# 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 Advi sory Panel Meeting

Held at the

Environmental Protection Agency Conference Center Arlington, VANOTICE

These meeting minutes have been written as part of the activities of the Federal Insecticide, Fungicide, and Rode nticide Act (FIFRA), Scientif ic Advisory Panel (SAP). The meeting minutes represent the views a nd recommendations of the FIFRA SAP, not

the United States Environmental Protection Agency (Agency). The content of the meeting minutes does not represent informati on 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 n ecessarily 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 comm ercial products constitute a recommendation

for use.

The FIFRA SAP is a Federal advisory committee operating in accordance with the Federal Advisory Committee Act and esta blished under the provis ions of FIFRA as amended by the Food Quality Protection Act (F QPA) of 1996. The FIFRA SAP provides advice, information, and recommendations to the Agency Administrator on pesticides and pesticide-related issues regarding the imp act of regulatory actions on health and the environment. The Panel serves as the primar y scientific peer review mechanism of the Environmental Protection Agency, Office of Pe sticide Programs (OPP), and is structured to provide balanced expert assessment of pe sticide 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 th e 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 Designa ted Federal Official, via email at bailey.joseph@epa.gov

.

In preparing these meeting minutes, the Pa nel carefully considered all information provided and presented by EPA, as well as in formation presented by public commenters.

iTABLE 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

ii2Federal Insecticide Fungicide and Rodenticide Act Scientific Advisory Panel Meeting September 14 - 17, 2010

PARTICIPANTS

FIFRA SAP Chair

Steven G. Heeringa, Ph.D. , Research Scientist & Dire ctor 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, Nort h Carolina State University, Raleigh, NC

Cary N. Pope, Ph.D ., Professor, Head & Sitlington Ch air of Toxicology, Department of Physiological Sciences, Oklahoma State Univ ersity 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 Scienc es, University of California, Riverside,

CA

FIFRA Science Advisory Board Members

Susan F. Akana, Ph.D. , Associate Professional Research er, Department of Physiology University of California San Francisco, San Francisco, CA

3John 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/Associ ate Water Science Director, U.S. Geological Survey, Pearl, MS

Kenneth Barry Delclos, Ph.D., Pharmacologist, FDA, Nationa l 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 Ca ncer 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 (E ndocrinology & Metabolism), Program in Systems Biology and Physiology, Univers ity of Cincinnati , Cincinnati, OH,

Kannan Krishnan, Ph.D., Professor, Department of O ccupational and Environmental Health, University of M ontreal, Montreal, CANADA

Herbert K. H. Lee, Ph.D., Professor, Department of App lied 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 Reproduc tive 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

4Moiz M. Mumtaz, Ph.D., Science Advisor/Senior Toxicolo gist, Division of Toxicology and Environmental Medicine, Agency for Toxi c Substances & Disease Registry, Centers for Disease Control & Prevention, Atlanta, GA

Katherine Roby, Ph.D., Director, Reproductive Endocrino logy Laboratory, Center for Advanced Reproductive Medicine, University of Kansas Medical Ce nter, Kansas City,

KS

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

5INTRODUCTION

The Federal Insecticide, Fungicide and R odenticide 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 Drinki ng Water Monitoring Frequency.

Advance notice of the mee ting was published in the Federal Register on June 11, 2010. The review was conducted in an open pa nel 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 prel iminary reviews of several epidemiologic

studies of the relation of atra zine to birth outcomes and de scribed a plan to evaluate atrazine epidemiology data from the Agri cultural 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 m ode of action along w ith 2) statistical approaches for evaluating monitoring freque ncy 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 toxico logy 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 th e non-cancer WOE analysis integrating experimental toxicology and epidemiology studi es with statistical analysis for determining whether or not adjustments are necessary in the samp ling frequency of CWS water monitoring.

Opening remarks at the meeting we re provided by Steven Bradbury, Ph.D., Director, Office of Pesticide Programs and Ti na 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 Frankenbe rry, Environmental Fate and Effects Division, all of the Office of Pesticide Program s. In addition, a presentation was given by Suzanne Fenton, Ph.D., of the National Instit ute of Environmental Health Sciences,

National Institutes of Health.

6PUBLIC COMMENTS

Oral Statements were presented as follows:

Janis McFarland, Ph.D., Charles Brecke nridge, Ph.D., Russell C. Hovey, Ph.D. (University of California - Davis), Robert Handa, Ph.D. (The Univ ersity 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 Calif ornia - 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-cance r outcomes is thoughtful and comprehensive; however, the Panel did not agre e 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 includ ing 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 indepe ndently measure atrazine or triazines, but this was not well justified by EPA; 4) the 46 ex posure assessment studies identified in the

literature search should be reviewed and summar ized in any future cancer risk evaluation, and it was suggested that the review a nd summary be provided to the SAP for consideration; 5) a system of scoring st udy 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) a dditional studies other than th ose 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 an alyses of the existing outcomes in these studies. In general, the Panel believed the 19 non-cancer outcome st udies 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 Epidem iology Database & Risk Assessment - Overall, the Panel agreed with EPA's conclusions that th e 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 ep idemiology and experimental toxicology information, the following are needed: 1) a st rong experimental toxicology database; 2) understanding of toxicological mode(s) of action; and 3) a strong epidemiology database. For atrazine, a robust experimental toxico logy database exists and understanding its modes of action is increasing. However, with regard to human epidemiologic studies, studies are needed that are specifically designe d 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 huma ns 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 assessm ent at the present time, but that the epidemiologic and toxicologic databases, in combination, can be used to inform h azard identification and characterization. Contrary to the Agency's opinion stated in the Issue Paper, the Panel

8believed 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 importa nt experimental design and methodology differences between the Rayner and Coder studies relate d to the following aspects: 1) composition of the diet cons umed 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 th e 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 asse ss the potential human risk of atrazine is not warranted at the present time.

Charge Issue 2.2 & 2.3: Enoch (2007) Mamm ary 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 suitab le 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 gla nd 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, replic ation of the study is needed. The observations of this study have not been corroborated using other mixtures of parent atrazine and metabol ites reflective of temporal a nd 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) a nd the proportion of metabolites would likely change temporally. A ny replication would need to evaluate the same endpoints measured in this study and, perhaps, other physio logical endpoints. The Panel concurred with the Agency’s conclu sion that the few available mammary gland

studies do not constitute a su fficient 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 c onclusion should not be construe d to mean that the mammary gland is not a legitimate target for further analysis and conclude d that the suggestive evidence in these studies argues strongly fo r 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 registra nt and make appropria te modifications. The Panel recommended that the Agency be more pr oactive in this area, and be more rigorous in addressing the uncertainties of extrapolat ion 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 alterna tive to the administered dose metric used in

9the 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 us e of area under the plas ma 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 extrapol ating 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 14 C-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 appro ach taken by the Agency is correct, but requires better data than those currently av ailable. The pharmacokinetic study in progress as described in the regist rant’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 tig ht definitions of the PK profiles of the individual compounds (parent co mpound and analytes). This type of study lends itself to analysis of relationships between the AUCs of the different compounds and biological responses. This information combin ed with good estimates of plasma partition coefficients should allow the developmen t 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 develope d for the rat strain in which the PoD or dose-response is

characterized.

Pseudo steady state implies that the ab sorption 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 si nce 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 th e average levels. Perhaps if the samples had been taken at 12 h after dosing, the profile would have been di fferent. If the samples had been taken at random times after dosing, there would not ha ve been such a smooth plateau. The real profile will not have such small amplitude fl uctuations 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 St ate 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 da ta from two different rat strains across 15years of data collection from four laboratories. The Figure serves as a visual meta10analysis and assists in the unde rstanding of the atrazine dos e-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 ra t 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 a nd 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 st ate 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 ra nge of data and the separate evaluation of data from two rat stra ins. All these aspect s 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 da ta, 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 res ponse, a causal association between the modest, yet statistically significant, observed change s 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 s hould continue to be investigated.

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

benchmark value remains to be addressed robus tly 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 he lpful, then, to develop a variety of potential points of de parture for relevant endpoints with a view to characterizing their degree of protection (in relati on 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 understa nding of the biologi cal significance of observed effects, but also as a tool to compare and contrast the uncertainties associated with various options. Comparative uncertainty analysis would incl ude consideration of appropriate interspecies and in traspecies adjustments, in the context of their associated degree of uncertainty.

The existing data on th e dose response of the at tenuation of the LH surge associated with exposure to atrazine (in rats ) are sufficient to permit robust analysis for the benchmark dose. These include significant da ta in several strain s exposed to a range of doses delivered either by oral gavage or in the diet. The Agency modeled four relevant candidate data sets, represen ting the four studies summari zed 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. Sel ection of this study as the basis for BMD modeling is appropriate, not only because of the range of doses used, and the lower

11variability of the data, but also owing to the inclusion of data on animals exhibiting a statistically defined LH surge.

C harge Issue 3.2b: Benchmark Resp onse for LH Attenuation - A solid understanding of a benchmark response is needed for BMD an alysis. 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 characteri stics. Thus, it is a suitable benchmark response. Even so,

inherent difficulties exist. These include th e 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 ampl e 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 th e Issue Paper, but impo rtantly, there are not sufficient data in the broad body of literatu re (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 conclude d that the available data are in sufficient 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 designi ng a drinking water monitoring study, the Panel noted that if the true purpose of a mon itoring program is to generate valid “human exposure estimates,” then sampling the fini shed 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 crite ria for inclusion in the monitoring program should be carefully considered to avoid undere stimating true atrazi ne concentrations. The Panel noted that monitoring programs need flexibility to accommodate year to year variability in agricultural practices. The Pane l 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 s uggested use of an equation that considers

12atrazine application timing and amounts along with hydrology of the site. Further, the

Panel indicated that reser voirs warranted separate consideration when designing a monitoring program. Basing sampling frequenc y on the toxicological exposure duration of concern, as proposed by EPA, would be appropriate if there was agreement on what constitutes toxicological exposures of c oncern. Some Panel members expressed the opinion that there was not st rong evidence of adverse hea lth 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 agr eed that it is important to continue to

monitor atrazine to better unde rstand 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 ch emograph shapes over time at a site, using the chemograph shape as the mechanism to link CWS sites a nd the high sampling frequency sites, may be overly difficult and may provide a false sense of confidence in te rms 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 alte rnative approach, the Pa nel 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 Da tasets for Analysis of Flowing Water

and PRZM/EXAMS for Lakes and Reservoirs - When developing methodology, it is important to have true daily data, as bot h the Heidelberg and AEEMP datasets do. The Heidelberg dataset is of hi gh quality and reflects fre quent sampling, but it does not represent geographical diversity. Therefore, ge neralization from this dataset to the data

from other watersheds would need to be done with care to avoi d less than accurate predictions. The Perry Lake dataset woul d be more relevant to reservoirs. PRZM/EXAMS modeling is also useful, providi ng calibrated curves with simulated daily values; however, it should be noted that th ese 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 wa tershed. 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 m odels to less frequently-sampled datasets. Data from intensely sampled sites would be e xpected to give much more accurate results than a linear or stair-step in terpolation of less frequently sampled data (such as weekly data). The Panel noted that there is substantia l literature on the use of computer models to augment physical data, and when properly calib rated (i.e., adjusted to match the available sparser real data), the appr oximations can be very good.

