FIFRA SCIENTIFIC ADVISORY PANEL ( SAP)

OPEN MEETING POTENTIAL DEVELOPMENTAL EFFECTS OF ATRAZINE ON AMPHIBIANS June 17, 2003 [ 8: 30 a. m.]

Crowne Plaza Hotel Washington National Airport 1489 Jefferson Davis Highway Arlington, Virginia 22202 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2 PARTICIPANTS FIFRA SAP Session Chair Stephen M. Roberts, Ph. D.

Designated Federal Official Paul Lewis FIFRA Scientific Advisory Panel Members Steven Herringa, Ph. D.

Gary E. Isom, Ph. D.

Fumio Matsumura, Ph. D.

Mary Anna Thrall, DVM, MS FQPA Science Review Board Members Joel Coats, Ph. D.

Peter Delorme, Ph. D.

Robert J. Denver, Ph. D.

James Gibbs, Ph. D.

Sherril L. Green, DVM, Ph. D.

Darcey B. Kelley, Ph. D.

Werner Kloas, Ph. D.

Gerald A. LeBlanc, Ph. D.

Carl Richards, Ph. D.

David Skelly, Ph. D. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 3 DR. ROBERTS: The topic for this session is going to be the potential developmental effects of atrazine on amphibians. I would like to begin the meeting by introducing our designated federal official, Mr. Paul Lewis, and ask if he's got any announcements for us.

MR. LEWIS: I thank you Dr. Roberts. And I want to first thank Dr. Roberts for serving as our incoming chair for the FIFRA SAP, looking forward to working with him. And also to acknowledge our permanent panel members, Dr. Gary Isom and Dr. Steven Herringa. Dr. Handworker, also another permanent panel member, is unavailable at this meeting today but will be here in July.

I am Paul Lewis and I will be serving as a designated federal official to the FIFRA SAP for this meeting over the next four days. I want to thank both the members of the panel and the ad hoc members for agreeing to serve the next four days for what I think we'll find a very challenging and interesting discussion that we'll be having.

Reviewing the potential developmental effects of atrazine on amphibians, we appreciate the time and the effort of the panel members in reviewing the materials and preparing their remarks,

taking into account their busy schedules.

By way of background, the FIFRA SAP is a federal advisory 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 4 committee that provides independent scientific peer review and advice to the Agency on pesticides and pesticide related issues regarding the impact of proposed regulatory actions on human health and the environment. The FIFRA SAP only provides advice and recommendations to the Agency, that is, making implementation authority remains with the Agency.

FIFRA established what is called a permanent panel which consists of seven members. The expertise of the panel is also augmented through a science review board and science review board members serve as ad hoc temporary members of the SAP providing additional scientific expertise to assist in reviews conducted by the panel.

As the designated federal official for this meeting, I serve as liaison between the panel and the Agency. And I'm also responsible for insuring provisions of the Federal Advisory Committee Act are met.

The Federal Advisory Committee Act establishes a system for governing the creation, operation, termination of executive branch advisory committees. It highlights the consideration of federal advisory committees on the FACA as follows: The committees are chartered. They are governed by uniform procedures. They provide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 5 only advice, are open to public scrutiny.

In addition, FIFRA SAP meetings are also subject to FACA requirements. And these include public meetings, timely public notice of the meetings, and document availability. In that respect, we have documents for this meeting. The background paper, public comments, and the final meeting will be available through the Office of Pesticide Programs docket.

In terms of financial conflicts of interest, as the designated federal official of this meeting, a critical responsibility is to work with appropriate Agency officials to ensure all appropriate ethics regulations are satisfied. In that capacity, panel members are briefed with provisions of the federal conflict of interest laws. Each participant has filed a standard government financial disclosure report.

I along with our deputy ethics officials for the Office of Prevention of Pesticide and Toxic Substances, in a consultation with the office general counsel, have reviewed the report to ensure all ethics requirements are met. And a sample copy of the form is available on our FIFRA SAP web site.

The panel will review challenging science issues over the next several days. We have a full agenda and meeting times are 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 6 approximate. This may not keep to the exact times as noted due to panel discussions and public comments. We strive to ensure adequate time for Agency's presentations, public comments being presented,

and panel deliberations.

For panel members and public commentors, please identify yourselves and speak into the microphones provided since the meeting is being recorded. Copies of all presentation materials, public comments, will be available in the Office of Pesticide Program docket, as I mentioned previously, within the next few days.

For members of the public requesting time to make a public comment, please limit your comments to five minutes unless prior arrangements have been made. For those that have not preregistered,

please notify myself or a member of the FIFRA SAP staff who are sitting just to the right of me here.

