As noted above, the Panel identified several significant procedural and 

methodological differences in the Rayner and Coder studies. They are analyzed in detail 

below: 

(1) Diet. The compositional differences in the diets used in the Rayner and Coder 

studies may have affected the outcomes of atrazine exposure. The Coder et al. 

study report indicates “The basal diet used in previous studies conducted with 

atrazine (Rayner, 2004; Rayner, 2005) was PMI Nutrition International, LLC, 

Formulab Diet® 5008. Compositional differences between the diet used in the 

previous studies, and that used in the current study, were slight and were not 

expected to impact animal health or the outcome of the study” (Coder (2010), 

Page 36 of 2827). However, no data are presented to support this statement. Both 

Rayner et al. studies used Purina 5008 while the Coder study used Purina 5002. 

These are both natural ingredient diets with relatively high soy content and high 

and variable phytoestrogen levels. However, they differ in their fat source 

37 
(animal fat for 5008, soy oil for 5002), their total protein, fat, and carbohydrate 

contents, and in some vitamins. There is substantial literature on the effects of 

soy and phytoestrogens on the mammary gland, as well as on puberty and estrous 

cycles. While the Organization for Economic Co-operation and Development 

(OECD) conducted a study of diets of varying phytoestrogen content used in the 

uterotrophic assay as an endocrine disruptor screening tool to determine their 

impact on the validation results (Owens et al., 2003), it is unclear if this type of 

comparison has been extended to studies of other endocrine-based endpoints. 

Based on values reported by others (Brown and Setchell, 2001; Thigpen et al., 

2003, 2004, 2007) for the isoflavone content of the diets used in the Rayner and 

Coder studies, the dietary isoflavone levels in the Rayner and Coder studies may 

have exceeded the levels that Owens et al. (2003) concluded could be 

accommodated without impairing the responsiveness of the test system being 

validated (i.e., the rat uterotrophic bioassay). There is some disagreement in the 

literature as to the ability of these diets to cause effects on the timing of puberty, 

but it may depend on the strain of rodent used. There are sporadic reports in the 

literature where diet changes have been implicated in altering effects of treatment 

with endocrine active compounds, and in some cases the diet effect has been 

attributed to the phytoestrogen content (Boettger-Tong et al., 1998; Muhlhauser et 

al., 2009; Thigpen et al., 2003; Wang et al., 2005). However, there does not 

appear to be published information on whether the dietary content of soy and/or 

its component phytoestrogens can shift the dose responses for atrazine. 

Nonetheless, when looking at the subtle effects on mammary gland development 

and onset of puberty, these could be important factors to consider. 

(2) Light-Dark Cycles. Differences in photoperiods under which the rats in the 

Rayner (14/10 cycle) and Coder (12/12 cycle) studies were housed have the 

potential to affect the reproductive system and mammary gland development. 

Development of reproductive function is known to be influenced by pineal gland 

activity and there is evidence in the literature that altering light/dark cycles 

influences the timing of vaginal opening in rats (Lehrer, 1986). There is also 

literature linking altered photoperiod to elevated breast cancer risk in women 

(Kakizaki et al., 2008) and rodents (Tamarkin et al., 1981) and to mammary gland 

development alterations in prepubertal mice (Mediavilla et al., 1992). However, 

there does not appear to be published information about whether photoperiod 

alterations affect atrazine toxicity in mammals. Nevertheless, when considering 

subtle differences in the effects of atrazine on puberty and mammary gland 

development in rats, photoperiod differences may be important factors to 

consider. 

(3) Pair-feeding. All of the Rayner and the Coder studies found significant effects 

of atrazine on dam and pup weights. The Coder study found that atrazine reduced 

food consumption in the dams in the 100 mg/kg group. They included a pairfeeding control for this dose group which was not included in the Rayner studies. 

It is increasingly being recognized that maternal nutrition and/or metabolism 

during gestation can have long-term effects on mammary gland development and 

38 
breast cancer risk of offspring. Given that differences in the protein/fat content 

and composition of maternal diets have been shown to affect offspring mammary 

gland development (Alvarez-Sanz et al., 1986; Eason et al., 2004; Hilakivi-Clarke 

et al., 1996, 1998), inclusion of dietary controls is necessary to accurately 

interpret potential toxic effects of atrazine. Two problems exist in this regard 

when interpreting the results of the Rayner and Coder studies. First, in 

combination with reduced food intake at the high dose during gestation, the 

differences noted in the composition of the diets employed by these two 

laboratories have the potential of differentially affecting offspring outcomes. 

Second, conclusions about the effects of atrazine on mammary gland development 

are complicated by significant differences in the body weights of atrazine-treated 

dams and their pair-fed controls (Coder (2010), Figure 2), which have the 

potential of independently altering fetal nutrition and subsequent mammary gland 

development. Moreover, it is likely the pair-fed controls had major shifts in 

endocrine and metabolic rhythms in response to the timing of access to their daily 

food allotment at the beginning of the light-cycle. 

(4) Differences in analytical methods. The Rayner studies used a semiquantitative scoring system that ranked various parameters of mammary gland 

development including ductal length, ductal branching pattern and complexity, 

and end-bud number to assess the effects of atrazine. The use of this scoring 

system has a number of strengths that include: (a) reproducibility, as evidenced by 

multiple corroborating studies over several years; (b) correlation with known 

effects of atrazine on vaginal opening that support the efficacy of treatment; and 

(c) the use of a holistic, or integrative, measure to evaluate mammary gland 

development. While useful as an approach in a single laboratory with constancy 

in practice and training, this scoring system lacks the generally accepted standards 

by which to judge mammary gland development, such as those routinely used by 

human and animal pathologists. Consequently, inter-laboratory comparisons are 

difficult and data interpretation is problematic. 

The Coder study tried to avoid these problems through the use of quantitative 

image analysis approaches to evaluate the same development parameters analyzed 

in the Rayner studies. Although much was made of the differences in these 

approaches by the Coder study authors, it is surprising that they did not employ 

both qualitative and quantitative scoring measures of mammary gland 

development, which would have provided a definitive inter-study comparison. It 

is plausible that small differences between treatment and control groups existed in 

the morphometric analyses that, individually, were deemed nonsignificant, but in 

combination may have impacted scoring, as noted by the Rayner studies. Thus the 

EPA might consider requesting that the study sponsor have the slides generated in 

the Coder et al. study scored using the Rayner scoring system. Ideally, the same 

histologists that scored the Rayner slides could score the slides in the Coder study. 

Direct comparison of results derived from the two methods would be very 

informative in terms of establishing the biological and toxic effects of atrazine. 

39 
 (5) Stress. Maternal stress during late gestation has been reported to alter 

reproductive development in offspring of rodents and primates, including delays 

in onset of puberty (Harvey and Chevins,1987; Vom Saal et al., 1991; Zehr et al., 

2005). Although maternal stress during gestation has not been linked directly to 

alterations in mammary gland development in offspring, normal mammary gland 

development during puberty is dependent upon immune cell function (Lin et al., 

2002), which is altered in rats born to dams exposed to stress during pregnancy 

(Klein and Rager, 1995). Evidence was presented in the Coder study that dams 

exposed to atrazine were aggressive and cannibalized their young. This type of 

unusual behavior was not observed in the rats in the Rayner studies, suggesting 

that they were experiencing better control conditions than those in the Coder 

study. The possibility that stress-related effects account for differences in the 

results of the Rayner and Coder studies is further supported by the observation 

that vaginal opening in control offspring of rats in the Coder study was delayed 

relative to those in the Rayner study, and that atrazine exposure did not produce 

the expected delay in vaginal opening in the Coder study whereas it did in the 

Rayner study. Thus, considerations of differences in maternal stress are important 

when attempting to reconcile the inter-laboratory differences in the biological 

affects of atrazine. 

Question 2.2 

In the three studies considered in Question 2.1 (Rayner et al., 2004 and 2005; Coder, 

2010), effects have only been observed at an atrazine dose of 100 mg/kg/day. In 

contrast, Enoch et al. (2007) report delays in mammary gland development as low as 

0.09 mg/kg/day following exposure to atrazine and a mixture of several 

metabolites/degradates (DEA, DIA, DACT, hydroxy-atrazine). The Agency’s review of 

this mixture study is provided in Appendix A (Section A.10) Please comment on the 

Enoch et al. (2007) study and the degree to which the Agency’s review accurately reflects 

the strengths and limitations of the study. Please include in your comments a 

consideration of the study design of the mixtures experiment and how this design impacts 

the interpretation of the results. 

Panel Response 

 The Enoch et al. (2007) study was designed to evaluate exposures to 

environmentally relevant mixtures. The study goal was to determine if a mixture of 

atrazine parent, its chlorinated metabolites and hydroxyatrazine, in proportions found in 

the aquatic environment, when administered orally, produces developmental effects in 

Long Evans rats. Mammary gland development, as assessed by a visual scoring system, 

was studied in female offspring of dams exposed on gestation days 15-19 to 0.09, 0.87 

and 8.73 mg/kg levels of the mixture. Some of the results from their prior studies were 

used to strengthen the experimental design of this study. Noteworthy is the 

administration of the mixture at half doses twice daily on GD 15-19. This time period 

was identified as the critical window in gestation for atrazine’s effect on delaying 

mammary gland development (Rayner et al., 2005). The conclusion of the Enoch study 

40 
was that acute exposure to mixtures of as low as 0.09 mg/kg causes persistent alterations 

in mammary gland development of female offspring, and that these effects do not appear 

to be related to body weight or timing of onset of puberty. 

 Findings included increased GD20 fetal and PND4 and PND 60 offspring body 

weights; these may be an example of prenatal metabolic programming (Plagemann, 

2005). These observations could be consistent with the Rayner and Stanko (2010) 

studies at the higher doses if the atrazine parent/metabolites are disturbing a developing 

brain neurocircuitry. During the public comment period at the meeting Syngenta 

presented data in adult rats showing that atrazine decreased activation of GnRH neurons 

in the hypothalamus without affecting paraventricular nucleus (PVN) c-Fos 

immunoreactivity. Whereas the (presumptively, corticotrophin-releasing factor; CRF) 

cells of the PVN are integrally involved in the stress response, the distributed CRF 

system also has CRF cells in the medial preoptic area (mPOA) with synaptic connections 

to the GnRH cells which could be the vulnerable brain site in atrazine-exposed fetuses 

(MacLusky et al., 1988). 

 

 However, there are several limitations in the study design that are relevant to the 

testing of a mixture. Dose-response data for individual components could have helped 

clarify the interpretation of the mixture data. The observations were based on 

administered dose because absorption and bioavailability values were not determined and 

are unknown. The dose response as a function of 100-fold change in the administered 

dose of the mixture was weak. No data on chemical analyses of the mixture were 

provided to determine if the concentration of chemical(s)/metabolite(s) was constant as a 

function of time (see Panel response to question 3.1). 

 EPA’s concern with the statistical treatment of the data (individual pups versus by 

dam or litter) was resolved by Dr. Fenton. Similarly, concern for the qualitative scoring 

of mammary gland development also was ameliorated by Dr. Fenton’s detailed 

explanation of the methodology, noting the long history and widespread use of these 

measures. It is surprising that the Enoch et al. methodology did not include external 

standards. A head-to-head comparison with multifactor imaging analysis would be a 

welcome exercise. 

 The Panel agreed with EPA’s conclusion that the Enoch study is not suitable for 

use in quantitative risk assessment. However, the study does raise some interesting points 

and dilemmas. Certainly, the very low doses at which effects on mammary gland 

development were reported could be of importance in assessing the risks of atrazine 

exposure. However, since these effects occurred at doses so much lower than observed in 

other studies, there needs to be replication of the study. The observations of this study 

have not been corroborated using other mixtures of parent atrazine/metabolites reflective 

of temporal and spatial occurrence or for atrazine parent at equivalent doses. Atrazine 

itself will, once absorbed, yield a mixture of free and conjugated metabolites (but, little or 

no hydroxyatrazine) and the proportion of metabolites would likely change temporally. 

Any replication would need to evaluate the same endpoints measured in this study and, 

perhaps, other physiological endpoints 

41 
Question 2.3 

As described in the Introduction of the draft Issue Paper (Section 1.0), the Agency’s 

problem formulation is being conducted in a step-wise manner. One important 

component of this problem formulation is the evaluation of the overall database and the 

quality of the available experimental toxicology studies. In the case of mammary gland 

development, the Agency is proposing to acknowledge these four studies (Rayner et al., 

2004; Rayner et al., 2005; Enoch et al., 2007; Coder, 2010) in the hazard 

characterization but place little emphasis on them in its proposed updates to the doseresponse assessment for atrazine. The only findings reported in the Rayner et al., (2004), 

Rayner et al.(2005, and Coder (2010) studies, occurred at a high dose (100 mg/kg/day) 

which is approximately 50-fold higher than the PoD of 1.86 for LH attenuation being 

proposed by the Agency. In the Enoch et al. (2007) study, there are significant 

limitations with the conduct and reporting the Agency believes which preclude the study 

from use in quantitative risk assessment. 