13Charge Issue 4.3: Regression-based Mode ling 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 th e most promising approach to deal with sparse data in flowing water (the scenario for which these models were designed). This approach makes use of in formation about the pathways and aggregate data that are not incorporated by purely stat istical methods such as kriging or neural networks. Combining a model such as WARP w ith 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 sh ape 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 eas ier 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 c onsideration 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 tocollect and analyze water samples in their output stream (drinking water) at some predefined frequency (e.g., daily or weekly in the ca se of some sites), it should be possible to use the models to facilitate targeting sa mpling to periods of time most likely to experience an exceedance. The Panel suggested a sampling plan that closely follows sampling protocols typically used for obtai ning 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 seei ng measurable concentrations.

Charge Issue 5: Potential Sensitivity of Infants and Children - Consideration of the value of the FQPA safety factor is seem ingly best predicated on transparent and systematic consideration of the most importa nt qualitative and quant itative uncertainties associated with both exposure and effect, releva nt to susceptible life stages, in a context consistent with that for other pesticides. Wh ile considerable information related to water monitoring is available, diet ary intake of atrazine in young children or adolescents remains somewhat uncertain and at the presen t time, it remains unclear what the relevant duration of dosing for either adults or deve loping organisms may be. There is a general

consensus from the results of the animal studies that atrazine can influence the gonadotropin-releasing hormone (GnRH)-media ted 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 st udies seem to be pointing in the right

direction to characterize these values. An extension of the da ta on internal dose is needed for developing a PBPK model th at will better estimate the internal concentrations in people. This activity seems to be unde rway 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 ad ults related to changes in LH surge. Thus,

14it remains unclear whether existing data c oupled with as yet unf inished 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 bigge st uncertainties at the present time is the validity of the very low dose effects obser ved on mammary gland development in the

Enoch et al . (2007) paper which suggests that alte rations in development occur following repeated exposures to a mixture of atrazi ne and metabolites/degradates. It should be stressed that earlier studies also reporte d 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 unkno wn, 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 highe r sensitivity in developing organisms. The (re)-consideration of the FQPA 10X Safety Factor should start with documentation and conclusions regarding the ad equacy of the data describing atrazine’s toxicity profile in adults in order to make intelligent c onclusions regarding comparative sensitivities. Once that is completed, the data for earlier lif e stages can be presented and more clearly interpreted.

Charge Issue 6: Implications of MOA a nd 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 discus sed during the SAP meeting. There are some assumptions and extr apolations that contribute to the proposed critical window of human exposure but give n the collective uncertainties that these assumptions introduce, the imprecision in th e Agency’s proposed sampling frequency seems justified. This may be about as preci se an estimate as can be obtained when starting with the experimental animal data and the exposur e requirements for LH surge suppression as opposed to using outcomes th at are more unequivocally adverse. One question that clearly needs furt her consideration is whether there is a critical exposure (such as a minimum AUC at the target site) th at 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 Ag ency has appropriately concluded that the limited epidemiological human evidence is insu fficient to establish causality and does not provide sufficient quantitative exposure info rmation to use in a risk assessment. However, what if one assumes that the repor ted human health outcome s are, in fact, due to current levels of exposure to atrazine? The patterns of atrazine c oncentrations in water could then be used to provide reasonable es timates of the extent and duration of human consumption of atrazine following agricultu ral 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 c oncentrations observed in the field, given the expected maximum water consumption. Where spikes in water concentration are short

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

blood levels will not be achieved. In this in stance, one might consider what would be average levels of exposure, relate this to th e equivalent internal e xposure 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 wa ter that may represent

risks to human health. These ri sks could be compared, certai nly 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 th at 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 Ag ency 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 me tabolites/degradates, there is va lue in doing a better job of establishing the relationship between the m easured concentrations at the community level, and the resulting absorbed dose in humans.

16PANEL DELIBERATIONS AND RESPONSE TO CHARGE

As part of the re-evaluation of the health effects of atra zine, three meetings of the FIFRA Scientific Advisory Panel (SAP) were scheduled in 2010. The first two meetings were held in February and April. The pur pose of the September meeting was to solicit feedback from the SAP on the status and overal l scientific directi on 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 toxicol ogy data. The Agency also sought the Panel’s feedback on the proposed updates to the dose-response assessm ent 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 ge neral framework for designing monitoring studies along with basic approaches for analyz ing 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 implic ations 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 Is sue Paper provide the Agency reviews and synthesis of the non-cancer epidemiology st udies available for atrazine. These include studies on a variety of topi cs, notably female and male reproductive outcomes and birth outcomes in addition to other topics. S ection 4.0 integrates the findings of the epidemiology and experimental toxicology studies.

Question 1.1

Please comment on the sufficiency of the Agen cy’s non-cancer epidemio logy reviews with respect to identifying the major stre ngths and limitations of each study.

Panel Response

The Panel congratulates EPA on its inclus ion and evaluation of the epidemiologic literature as a component of the risk asse ssment 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 be haviors 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 out comes, 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 Adviso ry Panel review but do not appear to have been heeded in the present draft of the Issu e Paper. We encourage EPA to incorporate the earlier advice regarding evaluati on of epidemiologic studies.

17The 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 c overed 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 belo w. The cancer study database will be larger and more relevant for men. Women are ge nerally under-represent ed 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. S econd, EPA should include an appendix with a full citation and abstract for each paper ex cluded from full evaluation and a summary describing why the paper was excluded so th at 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 b ecause they did not independently measure atrazine or triazines. This is not well jus tified in the text. Ot her studies that were included, either ecologic or observational in design, did not report any semi-quantitative

measures of atrazine or triazi ne exposure. For example, s eason 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 triazi ne 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 epid emiologic review of reproductive and other effects, these are not n eeded, 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 s uggested that the review and summary be provided to the SAP for consideration. When the epidemiologic database is sufficiently strong to support dose-response assessment, su ch studies can be used to connect the exposure assessment data with the questionnai re and biomarker data, given knowledge of

the toxicokinetics of the herbicide.

Fifth, it may be efficient to develop a sc oring system for the quality of studies (i.e., how well do they meet the inclusion crit eria) to show how each paper addresses the key factors identified by the EPA and the Fe bruary 2010 SAP Panel (statistical power, sample size, generalizability, accurate and precise exposure and outcome assessment methods, sophistication of statistical analys is, control for confounding, minimized participation and selection bias es, etc.). These have been developed for other types of epidemiologic analyses (e.g., meta analysis, Cochrane reviews) a nd could be applied easily by EPA. Also, in future reviews of ot her pesticides and for larger epidemiologic databases, EPA may want to have two revi ewers 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

18outcome, 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 we ight 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 drinki ng water contamination levels) for the group level data is available, the studies that include such expos ure measures should be given more weight than those that have no meas ures or proxies (e.g. , season of birth).

In general, EPA has done a good job of im proving an Issue Paper that was already strong. Four problems are typica lly 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, me thods, settings, etc.); 3) the data are generally of poor quality, and 4) the data ma y just not be what a ssessors would like to have (e.g., high-dose lifetime animal ingest ion 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 th at EPA has largely attempted to consider and address these problems in evaluating the literature.

The Panel’s evaluation of the releva nt 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 highli ghting the strengths and limitations of the

Farr et al. (2004) analytic approach to evalua ting the relationship of atrazine exposure to menstrual cycle characteris tics in the Agricultural Health Study (AHS). EPA was correct in noting th at lifetime exposure is assessed while menstrual cycle outcomes are captured eith er within the past 12 months or as average cycle characteristics, wh ich 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 c ohort study, it was analyzed with odds ratios (OR), which might enhance estimate s 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 analys is, based on data from the AHS, was only 57%, which raises the potential for part icipation bias (e.g., hypothetically, women

with menstrual abnormalities or concerns about pesticide exposure might have been more likely to participate, making th e data less representative and potentially overestimating the relationship of atrazine to long menstrual cycles and missed periods, the strongest associat ions 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 genera lizability of the findings from this study is somewhat

19limited, 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 stre ngths 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 indicat or of subsequent disease risk and longevity (i.e., later age at menopause is associated with re duced risks to, and mortality from, a number of chronic di seases 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 inco rrect; a hazard ratio of 0.79 for atrazine exposure does not constitu te a “greater” associati on 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 whethe r the hazard ratio is less than or greater than 1.0), the smaller the asso ciation. 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 th e outcome could not readily be addressed in this study. The Issue Paper should menti on that the initial pa rticipation rate in this portion of the AHS wa s relatively low (59.5%), thus again creating the potential for participation bias, albeit pr obably not great for this outcome. Also, as before, the generalizability of the results from the AHS is somewhat limited

due to the lack of hetero geneity in race/ethnicity, so cioeconomic 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 gestati onal diabetes. EPA might add to the next version of the Issue Paper the point th at while the relationship of atrazine exposure to gestational diabetes is depi cted only graphically, the adjusted odds ratio is in excess of 1.0, and the 95% c onfidence 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 cycl es, more missed periods, later age at menopause, and gestational diabet es) 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 w ith those of the outcomes, and use of nonstandard questions for some outcomes. Als o, the use of odds ratios may overestimate relative risks and, thus, the magnitude of associ ation. Some of these issues regarding the

limitations of the AHS were raised in th e February 2010 SAP’s report on the use of epidemiologic studies in risk assessment, but ap pear not to have been incorporated in the

20critiques included in this Issu e Paper. If EPA disagrees wi th the Panel’s comments, the

next version of the Issue Pa per 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 de signed to assess menstrual cycle characteristics and age at menopause, have collected biologic samples th at could be assessed for atrazine exposure, and/or have better representa tiveness 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 th e Midlife Women’s Health Study (Bromberger et al ., 1997) or the Australian lo ngitudinal study of midlife women (Burger et al., 1995), the Semi conductor 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 w ould make the findings more generalizable, and would permit examination of potentially important modifications of the effects by race/ethnicity, lifestyle or health-relatedfactors, which are important in geneenvironment interactions and potential epigen etic effects. Additionally, the concern expressed in the EPA criti que regarding adequate cont rol 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 pestic ides. Results for atrazine then are presented in comparison with the results for all pesticides. This is in correct; the result of such an analysis is to reduce the apparent strength of evidence fo r 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 shoul d 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 a nd the ability to examine interactions. In addition, the participati on 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 im portant strengths. A small correction

should be made in the second line of the cr itique on page 362 to indicate that this study was of non-persistent pesticides (ra ther than persistent, as currently indicated on this page). The study aut hors found that high atr azine levels were associated with poor semen concentration, morphology and motility.