As I mentioned previously, there is a public docket for this meeting. All background materials, questions posed to the panel by the Agency, and other documents related to this SAP meeting are available in the docket. Overheads will be available in the next few days.

Finally, background documents are also available on the SAP web site. And the agenda for this meeting lists contact information 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 7 for that type of material.

For members of the press, we have a press contact from our office, Mr. David Deacon, of the Office of Media Relations, is available to answer your questions. Mr. Deacon, please stand. Thank you. Any interested people who have questions from the press, please refer them to Mr. Deacon.

At the conclusion of the meeting, the FIFRA SAP will prepare a report as response to questions posed by the Agency, background materials, presentation, and public comments. The report serves as meeting minutes, and we anticipate the minutes to be available in approximately two to four weeks and will be posted on our web site and in the OPP docket.

Finally, due to unforeseen circumstances, this Friday's meeting will move from this location to the Holiday Inn National Airport,

2650 Jefferson Davis Highway in Arlington, Virginia. The meeting address for this Friday's meeting is noted on the meeting agenda outside this room, and you'll notice some placards in the hallway when you first enter the room. The Holiday Inn is approximately 8 to 10 blocks from our present location and parking is available. This meeting change will only be for this Friday, June 20. All other days,

our meeting will be occurring here. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 8 We apologize for any inconvenience that may occur and are making special arrangements for this change of meeting location this Friday. If you require any assistance to attend the Friday meeting,

including maps or shuttle service to the Holiday Inn hotel, please visit a member of the FIFRA SAP meeting.

Thank you. Dr. Roberts.

DR. ROBERTS: Thank you, Paul. The SAP staff has assembled an outstanding panel of experts to deal with this topic, and I would like to introduce that panel now and do so by asking each member of the panel to state their name, their affiliation, and their area of expertise. And I think we'll just go around the table counter clockwise starting with Dr. LeBlanc on my immediate right and then for each member of the panel around the table to introduce themselves. Dr. LeBlanc.

DR. LEBLANC: Thank you. My name is Gerry LeBlanc. And I'm a professor in the Department of Environmental and Molecular Toxicology at North Carolina State University. My area of expertise is awake toxicology.

DR. KELLEY: I'm Darcy Kelley. I'm professor of Biological Sciences and a member of the Center for Environmental Research and Conservation at Columbia University. And my area of expertise is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 9 sexual differential of the amphibian Xenopus laevis.

DR. KLOAS: My name is Werner Kloas. I'm professor for endocrinology at University of Berlin. And I'm also heading the Inland Fisheries Department of Leibniz Institute of Freshwater Ecology and Inland Fisheries. My working group is the research focused on awake disruption in amphibians, especially addressing sexual differentiation and also the thyroid system.

DR. GREEN: My name is Sherril Green, and I'm an associate professor in the Department of Comparative Medicine at Stanford University. My area of interest and expertise is in laboratory Xenopus laevis, Xenopus laevis and Rana pipiens specifically as a laboratory animal model.

DR. COATS: My name is Joel Coats. I'm professor of entomology and toxicology at Iowa State University. My areas of expertise are in pesticides, especially environmental toxicology and environmental chemistry.

DR. DENVER: My name is Robert Denver. I'm associate professor of molecular, cellular, and development biology at the University of Michigan. And my area of expertise is developmental neuroendocrinology of amphibians.

DR. ROBERTS: Let's jump over to Dr. Gibbs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 10 DR. GIBBS: My name is James Gibbs. I'm an associate professor of conservation biology at the State University of New York's College of Environment Science and Forestry in Syracuse,

New York. And my area of expertise is amphibian demography and population dynamics.

DR. RICHARDS: My name is Carl Richards. I'm a professor of biology at University of Minnesota Duluth and director of the Minnesota Sea Grant College Program. My expertise is that of an aquatic ecologist and landscape ecologist.

DR. DELORME: My name is Peter Delorme. I'm a senior risk assessor with the Canadian Government working on risk assessments of pesticides. My area of expertise is aquatic ecology.

DR. SKELLY: My name is David Skelly. I'm an associate professor of ecology at Yale University. And my area of expertise is population and community ecology of amphibians.

DR. MATSUMURA: My name is Fumio Matsumura. I'm at the Department of Environmental Toxicology. My area of expertise is molecular toxicology. I'm interested in the frogs, too.

DR. THRALL: I'm Mary Anna Thrall. I'm a professor of veterinary pathology in the College of Veterinary Medicine at Colorado State University. And my area of expertise is veterinary 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 11 clinical pathology.