In light of Panel discussion on Questions 2.1 and 2.2, please comment on the manner in 

which the Agency has proposed to use the mammary gland development studies in the 

hazard assessment for atrazine. 

Panel Response 

 The Agency proposed to “place little emphasis” on the mammary gland 

development studies that were summarized in the Issue Paper, and presented during the 

meeting. Based on the Panel’s own analysis, as detailed in the responses to Questions 2.1 

and 2.2, this does seem to be the most appropriate way of handling these studies 

currently. The changes in mammary gland development were observed only at relatively 

high doses (100 mg/kg/day), doses to which humans are unlikely to be exposed. No clear 

dose-response relationship or mode of action has been defined for these mammary gland 

effects. There are only four papers in the current literature, one of which has significant 

limitations which make it unsuitable for quantitative risk assessment. Two of the 

remaining three reports indicate that atrazine delays vaginal opening and mammary gland 

development until adulthood, based mostly upon subjective endpoints. The third report 

finds no effect of atrazine on time to vaginal opening or mammary gland development 

after PND1, and utilizes mostly quantitative endpoints. The results of this latter study, 

however, are highly variable. Some of the mammary gland responses may be transient; 

the observation of carryover of effects into the F2 generation needs replication. 

 The conclusion that the mammary gland effects do not provide a useful PoD for 

quantitative risk assessment should not be construed to indicate, however, that the 

mammary gland is not a legitimate target for further analysis. There are good reasons to 

believe that mammary gland development and mammary gland function are important 

concerns for environmental agents. These preliminary studies clearly raise the possibility 

that there may be deleterious effects of atrazine on mammary gland development. 

Therefore, further study is needed at additional (i.e., lower) doses, with added emphasis 

42 
on possible cross-generational effects of parent atrazine and its metabolites, including the 

hydroxy-metabolites. The mixture study (Enoch et al., 2007) is potentially important, but 

replication of this study and investigation of the individual components of the mixture 

should be pursued. This mixture study can be explained by any of the following several 

possibilities: 

1) The observed effects are artifacts, owing to a flaw in the study design and/or 

execution. 

2) Only a single very active metabolite is responsible for the observed effects. 

3) Multiple very active metabolites may act in a dose-additive fashion. 

4) There is significant interaction among the metabolites (i.e., synergy). 

 The likelihood of significant interaction among these compounds would be slim, 

but interactions resulting in an order-of-magnitude or greater increases in the toxicity are 

not outside of the range of possibility. The Panel pointed out that the likelihood of 

artifact can be evaluated in follow-up studies. If the effects are found not to be artifactual, 

then additional future studies should address the possibility that one or more very active 

metabolites exist. Hydroxyatrazine(s) were discussed as candidate active metabolites 

although they are not significantly produced by mammals and would, therefore, 

contribute little to the toxicity profile when atrazine is administered in animal models. 

Overall, these effects are particularly important because they involve developmental 

exposure and they occur at doses lower than reported for LH surge effects. And, like the 

LH surge effects, the mammary gland reactions to atrazine are likely to be indirect, 

involving effects on the hypothalamus or other brain sites. 

 It remains true that particular features of mammary gland development suggest 

that it is a valid, and potentially sensitive, target for environmental agents, including 

potentially toxic compounds such as atrazine. These features include the fact that 

mammary gland development occurs over several distinct phases of life, including 

prenatal, pubertal, gestation, lactation and aging; and the breast is a very common cancer 

target. In addition, the importance of breast milk for healthy development of the young, 

and the importance of breastfeeding for healthy metabolism of the mother are now welldocumented. An additional consideration discussed by the Panel is that the biological 

mechanisms controlling mammary gland development in the rodent and primates 

(including the human) map more closely between the species than do those controlling 

the hypothalamus-pituitary-gonadal (HPG) axis. Concerns raised during the meeting 

about the applicability of rodent HPG axis findings to humans may, therefore, be less 

important for mammary gland endpoints. 

 The comments above argue that developing accepted methodologies and Standard 

Operating Procedures (SOPs) that would inform useful endpoints related to mammary 

gland development and physiology will be important. These methods and procedures 

should address actual lactation endpoints, both qualitatively and quantitatively. This task 

presents an obvious set of challenges. However, very precise and comprehensive 

lactation endpoints are routinely measured and used for specific goals in the dairy 

sciences and industry, where very small differences in lactation outcomes have major 

43 
economic consequences. It should be possible to use this rich literature and 

methodological resource to establish approaches to characterize effects of environmental 

agents, such as chemical contaminants, on mammary gland outcomes. These methods 

should, and could, take into account the importance of gathering both animal 

experimental data and human data that can be meaningfully integrated, and which 

address cross-generational outcomes. Cancer outcomes were not within the scope of this 

SAP, but remaining vigilant about breast cancer outcomes is obviously still important. 

 

 It is important to consider the weight of evidence in selecting relevant studies as a 

basis for the evaluation of dose-response relationships for the effect considered critical. 

Factors to be taken into account include not only the quality of individual studies, 

including internal consistency of observations, but also the collective weight of evidence. 

For individual studies, it is always desirable to have several complementary measures of 

effect, particularly where some are subjective, as a basis for the measurement of internal 

consistency of the observations. Similarly, consistency of results across studies in the 

database is an important criterion in assessing weight of evidence for dose-response for 

critical effects. Effects observed by one research group at doses that are not consistent 

with the weight of evidence should not unduly influence selection of critical doseresponse relationships for the point of departure. This does not imply that the results 

should not be investigated further, particularly where effects are found at very low doses 

and where mixtures of compounds that may be synergistic are involved. It is advisable 

that generic criteria for internal consistency and those relevant to weight of evidence for 

dose-response for critical effects (consistency, specificity and biological plausibility) are 

defined a priori. 

 In summary, the Agency’s Issue Paper concluded that the few available mammary 

gland studies do not constitute a sufficient body of evidence to be used in a quantitative 

risk assessment (i.e., for establishing a PoD). The Panel concurred with this finding. The 

suggestive evidence in these studies does, however, argue that developing useful and 

standardized methods for assessing mammary gland functional outcomes remains an 

important task. 

Question 3.0: Proposed Updates to the Dose-Response Assessment 

At the April, 2010 SAP, the Panel made two key recommendations related to doseresponse assessment: 1) consider pursuing a dose-response analysis based on an 

internal dose metric, as an alternative to the administered dose metric used in the 

previous risk assessment; and 2) identify the sensitive endpoint associated with functional 

impairment and perform benchmark dose (BMD) modeling. The Agency conducted both 

recommended analyses (Section 5.0 of the draft Issue Paper) in response to these 

recommendations. LH attenuation, as a key event in atrazine’s MoA, is the most sensitive 

internal dose metric that is associated with a number of endocrinopathies 

44 
Question 3.1 

Given that there is no robust pharmacokinetic model yet available, a noncompartmental analysis of the existing pharmacokinetic data was conducted and 

described in Section 5.2 of the draft Issue Paper. EPA thinks this represents the best 

method to estimate an internal dose metric without exceeding the limits of the available 

data. 

a. Please comment on the Agency’s analysis of the pharmacokinetic data contained in 

Thede (1987) to estimate internal dose reflective of atrazine and its metabolites in rat 

plasma. 

b. Based on Figure 5.5 in the draft Issue Paper, the Agency has preliminarily concluded 

that plasma levels of atrazine and its metabolites reach (or nearly reach) 

pharmacokinetic pseudo-steady state in the rat plasma after approximately four daily 

oral exposures over a wide range of doses. Please comment on the extent to which the 

data do and do not support this finding. 

Panel Response 

The Panel’s responses to Question 3.1a and 3.1b have been combined. 

 The key uncertainty in the characterization of exposure for dose-response analysis 

is the extrapolation to low dose in humans from high dose studies in animals. The “best 

available science” for doing these extrapolations is physiologically-based 

pharmacokinetic (PBPK) modeling, particularly when a specific target organ has been 

identified (e.g., the hypothalamus-pituitary complex, for which there are adequate data to 

identify the relevant dose metric associated with the critical effect). PBPK models are 

clearly not the quickest or easiest tool to apply. While the Panel was cognizant of the 

time and resource constraints on the Agency with regard to conducting risk assessments 

of various pesticides for re-registration or other risk management activities, the Panel 

found it disappointing that PBPK model refinement for atrazine has not been undertaken 

since 2007 when the McMullin model was published in the peer-reviewed literature. 

PBPK models have been or are being developed for other pesticides (e.g., synthetic 

pyrethroids, chlorpyrifos), and for atrazine by the registrant. It would be prudent for the 

Agency to work with the current model described by the registrant and make appropriate 

modifications. It is recommended that the Agency be more proactive in this area, and that 

it begin to address more rigorously the uncertainties of extrapolation from rodent studies 

to low dose regimens in humans. 

 In the absence of a functional PBPK model and appropriate data, the Panel very 

much supported the work of the Agency in pursuing a dose-response analysis based on an 

internal dose metric, as an alternative to the administered dose metric used in the 

2003/2006 risk assessment. This should reduce the uncertainty associated with interroute, interspecies and intraspecies (inter-strain) extrapolations. In the noncompartmental analysis, the Agency has attempted to make maximal use of the limited 

45 
data that are available. EPA has made mainly conservative choices in the absence of 

empirical information and verified estimates to the extent that is possible. 

The Panel agreed with the Agency that, on the basis of the currently available 

data, plasma appears to be a reasonable biological compartment that is reflective of tissue 

dose, and that use of area under the plasma concentration time curve (AUC) provides an 

appropriate measure of internal exposure. It is the best way of approaching the problem 

of relating the exposure of rats to atrazine to the magnitude of resulting biological 

responses, and extrapolating to the prediction of human exposure levels. This is 

particularly useful in the absence of knowledge of the exact site of action since it 

provides a measure of the opportunity for distribution to all tissues (target and nontarget). This conclusion is supported by the work of Stoker and Cooper (2007). The 

studies of Dooley and co-workers demonstrating the covalent binding of DACT to 

proteins with available cysteine residues in all parts of body (19 in rat pituitary (Dooley, 

et al., 2008)) 30 in rat brain (preoptic area, medial basal hypothalamus, and cortex; 

Dooley, et al., 2010) indicate the potential impact of DACT on a range of proteins 

(enzymes and structural components). However, there is currently no evidence to identify 

the consequences (inhibition or activation) of binding on the activity of individual 

proteins. 

In the absence of good data on the plasma concentrations of parent compound and 

individual metabolites at various elapsed times after dosing, the use of total chlorotriazine 

based on total 14C-compounds is a reasonable first step (particularly in absence of 

information on the pharmacodynamic activity of the parent compound and individual 

metabolites). However, the data of Thede (1987) has some limitations, and these should 

be kept in mind when interpreting any pharmacokinetic analysis. The plasma 

measurements are based on total radiolabel, and the samples were taken at 24 hours after 

each of the repeated doses. This means that the plasma profile is poorly defined, and, 

because of the widely separated sample points, will appear smooth. There are too few 

measurement points in the washout period to obtain sound estimates of elimination 

parameters. The Agency’s interpretation of the Thede data includes the assumption that 

the parent compound and individual metabolites (DACT, DEA, and DIA) are all equally 

active toxicologically. Considering that atrazine is short-lived and that the glutathione 

conjugates are likely to be inactive, several possible dose metrics are available. These 

include sum of total metabolism of atrazine, total metabolism minus glutathione 

conjugates, total of DIA, DEA and DACT, total of DIA and DEA. 

 Atrazine and its two mono-dealkylated analogues are rapidly depleted by primary 

detoxication; the major end product (DACT) dominates the plasma profile. There is an 

assumption in the interpretation that none of the pharmacokinetic processes (absorption, 

distribution, metabolism and elimination) is saturated, and so the proportions of the 

parent compound and metabolites are independent of dose. The composition of the total 

radiolabel will change with time after each administration by oral gavage. Immediately 

after dosing, the concentrations of atrazine, DEA and DIA will be high, and will fall over 

the following hours, and after 24 hours (the time of the next dose) more than 90% of the 

AUC will again be made up of DACT. In this context, it is recommended that the 

46 
differences in molecular weight between atrazine and its metabolites (215.7, 145.5, 

173.7, and 187.7 for atrazine, DACT, DIA, and DEA, respectively) are accounted for in 

the calculation of milligram equivalents of atrazine. The factors involved (1, 1.48, 1.24, 

1.15) will be important, especially at later times. Evidence in Laws et al. (2003), Stoker 

et al. (2002) and McMullin et al. (2004) might be used to clarify the Agency’s approach 

of assuming equal toxicological potency for atrazine and all of its metabolites. 