This single study examining the relations hip of atrazine exposure to reduced sperm quality was suggestive with the important strengths of being hypothesis-driven and

21using 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 appropriat e with the exception of the exposure assessment issue described below. The Issue Paper should note that the Ontario Farm Family study used a historic al cohort design. Data were analyzed with odds ratios, which may overestimat e relative risk, with out adjusting for confounders. Some analyses appeared to use post-conception atrazine or other herbicide exposure or later preconcepti on 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 uncl ear about how multiple pregnancies from the same woman, which represent lack of statistical independence, were handled statistically. (Tests for n on-independent outcomes exist, but were not used here.) The Issue Paper also should emphasize th at the associations of preconception atrazine exposure with early spontaneous abortions were modest in magnitude and not statistically significan t (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 risk s 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, mi ght represent a much better effort at historical pesticide exposure reconstructi on than the self-report ed measures used in other observational studies (e.g., the AHS ever/never classification). The Issue Paper correctly notes that women who we re 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 repor ted preconception use of both triazines

and thiocarbamates had an OR=7.5 for spontaneous abortion, all of which suggests that older maternal age may m odify 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 w ith 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 so me 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 like lihood of detecting differences as statistically significant. Paternal use of atrazine on crops had an

22adjusted 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-g estational-age of 0.5 (95% CI 0.2-1.3).

In summary, these two studies of the rela tionship of atrazine exposure to fetal and perinatal outcomes were suggestive, with la rgely positive but non-signi ficant associations that were modest in magnitude. They also possessed a number of limitations, including

use of odds ratios instead of relative risk s and small sample sizes for some outcomes, resulting in wide confidence inte rvals, lack of adequate statis tical power, lack of clarity in the handling of lack of independence of multip le 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 co rrelation 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 wa ter with elevated atrazine.

(2) Hornemann et al . (2009) (pages 35 and 340 of the Issue Paper): The EPA critique appropriately notes the limitati ons of this ecologic study by Hornemann

et al . (2009). These authors examined seas onality of omphalocele prevalence rate during a time when atrazine was banned fr om use in agriculture in Germany so that they had little likel ihood of observing differences in prevalence rates by season. Also, the ecologic design would not permit assessment of actual individual maternal prenat al atrazine exposure in relation to o ccurrence 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, wh ich does not permit assessment of the individual maternal exposu res 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 th e major limitation of the Ochoa-Acuna (2009) study as being the use of residen ce near corn fields as a surrogate of potential chemical exposure, so that act ual individual maternal exposures were not measured. In addition, the sample sizes were small for several types of birth defects, and it was likely th at 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

23soybean fields, other chemicals such as fe rtilizer 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-cont rol study of gastro schisis reported by

Waller et al . (2010) is quite appropriate in noti ng the strengths of the case-control approach, including individual informati on on exposures and outcome, compared with that of an ecologic design, and th e 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 monitori ng site, rather than actual maternal tap water exposure); b) th e 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 mo thers relocating residence between early pregnancy and delivery; and d) the lack of clarity of selecti on of confounders to control in multiple logistic regression mode ls. 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 author s concluded that the occurrence of gastroschisis was significan tly 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 exposure s 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 i ndividual maternal exposures to atrazine and the other used a largely ecologic measur e, rather than an individual measure of atrazine exposure. Additional limitations incl uded: 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-Ac una); and confounding variables were not adequately described and adjust ed 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, hi storical 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-ges tational-age infants appropriately noted the strengths of using a more refined exposure measure of atrazine based on monitoring data and adjustment fo r multiple individual-level confounding variables. The Issue Paper, however, al so 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

24misclassification of exposure. No a ssociation was found between atrazine concentrations and preterm births, but a significant positive association (a 20 % increase) was found with sm all-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 at razine 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 significa nt 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-g estational-age. Indeed, no significant

associations were detected between atrazi ne concentrations in drinking water and these three pregnancy outcomes. The I ssue Paper also shou ld note that the findings are suggestive of elevated odds of low birth weight (which was not statistically significant) and small-for-ges tational-age in pregnancies whose third trimester overlapped high atrazine use periods (50% increase) and, similarly, for preterm deliveries whose first trimester ove rlapped 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, prematur ity, 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 stud y did not examine individual maternal drinking water exposures and controll ed 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 conf ounders 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 cont rol group similar to the cases, ascertainment of birth weight from medi cal records, paternal confirmation of pesticide exposure reported by mothers and adjustment for multiple confounding

25variables. The critique also highlights th e major limitation that the analysis is not specific for atrazine exposure, and the sample size was too small to examine specific pesticides. The results indicate d 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 wei ght 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 di fferences while adjusting for multiple confounding factors. Indeed, the study f ound 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 compri sed of a largely white population sample, which limits the generalizability of the findings and the ability to examine any modification of effects by race/ethnicit y, 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 individua l 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, al though individual maternal water consumption was not assessed to improve estimates of i ndividual exposures. The findings suggest a relation of atrazine exposure to small-for-g estational-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 th an 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 e ffect on the fetus at some time or in some settings and work through the moth er’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 th is paper reporting on a cross-sectional

association of wheeze with pesticide use in the AHS showed a modest but significant association of atrazine w ith 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 limit ations 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

26modification 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 (aller gic and non-allerg ic asthma) and controlling for additional potential confounding factors resu lted in no significan t 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 identi fies the limitations of th e cross-sectio nal study by

Kossman et al . (1997), including the inability to ev aluate the specific relationship of atrazine exposure to imp aired respiratory function a nd the lack of control of confounding factors (particularly importan tly, smoking). In addition, the Issue Paper should note that the sample size was small and would not have provided adequate statistical power to detect meani ngful 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 cr oss-sectional study to include the inability to evaluate the specific relationship of atrazine exposure to immune parameters that may be biomarkers of im paired respiratory function and the lack of control of confounding factors (particu larly smoking). In addition, the Issue Paper should note that the sample size was small and would not have provided adequate statistical power to detect meani ngful 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 limitation s, including sample sizes inadequate to provide sufficient statistical pow er to detect meaningful eff ects associated with specific chemicals while controlling for confounding vari ables; 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 s hould be well-designe d 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 a nd outcomes and more representative and diverse study samples. New research shoul d 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 individua ls with likely or known drinking water exposures), perhaps in appropr iate agricultural areas. Such studies will need to be supported by laboratory data bear ing on important public health issues so that significant effects or observations of no significant a ssociation(s) in humans can be appropriately interpreted.

27One Panelist expressed concern about th e 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 in terest 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 upp er 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 sophistic ated ways are available to approach the

LOD issue.

Question 1.2

At this time, atrazine’s non-cancer epidemiologic dat abase 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 th e 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 th e qualitative similarities and differences between the epidem iologic findings and the experim ental toxicology database.
In short, the observational st udies – particularly those relate d to reproductive effects in adult females, as well as small for gestationa l age in newborns – lend further support for the human relevance of the laboratory animal findings.

a. Please comment on the scientific info rmation 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 data base 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 identific ation, dose-response assessment and hazard characterization, but for purposes of qua ntitative risk assessment (i.e., risk characterization), the experimental toxicology data (particularly, in vivo data from the rat) will be used for endpoint selecti on and dose-response assessment.

EPA has conducted a thorough study- by-study review of the non-cancer epidemiologic papers; the SAP critique of the Ag ency 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 studi es) 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 observat ional data. Furthermore, many of the 19 studies are ecologic in desi gn and should be given little weight for risk assessment

purposes.

28Some general comments on the broader issues regarding in terpretation of the integrated data were presented and de serve 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 atten tion 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 li kelihood that critical (apparently) negative results might be reve rsed by better and stronger designs and, especially, larger studies (i.e. with greater statistical power), wh ether in humans or animals and whether findings, even when non-significant, show consistency across studies with different designs pe rformed in different populations.

Similarly, the “multiple comparison problem” is not sufficiently appreciated. This problem is widely recognized as it applies to single studie s; 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 traditiona l default 5% level for statistical significance in scientific analyses, an averag e of one in 20 tests, in which no effect truly exists, will be labeled “statistically significan t.” This 5% approach (or 95 %, in the case of confidence bounds) has been very productive for many decad es across all areas of science, as important results can be evaluated in a broa d 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 numbe rs of reports. The que stion in such reviews is not whether some result is statistically significant, but whether the overall pattern of results labeled significant actua lly lies outside the expected range. Roughly speaking, if the literature reported 10 0 tests of data in which no true effect exists, one should expect about 5 to be “significant.” If say, 10 are sign ificant, 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 le ngth on the plausibility that such findings are a result of multiple comparisons.

One more point is that statistical co nfidence bounds and p-values measure the effects of random error, and random error onl y. Bias increases the uncertainty, so that calculated p-values are too small and conf idence 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 participa tion biases in the de sign and implementation of the study.

These points can be summarized to say th at 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 mechan ical rules about what is a

real effect. It would be helpful to have more explicit acknowledgment of points for which judgment (informed by statistical an alysis) still has an important role.

29The 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 ha s operated a very broad-based registry for many years which could be used in geographi c 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 di fferent 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 defect s). Thus, an exclusive focus on exposures occurring based only on the first trimester of gesta tion is not recommended.

b. Please comment on scientific inform ation that does and does not support the integrative analysis and conclusions, contai ned in Section 4.0, w ith respect to the similarities, differences, and uncertaintie s of the experiment al 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 improveme nts to the framework were provided. The work presented by the EPA at the Septembe r 2010 atrazine meeting and summarized in Appendices B1 and B2 reflects consideration of the majority, but not all, of these

comments/recommendations.

Well-designed observationa l studies in humans that test hypotheses which are developed, in part, based upon the advers e effects observed in animals and an understanding of the toxicological mode(s) of ac tion are needed to be able to incorporate the epidemiologic data into the risk a ssessment 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 mate rials that were provided for the September 14-17 meeting and, to a lesser degr ee, the additional data /studies that were presented in the public comm ent session on September 15, 2010.

To discuss the integrative analysis of the epidemiology and to xicology 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).

30While some major uncertainties in th e toxicology database (which includes hundreds of studies) were identified in the SAP meeting, atrazine exposure disrupts the regulation of pituitary luteinizing horm one 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 ev ents (depending, in part, upon life stage of the exposed individual), and might lead to altered reproductive f unction. The available

data did not allow a clear consensus to emerge on the necessary percent attenuation needed to yield adverse developmental and re productive effects. The form of the dose, route of dosing, and frequency of administrati on 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% atte nuation 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 pharmacoki netic 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 admini stered by feeding and oral gavage. A dose by the latter route that produced reproductive toxicity was without effect when administered in the food (accordi ng 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 pr oblems, but clearly there is a need for

data that are relevant to human exposure via drinking wate r. Moreover, some of the toxicologists speculated that the rodent may not be a good mo del for the preovulatory LH surge in humans. All of these factors are im portant for the interpretation and design of both the animal and epidemiologic studies. Assuming a relevant MOA, it will be necessary to consider rout e and duration of exposure, as well as magnitude of exposure/dose, to assess the potential for ri sks 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; thes e exposures are not consistent within or between life stages and may be predominan tly oral/dietary (unl ess 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 ge nerally tested in animal experiments.