DR. ISOM: I'm Gary Isom, professor of toxicology at Purdue University. And my area is neural toxicology and neural degenerative diseases.

DR. HEERINGA: I'm Steve Heeringa, biostatistician and director of the statistics design group at the Institute for Social Research at the University of Michigan. My specialty is population based studies and design of population based studies.

DR. ROBERTS: And I'm Steve Roberts. I'm a professor with joint appointments in the College of Veterinary Medicine and College of Medicine at the University of Florida. I also serve as director of the Center for Environmental and Human Toxicology there. My areas of expertise are mechanisms of toxicity, particularly involving the liver and immune systems and also methods of risk assessment.

I would now like to welcome Mr. Merenda who is Director of the Office of Science Coordination and Policy. Good morning, Mr.

Merenda.

DR. MERENDA: Good morning, Steve, and welcome to all of the panelists as well as all of those who are participating in attending this session in the audience.

Within EPA, the concept of independent, external scientific 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 12 peer review plays a very important role in our evaluation of decision processes. And so this kind of event, while complex to organize, is a very important part of our job. In fact, it's an important part of the job of my office, the Office of Science Coordination and Policy in EPA's Office of Prevention Pesticides and Toxic Substances.

We find that these kinds of meetings with both the permanent panel members and a number of expert ad hoc members who are selected specifically for your expertise on the subject atrazine and is extremely valuable to the Environment Protection Agency in helping us to better understand where we've done things well and where may have missed some points or where we need to look further and dig deeper as we evaluate the data available to us and make risk management and regulatory decisions.

This is going to be, as Paul said, a very full program. Over the next four days, there are a number of complex issues for us to deal with. And we are quite pleased to have the expertise that is being brought to us by all of you and we're quite thankful for your willingness to take time from busy schedules and other commitments to spend these four days with us in helping us better understand these problems.

So welcome, and just to reemphasize what Paul said, that if you 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 13 have any needs for logistics or assistance with making this meeting work better or making your own arrangements work out while you're here at the meeting, please, do not hesitate to contact a member of our staff on the FIFRA Scientific Advisory Panel.

DR. ROBERTS: Thank you very much. I would also like to extend the panel's welcome this morning to Ms. Anne Lindsay, who is the Acting Deputy Director of the Office of Pesticide Programs. Good morning.

MS. LINDSAY: Good morning. You've taken my first line away. I was going to say I was Anne Lindsay, Acting Deputy Director for Programs in the Office of Pesticide Programs.

I'm actually here on behalf of Jim Jones who is our relatively new office director, though not new to the Pesticide programs. He is actually dealing with family responsibilities this week and asked me to welcome you to Washington, and in particular, to thank you for agreeing to serve on this scientific advisory panel and the sort of extensive meeting that we've got set up for the week. I'd actually hoped I was going to welcome you to a sunny Washington. But that doesn't look like that's going to happen.

The topic of this meeting, the Potential Developmental Effects of Atrazine on amphibians, is one that has generated an extraordinary 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 14 amount of interest since the first data appeared suggesting a link between atrazine exposure and development effects in frogs. It's not only a topic that's drawn a lot of interest, I think it's fair to say, that it's actually been extremely controversial.

Now for those of us who work at EPA on Pesticide regulation,

such controversy is actually often or frequently part and parcel of doing our job. It's not that we want it to be that way. But that is often how it is. And I think that's the case because the issues we deal with are usually very complicated and often, as I think the subject of this meeting, really falls at the cutting edge of science so the answers may not be clear cut.

We find ourselves dealing frequently with situations in which the answers are not obvious. And different groups also have strongly held views and strongly held contrasting views. Over the years, we've developed a deep appreciation, therefore, for the value of science as the basis of our work and to have that science guide our decisions.

And that's really, frankly, where all of you as a panel and as experts in your various areas come in. Your job is to help us figure out what the scientific information actually does tell us, where we can trust information, where there are questions about it. We want your advice to guide us as we move forward making our regulatory 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 15 decisions.

I've spent probably not only the largest part of my career in public service maybe the largest part of my life at this point in public service. I think when you do that, you have to be able to have a great deal of pride and draw a great deal of satisfaction in contributing back to the community and the country that you come from. And that is certainly, I think, how I and the other EPA folks who will be presenters here today feel about our work.

We hope that you, too, since at this point as members of the panel, you're part of the public service in effect, will take that sense of satisfaction for making a very real contribution to the civic life of our country. There's no doubt in my mind that the work that you will do as panel members has enormous value to us at EPA. And because of that, I believe it will have enormous value to the citizens of this country. So I want to thank you very much for making that contribution of your time and expertise.