Estimates of the elimination rate constants on the basis of the last three points 

(based on a single animal) are associated with some uncertainty. However, there is a 

very rapid fall from the level at the time of the last dose. Indeed, if more frequentlycollected samples were available, the concentration would have been seen to rise after the 

last dose before falling from the peak level. This pattern is indicative of two 

simultaneous processes, one very rapid that is virtually complete over 24 hours, and then 

a slower process. On the other hand, the data from the new registrant study (summarized 

in Breckenridge, et al., 2010), where there are more points available over the washout 

period, do not indicate double exponential elimination. It would be worthwhile to look 

closely at the washout data, since the estimates of the elimination rate constants will be 

important in any predictions or extrapolations to other dosing regimens. 

 If AUC of plasma concentration is to be used as the measure of internal dose of 

triazine equivalents, then consideration should be given to the interspecies differences in 

bioavailability, rate of glutathione conjugation, role of intestinal metabolism, and red 

blood cell/plasma partition coefficient. Furthermore, knowledge of the contribution of 

elimination processes to the internal exposure profiles is essential, and work is necessary 

to assess the uncertainties associated with this when extrapolating between species (e.g., 

rat to human). The difficulties involved are highlighted by known differences between 

strains within a species, as exemplified by the differences in the LH response, and 

biotransformation enzymes between Long Evans and Sprague Dawley strains of rats. 

Long Evans rats have lower overall CYP activities, which may lead to slower clearance 

(Jori et al., 1971). The Panel recommended that studies to provide parameterization for a 

Long Evans PBPK be initiated as soon as possible. This parameterization should include 

an intestinal biotransformation metric (either microsomal or Caco-2 cells). The 

hepatocyte data provided by the registrant were useful, particularly given the differences 

between rat and human metabolite profiles, and differences between microsomal (Joo et 

al., 2010) and whole hepatocyte results in the formation of DACT (the major metabolite 

produced in vivo). In vitro studies also may be conducted with mixtures of the 

metabolites in a range of concentrations that bracket those in the environment. Such data 

could be used in the next risk assessment. 

It was the opinion of the Panel that the approach taken by the Agency is the 

correct one, but it requires better data than those currently available. The pharmacokinetic 

study in progress as described in the registrant’s presentation at the meeting and in 

Breckenridge et al. (2010) will provide data of the required quality since the use of 

frequent sampling and mass spectrometric detection of the analytes is providing tight 

definitions of the PK profiles of the individual compounds (parent compound and 

analytes). This type of study lends itself to analysis of relationships between the AUCs 

47 
of the different compounds and biological responses. This information combined with 

good estimates of plasma partition coefficients should allow the development of PBPK 

models that can be used to extrapolate from rodent to other species, including humans. 

This would provide a sound basis for the setting of benchmark doses, and PoDs, 

providing that it is developed for the rat strain in which the PoD or dose-response is 

characterized. While it is clearly preferable to have a validated PBPK model, within the 

time constraints for regulatory programs, it may be necessary, at a minimum, to prioritize 

collection of data that would reduce some of the uncertainty associated with the current 

Agency analyses. Certainly, the outstanding issue is how to deal with the uncertainties 

associated with the constraints of the existing data. Despite the limitations of the existing 

data, the exercise has been extremely valuable in informing critical aspects relevant to the 

risk assessment and monitoring programs. 

Pseudo steady state implies that the absorption and distribution processes are 

matched by the elimination processes. The interpretation of the levels presented in 

Figure 5.5 of the Issue Paper is misleading since all plasma samples were taken at 24 h 

after the previous dose. The points defining the profiles in Figure 5.5 are the minima in 

the saw tooth profile, and not the average levels. Perhaps if the samples had been taken 

at 12 h after dosing, the profile would have been different. If the samples had been taken 

at random times after dosing, there would not have been such a smooth plateau. The real 

profile will not have such small amplitude fluctuations as illustrated in Figure 5.4 of the 

Issue Paper (see Figure 6 in Breckenridge et al. (2010)). This means that the AUC is 

underestimated. The bias will be approximately constant across the plateau area, and so 

it will not change the goodness of fit of AUC versus administered dose, and could give a 

false sense of security. 

In the PK experiments undertaken by Thede (1987), the same dose was used 

throughout, and under these conditions, the approach taken of estimating AUC provides a 

useful measure of exposure. The estimate of AUC in the region of pseudo-steady state 

exposure over the full 4 days is proportional to oral gavage dose, as shown in Figure 5.6 

of the Issue Paper. However, as described above, caution needs to be taken in the 

interpretation of the Thede (1987) data set because of the poor definition of the serum 

profile. The new registrant PK data sets show that there are large fluctuations in serum 

concentrations of atrazine and its metabolites with time after oral gavage dosing, and that 

a smoother profile is achieved with dietary administration. Despite the difficulties in 

estimating dose consumed in the diet compared with administration by oral gavage, it is 

worthwhile. Dietary administration provides a smoother plateau, is less stressful and 

disruptive than gavage, and is more realistic representation of human exposure through 

drinking water, depending on the presence of food in the digestive tract. 

c. Figure 5.8 of the draft Issue Paper shows a plot of LH attenuation data versus 

administered atrazine across a range of exposure durations (i.e., four days up to six 

months). The data contained on this figure were collected from several different 

laboratories and involved two modes of oral administration (dietary, oral gavage) and 

two different rat strains. Attenuation of LH, as measured by percent of control, is 

remarkably similar across studies, strains, laboratories, and most notably duration of 

48 
exposure. The Agency has preliminarily concluded that the findings on Figure 5.8 

strongly support the hypothesis that pseudo-steady state is achieved (or nearly achieved) 

in adult rats after four daily consecutive oral exposures. The Agency also believes that 

pseudo-steady state is strongly associated with the attenuation of the LH surge following 

atrazine exposure in rats. Please comment on the analysis shown in Figure 5.8 and the 

Agency’s preliminary conclusions related to these findings in rats. 

Panel Response 

 The Panel consensus was that Figure 5.8 is an imaginative distillation of data 

from two different rat strains across 15 years of data collection from four laboratories. 

The figure serves as a visual meta-analysis and assists in the understanding of the atrazine 

dose-related attenuation of the LH surge. It is remarkable that the overall response is 

similar given that there was such diversity in the study paradigms: different rat strains 

and different dosing regimens. The model presented in Figure 5.8 represents a good, 

initial approach to data integration and has great utility for exploring concepts and 

formulating approaches to test hypotheses further. 

 Overall, the Panel agreed that several aspects of the Figure need refinement in 

terms of the axis scaling, winnowing of LH data, and inclusion of the higher doses to 

incorporate the full range of data. All these aspects can be corrected and may yield a 

more accurate and sensitive model. 

 The first concern is the selection of a linear scale for the X and Y axis. In the 

Figure, the response data have been plotted as percentage of control against the linear 

dose (mg kg-1 d-1) of atrazine. A clearer and more standard approach would be to plot the 

response data (including confidence intervals) on a standard normal probability scale (i.e. 

in standard deviation units) and the concentrations on a log scale. This would then 

facilitate including the higher doses in some of the studies summarized here as well as 

other studies which included single doses (see Figure 5.2 from Cooper et al. (2000)). 

When interpreting this plot, it needs to be borne in mind that the points near the limits 

(0% and 100%) are associated with larger variance (and greater uncertainty) than points 

near the median. It would be useful to incorporate the higher doses to see where they fall. 

This should not be precluded on pharmacokinetic grounds, given that AUC is highly 

correlated with administered dose up to a concentration of 100 mg kg-1 d-1, with no 

indication of saturation of any of the pharmacokinetic processes (absorption, distribution, 

metabolism, and elimination). Further, the higher doses fitted on the Hill model (Figure 

5.11 of Issue Paper). If there is no effect of further increasing the dose, then this would 

imply that a limiting response has been reached; this would be of interest in setting 

bounds to the dose response space. Alternatively, if there is an effect of further increasing 

the dose, it is possible that two phases of PK dynamics can be characterized. Inclusion of 

the complete data set will help to further develop the hypothesis. This may allow for 

linking higher doses with more dramatic attenuation of the LH surge and more immediate 

effects of downstream fertility outcomes. 

49 
The second concern is the selection of 1800 h LH values as the basis for 

calculating “Percentage of control for the LH Surge Attenuation” for the Y axis rather 

than peak LH or AUC levels. Inspection of the raw data of Morseth and Minnema 

revealed that peak LH varied between 1600 – 2000 h in individual animals and treatment 

groups. For example, in the Morseth 1-month study (1996a), for both decapitation and 

repeated sampling studies, the peak of mean LH concentrations in the controls (0 

mg/kg/day) was at 1800 h, but in the 5 mg dose groups, the mean LH peak was at 1600 h 

in the decapitation study and 2000 h in the repeated sampling study. In the Morseth 6 

month study (1996b), LH appears to peak at 2000 h in controls and at 1800 h in the 25 

ppm group in repeatedly sampled rats, whereas in the decapitation study, both 0 and 25 

ppm groups peak at 1800 h. When assessing the LH peak, the best LH values to use, if 

serial samples are available, are the actual LH maxima, regardless of the time at which 

they occurred. 

 Third, of even greater concern is the variability of the LH data. Data from the 

Cooper (2010) 4-day study provide the major proportion of the dose response data. In 

this study, groups of Long Evans rats were sacrificed at various times in proestrus. 

Verification that the rats were in proestrus was based upon three criteria, namely, a 

proestrous vaginal smear, ballooned uterus, and an elevated serum progesterone 

concentration determined a posteriori. Only data from those rats that met all three 

criteria were used in the analysis. Additional data for dose levels between 1.5 and 5.0 

mg/kg/day were obtained from the Morseth (1996) and Minnema (2001) studies that used 

Sprague-Dawley rats. These data are much more variable than the Cooper data (see 

below); therefore, there are no apparent differences among the means, or among the 

NOAELs or LOAELs. 

 There are significant problems with the manner in which the LH data in the 

Minnema and Morseth studies were analyzed that can be corrected. Both of these reports 

employed all LH values from all samples that were assayed. However, precise 

determination of an LH surge (elevation of LH above the 95% confidence limits of 

baseline for at least 2 consecutive time points) was not exacted. Not all animals had an 

LH surge (including controls); therefore, there is a significant underestimation of LH 

values, means and overestimation of standard deviations. It was not possible to 

determine whether LH surges were defined in the McMullin report. 

 Correction of the Minnema and Morseth data sets such that only those animals 

that exhibited an LH surge are included in the analysis of LH surge peaks, will increase 

the mean LH levels, decrease the variation in the data, and might also alter the 

conclusions with regard to the NOAEL dose. Most importantly, correcting the data will 

affect the benchmark response (BMR) analyses. 

 A fourth concern is that the potential importance of the correspondence between 

the dose of atrazine and the proportion of animals with delayed/ lengthened cycles was 

overlooked. The delayed cycles are probably the result of insufficient or absent LH 

surges. For example, the percentage of atrazine-treated animals that did not meet the 

above criteria for an LH surge for each dose in the Minnema paper were: 0 mg, 34.2%; 

50 
2.5 mg, 30%; 5 mg, 50%; 40 mg, 67%; 200 mg, 58%. These proportions are high, i.e., 

about one-third each of the vehicle and 2.5 mg groups did not surge on the expected day 

of proestrus. More importantly, there was a trend towards increasing proportions of 

animals that did not surge with increasing doses of atrazine. Similar results were not 

reported by Morseth, but the data are available. This endpoint should be reported and 

analyzed separately for all studies. In this regard, a similar relationship was observed in 

the new Cooper study (2010) and registrant data, both of which indicated that the 

proportion of animals that failed to meet the proestrous criteria or have lengthened cycles, 

and therefore failed to surge, increased with increasing atrazine dose, although the 

proportions are much lower than in the Minnema data. This is a very important functional 

observation, especially as attenuation of the LH surge has no adverse effect on 

reproductive function and does not prevent ovulation until about 80% attenuation (Krieg, 

et al., 2000; Kirchick, et al., 1978). Therefore, the proportion of animals and the latency 

to exhibition of delayed cycles might constitute a better endpoint, or “adverse response” 

for determining the effect of atrazine than is attenuation of the LH surge.

Finally, data from two strains of rats with significantly different sensitivities to 

atrazine are combined, but they should be also be evaluated separately. Moreover, it is 

important to recognize that the appearance of the curve may change with data obtained 

from exposures at different stages of the life cycle (i.e., pre-pubertal/peripubertal and 

aged/peri-senescence). As additional data, particularly with high(er) atrazine doses, are 

added to the model, a mindful consideration of non-LH effects should still be considered. 

For example, food intake and body weight changes (as % control), described in the 

Morseth and Minnema studies, plummet at the high atrazine doses and generate a 

parallel, inverse curve. 