As EPA noted, in order to integrat e the epidemiology and experimental toxicology information, a strong experimental toxicology database, an understanding of toxicological mode(s) of action, and a str ong epidemiology database is needed. For

31atrazine, a robust experimental toxicology data base exists, including hundreds of studies, and understanding of its likely mode(s) of ac tion is increasing. To complement this toxicology database, we need human epidemio logic studies which have been specifically designed to evaluate relevant outcomes and that are based on defined mode(s) of action. Significant improvements in the epidemiologi c 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 desi gned to evaluate these outcomes in humans. These can be conducted at relatively low cost (as compared wi th 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 occu rrence), these types of studies could be conducted in occupational cohorts, such as farm or manufacturing workers, or alternatively, in women living on farms who ar e likely to have higher exposures than the general population (whose prim ary source of exposure is vi a drinking water). Ideally, one would obtain information on current (exter nal) exposures (via direct measurement, questionnaire, records, and/or diaries) a nd 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 ty pe of information would be

far more useful for risk assessment than the m easures extant in the epidemiologic atrazine

literature to date.

Currently, the epidemiology database c onsists of 19 non-cancer studies which met

EPA’s selection criteria for deta iled review. EPA states that th is 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) a nd are not of sufficient quality to include in a comprehensive risk assessment. Thes e 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 onl y for problem formulation. Epidemiologic data relevant for female or male reproduc tive 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 asse ssment were designed to be relevant for cancer outcomes (i.e., long latent periods, so me consideration for frequency (seasonal) and duration of exposure (years) and a few se lect factors that may modify exposures, such as protective clothing worn and equipmen t used but not genetic, lifestyle or other health-related factors). Furthe rmore, the AHS was not designed to and thus did not measure well other outcomes (such as menstr ual or reproductive outcomes) nor was its study population adequately divers e and large to be more ge neralizable and to examine effect modification adequately as noted in the previous sentence.

32The 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 avai lability 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 th e 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 assessm ent. 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 dis eases, it was not designed to identify accurately these speci fic reproductive outcomes. Th e critique of the exposure assessment methods is provided knowing that the study was not de signed to test hypotheses about pesticide or at razine-specific exposure in re lation to these reproductive outcomes and thus is not appropriate fo r assessing risk for these outcomes.

An example of an inappropriate “ever/n ever” 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 littl e relevance for most health outcomes. The more relevant question for a cancer outco me would be “Have you smoked at least 10 cigarettes a day for 10 or more years?” Fo r short-term effects, the question “Have you smoked 10 or more cigarettes/day over the pa st 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, releva nt mixtures, or chemical classes are far more relevant (e.g., triazines as a group) and categorizati on based on suspected mode of action (i.e., targets a specific gene or endocri ne 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-gestati onal-age, low birth weight) studies conducted hypothesis testing (Table 3.1, EP A Issue Paper), and two others had nonsignificant results. Unfortunately, inade quate exposure assessment was a major

limitation in these studies. The other studies reviewed, including the ecological st udies of group data, should be given little consideration in EPA's analysis. In addition, it should be very clear in the EPA reviews of individua l studies that the impor tant distinction be made between ecological studies and the “ecol ogical approach” for exposure assessment. For example, assigning an area value (i.e., averag e or median concentration of atrazine in finished drinking water from a community water system) to an individual study participant is not an ecol ogical study design. That woul d represent an observational study with individual assignmen t of exposure. As noted pr eviously, ecologic studies should be considered only for hypothesis generation.

33The Panel agreed that the integratio n of study findings, interpretations and conclusions in Section 4 of the Issue Paper is largely correct. Ho wever, Section 4 does not adequately address the si gnificant 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 toxi cology and the limited epidemiologic findings would differ with regard to these effects. Fu rthermore, as stated previously, other human studies of menstrual cycle char acteristics and/or age at menopa use 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 biol ogic specimens that could be measured for atrazine, its metabolites and other environmen tal 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 interpre ting 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 relate d 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 th at 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 miscla ssification, 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 “atrazin e” 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 thir d trimester, when fetal weight increase is generally greatest, is at least equally plausible.

An integrative analysis of the epid emiology and toxicology databases could be strengthened with additional information on the toxicokine tics and toxicodynamics in humans or an effective PBPK model, wh ich is expected by 2011. The registrant presented information in the public commen t session that humans primarily form the DEA metabolites, while rats primarily form the DIA metabolites. Similar formation of the DACT metabolites is expected. This inform ation is helpful for dose reconstruction in humans based on biological monitoring studi es. In addition, a thorough review of the human exposure data (includi ng biological monitoring studie s) 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.

34The EPA conclusions in Sec tion 4 of the Issue Paper in dicate that “the non-cancer epidemiology database provides important info rmation 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 epid emiology database, studies in which the potential for other non-causal explanations is comparativel y 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 studi es as discussed in th e Panel’s responses to Questions 1.1 and 1.2a, the potential for other no n-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 id entification and characterization. Contrary to the opinion stated in the Issue Paper, the Panel believed that the epidemiologic findings could be used for endpoi nt selection if the studies ar e well-designed (even if not achieving unattainable perfection) and e xperimental data are not available.

Thus, based on an understanding of the mode of action in animals anticipated to pertain to humans, additional research s hould 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 st atistical power to dete ct meaningful effect

sizes in the outcomes of intere st will be a major considerat ion in low-dose research, as will adjustment for appropriate known confoun ding factors for some outcomes, such as maternal age and smoking for low birth weight.

The concerns about effects not yet iden tified in humans, but seen in laboratory findings are recognized but further effort s in epidemiology will produce additional equivocal findings unless st udies are well-designed (i.e., have good exposure and

outcome measurement in individual men a nd women with adequate sample sizes, adjustment for confounding factors, and minima l participation and se lection biases) and evaluate health effects of doses that are relevant to real-life human exposures either in the occupational setting (e.g., farm workers) a nd real-life drinking water exposures (e.g., studying populations likely or known to cons ume atrazine-contaminat ed 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 Februa ry 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 exposure s. The proposed framework does not obligate the generation of new data but has the virtue of bringing out assumptions and judgments

35that might otherwise be harder to identif y. 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 At razine Ecologic and Retrospective Studies" by Breckenridge, Pastoor and Scialli (2010). While the Panel disagreed with a number of the critical judgments pr esented 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 la rgely 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 fo r 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 immunotoxic ity effects. At that time, the Agency deferred review of experim ental toxicology studies on mammary gland development to the September SAP meeting. Development of mammary tissue occurs in defined stages linked to s exual maturation and reproduction including embryonic, preand peripubertal periods, as well as pregnancy a nd lactation. Given t hat atrazine affects the hormonal environment and reproductive syst em, it is not unreasonable to hypothesize that it may also affect mammary gland d evelopment. The database of mammary gland development studies includes four studies---thr ee 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 t hat the key events leading to reported delays in mammary gland development in the young follow ing gestational exposure to atrazine are not known and none of these studies propos e a mechanism/mode of action.

Question 2.1

The studies from the two different re search 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 scor ing 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 prolifer ation in the mammary

tissue.

36Please comment on the quality, strengt hs, 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, mammar y gland development and age of vaginal opening of their female offspring. All three studies used the Long Ev ans strain of rats and oral gavage as the route of administra tion. Each study appeared to be well-executed. However, the studies differed in several as pects 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 fi ndings 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 da m and offspring body weights as well as mammary gland development in the offspring, the Rayner stud ies 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 na ture of the effects that atrazine produced on postnatal mammary gland development.

The Panel concluded that numerous si gnificant methodological and procedural differences in the Rayner and Coder studi es have confounded the understanding of the toxicological significance of atrazine exposure on certain as pects of female reproductive system development. It was the Panel’s opinion that unless/until these differences are resolved, understanding of the effects of at razine 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 Code r studies. They are analyzed in detail

below:

(1) Diet . The compositional differences in th e 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 us ed in previous studies conducted with atrazine (Rayner, 2004; Rayner, 2005) wa s 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 pres ented 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), th eir total protein, fa t, and carbohydrate contents, and in some vitamins. There is substantial literature on the effects of soy and phytoestrogens on the mammary gl and, as well as on puberty and estrous cycles. While the Organization for Economic Co-operation and Development (OECD) conducted a study of diets of va rying phytoestrogen content used in the uterotrophic assay as an e ndocrine 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 leve ls 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 bioassa y). There is some disagreement in the literature as to the ability of these diet s to cause effects on the timing of puberty, but it may depend on the strain of rodent us ed. There are sporadic reports in the literature where diet changes have been im plicated in altering effects of treatment with endocrine active compounds, and in so me cases the diet effect has been attributed to the phytoestr ogen 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 whet her the dietary content of soy and/or its component phytoestrogens can shif t the dose responses for atrazine. Nonetheless, when looking at the subtle effects on mammary gland development and onset of puberty, these could be important factor s 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 re productive system and mammary gland development. Development of reproductive function is known to be influenced by pineal gland activity and there is evidence in the lite rature that altering light/dark cycles influences the timing of vaginal opening in rats (Lehrer, 1986). There is also literature linking altered photoperiod to el evated 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 publishe d information about whether photoperiod

alterations affect atrazine toxicity in mammals. Neve rtheless, when considering

subtle differences in the effects of atrazine on puberty and mammary gland development in rats, photoperiod differe nces may be important factors to

consider.

(3) Pair-feeding . All of the Rayner and the Code r studies found significant effects of atrazine on dam and pup weights. The Coder study found that atrazine reducedfood 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 e ffects on mammary gland development and

38breast cancer risk of offspring. Given that differences in the protein/fat content and composition of maternal diets have b een 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 cont rols is necessary to accurately interpret potential toxic effects of atrazi ne. Two problems exist in this regard when interpreting the results of the Ra yner 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 poten tial 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 (C oder (2010), Figure 2), which have the potential of independently altering feta l nutrition and subsequent mammary gland development. Moreover, it is likely the pair-fed controls had major shifts in endocrine and metabolic rhythms in respons e 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, duc tal branching pattern and complexity,

and end-bud number to assess the effects of atrazine. The us e of this scoring system has a number of strengths that include: (a) reproducibil ity, as evidenced by multiple corroborating studies over severa l 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 integrativ e, measure to evaluate mammary gland development. While useful as an appro ach in a single laboratory with constancy in practice and training, this scoring system lacks the generally accepted standards by which to judge mammary gland developm ent, such as those routinely used by human and animal pathologists. Conseque ntly, inter-laborato ry comparisons are difficult and data interpretation is problematic.