I'd like in particular to acknowledge EPA's thanks to Dr.

Roberts, the chairman of the SAP. This is actually, I believe, your first meeting as chairman of the SAP. But you are well known and a highly regarded scientist who's served for a number of years on our panel and has presided as session chair at some of our most 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 16 controversial meetings with firmness and grace. So we're looking forward to a continuation of that firmness and grace. We thank you for contributing on the panel with these new responsibilities.

We'd also like to extend our appreciation to the other new permanent members of the panel who I think Paul has introduced but Dr. Steward Handworker, Steve Heeringa, and Gary Isom. It's a delight to have Dr. Heeringa back with us after serving as an ad hoc panel member on multiple occasions and also we welcome Dr. Isom as a permanent member of the SAP.

In addition, my thanks to all of the scientists who have agreed to serve as expert advisors on the atrazine issues. Your willingness to contribute your knowledge and expertise to sorting out pressing scientific issues is invaluable.

And then finally, Paul, I want to thank you and the other members of the SAP staff. You do a great job taking care of the panel members and a great job running the meeting. And for that, we're deeply appreciative.

So, finally, let me wish you the best for the upcoming meetings and we look forward to receiving your report.

DR. ROBERTS: Well, thank you for your kind remarks.

They're very much appreciated. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 17 Our discussion of the scientific issues is going to begin with presentations by the Agency. And the first presenter is Dr. Steven Bradbury of the Office of Pesticide Programs. Good morning, Dr.

Bradbury.

DR. BRADBURY: Good morning, thank you. I'd also like to extend my thanks to the SAP staff for helping to organize the meeting and to the panel for the discussions we'll be having over the next several days. Your input and advice will be greatly appreciated and very important part of the scientific analysis that we've embarked upon with the white paper.

What I'd like to do in my presentation is go over a few of the issues you'll be hearing in more detail from Tom Steeger and Joe Tietge later this morning. And to review a bit why we're here and what we hope to accomplish over the next several days, as I've mentioned in my opening remarks, we're looking forward to obtaining your recommendations on our analysis to date regarding the potential developmental effects of atrazine on amphibians. And as we go through the morning's discussions, we'll be walking through several topics of particular note.

One will be the integration of the available information as we've summarized in the white paper. We'll also be discussing 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 18 aspects of that information in the context of how it allows one to formulate risk hypotheses about the potential effects of atrazine on amphibian development. And from those risk hypotheses,

establishing a conceptual model for potential effects. And ultimately,

then taking a look at an analysis plan as to how to move forward in the context of that available information.

To provide a little background as to where we've been and how we've got here, let's go back to January 31, 2003. That's when EPA released an Interim Reregistration Eligibility Decision, an IRED.

And that document included an assessment of human health risk assessment issues as well as taking a look at ecological risk assessment issues.

In the January ' 03 ecological risk characterization, we followed sort of the basic tenets of the Agency's guidelines for ecological risk assessment and took a look at exposure issues and took a look at the physical chemical properties of atrazine and compared both modeling and monitoring and find general consistency between monitoring studies and the classes of watersheds and uses that atrazine is associated with.

The effects analysis or effects characterization in that document focused on the integrity of aquatic communities or the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 19 stability of aquatic communities structure and function as a risk assessment endpoint and came to the conclusion the likelihood of adverse effects would occur at approximately 10 to 20 micrograms per liter over recurrent or prolonged periods of exposure. And this analysis was similar to a somewhat independent analysis going on in the Office of Water as they develop their draft water quality criteria for atrazine.

In the document, we also discuss some of the uncertainties that are associated with this risk assessment and all risk assessments have different levels in context of uncertainty. Then we discuss some of the exposure characterizations uncertainties, including some data gaps in terms of being able to predict the atrazine concentration patterns and attributes across a full population of water body types in the United States. And we also discussed some of the uncertainties in terms of the spatial and temporal variability of atrazine and the ability to predict or model those patterns.

In terms of effects characterization, we discussed the challenge of converting steady state atrazine exposures to fluctuating or transient atrazine exposures and how to work through the dosimetry of that kind of an exposure scenario. We talked about the issues in terms of quantifying aquatic community recover or resistance to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 20 repeated exposures to the compound. And, finally, we also discussed the uncertainties concerning the potential developmental effects of atrazine on amphibians.