 It is also important to consider the pattern and duration of exposure to atrazine 

when considering the data from the various studies summarized in Figure 5.8. The Panel 

questioned the Agency's conclusion that pseudo-steady state is strongly associated with 

the attenuation of the LH surge following atrazine exposure in rats. Although the PK 

data indicate that a pseudo-steady state has been achieved over the four days of dosing in 

the Thede experiments, it is important that the idea of a pseudo-steady state being 

necessary for attenuation of the LH surge is not used in interpreting these data. The data 

do not support this interpretation since no other dosing pattern was used. In order to be 

sure that a pseudo steady state was necessary to produce an attenuation of the LH surge, 

it would be necessary to examine other dosing regimens (for instance a high dose on day 

1, a low dose of day two, no dose on day 3 and a high dose on day 4) which gave the 

same overall AUC of atrazine and metabolites as in the reported experiments. A series of 

such experiments in which the biological response corresponding to the different patterns 

of dosing was measured would provide evidence for or against the assumption that 

pseudo steady state was necessary to produce attenuation of the LH surge at low doses. 

Until such evidence is available, it is necessary to keep an open mind since it may not 

matter whether the system is in steady state or is subjected to concentrations fluctuating 

from day to day. There may be a critical window of exposure that is less than the four 

days used in the reported work, and providing that a minimum exposure (AUC) is 

maintained over this period, a reduction in the LH surge will result. It has been assumed 

51 
that the tolerance remains constant throughout the four days of dosing. If a range of 

higher doses (corresponding to higher AUC) is used, it may be possible to see whether 

there is an upper limit in exposure beyond which no increase in suppression of the LH 

surge occurs.

Question 3.2 

In Section 5.3, the Agency describes a benchmark (BMD) analysis conducted for LH 

attenuation studies. As described in the draft Issue Paper, the Agency initially 

considered a wide range of toxicological endpoints for inclusion in the BMD analysis. In 

considering all of the data, this analysis focused on studies measuring LH attenuation 

where female rats were exposed orally for four days and/or longer. This was not only the 

most sensitive endpoint but its temporal and dose response are well defined 

experimentally. The LH effect originates from atrazine’s effect on the hypothalamic 

control of pituitary function through its interference with GnRH neurotransmitters. Thus, 

protection from LH attenuation as a precursor event to several functional impairments 

would be protective of atrazine’s neuroendocrine effects. 

a. Please comment on the BMD analysis summarized in Section 5.3 of the draft Issue 

Paper with details provided in Appendix C. 

Panel Response 

Mode of Action; Biological Significance of the Selected Critical Effect/Degree of 

Protection - The Agency is to be commended for considering early key (i.e., precursor) 

events in hypothesized modes of action as the basis for characterization of the critical 

dose-response relationships as more relevant than the apical endpoint to prevent adverse 

effects in the human population. The data on these earlier precursor or key events 

(attenuation of the LH surge, in the case of atrazine) often lend themselves to better 

characterization of dose response owing to the availability of incidence data at more dose 

levels than have been traditionally included in standard longer term toxicity studies. The 

hypothesized MOA for atrazine’s effects on reproductive function is presumed to include 

suppression of GnRH pulses, consistent with an immediate downstream effect on the LH 

surge. 

 Based on the available data, a benchmark dose modeled from data on suppression 

of the LH surge appears to be protective for other endpoints, since this phenomenon 

occurs at doses lower than for the wide range of effects identified in a rather extensive 

toxicological database. The Panel agreed with the Agency’s conclusion that, at the 

present time, the most sensitive biological endpoint is attenuation of the LH surge. 

 It must be noted, though, that while attenuation of the LH surge over one estrous 

cycle represents a sensitive biological response, a causal association between the 

observed modest, yet statistically significant, changes in LH surge peak and adverse 

fertility measures has not been made. The relatively small attenuation of the LH surge 

52 
may well be a harbinger of a possible future adverse event, but at present this remains to 

be demonstrated and other possible endpoints should continue to be investigated. 

 Specifically, it should be noted that only about 20% of the LH that is released on 

proestrus is required for normal ovulation and fertility; therefore attenuation of the LH 

surge to about 40% of control levels, observed at doses up to 30 mg/kg bw/day (Figure 

5.8 of the Issue Paper), would not be expected to lead to subsequent loss of reproductive 

function. Indeed, there was no effect on estrous cyclicity in atrazine-treated groups at 

doses of 30 mg/kg bw/day or less. 

 The possibility exists that attenuation of the LH surge at a level less than that 

which prevents ovulation might not be protective for other reproductive effects, such as 

anovulation or decreased fertility. There are also some data seemingly inconsistent with 

the hypothesized mode of action. For example, suppression of GnRH pulses would be 

expected to lead to an increase in serum follicle-stimulating hormone (FSH) levels. There 

is no evidence to show that this is the case, even at high doses of atrazine (Cooper et al., 

2000; Foradori et al., 2009). Indeed, it appears to have been assumed that the effects of 

chronic exposure to doses less than those that would alter reproductive function may have 

adverse effects on other areas in the brain and be potentially deleterious to other 

functions. However, these sites of action and responses other than LH surge attenuation 

remain to be identified, perhaps using molecular approaches. For example, binding to the 

growth hormone releasing hormone (GHRH) receptor and decreases in GH release 

following exposure to atrazine has been suggested based on a recent report by Fakhouri et 

al. (2010). In relation to relevance to humans, changes in basal LH release, which also 

are directly regulated by GnRH secretion, might also be a sensitive endpoint. 

 

 The biological significance and degree of adversity of the effect used to derive the 

benchmark value remains to be addressed robustly and needs to be considered carefully 

in relation to other endpoints on continued exposure, based on the totality of data, 

including that from more traditional studies. This would permit consideration of the 

degree of protection offered by the proposed point of departure compared with those 

based on traditional apical toxicological endpoints, for which the database is 

considerable. This will need to be taken into account when, for example, selecting the 

appropriate uncertainty factors, along with the adequacy of the toxicokinetic and 

toxicodynamic data that will inform the inter- and intra-species extrapolations. 

 It may be helpful, then, to derive and array a variety of potential points of 

departure for relevant endpoints with a view to characterizing their degree of protection 

(in relation to biological significance of the observed effect) and associated uncertainty. 

This would serve as a critical step for the interpretation of the derived BMD, not just to 

provide understanding of the biological significance of observed effect, but also as a tool 

to compare and contrast the uncertainties associated with various options. Comparative 

uncertainty analysis would include consideration of appropriate interspecies and 

intraspecies adjustments, in the context of their associated degree of uncertainty. 

53 
Adequacy of Data for the Benchmark Dose (BMD) Analysis - The advantages of 

deriving and using benchmark doses rather than the historical effect/no-effect levels are 

well known and documented; BMDs reflect the use of all of the dose-response data in a 

much more robust fashion, accounting for dose selection, dose spacing and sample size. 

They also offer potential advantage from the perspective of integration of data from 

multiple studies. 

 The existing data on the dose response of the attenuation of the LH surge 

associated with exposure to atrazine (in rats) are sufficient to permit robust analysis for 

the benchmark dose. These include significant data in several strains exposed to a range 

of doses delivered either by oral gavage or the diet. 

Selection of the Critical Study for BMD Modeling - The Agency modeled four 

relevant candidate data sets, representing the four studies summarized in Figure 5.8 and, 

appropriately, selected the “new” (unpublished) NHEERL 4-day data as the most 

appropriate for deriving a PoD (i.e., Cooper, 2010 (unpublished). 

 Selection of the Cooper (2010) study as the basis for benchmark dose modeling is 

appropriate, not only because of the range of doses used, and the lower variability of the 

data, but also owing to the inclusion of data on animals experiencing a statistically 

defined LH surge. This was not the case for the Minnema and Morseth studies. For these 

studies, if one were to reassess the data based only on animals having a statistically 

defined LH surge, the overall variation in the results would decrease and the average LH 

values would increase. For example, in the Minnema data set, the LH levels at 1800 h in 

the control, 2.5 and 5.0 mg/kg/day dose groups are reported as 2.73, 2.95 and 3.38 ng/ml; 

if corrected, they would be 4.08, 3.78 and 4.43 ng/ml, respectively, with less variation 

about the means. The latter values were calculated using data only from animals whose 

serum LH levels increased greater than 2 standard deviations (SD) above baseline levels 

for at least two consecutive times, a commonly used definition of an LH surge. 

 Prior to the meeting, in public comments submitted by the registrant, certain 

aspects of the new Cooper et al. (2010) data were subjected to criticism which, if 

accurate, could have negated the value of the study. The Panel had the opportunity to 

hear Dr. Cooper’s response to these criticisms. One of the criticisms was that the 

statistical analysis did not include the control data from Study Block III. Dr. Cooper 

clarified that this assertion was untrue, and that this data set was, in fact, included in the 

analysis. A second criticism was that the age range of the animals studied was too great. 

Dr. Cooper clarified this issue when he explained that the age range covered was valid in 

that each animal fitted the selection criterion of maintaining an estrous cycle over the 

course of the study period. The third criticism was that each of the three study blocks was 

separated by too much time. Because of this, the three data sets should not be analyzed 

together. The Agency BMD analysis incorporated the data from all three study blocks. 

Since a purpose of these three studies, taken together, was to identify (initially) effect 

levels, and then, ultimately, no-effect levels (which were identified in Study Block III), 

an approach that could be taken to address the third point would be to attempt a BMD 

analysis using only the data from Block III and compare the outcome of this analysis with 

54 
that which considered the data from all three study blocks combined. The Panel 

concluded that these new data were appropriate for use in benchmark dose analysis. 

Other Aspects - The only data that supposedly were used in the modeling of the dose 

response curve for attenuation of the LH surge by atrazine were from the new Cooper et 

al. study. However, in Figures 5.9 - 11 of the Issue Paper, data from other studies were 

presented, but their source is unclear. In Figure 5.9, the highest dose is graphed at 75, but 

the Cooper et al. highest dose was 25. The BMD is about 5. In Figure 5.10, the lowest 

dose is <1, a dose not used in the Cooper et al. study and the highest dose is about 13, 

which also was not a dose used by Cooper, et al.. The BMD level in this figure is about 

2.1. In Figure 5.11, the dose range extends to >300, the lowest dose only around 10, 

leading to a BMD of 50. Documentation for these additional data sources should be 

included in the Figure legends and accompanying description. 

 Traditionally, the 95% confidence limits, which is 2 SD from the mean, are used 

to determine whether there is a difference in LH levels from the controls. In this case, the 

BMDL was selected for use as the PoD; it was defined as the 95% lower confidence 

limit, which appears to be in agreement with traditional approaches. 

 In Figures 5.9 - 11, it is not totally clear whether the BMDL, which is the 95% 

lower confidence limit, is, in fact, any different from the BMD, which is defined as 1 SD 

(which would be the 67% limit) from the mean of the controls. In each figure, the BMD 

and BMDL are equidistant from the mean. 

b. The selection of a suitable benchmark response (BMR) is an important component of 

conducting a BMD analysis. As described in Section 5.3, for continuous endpoints, like 

LH attenuation, the BMR most often represents an X% change from background levels 

(or untreated controls). Typically, the BMR is selected on the basis of a combination of 

biological (mode of action, quantitative link between key events and adverse outcomes, 

historical/concurrent controls) and statistical considerations (sample size, variability, 

etc). LH attenuation may be potentially associated with several adverse outcomes and the 

level of attenuation of the LH surge considered to be adverse is a function of several 

factors including the functional outcome and the life-stage. There is an absence of 

information concerning the level of response (or % change) associated with the various 

functional impairments. Also, the differences in reproductive cycles/aging between 

rodents and humans add an additional level of complexity to establishing a specific BMR 

value. When an X% change can not be defined, the Agency’s draft BMD guidance 

suggests that the BMD and BMDL corresponding to a change in the mean response equal 

to one standard deviation from the control mean be used as the BMR. This approach was 

applied to atrazine. Please comment on the scientific factors important for establishing a 

BMR for LH attenuation as part of the BMD analysis for atrazine. 

Panel Response 

 

A solid understanding of a benchmark response is needed for BMD analysis. In 

this case, attenuation of the LH surge is a sensitive measure of atrazine exposure. It 

55 
directly relates to mode of action, being one of the key precursor events to the ultimate 

endpoint(s) of toxicity and it exhibits dose response characteristics. Thus, it is a suitable 

benchmark response. 

 Even so, inherent difficulties exist. These include the wide range in LH surge 

amplitude normally observed in both rodents and women, and the variability in the exact 

time of day the actual peak in LH surge occurs. It should be cautioned that, when 

determining the BMR, inclusion of highly variable data, such as averaging data from 

animals exhibiting a surge with those not exhibiting a surge, could lead to an 

overestimate of the BMD. With regard to demonstration of a dose response of inhibition 

of the LH surge to atrazine exposure, several replicates of the dose response curve, 

ideally in each strain of rat tested, would help to validate the precision of the BMR 

determination. It has been well documented that the Long Evans strain is more sensitive 

than the Sprague Dawley strain, and therefore data from these two strains should not be 

combined for the determination of a BMR. Their differences should be assessed, 

acknowledged and taken into consideration, but their data should be analyzed separately. 