The Coder study tried to avoid these pr oblems through the use of quantitative image analysis approaches to evaluate the same development parameters analyzed in the Rayner studies. Although much wa s made of the differences in these approaches by the Coder study authors, it is surprising that they did not employ both qualitative and quan titative scoring measur es 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 st udy sponsor have the slides generated in

the Coder et al . study scored using the Rayner sc oring 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 bi ological 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 (Harve y and Chevins,1987; Vom Saal et al ., 1991; Zehr et al

., 2005). Although maternal stre ss during gestation has not been linked directly to alterations in mammary gland developm ent 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 pr esented 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 cont rol 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 Ra yner 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 diffe rences in maternal stress are important when attempting to reconcile the inter-l aboratory differences in the biological

affects of atrazine.

Question 2.2

In the three studies considered in Qu estion 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, hydr oxy-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 whic h 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 mi xtures 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 st udy goal was to determin e if a mixture of atrazine parent, its chlorina ted metabolites and hydroxyatrazi ne, in proportions found in the aquatic environment, when administered orally, produces developmental effects in Long Evans rats. Mammary gland development, as assessed by a vi sual 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 dos es twice daily on GD 15-19. This time period was identified as the critical window in ge station for atrazine’s effect on delaying mammary gland development (Rayner et al ., 2005). The conclusion of the Enoch study

40was that acute exposure to mixtures of as low as 0.09 mg/kg causes persistent alterations in mammary gland development of female offs pring, and that these effects do not appear to be related to body weight or timing of onset of puberty.

Findings included increased GD20 feta l and PND4 and PND 60 offspring body weights; these may be an example of pr enatal metabolic programming (Plagemann, 2005). These observations could be consis tent with the Rayner and Stanko (2010) studies at the higher doses if the atrazine parent/metabolites are disturbing a developing brain neurocircuitry. Duri ng the public comment period at the meeting Syngenta presented data in adult rats showing that atrazine decrease d activation of GnRH neurons in the hypothalamus without affecting paraventricular nucleus (PVN) c-Fos immunoreactivity. Whereas the (presumptivel y, 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 pre optic area (mPOA) with synaptic connections

to the GnRH cells which could be the vulnera ble brain site in atra zine-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 fo r individual components could have helped clarify the interpretation of the mixtur e data. The observations were based on administered dose because absorption and bioava ilability 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 c oncentration of chemical(s)/meta bolite(s) was constant as a function of time (see Panel response to question 3.1).

EPA’s concern with the stat istical 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 wa s ameliorated by Dr. Fenton’s detailed explanation of the methodology, noting the l ong 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 impor tance 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 observat ions of this study have not been corroborated using other mixtures of parent atrazine/m etabolites reflective of temporal and spatial occurrence or for atra zine parent at equiva lent doses. Atrazine itself will, once absorbed, yield a mixture of free and conjugated metabolites (but, little or no hydroxyatrazine) and the propor tion of metabolites would likely change temporally. Any replication would need to evaluate th e same endpoints measured in this study and, perhaps, other physiological endpoints

41Question 2.3

As described in the Introduction of the dr aft 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 toxicol ogy 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 findi ngs 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 co nduct 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, a nd 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 a ppropriate way of handling these studies currently. The changes in mammary gland deve lopment were observed only at relatively high doses (100 mg/kg/day), doses to which hum ans are unlikely to be exposed. No clear dose-response relationship or mode of acti on has been defined for these mammary gland effects. There are only four pa pers in the current literature , one of which has significant limitations which make it unsuitable for quan titative 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 vagi nal 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 in to 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 legitima te 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 pre liminary studies clearly raise the possibility that there may be deleterious effects of atrazine on mammary gland development. Therefore, further study is needed at additi onal (i.e., lower) doses, with added emphasis

42on possible cross-generational effects of parent atrazine and its metabolites, including the hydroxy-metabolites. The mixture study (Enoch et al ., 2007) is potentiall y important, but replication of this study and investigation of the individua l 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 re sponsible for the observed effects. 3) Multiple very active metabolites may act in a dose-additive fashion. 4) There is significant interaction am ong the metabolites (i.e., synergy).

The likelihood of signifi cant interaction among these compounds would be slim, but interactions resulting in an order-of-magn itude 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 fo llow-up studies. If the effects are found not to be artifactual,

then additional future studies should address th e possibility that one or more very active metabolites exist. Hydroxyatrazine(s) were discussed as candidate active metabolites although they are not significantly produ ced by mammals and would, therefore, contribute little to the toxicity profile when atrazine is ad ministered in animal models. Overall, these effects are particularly im portant 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 hypothala mus or other brain sites.

It remains true that particular featur es of mammary gland development suggest that it is a valid, and potentially sensitive, target for environm ental agents, including potentially toxic compounds such as atrazi ne. These features in clude the fact that mammary gland development occurs over se veral distinct phase s 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 betw een 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 developi ng accepted methodologies and Standard Operating Procedures (SOPs) that would in form useful endpoints related to mammary gland development and physiology will be impo rtant. These methods and procedures

should address actual lactation endpoints, both qualitatively and quantitatively. This task presents an obvious set of challenges. Ho wever, very precise and comprehensive lactation endpoints are routinely measured and used for specific goals in the dairy sciences and industry, where very small diffe rences in lactation outcomes have major

43economic consequences. It should be possi ble to use this ri ch literature and methodological resource to esta blish approaches to characte rize 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 seve ral complementary measures of effect, particularly where some are subjective, as a basis for the measurement of internal consistency of the observations. Similarly, c onsistency of results across studies in the database is an important criterion in asse ssing weight of evidence for dose-response for critical effects. Effects observed by one rese arch 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 depart ure. This does not imply that the results should not be investigated furt her, particularly where effect s are found at very low doses and where mixtures of compounds that may be synergistic are involve d. It is advisable that generic criteria for internal consistency and those relevant to weight of evidence for dose-response for critical effects (consistenc y, specificity and biologi cal 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, how ever, argue that developing useful and standardized methods for assessing mamma ry 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 pursu ing 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 dra ft 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 associat ed with a number of endocrinopathies

44Question 3.1

Given that there is no robust pharmaco kinetic model yet available, a noncompartmental analysis of the existin g 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 wi thout 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 reflecti ve of atrazine and its metabolites in rat plasma.

b. Based on Figure 5.5 in the draft Issue P aper, the Agency has pr eliminarily concluded that plasma levels of at razine and its metabolites reach (or nearly reach) pharmacokinetic pseudo-steady state in the ra t 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 comp lex, for which there are adequate data to identify the relevant dose metric associated with the critical eff ect). PBPK models are clearly not the quickest or eas iest tool to apply. While th e 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 PB PK model refinement for atrazine has not been undertaken since 2007 when the McMullin model was publ ished in the peer-reviewed literature. PBPK models have been or are being deve loped for other pesticides (e.g., synthetic pyrethroids, chlorpyrifos), and for atrazine by the registrant. It w ould be prudent for the Agency to work with the current model desc ribed 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 uncerta inties of extrapolation from rodent studies to low dose regimens in humans.

In the absence of a functional PBPK m odel 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 (int er-strain) extrapolat ions. In the noncompartmental analysis, the Agency has atte mpted to make maximal use of the limited

45data that are available. EPA has made main ly conservative choices in the absence of empirical information and verified estimat es 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 biologi cal 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 a pproaching the problem of relating the exposure of rats to atrazine to the magnitude of resulting biological responses, and extrapolating to the predic tion of human exposure levels. This is particularly useful in the absence of know ledge of the exact s ite of action since it provides a measure of the opportunity for di stribution to all tissues (target and nontarget). This conclusion is supported by th e work of Stoker and Cooper (2007). The studies of Dooley and co-workers demons trating the covalent binding of DACT to proteins with available cystei ne 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 ac tivity 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 14 C-compounds is a reasonable first st ep (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, w ill 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 metabolite s (DACT, DEA, and DIA) are all equally active toxicologically. Considering that atrazi ne is short-lived and that the glutathione conjugates are likely to be inactive, severa l possible dose metrics are available. These

include sum of total metabolism of atr azine, total metabolism minus glutathione conjugates, total of DIA, DEA and DACT, total of DIA and DEA.

Atrazine and its two mono-dealkylated an alogues are rapidly depleted by primary detoxication; the major end product (DACT) do minates 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 th e 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 th is context, it is recommended that the

46differences in molecular weight between atrazine and its metabolites (215.7, 145.5, 173.7, and 187.7 for atrazine, DACT, DIA, and DE A, 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 constant s 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. Indee d, if more frequentlycollected samples were available, the concentrat ion would have been seen to rise after the last dose before falling from the peak le vel. 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 poi nts available over the washout period, do not indicate double expon ential elimination. It woul d be worthwhile to look closely at the washout data, si nce the estimates of the elimin ation rate constants will be important in any predictions or extra polations to other dosing regimens.

If AUC of plasma concentration is to be used as the measure of internal dose of triazine equivalents, then cons ideration should be given to th e interspecies differences in bioavailability, rate of glut athione conjugation, role of in testinal metabolism, and red blood cell/plasma partition coefficient. Furthe rmore, 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 extrapolati ng 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 activi ties, which may lead to slower clearance (Jori et al ., 1971). The Panel recommended that st udies to provide parameterization for a Long Evans PBPK be initiated as soon as possible. This parameterization should include an intestinal biotransformation metric (e ither microsomal or Caco-2 cells). The hepatocyte data provided by the registrant we re 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 conducte d with mixtures of the metabolites in a range of concentrations that br acket those in the environment. Such data

could be used in the next risk assessment.

It was the opinion of the Panel that th e 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 registra nt’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 dete ction 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

47of the different compounds a nd biological responses. This information combined with good estimates of plasma partition coefficien ts should allow the de velopment of PBPK models that can be used to extrapolate from rodent to other speci es, including humans. This would provide a sound basis for the setting of benchmark doses, and PoDs, providing that it is develope d for the rat strain in which the PoD or dose-response is

characterized. While it is clearl y preferable to have a valida ted PBPK model, within the time constraints for regulatory programs, it ma y be necessary, at a minimum, to prioritize

collection of data that would reduce some of the uncertainty associated with the current Agency analyses. Certainly, th e outstanding issue is how to deal with the uncertainties

associated with the constraints of the existi ng data. Despite the limitations of the existing data, the exercise has been extremely valuable in informing critical as pects relevant to the risk assessment and monitoring programs.