At the time that the IRED was being prepared, there were several studies that were addressing the potential effects of atrazine on amphibian development and were being published at about the same time or during the period of time that we were preparing that January ' 03 document.

As a consequence, we really didn't have time to perform a rigorous evaluation of these data for inclusion in that January IRED.

And consequently we agreed with NRDC that we would proceed with our analysis of this issue of the potential effect of atrazine on amphibian development during the time period that we're all in now.

And we set course on a path to review the available information through February 28 of 2003 and then convene a panel as we're doing today to discuss the information at hand and to gain your insights and comments on the conclusions we've reached to date concerning that information.

More specifically, what we wanted to set out in terms of the agreement between January and October, was to take a look at the significance of the amphibian risk data and determine whether there 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 21 was a need for additional data to characterized more fully atrazine's potential risks to amphibian species, and if so, what data should be developed to further reduce the uncertainties associated with this question.

What I'd like to do over the next few minutes is just provide a bit of a roadmap and some of the milestones for the rest of the morning's presentations by the EPA folks. And the white paper actually sort of sets up the problem. It helps formulate the problem before us. And I've used the words " formulate the problem" in the context of the Agency's 1998 ecological risk assessment guidelines.

And the roadmap that we're going to use today to summarize the highlights of the White Paper to in fact use the Agency's guidelines as a framework for the presentations and the logic train that we went through in interpreting the information that's currently available.

I think you're all aware of Agency's guidelines, but I'd like to spend just a few minutes touching upon what I feel are some of the important aspects of the Agency's guidelines and the role of science in risk management and regulatory decision making.

Obviously, I believe, ecological risk assessments are the science part of the overall decision making process. And in our risk assessments, which is the box in that figure on the screen, is where 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 the science of ecological risk assessment occurs. And through the development of an ecological risk assessment, clarifying the uncertainties, establishing what we know and what we don't know the assumptions behind the analyses helps inform the overall decision making process which blends in, of course, to many other considerations other than the science.

So the key over the next few days is to take a look at the science associated with this issue and determine where our certainties are, where are uncertainties are, and how that can be helpful in helping to inform the process of overall decision making with regard to atrazine and its potential effects on amphibians. So the focus is on the science.

Over the course of the next several presentations, or my presentation, Tom's and Joe's, we're going to focus on especially the problem formulation phase of an ecological risk assessment because,

in fact, that's sort of where we are right now. We're in the stage of formulating the problem, starting to define what we know, what we don't know, what types of risk hypotheses can we formulate with the existing information, and to develop a pathway for moving forward.

So we're in the process of generating and evaluating preliminary hypotheses about why effects may occur or could be 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 occurring and to articulate the uncertainties that would be associated with moving forward in a risk assessment. I think it's also important to remember that the guidelines for ecological risk assessments acknowledge an iterative process. And that as the science evolves and risk management issues evolve, the risk assessments can evolve too. And so in a sense, we're in an iterative process of the overall risk assessment for atrazine.

As I mentioned earlier, in the January document we articulated one uncertainty that was associated with the potential effects of atrazine on the development of amphibians. And in a sense, we're going through an iteration of the risk assessment for atrazine; and we're at the stage of looking at problem formulation.

So what does that mean? What I've done in the next slide is expanded on the concepts of problem formulation and what some of the key aspects of this phase of a risk assessment entail. And this will be the focus, this is really the focus of the White Paper and it is the focus of the presentations we will be making today.

Through the problem formulation, one sets the stage for moving forward in the overall risk assessment. Outcomes of a problem formulation could include a decision that there's no need to go forward, that, in fact, there isn't an issue that requires a risk 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 24 assessment. Another option, talking about broad pathways, one can move forward after problem formulation would be that, in fact, there is sufficient data to move ahead with the risk assessment with specified levels of certainty. And another option would be that, while it's possible to form risk hypotheses, there are certain uncertainties that are associated with the risk assessment; and the analysis plan,

therefore, may call for or suggest additional data that could be needed.

The key in problem formulation is associated with box the on my left and the arrows that are going both ways. And that's the dialogue between the risk managers and the risk assessment team.

The risk assessment is science, but it's not science in a vacuum. It's science to inform regulatory decision assessment and risk management. And so the decisions with regard to certainty and uncertainty certainly have a scientific basis, but they also have a context. And the context is in the context of how much certainty is required to make a regulatory decision with a specified level of confidence.

The role of the risk assessor is to provide the risk manager insights into the uncertainties, the certainties, the risk hypotheses,

and to engage in a dialogue as to the pathway and moving forward. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 25 The White Paper is a problem formulation. The White Paper is designed to help inform all of us and you and gain your insights as well to inform the broader community as to the potential pathways that make sense given the information that we currently have.