 There are ample data in the literature to demonstrate that complete (or nearly 

complete, i.e. ~ 80%+) inhibition of the LH surge does directly correlate with adverse 

fertility outcomes, including altered cyclicity and inhibition of ovulation. Several studies 

presented in the Issue Paper, using both oral and dietary exposure to atrazine, have 

demonstrated very significant reductions of the LH surge and resultant effects on fertility 

measures, such as cyclicity, tightly linked in time. These tightly linked changes in LH 

endpoint measurements (decreased LH surge amplitude or AUC) and associated 

modification of fertility measures were observed with high doses of atrazine. 

 Outside of these very dramatic changes in LH surge (at or near complete 

abrogation), data correlating more modest, yet statistically significant, changes in LH 

surge characteristics with key fertility events are lacking. These data are not only lacking 

from the series of studies presented in the Issue Paper, but importantly, there are not 

sufficient data in the broad body of literature (rodent or human) related to reproduction 

and fertility to directly link small or moderate changes in LH surge characteristics with 

fertility outcomes. This does not negate the possibility that they do exist. Furthermore, a 

moderate change in LH surge peak amplitude in the context of one cycle may impart 

minimal to no effect on overall lifetime fertility measures. However, it is important to 

note, modest changes in LH surge amplitude over long durations of the reproductive 

lifespan are likely to have an effect on overall fertility measures. This potential should be 

addressed experimentally. 

 Specific data which examine long-term diminution of the LH surge over the 

reproductive lifespan and resultant effects on fertility have not been studied with atrazine. 

In fact, there is little or no understanding of the long term effects of LH surge diminution 

and fertility outcomes following exposure to any compound or agent known to modulate 

the LH surge. In this regard, the responsiveness of the hypothalamic-pituitary-gonadal 

axis to increasing estradiol and the ability of increasing estradiol to trigger the ovulatory 

surge of LH are reduced as the aging process progresses in both rodents and women. In 

56 
this context, changes in the characteristics of the LH surge including decreased amplitude 

of the surge in both rodents and humans precede cessation of cyclicity. Thus, modest 

changes in LH surge amplitude elicited by atrazine exposure together with the normal 

biological changes occurring during the aging process may result in overall enhanced 

sensitivity to the effects of atrazine. Thus, atrazine exposure during the reproductive 

aging phase of the life cycle may elicit greater effects on the LH surge and fertility. 

Studies specifically addressing the effects of atrazine during all key phases of the 

reproductive lifespan are lacking. Additional uncertainty arises with the significance of 

the duration of exposure. 

 Data are clear in identifying that a greater-than-one pulse of exposure to atrazine 

is necessary for attenuation of the LH surge. For example, single high doses (over 100 

mg/kg) administered on the morning of proestrus did not alter characteristics of the LH 

surge occurring later the same day. Additional data clearly demonstrate a once daily dose 

for 4 days and beginning on estrus can induce significant inhibition of the LH surge peak. 

In this instance, a dose response is observed. However, what is not clear is if less than 4, 

but greater than 1 days’ exposure is sufficient to alter the LH surge. Further complicating 

the matter, it is not clear if a 4-day exposure, beginning on a different day of the cycle, 

will result in changes in the LH surge similar to those when dosing begins on the morning 

of proestrus. Understanding of the relationship between duration of exposure and phase 

of the cycle will be key in translating rodent data to humans for risk assessment purposes. 

 It should be understood further that the significance of the duration of exposure 

and phase of the cycle may be difficult to define in the rodent system. Although there are 

similarities between elements of the estrous cycle in a rodent and the menstrual cycle in 

women, differences do exist also. The overall duration of the estrous cycle (4 days) may 

be too short to define specific cycle-phase related sensitivities. 

 Thus, based on the Panel's current understanding of effects related to atrazine 

exposure together with its level of understanding of long-term effects of modest changes 

in LH surge and fertility, they concluded that the available data are insufficient to directly 

relate a specific percent change in either amplitude or AUC of the LH surge with fertility 

outcomes. 

 The alternative, as presented by the EPA, to use the mean and standard deviation 

approach is basically a statistical means to define a significant change. This approach 

represents general, default guidance. It must be remembered that use of this approach 

inherently has the same limitations related to the inability to identify a specific percent 

change in the LH surge as a key benchmark response. Furthermore, there is some 

question if the use of one or two SD would be appropriate. The Agency could examine 

the variation in historical LH surge data to determine the appropriate SD to define 

change. Overall, use of the mean and SD approach is a sound and logical approach by the 

Agency at this time and will allow the Agency to meet current time constraints. 

 However, given the protective capacity of the response, the uncertainties 

associated with LH signaling differences between rats and humans, as well as the 

57 
uncertainties associated with “how much diminishment” is necessary for the occurrence 

of an adverse reproductive or developmental effect, calibration of this response with 

“down-stream” apical reproductive/developmental endpoints should be carried out to 

provide a linkage between this “early event” and the adverse effect. In this way, a 

putative numerical reduction in LH surge (i.e. ~30%) could be related to an apical 

endpoint of reproduction or development. These calibration studies should be conducted 

with an appropriate positive control (i.e., a known GnRH antagonist). Additionally, route 

of exposure should be by diet to more closely mimic the most relevant pattern of 

exposure in humans. 

 At the present time, use of data on attenuation of the LH surge is a suitable 

benchmark and directly relates to the present understanding of mode of action. However, 

additional endpoints including but not limited to growth hormone activity should not be 

overlooked. Furthermore, consideration of additional endpoints and/or additional modes 

of action/key events may allow for integration of both cancer and non-cancer health 

effects of atrazine. 

Question 4.0: Approaches To Evaluating Water Sampling Strategies And Frequency 

Of Monitoring 

The ultimate utility of pesticide monitoring data for drinking water exposure estimations 

in human health risk assessments depends on how well the monitoring data characterize 

the spatial and temporal variability of pesticide concentrations in drinking water, with an 

emphasis on the upper end of the exposure distribution. In addition, the method used to 

estimate concentrations from monitoring data depends on the duration of concern and 

how critical it is to estimate peak concentrations. 

Question 4.1 

A well-designed drinking water monitoring study takes into account both spatial and 

temporal patterns of exposure. Please comment on the USEPA’s recommended 

framework for designing a monitoring study by targeting the most vulnerable areas, 

targeting seasonal times for more intensive sampling, basing sampling frequency on the 

toxicological exposure duration of concern, and using autosamplers to supplement 

monitoring data (see Section 7.2 of the draft Issue Paper). 

Panel Response 

 The Panel noted that if the true purpose of a monitoring program is to generate 

valid “exposure estimates,” then sampling the finished water of a community water 

system (CWS) is more appropriate than sampling its raw input water. Studies that have 

examined the transport of atrazine through a water treatment plant sometimes show a 

statistically significant reduction in atrazine between the source water and finished water; 

however, complete removal is rare and some water treatment plants show no reduction 

(Coupe and Blomquist, 2004; Kingsbury et al., 2008). This suggests that the material 

covered in Section 7.3.2 of the Issue Paper might be better used in Section 7.2 where the 

58 
“General strategy for designing a monitoring study to characterize DW exposure” is 

discussed. However, if understanding the fate and transport of atrazine to a CWS is 

desirable, then sampling the raw water source is more appropriate. 

 There is a clear need to group drinking water facilities into categories according 

to how atrazine is delivered to the CWS. The distribution of atrazine in a water body 

changes significantly depending upon from where the CWS draws its raw water, whether 

delivery to the system incorporates some form of holding pond or reservoir or is directly 

from a stream or river, and whether mixing of raw water sources occurs. A one-size-fitsall monitoring network does not seem appropriate to address the estimation needs given 

the various types of CWS in the Midwest. 

The Panel addressed the four elements of the Agency’s recommended framework 

separately below. 

(1) Targeting Monitoring to the Most Vulnerable Areas - The Panel agreed that 

targeting the most vulnerable areas is appropriate. Caution was urged in how areas are 

defined as “vulnerable.” One wants to insure that all CWSs with the potential for atrazine 

in their source water are included. The current method that uses an exceedance of 1.6 

μg/L of atrazine in Safe Drinking Water Act (SDWA) samples as the criterion for 

inclusion of the system in the Atrazine Monitoring Program (AMP) is a good place to 

start. However, it was noted that the atrazine value from the SDWA sampling is an 

average of four quarterly values and it has been aptly demonstrated by EPA and the 

registrant that sampling at this frequency has the potential to underestimate the true 

atrazine concentration by a large amount. The use of the WARP model by EPA to 

determine if a CWS might be vulnerable to atrazine exceedances is an excellent second 

screen/trigger for inclusion in the monitoring program. 

 The Panel noted that agriculture is not a static enterprise and any monitoring 

program needs to have the flexibility to accommodate the large year to year variability in 

agricultural practices that results from market forces, regulation, changes in technology 

and other factors.

2) Targeting Intensive Sampling to the High Occurrence Period - The Panel agreed 

that intensively sampling during periods of expected high occurrence of exceedances is 

certainly appropriate. In explaining the occurrence of atrazine in surface waters over 

time, some Panel members suggested use of the following function: 

Atrazine_Occurrencesurface water(t) = Source_StrengthATRAZINE(t) + Hydrology(t) 

 The two components are Source_Strength and Hydrology. Source_Strength 

relates to the amount of atrazine applied in a basin and that it is a function of when the 

application occurred in time. Hydrology refers to the fact that to move atrazine off site, 

water is needed, and this, too, is a function of time. From this equation, it is clear that 

intensive sampling of a runoff event before atrazine is applied in a basin is of limited 

59 
usefulness in describing Atrazine_Occurrence as would be intensive sampling after 

atrazine application when there has been no runoff to move atrazine into surface water. 

 Reservoirs need to be considered separately when designing a monitoring 

program. It can make a large difference in exposure whether the CWS uses a small runoff-river reservoir with a retention time of days, a large reservoir with a retention time of 

months to years, or an off-stream storage reservoir where water is pumped from the 

stream for later use by the water supply system. The USGS has done research on 

predicting the concentration of atrazine at the outflow of a number of reservoirs in the 

Midwest (Battaglin and Goolsby, 1998). 

(3) Basing Sampling Frequency on the Toxicological Exposure Duration of 

Concern - The Panel believed that this approach would be appropriate if there were 

agreement among scientists on the toxicological exposure duration of concern. Some on 

the Panel expressed the opinion that there doesn’t seem to be strong evidence of adverse 

health impacts from atrazine exposures at the levels found in surface waters; because of 

this, these Panel members believed it was unfair to ask the registrant to increase their 

sampling efforts. On the other hand, the fact that there is so much atrazine used in the 

United States, and that it is found in every environmental compartment (i.e., ground 

waters, surface waters, rainfall, lakes, reservoirs and some drinking water supplies) raises 

the importance of further monitoring. There may be subtle human health or 

environmental effects that currently remain unidentified or not well understood. The 

Panel believed that it is important to continue to monitor for atrazine in the environment, 

to better understand exposure levels and ensure that they continue to decline.

(4) Use Autosamplers to Collect Data for Exposure Periods of Interest - The Panel 

agreed that this is appropriate. Autosamplers currently are used successfully by many 

researchers who are studying the fate and transport of pesticides including atrazine. 

Autosamplers can better capture transient events that happen during periods when 

personnel are not available to do the sampling. Getting good reliable information from 

autosamplers also requires having good quality assurance and quality control programs in 

place. 

 EPA should look at other methods and procedures for collecting water samples. 

For instance, water treatment plant operators should be able to collect samples from their 

system's raw water intake and/or finished water outflow every day. These samples could 

be refrigerated or frozen with the decision to analyze any single or composite samples 

made at some later date. 

Question 4.2 

The April 2010 SAP recommended that simulations of candidate monitoring strategies 

and evaluations of the utility of different exposure estimation methods be benchmarked 

against intensive empirical data that cover a representative range of sites. In response, 

the USEPA proposes using more intensively sampled ambient water monitoring for 

60 
flowing waters and PRZM/EXAMS modeling for static water bodies, matching them to 

the CWS based on similar chemograph shapes (see Section 7.3). 

a. Please comment on the Agency’s proposal to use chemograph shapes (number, 

duration, and magnitude of peaks) to match CWS with more intensively monitored 

datasets. In particular, the Agency is interested in recommendations on approaches for 

matching chemograph shapes given the loss of short-duration peaks that occurs with less 

frequent sampling. 