Pseudo steady state implies that the ab sorption 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 si nce 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 th e 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 fl uctuations 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 appr oach 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 describe d 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 ar e large fluctuations in serum

concentrations of atrazine and its metabolites with time after oral gavage dosing, and that a smoother profile is achieved with dietar y administration. Despite the difficulties in estimating dose consumed in the diet compared with administration by oral gavage, it is worthwhile. Dietary administration provide s a smoother plateau, is less stressful and disruptive than gavage, and is more realisti c representation of hu man exposure through drinking water, depending on the presen ce 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 figur e 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 studi es, strains, laboratories, and most notably duration of

48exposure. The Agency has preliminarily concluded that the findings on Figure 5.8 strongly support the hypothesis that pseudo-st eady 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 wi th the attenuation of the LH surge following atrazine exposure in rats. Please commen t 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 collec tion from four laboratories. The figure serves as a visual meta-analysis and assists in the understa nding 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 in tegration and has gr eat utility for expl oring 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 thes e 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 cont rol against the linear dose (mg kg -1 d -1 ) of atrazine. A clearer and more sta ndard 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 concentr ations 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 singl e 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 vari ance (and greater uncertainty) than points

near the median. It would be useful to incor porate the higher doses to see where they fall. This should not be precluded on pharmacoki netic 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 pharm acokinetic 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 increasin g 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 tw o 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.

49The 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-m onth 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 re peated sampling study. In the Morseth 6 month study (1996b), LH appears to peak at 200 0 h in controls and at 1800 h in the 25 ppm group in repeatedly sampled rats, wher eas 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 vari ability 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 sacr ificed at various times in proestrus.

Verification that the rats we re 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. Add itional data for dose levels between 1.5 and 5.0 mg/kg/day were obtained from the Morseth (1 996) and Minnema (2001) studies that used Sprague-Dawley rats. These data are much mo re variable than the Cooper data (see below); therefore, there ar e 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 su rge (elevation of LH above the 95% confidence limits of baseline for at least 2 consecutive time point s) was not exacted. Not all animals had an LH surge (including controls); therefore, th ere is a significant underestimation of LH values, means and overestimation of standa rd deviations. It was not possible to determine whether LH surges were defined in the McMullin report.

Correction of the Minnema and Morseth da ta sets such that only those animals that exhibited an LH surge are included in th e analysis of LH surge peaks, will increase the mean LH levels, decrease the variati on 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 propor tion of animals with delayed/ lengthened cycles was overlooked. The delayed cycles are probably th e result of insufficient or absent LH surges. For example, the percentage of atr azine-treated animals that did not meet the above criteria for an LH surge for each dos e in the Minnema paper were: 0 mg, 34.2%;

502.5 mg, 30%; 5 mg, 50%; 40 mg, 67%; 200 mg, 58%. These proporti ons 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 wa s a trend towards increasing proportions of animals that did not surge with increasing dos es of atrazine. Sim ilar results were not reported by Morseth, but the data are availabl e. 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 pr oestrous 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 surg e has no adverse effect on reproductive function and does not prevent ovu lation 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 consti tute 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 ch anges (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 pa ttern 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 strong ly associated with the attenuation of the LH surge following atrazine exposure in rats

. Although the PK data indicate that a pseudo-steady state has b een achieved over the four days of dosing in the Thede experiments, it is important th at the idea of a pseu do-steady state being necessary for attenuation of the LH surge is not used in interpreting these data. The data do not support this interpretati on 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 dosi ng regimens (for instance a high dose on day 1, a low dose of day two, no dose on day 3 a nd 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 re sponse corresponding to the different patterns of dosing was measured would provide evid ence for or against the assumption that pseudo steady state was necessary to produce at tenuation 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 con centrations fluctuating from day to day. There may be a critical windo w of exposure that is less than the four days used in the reported work, and pr oviding that a minimum exposure (AUC) is maintained over this period, a re duction in the LH surge will result. It has been assumed

51that 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 wh ich no increase in suppression of the LH

surge occurs.

Question 3.2

In Section 5.3, the Agency describes a be nchmark (BMD) analysis conducted for LH

attenuation studies. As described in the draft Issue Paper, the Agency initially considered a wide range of toxicological endpo ints for inclusion in the BMD analysis. In considering all of the data, th is analysis focused on studi es measuring LH attenuation where female rats were exposed orally for f our 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 fr om atrazine’s effect on the hypothalamic control of pituitary function through its interf erence with GnRH neurotransmitters. Thus, protection from LH attenuation as a precurso r event to several functional impairments would be protective of atrazi ne’s neuroendocrine effects.

a. Please comment on the BMD analysis su mmarized 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 c onsidering 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 av ailability 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 functi on is presumed to include suppression of GnRH pulses, consistent with an immediate downstream effect on the LH

surge.

Based on the available data, a benchmar k 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 e ndpoint is attenuation of the LH surge.

It must be noted, though, that while atte nuation of the LH su rge over one estrous cycle represents a sensitive biological response, a causal association between the observed modest, yet statistica lly significant, changes in LH surge peak and adverse fertility measures has not been made. The relatively small attenuation of the LH surge

52may well be a harbinger of a possible future a dverse 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 at tenuation of the LH surge to about 40% of control levels, obser ved 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 atrazi ne-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 repr oductive effects, such as

anovulation or decreased fertilit y. There are also some data seemingly inconsistent with the hypothesized mode of action. For exampl e, 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 appear s to have been assumed that the effects of chronic exposure to doses less than those th at 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 a nd responses other than LH surge attenuation remain to be identified, perhaps using mol ecular 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 releva nce to humans, changes in basal LH release, which also are directly regulated by GnRH secreti on, 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 robus tly and needs to be considered carefully in relation to other endpoints on continued exposure, based on the totality of data, including that from more tr aditional studies. This woul d 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 in ter- and intra-species extrapolations.

It may be helpful, then, to derive an d array a variety of potential points of departure for relevant endpoints with a view to characterizi ng their degree of protection (in relation to biological signi ficance 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 biol ogical significance of observed effect, but also as a tool to compare and contrast the uncertainties asso ciated with various options. Comparative uncertainty analysis would include consid eration of appropriate interspecies and intraspecies adjustments, in the context of their associated degree of uncertainty.

53Adequacy 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 us e 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 integr ation of data from multiple studies.

The existing data on th e dose response of the at tenuation of the LH surge associated with exposure to atrazine (in rats ) are sufficient to permit robust analysis for the benchmark dose. These include significant da ta in several strain s 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 th e four studies summarized in Figure 5.8 and, appropriately, selected the “new” (unpublis hed) 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 fo r 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 th e 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 gr oups 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 ca lculated using data only from animals whose serum LH levels increased grea ter than 2 standard deviations (SD) above baseline levels for at least two consecutive times, a comm only used definition of an LH surge.

Prior to the meeting, in public comment s submitted by the registrant, certain aspects of the new Cooper et al . (2010) data were subjec ted 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 critic isms. One of the criticisms was that the statistical analysis did not include the c ontrol 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 ag e 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 th ird 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 incorporat ed 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 addre ss 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

54that which considered the data from all three study blocks combined. The Panel

concluded that these new datawere 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 Is sue Paper, data from other studies were presented, but their source is unclear. In Fi gure 5.9, the highest dose is graphed at 75, but the Cooper et al . highest dose was 25. The BMD is a bout 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. Do cumentation for these additi onal 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 cl ear whether the BMDL, which is the 95% lower confidence limit, is, in fact, any differe nt 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 con tinuous endpoints, like LH attenuation, the BMR most often represen ts 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 associ ated 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 differen ces in reproductive cycles/aging between rodents and humans add an additional level of co mplexity 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 sc ientific 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

55directly relates to mode of action, being one of the key precursor events to the ultimate endpoint(s) of toxicity and it exhibits dose resp onse 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 o ccurs. It should be cautioned that, when determining the BMR, inclusion of highly vari able 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 de monstration of a dose re sponse of inhibition of the LH surge to atrazine exposure, seve ral replicates of the dose response curve, ideally in each strain of rat tested, woul d 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 therefor e data from these two strains should not be

combined for the determination of a BM R. 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 inhibiti on of ovulation. Several studies presented in the Issue Paper, using both or al 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 change s 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 th e Issue Paper, but impo rtantly, there are not sufficient data in the broad body of literatu re (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 po ssibility 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 fer tility 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. Th is potential should be addressed experimentally.

Specific data which examine long-term diminution of the LH surge over the reproductive lifespan and resultant effects on fer tility have not been st udied 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 rega rd, the responsiveness of th e 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 pro cess progresses in both rodents and women. In

56this context, changes in the characteristics of the LH surge includi ng decreased amplitude of the surge in both rodents and humans pr ecede cessation of cyclicity. Thus, modest changes in LH surge amplitude elicited by atrazine exposure together with the normal biological changes occurring during the agi ng process may result in overall enhanced sensitivity to the effects of atrazine. T hus, atrazine exposure during the reproductive aging phase of the life cycle may elicit gr eater 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 uncer tainty arises with the significance of the duration of exposure.

Data are clear in identifying that a great er-than-one pulse of exposure to atrazine is necessary for attenuation of the LH surg e. For example, single high doses (over 100 mg/kg) administered on the morning of proest rus did not alter charac teristics 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’ exposur e is sufficient to alter the LH surge. Further complicating the matter, it is not clear if a 4-day exposur e, 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 dur ation of exposure and phase of the cycle will be key in translating rodent data to humans for risk assessment purposes.

It should be understood furt her that the significance of the duration of exposure and phase of the cycle may be difficult to de fine in the rodent sy stem. 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 dur ation of the estrous cycle (4 days) may be too short to define specific cycle-phase related sensitivities.

Thus, based on the Panel's current unders tanding of effects re lated to atrazine exposure together with its level of understand ing 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 am plitude 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 woul d 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 a pproach 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

57uncertainties associated with “how much di minishment” is necessary for the occurrence of an adverse reproductive or developmental effect, calibra tion of this response with “down-stream” apical reproductive/developm ental 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. Th ese 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 cl osely mimic the most relevant pattern of exposure in humans.

At the present time, use of data on at tenuation of the LH surge is a suitable benchmark and directly relates to the presen t 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 integr ation of both cancer and non-cancer health

effects of atrazine.

Question 4.0: Approaches To Evaluating Wa ter 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 variabili ty of pesticide concentrations in drinking water, with an emphasis on the upper end of the exposure dist ribution. In addition, the method used to estimate concentrations from monitoring data depends on th e duration of concern and

how critical it is to estimat e 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 intensi ve sampling, basing samp ling frequency on the toxicological exposure duration of con cern, 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 purpos e of a monitoring program is to generate valid “exposure estimates,” then sampling th e 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 wa ter 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 at razine in a water body changes significantly depending upon from wh ere 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 appropria te 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 curr ent method that uses an exceedance of 1.6

μ g/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 atra zine value from the SDWA sampling is an average of four quarterly values and it ha s 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 at razine exceedances is an excellent second screen/trigger for inclusion in the monitoring program.