The key products in a problem formulation are the items in the circles on the figure. The clarification of the risk assessment endpoints and the measures of effects is an important outcome of problem formulation as is the conceptual models which is essentially as series of one or more risk hypotheses as to how one may envision or hypothesize that atrazine, in this case, could have potential developmental effects on amphibians.

And then an analysis plan, given the current body of information, a plan in moving forward. How are we going to use the available information, if additional information could be gained, what kind of information would contribute to closing what knowledge gaps.

And again, before moving forward into actual risk characterization dialogue with the risk management community to ensure that the regulator decision makers in the agencies have a clear understanding of the uncertainties, and the issues associated with moving forward with different levels or amounts of data. The risk assessment is designed to inform regulatory decisions; it doesn't make regulatory 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 26 decisions.

Let me just spend a few minutes, then, with this roadmap as background, hit a few of the milestones on the journey we'll take over the next couple of hours in reviewing the White Paper.

As I indicated in that previous slide, one of the most critical steps actually in problem formulation is integrating the available information. What do we know, what don't we know, and how do we use that information to draw conclusions concerning risk assessment endpoints, measures of effects; how do we use that information to help establish risk hypotheses, and ultimately establish the analysis plan for moving forward.

Tom Steeger will be providing an overview of the key studies that were discussed in the White Paper in some detail. And as you'll recall, there were 17 studies available for analysis by the Agency as of February 28, 2003. Seven of these studies were laboratory based studies; and 10 of the studies were field experiments.

The White Paper describes how we looked at the study attributes, experimental designs, the various protocols that were used,

and how we looked across those studies to take a look at the body of knowledge, looking at the consistency across the studies, how the studies as a whole provide insights into the strength of cause effect or 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 27 dose response relationships, the extent to which the body of knowledge provides insights on mechanistic plausibility concerning the potential effects, and issues regarding ecological relevancy.

And, of course, throughout the White Paper and throughout the process of developing a problem formulation, one keeps track of the certainties and uncertainties as one integrates and evaluates the existing information.

Tom, in his talk, will go over the body of the information and sort of a synthesis mode. Tom's not going to go through each individual study because the White Paper provides that level of analysis. And rather Tom's going to summarize the synthesis and the integration of the information. And so as he talks about study protocols and design, it's designed to be reflective of the entire body of studies and not necessarily a specific comment for a specific study.

During Thursday or Friday, if you'd like to talk in more detail on specific studies, of course, we'd be happy to do so.

One of the outcomes of problem formulation, which I mentioned previously, is establishment of the risk assessment endpoints; and they draw in part from the available information, but they'd also have to be connected to the Agency, in this case, EPA's mission. What is it all about in terms of protecting the environment and human health. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 28 And so you have a train of logic that needs to connect environmental management goals to a risk assessment endpoint and then to the measures of effects which will be used to estimate how those risk assessment endpoints may change based on different exposure scenarios.

So in this context, we're talking about environmental management goal which is the viability of anuran populations. So the analysis of the studies that Tom and Joe will talk about, we're focusing on a risk assessment endpoint which involves the reproduction and recruitment of native anurans.

Again, a risk assessment endpoint needs to be a ecological entity and the attributes of the entity. So the entity is anuran, native anurans of North America, native anurans, and the attribute reproduction and recruitment.

Through the analysis of the existing information that Tom will describe as further highlighted in the discussion that Joe will provide,

there's a whole family of measures of effects that have been reported in the literature and it can be useful in terms of estimating how the risk assessment endpoint may change based on different atrazine exposures.

And on the slide, I've listed many of the measures of effects 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 29 that were included in those studies; and Tom will be talking about the interpretation of that information in the context of estimating the risk assessment endpoint behavior.

A second major output of the problem formulation stage and of the White Paper is the conceptual model. The conceptual model is a way of pulling together the lines of evidence, the information that's available, to formulate risk hypotheses and to provide insights into how we need to go forward. As I said, I'm going to provide some of the milestones on the path; and Tom and Joe will provide more of the details of how we got to some of the milestones that are shown on this slide.

We went through the available information and as we discussed in the White Paper we concluded that the lines of evidence did not show a consistent, reproducible effect of atrazine across the exposure concentrations in the amphibian species tested. But we also noted that there were issues concerning the study protocols, the experimental designs, and inherent uncertainties in the issue at hand to make it difficult to fully interpret the information. And consequently and as a result of the strength of the studies, we did come to the conclusion that the available data is of sufficient quality to establish a risk hypothesis that atrazine could cause developmental 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 30 effects in amphibians.