Panel Response 

 As noted in the question itself, the chemographs from the less frequently sampled 

sites may have short-duration concentration peaks that were not sampled. Matching a 

chemograph that is missing short-duration concentration peaks to chemographs from high 

frequency sampling sites will not show that short-duration concentration peaks are 

missing from the lower sampling frequency data. Instead, this approach will give a false 

confidence that the short-duration concentration peaks are not missing in the less 

frequently sampled data. In addition, chemograph shapes are influenced by factors such 

as pesticide use (amount and geographic distribution) and precipitation, as well as static 

watershed characteristics. A chemograph shape for a site can vary temporally because 

pesticide use and precipitation are temporally variable. Given the likelihood of missing 

short-duration concentration peaks and the variability of chemograph shapes over time at 

a site, using the chemograph shape as the mechanism to link CWS sites and the high 

sampling frequency sites may be overly difficult and may provide a false sense of 

confidence in terms of assessing the adequacy of sampling frequency for a site. 

 Functional Data Analysis (Ramsey and Silverman, 2002; 2005) may provide a 

potential approach to match sites based on chemograph shapes; however, care must be 

taken in accounting for missing peak concentrations when matching. 

A suggested alternative approach is to match high frequency sampling sites to 

CWS sites based on water body and watershed characteristics. First, sites should be 

grouped by their source water type, such as static, flowing, or other. Within the source 

water groups, sites then can be further classified or grouped based on their watershed, 

pesticide-use, and climate (precipitation) characteristics, as well as their geographic 

locations. In terms of static or non-flowing waters, hydrologic characteristics such as 

retention time also should be used in this grouping exercise. 

b. Please comment on the strengths and weaknesses of using more intensively sampled 

datasets from Heidelberg and AEEMP for analysis of flowing water and PRZM/EXAMS 

for lakes and reservoirs. 

Panel Response 

 Appendix D very nicely shows the problems with trying to draw inference for an 

exposure when the interval is sampled only with a single point per interval. Sometimes 

one gets lucky with the timing of the sample, and sometimes one misses the peak 

61 
completely, thus grossly underestimating exposure. Thus, it is important to have true 

daily data when developing a methodology, as both the Heidelberg and AEEMP datasets 

do. PRZM/EXAMS modeling is also useful and provides calibrated curves with 

simulated daily values. While it should be noted that these models are not necessarily 

true data, and thus any conclusions drawn using them should be taken as approximations, 

they would be expected to give much more accurate results than a linear or stair-step 

interpolation of less frequently sampled data (such as weekly data). There is a substantial 

literature on the use of computer models to augment physical data, and when properly 

calibrated (i.e., adjusted to match the available sparser real data), the approximations can 

be very good (Kennedy et al., 2001; Higdon et al., 2004; Williams et al., 2006). 

 The Heidelberg dataset is high quality and represents frequent sampling, and it 

has the benefit of spanning multiple years, allowing for the understanding of some of the 

relationships with external driving conditions such as rainfall. However, there are 

definite limitations in its use. It does not represent geographical diversity (as the AEEMP 

datasets do) as its data are concentrated in a single region in Ohio. The stream 

characteristics (e.g., watershed parameters, pesticide usage patterns, climate) also are not 

necessarily representative of the broader range of flowing water sources in other parts of 

the country. It does not exhibit the scale effects that many water sources do. 

Generalization to other watersheds would need to be done with care to avoid incorrect 

predictions which could lead to a false sense of security in their accuracy. 

The Perry Lake dataset would be more relevant for reservoirs. SEAWAVE was 

designed for flowing water, rather than for reservoirs, and PRZM/EXAMS for a 

somewhat different scenario. Furthermore, PRZM is a very detailed model, not the 

easiest to use in practice, and needs to be tailored to each individual watershed. SWAT is 

another model with similarities in design intent and need for specificity of application. 

 Each reservoir may be different, depending on drainage basin characteristics (e.g., 

size), capacity, turnover time, and other location-specific variables. A smaller streambased reservoir may exhibit similar behavior to flowing water. However, a larger 

reservoir will generally have multiple input sources and slow turnover times, and thus 

pesticide concentrations will be much more smoothed (attenuated peaks) than for flowing 

streams. In such a case, sparser (e.g., weekly) sampling might be sufficiently 

representative of the overall chemograph, as demonstrated with the Perry Lake example. 

Working with flowing water could be seen as a conservative approach. Predicting peaks 

in reservoirs may be easier than for flowing water, since the peaks should be more broad 

and easier to catch with less frequent samples. 

There was some sentiment among the Panel members that a more practical use of 

resources could be the collection of more varied, intensely-sampled datasets (particularly 

for reservoirs), rather than attempting to fit complex, labor-intensive, site-specific models 

to less-frequently sampled datasets. 

62 
Question 4.3 

Once the magnitude and duration of exposure of toxicological concern are identified, the 

USEPA will determine which method(s) to use to analyze the existing atrazine CWS 

monitoring data for use in drinking water exposure estimates. Based on the April 2010 

SAP recommendations, the Agency is focusing on regression-based modeling combined 

with random function modeling (for example, a revised WARP model coupled with 

kriging or the USGS SEAWAVE-Q model). 

Please comment on the strengths and weaknesses of these proposed approaches and 

provide any recommendations for improving the model applications. 

Panel Response 

 The easiest approach would be to have sampling twice during each period of 

interest. The current sampling scheme would be appropriate for a period of interest of 14 

days or longer; otherwise, more frequent sampling would be recommended. Sampling 

could be manual or via autosamplers. This approach would give direct information about 

exposure over an interval, without the need for complex interpolation. 

 Combining a deterministic model, like SEAWAVE-Q or PRZM/EXAMS, with a 

regression-based model, like WARP, appears to be the most promising approach to deal 

with sparse data in flowing water (the scenario for which these models were designed). 

This approach makes use of information about the pathways and aggregate data that are 

not incorporated by purely statistical methods such as kriging or neural networks. 

However, the April 2010 SAP noted that, while exposure estimates generated through 

inference from other sites (such as WARP and other models) are appropriate for study 

design and screening-level evaluation, the uncertainty may be unacceptably high with 

these methods in terms of evaluating atrazine levels in order to implement regulatory 

actions. The purpose of models such as WARP is to provide concentration estimates and 

probabilities in order to guide more intensive monitoring and assessment. Similarly, 

WARPsim (Vecchia and Crawford, 2006) was developed to simulate daily pesticide 

concentrations based on WARP coupled with time-series data on precipitation and 

temperature, and includes a seasonal component to simulate the time-series 

concentrations. However, it was not intended to be used to predict actual concentrations 

for a specific day or location. 

Combining a model such as WARP with a purely statistical method will give 

better interpolation than simple methods such as linear interpolation, but it would fail to 

make use of information about the expected shape of concentration curves that could be 

predicted by a deterministic model. However, there is a trade-off in the difficulty of 

implementing these methods, and it may be easier to implement a combination of WARP 

and a relatively simple statistical approach such as kriging, compared with combining 

WARP with a deterministic model. Such a consideration might have ramifications in the 

field. 

63 
 Kriging does allow reasonable estimation of confidence bands, which could be 

useful for guessing what might be happening in between observations. (Please note that 

empirical variograms are highly variable). Slide 94 of the Agency’s presentation clearly 

shows the need for joint estimation of variograms in order to get the confidence intervals 

right (one can get really wide confidence bands on the 16-day sampling). The correlation 

structure is clearly non-stationary and Gaussian may fit well overall, mainly because of 

the many small values. Around the peak(s), the structure is changing much more rapidly 

and does not look like a Gaussian correlation structure. Using a Gaussian correlation will 

seriously underestimate the peaks. One possibility to avoid fitting complex nonstationary models would be to use only the portions of the chemographs closer to the 

peak areas for fitting the variogram structure. While this may over-estimate variability in 

the low-level parts of the chemograph, those are generally not the regions of concern. 

WARP has been developed as a nationwide model. In the case of the atrazine 

evaluation, it would be better to tailor it to the Corn Belt, which could require changes in 

the variables selected for inclusion in the model. 

 One Panelist noted that there is a need for a quality control plan. As currently 

there are no replicates collected, one doesn’t have a good idea about sampling variability 

or measurement error. In the presence of both data and model uncertainty, one should be 

wary of overestimating the confidence level of any model predictions. 

 The Panel also discussed how this exercise will inform broader monitoring efforts 

around atrazine in the environment. At the April 2010 SAP, the Panel recommended the 

use of statistical models incorporating environmental explanatory variables and 

geostatistical correlations to simulate chemographs for input waters for CWSs. With such 

a system, the exposure end-points of interest can be estimated and proposed sampling 

procedures evaluated for their statistical properties. It is clear from the presentations 

made to this Panel that both EPA and the registrant continue to pursue this approach. It 

should be noted that it is possible to generate simpler empirical models of more complex 

simulation model output. These simpler models can be used in place of the more 

computationally intensive simulation models for rough predictions for larger geographic 

areas making national prediction possible. This model might be as simple as a linear 

regression, or more sophisticated like a Gaussian process emulator (Kennedy and 

O'Hagan, 2001; Santner et al., 2003; Gramacy et al., 2008). 

 It seems that a lot of time and effort is being concentrated on properly modeling 

the magnitude of concentrations in source (raw) waters. Realizing that source waters will 

eventually be modified through the CWS treatment process and that the ultimate 

exposure metrics for the human risk assessment are derived from the levels of atrazine in 

CWS output (drinking) waters, it is comforting to see that both EPA and the registrant are 

making advances in characterization of CWS water treatment processes as they affect 

atrazine concentrations. 

 As EPA and the registrant continue to explore sampling plans for monitoring, 

some thought should be given to actually using the simulation models and CWS 

64 
characterizations as part of the monitoring process. In particular, it is feasible that models 

will eventually be accurate enough to provide predictions of atrazine concentrations in 

source waters to a CWS for the coming crop season. Instead of requiring a CWS to 

collect and analyze water samples in their output (drinking water) stream at some predefined frequency, say daily or weekly in the case of some sites, it should be possible to 

use the models to facilitate targeting sampling to periods of time most likely to 

experience an exceedance. 

 Description of a possible sampling plan follows. Prediction of expected 

concentrations for raw and drinking water for a CWS would be run at the beginning of 

the crop year using available long-term climate predictions for the catchment area of the 

CWS, the expected cropping practices in this area, and CWS site-specific characteristics. 

It is not required that one know the exact days of rainfall or atrazine application; just 

having distributions for the expected number of heavy rainfall events and expected 

duration and likelihood that these events would coincide with new atrazine in the field 

would be sufficient data for this exercise. For most CWSs, the expectation would be that 

there would be no predicted exceedances. Sites with a high likelihood of exceedances 

might be required to implement interventions under specified conditions to mitigate 

expected exceedances. The CWS would be required to collect water samples on a 

periodic basis, for instance, daily, and store them in a form that would preserve atrazine 

concentrations. At the end of the season, the model would be run again, but this time 

with actual rainfall patterns and crop/ag chemical application/use information. The postseason model run would identify periods in the season just past where high atrazine 

concentrations would have been predicted. The stored water samples for this period 

would then be retrieved and analyzed to get actual atrazine concentrations in CWS output 

waters. This information could then be used to assess CWS performance and the need for 

further system management changes to ensure atrazine concentrations in the future are 

below maximum allowable concentrations. 

 The described plan closely follows sampling protocols that are typically used to 

obtaining information on rare events. A benefit is that sampling effort can be more easily 

controlled and planned. CWS managers would know that they would be responsible for 

only X water sample analyses in any one year, but would not know exactly which time 

periods would be analyzed. They would be responsible for collecting and storing water 

samples for the season. During this meeting’s public comment period, the Panel heard 

that the economic impact of the sampling frequencies required is of high concern for 

CWS managers. This plan would control the sampling effort and, at the same time, focus 

sampling on periods with the highest likelihood of actually detecting measurable 

concentrations. It acknowledges the fact that for most CWSs and, for most times, there is 

little to no detectable atrazine concentration in output waters. It also allows for random 

selection and analysis of stored water samples to estimate the extent to which models are 

missing other peak periods, if any. 

 It is recognized that this method of organizing sampling effort deviates from the 

traditional approach which specifies that the sampling effort be distributed uniformly 

over the time period of interest. However, it has been made clear in the presentations to 

65 
the Panel that traditional thinking will lead to intensive and expensive sampling at high 

frequency with the expectation that the vast majority of analyses will yield “no detect” 

concentrations. While this may provide confidence to many that there is little or no 

atrazine in CWS output waters, this is an unnecessarily expensive approach that is not 

likely to provide better results than the more focused, less expensive sampling plan 

suggested here. 