The Panel noted that agriculture is no t a static enterprise and any monitoring program needs to have the flexibility to accomm odate the large year to year variability in agricultural practices that re sults from market forces, re gulation, 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 occu rrence of atrazine in surface waters over time, some Panel members suggested use of the following function:

Atrazine_Occurrence surface water (t) = Source_Strength ATRAZINE (t) + Hydrology(t)

The two components are Source_Stre ngth 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 equa tion, it is clear that intensive sampling of a runoff event before at razine is applied in a basin is of limited

59usefulness in describing Atrazine_Occurren ce as would be inte nsive 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 reten tion time of days, a large reser voir with a retention time of months to years, or an off-stream storag e 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 toxicologi cal 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 surf ace waters; because of this, these Panel members believed it was unf air to ask the registrant to increase their sampling efforts. On the other hand, the fact th at there is so much atrazine used in the United States, and that it is found in ev ery environmental compartment (i.e., ground waters, surface waters, rainfall, lakes, reservoi rs 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 importa nt 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. Autosa mplers currently are used successfully by many researchers who are studying the fate and tr ansport of pesticides including atrazine. Autosamplers can better capture transient events that happen during periods when personnel are not available to do the samp ling. 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 pr ocedures for collecting water samples. For instance, water treatment plant operators sh ould 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 simulati ons of candidate monitoring strategies and evaluations of the utility of different exposure estimation methods be benchmarked against intensive empirical data t hat cover a representative range of sites. In response, the USEPA proposes using more intensivel y sampled ambient water monitoring for

60flowing waters and PRZM/EXAMS modeling for st atic water bodies, matching them to

the CWS based on similar chem ograph shapes (see Section 7.3).

a. Please comment on the Agency’s pr oposal to use chemograph shapes (number, duration, and magnitude of peaks) to match CWS with more intensively monitored datasets. In particular, the Agency is inte rested in recommendations on approaches for matching chemograph shapes given the loss of s hort-duration peaks that occurs with less frequent sampling.

Panel Response

As noted in the question itself, the ch emographs from the less frequently sampled sites may have short-duration concentration peaks that were not sampled. Matching a chemograph that is missing short-duration con centration 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 concen tration peaks are not missing in the less frequently sampled data. In addition, chem ograph shapes are influenced by factors such as pesticide use (amount and geographic distri bution) and precipitati on, as well as static watershed characteristics. A chemograph shap e for a site can vary temporally because pesticide use and precipitation are temporally variable. Gi ven the likelihood of missing short-duration concentration pe aks and the variability of ch emograph 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 ade quacy of sampling frequency for a site.

Functional Data Analysis (Ramsey a nd 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 ch aracteristics. First, sites should be grouped by their source water type, such as stat ic, flowing, or other. Within the source water groups, sites then can be further cl assified or grouped ba sed on their watershed, pesticide-use, and climate (p recipitation) characteristics, as well as their geographic

locations. In terms of static or non-flowi ng waters, hydrologic char acteristics such as

retention time also should be used in this grouping exercise.

b. Please comment on the strengths and weak nesses 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 problem s with trying to draw inference for an exposure when the interval is sampled only with a single point pe r interval. Sometimes one gets lucky with the timing of the sa mple, and sometimes one misses the peak

61completely, thus grossly underestimating exposur e. 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 us ing 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 (s uch as weekly data). There is a substantial literature on the use of comput er models to augment physical data, and when properly calibrated (i.e., adjusted to match the availabl e 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, a llowing for the understanding of some of the relationships with external driving conditions such as ra infall. 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, pest icide usage patterns, c limate) also are not necessarily representative of the broader rang e of flowing water sour ces in other parts of the country. It does not e xhibit the scale effects that many water sources do.

Generalization to other waters heds would need to be done with care to avoid incorrect predictions which could lead to a false sense of secu rity 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 ta ilored to each individual watershed. SWAT is another model with similarities in design intent and need for specifici ty of application.

Each reservoir may be different, dependi ng on drainage basin characteristics (e.g., size), capacity, turnover time, and other loca tion-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 sm oothed (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 appr oach. Predicting peaks in reservoirs may be easier th an for flowing water, since th e 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 collecti on of more varied, intensely-sa mpled datasets (particularly for reservoirs), rather than attempting to fit complex, labor-intensive, site-specific models to less-frequently sampled datasets.

62Question 4.3

Once the magnitude and duration of exposure of toxicological concern are identified, the USEPA will determine which method(s) to us e to analyze the existing atrazine CWS monitoring data for use in drinking water ex posure estimates. Based on the April 2010 SAP recommendations, the Agency is focusing on regression-based modeling combined with random function modeling (for exampl e, a revised WARP model coupled with kriging or the USGS SEAWAVE-Q model).

Please comment on the strengths and we aknesses of these proposed approaches and provide any recommendations for im proving the model applications.

Panel Response

The easiest approach would be to ha ve sampling twice during each period of interest. The current sampling scheme would be appropriate fo r a period of interest of 14 days or longer; otherwise, more frequent sampling would be recommended. Sampling could be manual or via autosamplers. This a pproach 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 scenar io for which these models were designed). This approach makes use of information abou t the pathways and aggregate data that are not incorporated by purely sta tistical methods such as krig ing or neural networks. However, the April 2010 SAP noted that, wh ile exposure estimate s generated through inference from other sites (suc h 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 eval uating atrazine levels in or der to implement regulatory actions. The purpose of models such as WARP is to provide concentration estimates and probabilities in order to guide more intens ive monitoring and assessment. Similarly, WARPsim (Vecchia and Crawford, 2006) was de veloped to simulate daily pesticide concentrations based on WARP coupled w ith time-series data on precipitation and temperature, and includes a seasonal co mponent 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 sh ape 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 eas ier 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 c onsideration might have ramifications in the

field.

63Kriging 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). Slid e 94 of the Agency’s presentation clearly shows the need for joint estima tion 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 Gaussi an may fit well overall, mainly because of the many small values. Around the peak(s), th e structure is changing much more rapidly

and does not look like a Gaussian correlation stru cture. Using a Gaussian correlation will seriously underestimate the peaks. On e possibility to avoid fitting complex nonstationary models would be to use only th e 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 nationwid e 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 collect ed, one doesn’t have a good id ea about sampling variability or measurement error. In the presence of both data and model uncertainty, one should be wary of overestimating the confiden ce level of any model predictions.

The Panel also discussed how this exercise will inform broader monitoring efforts

around atrazine in the environment. At th e 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-poi nts of interest can be es timated and proposed sampling procedures evaluated for their statistical prop erties. It is clear from the presentations made to this Panel that both EPA and the regi strant continue to pursu e this approach. It should be noted that it is po ssible to generate simpler empirical models of more complex simulation model output. These simpler mode ls 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 ar e derived from the levels of atrazine in CWS output (drinking) waters, it is comforting to see that bo th EPA and the registrant are making advances in characteri zation 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 actua lly using the simulation models and CWS

64characterizations 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 sa mpling to periods of time most likely to experience an exceedance.

Description of a possible sampling pl an follows. Prediction of expected concentrations for raw and drinking water fo r a CWS would be run at the beginning of the crop year using available long-term climat e predictions for the catchment area of the CWS, the expected cropping prac tices 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. Fo r 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 interventi ons under specified conditions to mitigate expected exceedances. The CWS would be required to collect water samples on a periodic basis, for instance, da ily, 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 ch emical 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 anal yzed 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 atr azine concentrations in the future are

below maximum allowable concentrations.

The described plan closely follows sampli ng 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 k now 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 collec ting and storing water samples for the season. During this meeti ng’s public comment period, the Panel heard that the economic impact of the sampling fr equencies required is of high concern for CWS managers. This plan would control the sa mpling effort and, at the same time, focus sampling on periods with the highest like lihood of actually detecting measurable concentrations. It acknowledge s the fact that for most CWSs and, for most times, there is

little to no detectable atrazine concentration in output wate rs. It also allows for random selection and analysis of stor ed 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 effo rt deviates from the traditional approach which specifies that th e sampling effort be distributed uniformly over the time period of interest. However, it ha s been made clear in the presentations to

65the Panel that traditional thi nking will lead to intensive a nd expensive sampling at high frequency with the expectation that the vast majority of analyses will yield “no detect” concentrations. While this may provide conf idence 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 Is sue Paper, the FFDCA, as amended by the FQPA (1996), requires the Agency to give spec ial attention to infants and children by placing emphasis on the availa bility of toxicology and expos ure information to estimate the potential risk to these age groups. Th e 2003 Registration Eligib ility 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 uncer tainty 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 appropr iate duration of concern. Based on the
newest studies, our unde rstanding of atrazine’s te mporal pharmacokinetic and pharmacodynamic characteristics has changed and it appears that s horter durations may be appropriate. In light of the potential need to shorten the critic al duration of exposure and as discussed by the Panel in Questions 4.1-4.3, the Agency is evaluating appropriate methods for assessing the drin king water monitoring data.

Previously, the Agency noted data gaps relate d 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 disr uption on specific early life exposure periods (e.g., peripubertal). Since th at time new studies have become available (Section 6.0) which add to the existing dat abase and support the findings of the older studies. In addition, new tissue dosimetry stud ies provide informati on on the transfer of
atrazine and its chlorinated metabolites to th e fetus and to the lacta ting 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 th e potential for diff erential life stage sensitivity.

In the coming months, as the drinking wa ter exposure analysis develops further and the on-going toxicology studies beco me available, the Agency will work towards completing the scientific analysis that w ill inform whether or not a ne w FQPA Safety Factor should be applied in the atrazine risk assessment.

The Agency requests the Panel to comment on im portant scientific factors for the Agency to consider in its analysis. Please include in your comments specific consideration of

66uncertainties in estimating drinking wate r exposures and remain ing 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 considerati on of the most import ant qualitative and quantitative uncertainties associated with both exposure and effect, relevant to susceptible life stages, in a context consistent with that for other pest icides. In view of the

fact that the database for atrazi ne 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 anal ysis, including the following:

1. For exposure, this could rela te to the likelihood of capturi ng the relevant period(s) of susceptibility or over- or underestimating expo sure for all life stages, with the proposed monitoring strategy (including, for example, consideration of determination of total chlorinated triazines (TCT) ra ther than atrazine alone).

2. For effect, some critical questions and/or aspects to be addre ssed in this context include the following:

a. To what extent does the database on h azard and kinetics inform us about the potential for increased susceptibi lity of infants and children?

b. Is the early key event or late adverse e ffect 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 prot ective for later adverse effects)?

d. What is the impact of the potential reliance on a benchmark dose (versus an empirically-observed effect/no effect leve l) 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 c onsidered 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 us ed to provide some idea of relative sensitivity of various age groups of the human population?