Between Tom's talk and Joe's talk, we'll review the White Paper in the context of how we reach that risk hypothesis and establish in more detail the conceptual mode of atrazine's mechanism of proposed mechanism of action and how that could lead to developmental effects or reproductive effects in the context of the risk assessment endpoint that I mentioned previously.

And, finally, the last major product from a problem formulation and as discussed in the White Paper, is the analysis plan. The analysis plan, again, is a series of options to have a dialogue with the risk managers in the Agency in terms of where to move forward. And here, again, I think it's important to come back to the interface between risk assessment and risk management.

In an analysis plan, options are created based on the scientific certainties and uncertainties available in the information. The decision as to how much uncertainty or how much uncertainty one can make a decision is part of that interface between the risk assessor and the risk manager. And so the analysis plan lays out concepts for future studies, lays out a roadmap for future studies. But they're in the context of whether or not greater certainty is required to make the regulator decision. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 31 There's some science and then there's policy in FDM that goes on. And the White Paper is discussing the scientific certainties and uncertainties that EPA feels exists in the current body of information and provides some thoughts and some concepts and plans as to how those uncertainties could be closed if they need to be closed to make a regulatory decision.

So the analysis plan then, based on our risk hypotheses and the conceptual model, are designed to enhance or improve the clarity of potential causality in terms of atrazine's potential effects on amphibians as well as to further characterize the potential dose response relationship between atrazine exposure and developmental effects.

In further phases of the analysis plan, it talks about making connections to mechanisms of action as well as ecological relevancy.

I think that's another important point to bring out is that, as I mentioned earlier, risk assessments can be iterative, they can be phased, they can be tiered, and in fact, an analysis plan in an ecological risk assessment can lay out a phased or tiered approach to reducing uncertainties, incremental gains in knowledge as a basis of needs for informing the risk managers in the decisions they need to make. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 32 And as you'll note in the White Paper and as we'll summarize this morning, the analysis plan lays out a phased or tiered approach to looking at specific uncertainties in sort of cascade approach.

Again, the decisions to move through the phases in the analysis plan would be tied to risk management decision criteria. But how is the science in terms  what's the state of the science in informing the risk managers for different decisions they may need to make.

So in conclusion from my talk, I want to stress that the White Paper reflects our conclusions to date based on our analysis of the information and our interpretation of this information in the context of the Agency's risk assessment guidelines. Now we're at the important stage of gaining insights and advice and counsel from our scientific peers in terms how we've taken a look at the data, how we've integrated the available information, gain your insights and advice and counsel on how we evaluated the studies, how we characterize the available studies, and the conclusions that we drew from the available body of information.

Of course, we're also looking forward to your thoughts and opinions in terms of the risk assessment endpoints and the measures of effects. And then ultimately the conceptual model and the analysis plan. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 33 So with that, I'll close. And I'll be happy to answer any clarifying questions at this point.

DR. ROBERTS: Thank you, Dr. Bradbury, for laying out the task in front of us. Let me ask the panel if they have any questions or clarifications for you.

I see no questions, so thank you very much.

Let's go on then next to Dr. Steeger's presentation on an overview of the atrazine studies. Good morning, Dr. Steeger.

DR. STEEGER: Good morning. Thank you for the opportunity to discuss some of the recent literature that has become available regarding the potential effects of atrazine on amphibian development.

As indicated, the Agency has developed a White Paper intended to review recent studies conducted on the effects of atrazine on amphibian development. This presentation will provide an overview of the study reviews and attempt to integrate the information to answer whether there is sufficient information to substantiate claims that atrazine exposure results in development effects in amphibians.

Panel members have had an opportunity to review each of the studies for themselves. This presentation will look at the studies collectively rather than focus on individual studies.

As early as 1998 and continuing through this year, a series of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 34 studies have been published indicating the variable lengths of exposure to atrazine is associated with gonadal effects in amphibians based on two studies published by Tevera Mendoza in 2001, three studies published by Hayes in 2002, research described in a poster presented by McCoy, et al., in 2002 at the meeting of the Society of Toxicology and Enviornmental Chemistry; and a study by Carr, et al.,

in 2003; there is sufficient information to formulate a plausible hypotheses that atrazine exposure may result in development effects in amphibian gonads and that these effects may impact secondary sexual characteristics in these animals.

However, also based on these studies reported in the open literature, there is a lack of consistency in the type of effect produced and the concentration of atrazine required to produce that effect.