Question 5.0: Scientific Considerations in Potential Sensitivity of Infants & Children 

As described in Section 6.0 in the draft Issue Paper, the FFDCA, as amended by the 

FQPA (1996), requires the Agency to give special attention to infants and children by 

placing emphasis on the availability of toxicology and exposure information to estimate 

the potential risk to these age groups. The 2003 Registration Eligibility Decision (RED) 

notes that the Agency reduced the FQPA Safety Factor for those localities which are part 

of the drinking water monitoring program conducted by Syngenta, as required by EPA. 

This reduction in the Safety Factor was based on more intensive monitoring required in 

the monitoring program that reduced the uncertainty for estimating concentrations of 

atrazine and its metabolites in drinking water for the 90-day rolling average identified in 

the previous risk assessment as the appropriate duration of concern. Based on the 

newest studies, our understanding of atrazine’s temporal pharmacokinetic and 

pharmacodynamic characteristics has changed and it appears that shorter durations may 

be appropriate. In light of the potential need to shorten the critical duration of exposure 

and as discussed by the Panel in Questions 4.1-4.3, the Agency is evaluating appropriate 

methods for assessing the drinking water monitoring data. 

Previously, the Agency noted data gaps related to “information on Atrazine concerning 

exposure throughout all critical developmental periods (i.e., gestation through puberty in 

both sexes), in particular, early in development.... (emphasis added, FQPA memo, 

2002).” At the time of the previous risk assessment, there were only a few available 

studies which included measures sensitive to endocrine disruption on specific early life 

exposure periods (e.g., peripubertal). Since that time new studies have become available 

(Section 6.0) which add to the existing database and support the findings of the older 

studies. In addition, new tissue dosimetry studies provide information on the transfer of 

atrazine and its chlorinated metabolites to the fetus and to the lactating pup. Further, a 

study evaluating exposure to atrazine from multiple critical developmental periods 

identified previously by the Agency as a data gap is on-going. The results of this 

research will inform our understanding of the potential for differential life stage 

sensitivity. 

In the coming months, as the drinking water exposure analysis develops further and the 

on-going toxicology studies become available, the Agency will work towards completing 

the scientific analysis that will inform whether or not a new FQPA Safety Factor should 

be applied in the atrazine risk assessment. 

The Agency requests the Panel to comment on important scientific factors for the Agency 

to consider in its analysis. Please include in your comments specific consideration of 

66 
uncertainties in estimating drinking water exposures and remaining uncertainties in 

atrazine’s toxicological profile across life stages, particularly as they pertain to 

assessing risk to infants and children. 

Panel Response 

 Consideration of the value of the FQPA safety factor is seemingly best predicated 

on transparent and systematic consideration of the most important qualitative and 

quantitative uncertainties associated with both exposure and effect, relevant to 

susceptible life stages, in a context consistent with that for other pesticides. In view of the 

fact that the database for atrazine relevant to the selection of this factor is still evolving, 

reference here is to some of the generic aspects that might be considered explicitly based 

on outcome of additional analysis, including the following: 

1. For exposure, this could relate to the likelihood of capturing the relevant period(s) of 

susceptibility or over- or underestimating exposure for all life stages, with the proposed 

monitoring strategy (including, for example, consideration of determination of total 

chlorinated triazines (TCT) rather than atrazine alone). 

2. For effect, some critical questions and/or aspects to be addressed in this context 

include the following: 

a. To what extent does the database on hazard and kinetics inform us about the 

potential for increased susceptibility of infants and children? 

b. Is the early key event or late adverse effect for the critical effect sufficiently 

protective for all age groups, based on hazard characterization (including 

knowledge of mode of action)? 

c. How protective is it (e.g., is an early key event protective for later adverse 

effects)? 

d. What is the impact of the potential reliance on a benchmark dose (versus an 

empirically-observed effect/no effect level) in relation to uncertainty in the 

characterization of the relevant dose-response relationship? 

e. Does the degree of conservatism associated with use of the lower confidence limit 

for a benchmark dose increase confidence? 

f. While the epidemiological data are not considered sufficiently robust for inclusion 

in a quantitative risk assessment at this time, can data from any of the studies that 

are considered of highest quality be used to provide some idea of relative 

sensitivity of various age groups of the human population? 

 Even though considerable information related to water monitoring for atrazine is 

available, the overall potential for dietary intake of atrazine in young children or 

adolescents remains somewhat uncertain. It is recognized that information on atrazine 

and its degradates in source water is valuable. Certain water treatment technologies can 

reduce or eliminate atrazine; thus, finished drinking water data should be used in the 

determination of exposure via this source. The existing risk assessment for atrazine uses a 

90 day rolling average to estimate exposures. The Agency suggested that better 

67 
estimation of the duration of dosing most relevant for inducing developmental toxicity 

would be useful, but it remains unclear at the present time what the relevant duration of 

dosing for either adults or developing organisms may be. 

There is a general consensus from the results of the animal studies that atrazine 

can influence the GnRH-mediated LH surge following oral exposures leading to 

reproductive and/or developmental toxicity. The 2003 IRED (USEPA, 2003) for atrazine 

uses the 6-month dietary atrazine study (Morseth et al., 1998) as the critical study for 

establishing an intermittent/chronic RfD. Recent studies submitted by the registrant 

(Coder et al., 2010) suggest that prolonged dietary exposures to atrazine do not influence 

the LH surge. Such qualitative differences in response in studies using relatively similar 

dosing strategies is difficult to reconcile, although some factors (e.g., different strains of 

rats used) may contribute to the conflicting findings. 

 Toxicokinetic studies using radiolabel indicate that the levels of atrazine and its 

metabolites in the fetuses of treated dams are almost equal to those measured in the blood 

of the dams, suggesting that the placenta offers little barrier for these chemicals (Stoker et 

al., 2010; Kamel et al., 2010). On the other hand, the data show differential kinetics in 

the adult versus the pup during lactation with a significant drop-off in the concentration 

of atrazine and its metabolites as the chemicals move from the dam‘s mammary gland to 

milk in the pup stomach, to pup plasma and pup brain, such that the concentrations in the 

pup plasma and brain are approximately 10-fold (or more) less than that in the dam 

plasma (Stoker and Cooper, 2007; Stoker et al., 2010; Kamel et al., 2010). One of the 

most important uncertainties is the magnitude of the internal dose in the adults and the 

fetuses/pups. The newer kinetic studies are a step in the right direction for characterizing 

these values. The feeding regimen is a more realistic paradigm for delivering the dose, 

i.e., more reflective of periodic drinking water exposure. The indication from the data 

presented that a pseudo-steady state is occurring with repeated exposures is useful 

information for internal dose estimation and probably needs more verification. An 

extension of the need for data on internal dose is that it is needed for developing a PBPK 

model that will better estimate the internal concentrations in people. This activity seems 

to be underway and a PBPK model should be forthcoming. Some of the kinetic 

parameters for atrazine metabolism need to be determined for infants and children. 

While certainly a start, more than one human hepatocyte sample needs to be evaluated to 

develop metabolic parameters for the PBPK model. 

 The findings generally suggest, however, that there may not be selectively higher 

exposures to the developing organism during the prenatal or lactational period. A number 

of experimental studies have evaluated toxicity following prenatal, postnatal or 

peripubertal exposures to atrazine. With prenatal exposures (Rayner et al., 2005; 

Rosenberg et al., 2008), effects on endpoints including preputial separation and 

mammary gland development were noted with repeated exposures of 50-100 mg/kg/day. 

With combined prenatal/lactational exposures (Rayner et al., 2007), effects on preputial 

separation and prostatitis were observed with repeated 100 mg/kg/day exposures. Several 

studies evaluated effects of atrazine with peripubertal exposures (e.g., PND22-44, Stoker 

et al., 2000; Laws et al., 2000; Trentacoste et al., 2001; Friedmann et al., 2002; Pogrmic 

68 
et al., 2009). Preputial separation was affected at dosages as low as 12.5 mg/kg/day while 

testosterone levels were reduced at higher exposures (50 mg/kg/day). Thus, none of these 

studies indicate that the prenatally, lactationally, or peripubertally-exposed animal 

exhibits higher sensitivity to developmental disruption than the effects noted in adults 

related to changes in LH surge. 

 The Agency has noted in its recent atrazine assessments (i.e., the 2003 IRED and 

2006 RED) that the database available to support decision-making with regard to the 

FQPA 10X Safety Factor was not adequate to provide the necessary understanding of the 

potential for pre- and postnatal toxicity and/or exposure during these life stages, such that 

the Safety Factor could be reduced or removed in all settings. A reduction of the Safety 

Factor was deemed appropriate for assessments of those sites where the mandatory 

sampling survey was being conducted, in the belief that exposure to this subpopulation 

was being satisfactorily characterized. This decision will be revisited in the future, once 

the toxicology-related questions and related monitoring frequency issues are resolved. It 

remains unclear, however, whether existing data coupled with as yet unfinished studies 

will answer sufficiently the questions about the toxicity profile for all important life 

stages (gestational, lactational and peri-pubertal) and whether there are any life stagerelated differences in sensitivity. 

 One of the biggest uncertainties at the present time is the validity of the very low 

dose effects observed on mammary gland development in the Enoch et al. (2007) paper. 

The data suggest that alterations in development occur following repeated exposures to a 

mixture of atrazine and metabolites/degradates of atrazine (0.09-8.7 mg/kg/day from 

GD15-19; mixture of hydroxyatrazine (ring hydroxylation), deethyl atrazine, deisopropyl 

atrazine and diaminochloroatrazine). Certainly if these very low dose effects are real, 

they will need to be considered as representing a highly sensitive endpoint for risk 

assessment. However, because these mammary gland effects occurred at doses so much 

lower than have been observed for other endpoints, these data need to be replicated. 

These data arose from exposure to a mixture and the other data arose from atrazine 

treatment alone. However, if these data are reproduced in a new study, then further 

investigation of the effects of the mixture components will be warranted. 

 Several questions concerning the data in Enoch et al. (2007) were noted by the 

Panel. There was some continuing uncertainty about the scoring of histological lesions 

across studies. The ranked data appeared inappropriately evaluated using parametric 

analyses, although averaging values for pups within a group before analysis may alleviate 

those concerns. There were low mean scores in one of the control groups (PND33). There 

was generally little change in effect at higher dosages, although with later time-points 

(PND40 and 60) the general tendency was more extensive reduction in mammary gland 

development scores with higher dosing. It is reasonable, however, to assume that, even if 

the results do not show clear dose-related responses, a developmental alteration could be 

an all or none response across exposure levels, once a minimal threshold exposure was 

passed. 

69 
 One interesting piece of information that did not come out in the EPA 

presentations during the meeting was that the mammary gland accumulates considerable 

radioactivity following oral administration of radiolabeled atrazine (Stoker and Cooper, 

2007). The figure below taken from Stoker and Cooper (2007) shows the percentage of 

total radioactivity in various tissues three hours following oral administration of 

radiolabeled atrazine. Note the high concentration in the dam’s mammary glands 

compared with that in a variety of other tissues. 

Tissue radioactivity following 14C atrazine

(Stoker and Cooper, 2007)

Ant pituit

ovary

uterus

adrenals

ant. hypo

post. hypo

caudate

spleen

heart

lung

kidney

liver

mammary

0.0

0.5

1.0

1.5

2.0

2.5

-

Tissue

Percent of radioactivity

 It should be stressed that earlier studies also reported mammary gland effects with 

early in life atrazine exposure (Rayner et al., 2005), but with markedly higher levels of 

exposures. The study reported by Enoch et al. (2007) is the only study evaluating 

mixtures of atrazine and its degradation products. Thus, while no other studies have used 

a mixture of atrazine and degradation products to study developmental effects and those 

results need to be confirmed in further studies, there is some evidence to suggest that the 

mammary gland may be both highly exposed and sensitive to disruption by early atrazine 

exposures. 

 The inclusion of the environmental degradate 4-hydroxyatrazine (ring 

hydroxylation) in the Enoch et al. (2007) study may indicate that this particular 

contaminant is worthy of some focused evaluation. While not a product of mammalian 

metabolism of atrazine, 4-hydroxyatrazine is a substantial environmental contaminant 

resulting from atrazine degradation (approximately 20% of the sample used in the AMM) 

and it may warrant further study. There is a substantial number of toxicity studies on 4-

hydroxyatrazine that were not included in the materials made available to the Panel for 

the meeting. As for the parent atrazine and its chlorinated metabolites, data exist for 4-

hydroxyatrazine which evaluate the potential for genotoxicity/mutagenicity, acute 

toxicity, short-term, subchronic and chronic repeated oral dosing toxicity, developmental 

toxicity and carcinogenicity. EPA has established an RfD of 0.01 mg/kg/day for 4-

hydroxyatrazine, based upon the observation of histopathological lesions in the rat kidney 

in the chronic/carcinogenicity study (USEPA, 2006). The 2007 meeting of the 

FAO/WHO Joint Meeting on Pesticide Residues derived an acceptable daily intake (ADI) 

70 
of 0.04 mg/kg/day for this degradate, also based upon the renal toxicity observed in the 

same study (FAO/WHO JMPR, 2008). Furthermore, preliminary data suggest that 4-

hydroxyatrazine does not possess the neuroendocrine properties observed with atrazine 

and its chlorinated metabolites, i.e., up to a 400 ppm dietary exposure to 4-

hydroxyatrazine did not influence the LH surge (Eldridge et al., 2001). Based on these 

studies, 4-hydroxyatrazine does not appear to pose an additional hazard related to 

atrazine. 