Even though considerable information rela ted to water monitoring for atrazine is available, the overall potentia l for dietary intake of at razine in young children or

adolescents remains somewhat uncertain. It is recognized that information on atrazine and its degradates in source water is valuab le. Certain water treatment technologies can reduce or eliminate atrazine; thus, finished drinking water data should be used in the determination of exposure via th is source. The existi ng risk assessment fo r atrazine uses a 90 day rolling average to estimate exposures. The Agency suggested that better

67estimation of the duration of dosing most re levant for inducing developmental toxicity would be useful, but it remains unclear at th e present time what the relevant duration of dosing for either adults or developing organisms may be.

There is a general consensus from the resu lts of the animal studies that atrazine can influence the GnRH-mediated LH surg e 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 e xposures to atrazine do not influence the LH surge. Such qualitative differences in response in studies using relatively similar dosing strategies is difficult to reconcile, a lthough some factors (e.g., different strains of rats used) may contribute to the conflicting findings.

Toxicokinetic studies using radiolabel indi cate 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 th ese 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 br ain, such that the c oncentrations in the pup plasma and brain are approximately 10-f old (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 st udies 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 wa ter exposure. The indication from the data presented that a pseudo-steady state is occu rring with repeated exposures is useful

information for internal dose estimation a nd 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 conc entrations 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, th at there may not be selectively higher exposures to the developing or ganism during the prenatal or lactational period. A number of experimental studies ha ve evaluated toxicity following prenatal, postnatal or peripubertal exposures to atrazine. With prenat al exposures (Rayner et al ., 2005; Rosenberg et al ., 2008), effects on endpoints incl uding preputial separation and mammary gland development were noted with repeated exposures of 50-100 mg/kg/day. With combined prenatal/lact ational 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

68et 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 pr enatally, 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 atr azine 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 postnata l toxicity and/or exposure durin g these life stages, such that the Safety Factor could be reduced or remove d in all settings. A reduction of the Safety Factor was deemed appropriate for assessm ents 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 th ere are any life stagerelated differences in sensitivity.

One of the biggest uncertain ties at the present time is the validity of the very low dose effects observed on mammary gl and development in the Enoch et al . (2007) paper. The data suggest that alterations in devel opment occur following repeated exposures to a mixture of atrazine and metabolites/degradat es of atrazine (0.09-8.7 mg/kg/day from GD15-19; mixture of hydroxyatrazi ne (ring hydroxylation), deet hyl atrazine, deisopropyl

atrazine and diaminochloroatrazi ne). Certainly if these ve ry low dose effects are real, they will need to be considered as repr esenting 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 endpoi nts, these data need to be replicated. These data arose from exposure to a mixtur e 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 in appropriately evaluated using parametric analyses, although averaging values for pups w ithin 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, al though with later time-points (PND40 and 60) the general tendency was mo re extensive reduction in mammary gland development scores with higher dosing. It is r easonable, however, to a ssume that, even if

the results do not show clear dose-related re sponses, a developmenta l alteration could be an all or none response across exposure leve ls, once a minimal threshold exposure was passed.

69One interesting piece of informati on that did not come out in the EPA presentations during the meeting was that the mammary gland accumulates considerable radioactivity following oral ad ministration 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 th ree hours following oral administration of radiolabeled atrazine. Note the high con centration in the dam’s mammary glands compared with that in a variety of other tissues.

Tissue radioactivity following 14C atrazine (Stoker and Cooper, 2007)

A

nt

pituit

o v ary uteru s

ad

ren

als ant. hypo

po

st. hypo caud a te spleen hear t lung

kidn ey

liver

m

ammar y 0.0 0.5 1.0 1.5 2.0 2.5 -

TissuePercent of radioactivity

It should be stressed that earlier studies also reporte d 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 pr oducts. Thus, while no other studies have used a mixture of atrazine and degradation produc ts to study developmen tal effects and those

results need to be confirmed in further studie s, there is some eviden ce to suggest that the mammary gland may be both highly exposed and sensitive to disrupti on by early atrazine

exposures.

The inclusion of the environmen tal degradate 4-hydroxyatrazine (ring hydroxylation) in the Enoch et al . (2007) study may indicate that this particular contaminant is worthy of some focused ev aluation. While not a product of mammalian metabolism of atrazine, 4-hydroxyatrazine is a substantial environmental contaminant resulting from atrazine degrad ation (approximately 20% of the sample used in the AMM) and it may warrant further study. There is a su bstantial number of toxicity studies on 4- hydroxyatrazine that were not in cluded 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 potent ial for genotoxicity/mutagenicity, acute toxicity, short-term, subchroni c and chronic repeated oral dosing toxicity, developmental toxicity and carcinogenicity. EPA has esta blished an RfD of 0.01 mg/kg/day for 4- hydroxyatrazine, based upon the observation of hist opathological 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)

70of 0.04 mg/kg/day for this degradate, also based upon the renal toxicity observed in the same study (FAO/WHO JMPR, 2008). Furthermor e, preliminary data suggest that 4- hydroxyatrazine does not possess the neuroendocri ne properties obser ved with atrazine and its chlorinated metabolites, i.e., up to a 400 ppm diet ary exposure to 4- hydroxyatrazine did not influen ce 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 endpoi nts following gestational exposures to atrazine along with degr adation products (Enoch et al ., 2007). While th e mechanism for the non-cancer mammary gland effects follo wing 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 a nd extension (e.g., dosimetry, 4-hydroxyatrazine and other individual degradati on 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 repl icated, subsequent studies should specifically evaluate the role 4-hydroxyatraz ine 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 10 X Safety Factor should start with documentation and conclusions regarding the ad equacy 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 & T oxicity Profile on Water Monitoring

As described in the Introduction of the dra ft issue Paper (Section 1), the goals of the
current atrazine re-evaluati on 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 ap propriate, the frequency of drinking water monitoring needed. Two important issues related to achieving these goals are determining the point of departure and ident ifying 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 crit ical duration of concern, the frequency of drinking water monitoring is related to the te mporal 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) re quire a less frequent drinking water sampling design to approximate longer term exposures. However, as the duration of concern shortens, the frequency and timing of sampli ng become more important in determining how well the sample data capture short-dura tion exposures. Observation epidemiology studies raise hypotheses and suggest possi ble links between atrazine exposure and

71reproductive and developmental outcomes, but these epidemiology studies suffer from limitations which prevent firm conclusions. Although certain studi es some provide qualitative support for the human relevance of the endpoints identified through the experimental toxicological database, they prov ide little to no inform ation on the critical duration of exposure. In addition, the MO A and PK database are also lacking in human specific information on the effects of atra zine which could be used to quantitatively extrapolate between species. As such, the in formation available to evaluate the critical duration of exposure lacks quantitative precisi on. Thus, the critical duration of exposure is instead derived by inferring ge neric 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 poten tial durations of human exposure that would correspond to the exposure period of interest in rats li e 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 po tential 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 systemati cally addresses 1) th e use of LH surge suppression in the rat as the BMR, 2) the strength of evidence linking the BMR to a variety of endpoints identifie d 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 a nd humans, including kinetic and dynamic considerations, 4) the potential for water at razine concentrations exceeding a level of concern to be missed by current sampling pro cedures, 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 limita tions in both the data and in our scientific understanding of what the data are telling us.

The Agency has determined that the co llected 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 re quired of the registrant is once a week during the use season and once every two week s during the rest of the year. Several Panel members indicated that they felt th is sampling frequency was adequate. No one raised strong objections or c ould offer a rationale for an alternative sampling frequency

based on the collected information discussed during the SAP meeting.

72A series of assumptions and extrapolati ons contribute to the proposed critical window of human exposure and given the collec tive 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 une quivocally 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 pharmacoki netics and pharmacodynamics, and taking into account the broader database including th at 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 relevan ce of the information from the GnRH antagonist research

since a different mode of action was involve d and no data were provided on the minimum

concentration of atrazine that would be needed over the pe riod 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 cons ideration is whether there is a critical AUC (i.e., exposure of a target site) that lead s to a given level of suppression of the LH surge. This would be tenable if the rat es trous cycle was equally susceptible over all of the four days, but not if tolerance changed ove r 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 da ys, or 4 days? Indeed there could be a critical exposure time combined w ith 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 biologi cal 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 reachin g a pseudo-steady state. If the different patterns of dosing give the same toxicologi cal 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 answ er would still be little more than an educated guess.

Question 6 specifically requests alternativ e 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 hum an evidence is insufficient to establish causality and does not provide sufficient quant itative exposure information to use in a risk assessment. However, what if one assu mes that the reported human health outcomes are, in fact, due to current levels of e xposure to atrazine? Although the water sampling data may not be adequate to assure that atra zine peaks are captured in all water systems, clearly some of the patterns seen are based on rather comprehensive data sets. These patterns of atrazine concentrati ons in water could be used to provide reasonable estimates

of the extent and duration of human consumption of at razine following agricultural

73applications 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 bl ood levels will not be achieved. In this instance, one might consider what would be aver age levels of exposure, relate this to the equivalent internal exposure in the rat, a nd use the promised PBPK model to extrapolate

to humans.

This may represent a reasonable alternativ e 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 cons ervative 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 discusse d in greater detail in the Panel’s responses to Question #4, atra zine concentrations in source water and, subsequently, in the finished water suppl y of a CWS are dependent upon many factors that vary spatially and temporally. As noted, each CWS is un ique in factors that affect the delivery of atrazine such as drainage basin size, charac teristics of the soils, cropping pattern, slope, and whether the CWS source wa ter intake is directly in the stream, a reservoir, or an off stream storage facility. In addition, there are ma ny factors that affect the ability of a water treatment system to rem ove 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 th e source water of each CWS based on the characteristics of that particular water syst em, 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 prim ary source of environmental exposures to atrazine and its metabolites/degradates, ther e would be value in doing a better job of establishing the relationship between the m easured concentrations at the community level, and the resulting absorb ed dose in humans. This question could be first addressed using databases such as NHANES and c ould be supplemented with individual biomonitoring studies to determine the correl ations between water concentrations preand/or post-treatment, reside ntial tap samples and huma n urinary metabolites.

74To conduct well designed epidemiologic st udies in individuals in the future, additional individual-specific measures woul d be necessary. Important measures of water consumption and factor s that modify exposures w ould include the following: measures of water consumption from the hom e 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 im portant 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 incorpor ates 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 Hea lth 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 recomme nded that if the Agency chooses to go forward with an allometric scaling appr oach as a method for estimating a human equivalent dose that it employ the most curr ent 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 atrazi ne in the parental form is not hypothesized to be the principal/sole toxic moiety. Th e adjustment of clear ance based on body surface area adjustment is frequently applied to ensure equal bl ood 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, pa ragraph 2). In other words the use of an allometric dose adjustment f actor to calculate an HED would appear to

contradict the use of AUC fo r triazine-equivalents proposed to be used in the doseresponse analysis (Chapter 3 of the Issue Paper).

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