 The very low exposure levels (0.09-8.7 mg/kg/day) of the mixture suggest that 

mammary gland effects may be sensitive endpoints following gestational exposures to 

atrazine along with degradation products (Enoch et al., 2007). While the mechanism for 

the non-cancer mammary gland effects following atrazine/degradate exposures is 

unknown, these findings along with the apparently substantial accumulation of atrazine 

and/or its metabolites in the mammary gland provide some concern for higher sensitivity 

in developing organisms. Replication and extension (e.g., dosimetry, 4-hydroxyatrazine 

and other individual degradation product effects) of findings on altered mammary gland 

at low level exposures are necessary, however, to inform the quantitative risk assessment. 

If the Enoch et al. (2007) studies are replicated, subsequent studies should specifically 

evaluate the role 4-hydroxyatrazine may play, either alone or in environmentally-relevant 

mixtures, in the alteration of mammary gland development. There may also be a need to 

consider direct dosing of pups during lactation to determine the potential for (differential) 

sensitivity in that population. 

 The (re)-consideration of the FQPA 10X Safety Factor should start with 

documentation and conclusions regarding the adequacy of the data describing atrazine’s 

toxicity profile in adults in order to make intelligent conclusions regarding comparative 

sensitivities. Once that is completed, the data for earlier life stages can then be presented 

and more clearly interpreted. 

Question 6.0: Implications of MOA & Toxicity Profile on Water Monitoring 

As described in the Introduction of the draft issue Paper (Section 1), the goals of the 

current atrazine re-evaluation are: 1) to determine the extent to which new science 

indicates a need for the Agency to develop a revised human health risk assessment for 

atrazine and 2) to re-consider, as appropriate, the frequency of drinking water 

monitoring needed. Two important issues related to achieving these goals are 

determining the point of departure and identifying the critical effect and its associated 

duration of exposure. Proposed updates to the point of departure are considered in 

Questions 3.1 and 3.2. With respect to the critical duration of concern, the frequency of 

drinking water monitoring is related to the temporal pattern of the toxicological endpoint 

of concern used for the risk assessment. Generally, longer durations of toxicological 

concern (e.g., a long-term chronic effect) require a less frequent drinking water sampling 

design to approximate longer term exposures. However, as the duration of concern 

shortens, the frequency and timing of sampling become more important in determining 

how well the sample data capture short-duration exposures. Observation epidemiology 

studies raise hypotheses and suggest possible links between atrazine exposure and 

71 
reproductive and developmental outcomes, but these epidemiology studies suffer from 

limitations which prevent firm conclusions. Although certain studies some provide 

qualitative support for the human relevance of the endpoints identified through the 

experimental toxicological database, they provide little to no information on the critical 

duration of exposure. In addition, the MOA and PK database are also lacking in human 

specific information on the effects of atrazine which could be used to quantitatively 

extrapolate between species. As such, the information available to evaluate the critical 

duration of exposure lacks quantitative precision. Thus, the critical duration of exposure 

is instead derived by inferring generic knowledge from a variety of scientific disciplines. 

The Agency has used a variety of approaches to extrapolate findings from experimental 

animal data to humans including allometric scaling and human-specific information on 

the physiology of the menstrual cycle inferred from the IVF literature. According to the 

Agency’s analysis of the pharmaceutical data and allometric scaling of the rodent 

pharmacokinetics data, the potential durations of human exposure that would correspond 

to the exposure period of interest in rats lie between a few days to approximately 4 weeks 

of exposure. 

Please comment on the Agency’s analysis and preliminary conclusions contained in 

Section 8.0 of the draft Issue Paper as it relates to the potential critical windows of 

exposure. Please include in your comments additional or alternative approaches or data 

that may inform this issue. 

Panel Response 

 Section 8 in the Issue Paper systematically addresses 1) the use of LH surge 

suppression in the rat as the BMR, 2) the strength of evidence linking the BMR to a 

variety of endpoints identified in the human epidemiology and experimental animal 

studies, 3) the comparative timing and extent of exposures with respect to the potential to 

suppress the LH surge in animals and humans, including kinetic and dynamic 

considerations, 4) the potential for water atrazine concentrations exceeding a level of 

concern to be missed by current sampling procedures, and 5) the concept that sampling 

frequency can be meaningfully adjusted based on the potential for human health 

outcomes. The Agency has done a good job of summarizing the situation in each of these 

areas with respect to the uncertainties and limitations in both the data and in our scientific 

understanding of what the data are telling us. 

 The Agency has determined that the collected information suggests an optimal 

water sampling frequency would be between a few days and four weeks based on 

durations of exposure considered relevant with respect to potential human health 

outcomes. Currently, the sampling frequency required of the registrant is once a week 

during the use season and once every two weeks during the rest of the year. Several 

Panel members indicated that they felt this sampling frequency was adequate. No one 

raised strong objections or could offer a rationale for an alternative sampling frequency 

based on the collected information discussed during the SAP meeting. 

72 
A series of assumptions and extrapolations contribute to the proposed critical 

window of human exposure and given the collective uncertainties that these assumptions 

introduce, the imprecision in the Agency’s proposed sampling frequency seems justified. 

This may be about as precise an estimate as can be obtained when starting with the 

experimental animal data and the exposure requirements for LH surge suppression as 

opposed to using outcomes that are more unequivocally adverse. In this regard, the 

consideration by the Agency of the human relevance of the adversity of LH surge 

suppression on the basis of both pharmacokinetics and pharmacodynamics, and taking 

into account the broader database including that on pharmaceutical agents used to block 

the LH surge, is to be commended. However, one Panel member commented that it was 

difficult to assess the relevance of the information from the GnRH antagonist research 

since a different mode of action was involved and no data were provided on the minimum 

concentration of atrazine that would be needed over the period of exposure that would 

produce an equivalent reduction in GnRH level to produce a similar effect at the critical 

period. 

One question that clearly needs further consideration is whether there is a critical 

AUC (i.e., exposure of a target site) that leads to a given level of suppression of the LH 

surge. This would be tenable if the rat estrous cycle was equally susceptible over all of 

the four days, but not if tolerance changed over the critical period (for instance, if an 

AUC of 100 units on day one was sufficient, but only 50 was necessary on day 2). There 

appears to be a critical exposure time, but is this 2 days, 3 days, or 4 days? Indeed there 

could be a critical exposure time combined with a critical level. Although this would 

have the same units as AUC it would have a minimum exposure time (possibly a fixed 

exposure time). In order to be sure that AUC is related to biological response, it would 

be necessary to obtain the same AUC by using different dosing regimens (e.g., one small 

dose followed by a large dose followed by two small doses, or four moderate doses 

giving same AUC) but not necessarily by reaching a pseudo-steady state. If the different 

patterns of dosing give the same toxicological endpoint, then it is AUC that is the 

important measure of exposure. However, even if one could sort through this, and apply 

this information to estimates of the proposed range of human exposures responsible for a 

potential adverse outcome, the answer would still be little more than an educated guess. 

 Question 6 specifically requests alternative approaches. There is another way of 

approaching this that may be useful at least when setting the boundaries on exposures that 

may present a concern for human health effects. The current epidemiology database is 

characterized as providing suggestive evidence that the mechanisms of action thought to 

be operative in rats may be occurring in humans exposed to atrazine. The Agency has 

appropriately concluded that the limited human evidence is insufficient to establish 

causality and does not provide sufficient quantitative exposure information to use in a 

risk assessment. However, what if one assumes that the reported human health outcomes 

are, in fact, due to current levels of exposure to atrazine? Although the water sampling 

data may not be adequate to assure that atrazine peaks are captured in all water systems, 

clearly some of the patterns seen are based on rather comprehensive data sets. These 

patterns of atrazine concentrations in water could be used to provide reasonable estimates 

of the extent and duration of human consumption of atrazine following agricultural 

73 
applications for pre-emergent weed control. Simple models could be used to estimate 

human exposures corresponding to a range of times of exposure to the elevated 

concentrations observed in the field, given the expected maximum water consumption. 

Where spikes in water concentration are short lived and concentrations of atrazine change 

over a short time scale, then steady state blood levels will not be achieved. In this 

instance, one might consider what would be average levels of exposure, relate this to the 

equivalent internal exposure in the rat, and use the promised PBPK model to extrapolate 

to humans. 

This may represent a reasonable alternative approach to determining levels of 

atrazine in drinking water that may represent risks to human health. These risks could be 

compared, certainly on an order of magnitude scale, with those calculated from the 

animal data, and may provide a lower bound conservative floor from which to work, and 

provide a different perspective on the water sampling frequency problem. This would put 

the Agency in a much better position if, in fact, the Agricultural Health Study or other 

epidemiology studies provide further support for human health effects as the results 

continue to accumulate. 

 The other consideration when faced with the uncertainty over a critical exposure 

period of from a few days to 4 weeks is whether basing sampling frequency on human 

health effects is, in fact, the best course of action. As was discussed in greater detail in 

the Panel’s responses to Question #4, atrazine concentrations in source water and, 

subsequently, in the finished water supply of a CWS are dependent upon many factors 

that vary spatially and temporally. As noted, each CWS is unique in factors that affect the 

delivery of atrazine such as drainage basin size, characteristics of the soils, cropping 

pattern, slope, and whether the CWS source water intake is directly in the stream, a 

reservoir, or an off stream storage facility. In addition, there are many factors that affect 

the ability of a water treatment system to remove atrazine from water, such as the use of 

activated carbon and the type of oxidant used for treatment. It also has been shown that 

the amount of atrazine in a system can sometimes be related to the ongoing maintenance 

of the treatment plant. It may be more useful to consider a strategy that attempts to 

capture the pattern of atrazine in the source water of each CWS based on the 

characteristics of that particular water system, as opposed to a one size fits all approach 

based on the series of health-based considerations put forth by the Agency. Given the 

collective limitations of the health outcome-based approach, this would seem prudent, 

and would again put the Agency in a better position to take further action should the 

results of ongoing or future epidemiology studies prove more convincing. 

 In the meantime, since water is the primary source of environmental exposures to 

atrazine and its metabolites/degradates, there would be value in doing a better job of 

establishing the relationship between the measured concentrations at the community 

level, and the resulting absorbed dose in humans. This question could be first addressed 

using databases such as NHANES and could be supplemented with individual 

biomonitoring studies to determine the correlations between water concentrations preand/or post-treatment, residential tap samples and human urinary metabolites. 

74 
 To conduct well designed epidemiologic studies in individuals in the future, 

additional individual-specific measures would be necessary. Important measures of 

water consumption and factors that modify exposures would include the following: 

measures of water consumption from the home tap, whether the water is filtered, how 

long the water is run from the tap before taking a drink (known to be important for 

metals), bottled water consumption (very important for women), consumption at work 

and at home (different communities or sources of water), weeks spent traveling away 

from home (i.e., drinking from a different community source), water consumption from 

food (i.e., canned tomatoes) and other bottled beverages. 

Technical comments - On page 128 and in Table 8.1 of the Issue Paper EPA estimates 

human equivalent doses (HED) using an allometric scaling approach. The dosimetric 

adjustment factor incorporates an average female body weight of 60 kg (~132 pounds). 

However, the average body weight for a female in the U.S. has not been that low since 

about 1960, when it was 140 pounds (Ogden, et al., 2004). The most recent data 

published by the National Center for Health Statistics covers the period of 2003-2006 

(McDowell, et al., 2008). The mean body weight for an adult female > 20 years old is 

now up to 164.7 pounds or ~75 kg. It is recommended that if the Agency chooses to go 

forward with an allometric scaling approach as a method for estimating a human 

equivalent dose that it employ the most current value for the average adult female body 

weight. 

 One Panel member questioned the appropriateness of using body surface 

scaling to compute an HED, given that atrazine in the parental form is not hypothesized 

to be the principal/sole toxic moiety. The adjustment of clearance based on body surface 

area adjustment is frequently applied to ensure equal blood concentration of parent 

chemicals in both species; this approach cannot directly be used to compute an HED from 

the rat on the basis of total triazine-equivalents (page 128, paragraph 2). In other words 

the use of an allometric dose adjustment factor to calculate an HED would appear to 

contradict the use of AUC for triazine-equivalents proposed to be used in the doseresponse analysis (Chapter 3 of the Issue Paper). 

75 
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