As part of a consent decree between the Agency and the Natural Resource Defense Council, the Agency agreed to conduct and review the available literature regarding the effects of atrazine on amphibian development. The Agency reviewed a total of 17 studies that were submitted as of February 28, 2003. As Steve indicated, 12 of the studies were sponsored by the registrant where 5 were drawn from the open literature.

Registrant submitted studies received more scrutiny since more 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 35 data were available. Although none of the studies were conducted under good laboratory practice conditions, many of the studies had standard operating procedures and some level of quality assurance in place. Additionally, on studies where raw data were available, the data were re subjected to statistical analyses.

Since most of the published studies did not have standard operating procedures nor were raw data available for review on most of the studies, the open literature studies were evaluated at face value with the understanding that all these published studies would have been subject to some degree of scrutiny already through the journal's peer review process.

No formal guidelines existed for specifically examining the effects of atrazine on gonadal development in amphibians. Currently,

there are no guideline studies for amphibians and the Agency relies on other aquatic and terrestrial test species to serve as surrogates for estimating risks to amphibians.

Additionally, many of the measurement endpoints examined in the recent studies differ from those regularly utilized by the Agency to estimate acute and or chronic risk. However, the Agency is not confined to using guideline studies to identify potential hazards. The Agency routinely relies on open literature to provide additional 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 36 insights on the potential effects of pesticides and may use this information to request additional studies to address uncertainties.

The registrants Ingenta voluntarily undertook all of the studies submitted for the Agency review. The studies were prompted by concerns that atrazine exposure could potentially result in developmental effects in amphibians. Although over many years the registrant has completed both acute and chronic ecological effect testing on a range of species in both the laboratory and the field, we are focused today on the recently completed studies completed on amphibians.

As Steve indicated, a total of 17 studies were submitted by the agreed upon February 28, 2003, deadline. The deadline was imposed to allow sufficient time to review the studies and write a White Paper regarding the review for submission to this SAP. Seven of the studies were conducted exclusively in the laboratory, while 10 of the studies were conducted in the field. Field studies included Florida, Illinois,

Indiana, Iowa, Michigan, Nebraska, Utah, Wyoming, and South Africa.

When studies are submitted to the Agency, data evaluation records are completed on each of the studies. Typically, data evaluation records detail how and why the study was conducted, the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 37 results, and what the study's author concluded from the data. The Agency then analyzes the study's raw data and attempts to draw its own conclusions from the data. Reviewers identify any inconsistencies in study methods and results and then summarize their interpretation of the study results.

As noted earlier, most of the open literature did not have sufficient detail of the complete in depth data evaluation records.

And, in fact, data evaluation records are not typically completed on open literature. However, evaluation records were completed for the five open literature studies to capture as much of the methodology,

data, and results that were available in the published study.

All data evaluation records completed by the Agency undergo secondary review to verify the primary reviewers interpretation of the study. For each of the 17 studies reviewed in the White Paper, data evaluation records were reviewed by three secondary reviewers.

Copies of the data evaluation records for the amphibian effects studies have been provided to the panel members.

Reviewed were the studies' protocols and quality assurance, the strength of cause effect relationship, whether there was a dose response, whether the observed effects have a plausible mechanism of action that is consistent with what is known about the chemical, and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 38 finally, whether the measured effects are ecologically relevant.

A range of amphibian species were tested in the studies.

Although the laboratory studies may have relied on non native species, each of the field studies examined species within their native range; thus cane toads were studied in Florida, bull frogs were studied in Iowa, northern leopard frogs were studied in Wyoming, Utah,

Nebraska, and Indiana, green frogs were studied in Michigan, cricket frogs were studied in Illinois, and the African clawed frogs were studied in South Africa. Although most of the studies relied on tadpoles, field studies examined both larval and adult forms.

Endpoints measured in the laboratory and field studies included time to metamorphosis, growth in terms of length and weight,

presence of gonadal abnormalities, laryngeal muscle area, sex rations,

plasma steroid concentrations, and brain and gonad aromatase activity levels.

Gonadal abnormalities include misshapen gonads, for example,

discontinuous testes or multi lobe testes. However hermaphroditism was also observed. For the purposes of this presentation, the terms hermaphroditism, intersex, and ovotestes are used interchangeably to represent the co occurrence of testicular and ovarian tissue either in the same gonad or individual. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 39 Effects on the amphibian laryngeal dilator muscle were also described. Although a variety of methods were used to document this effect, generally the cross sectional area through the laryngeal dilator muscle was measured. Typically, male frogs have a larger dilator muscle than females.

No effort was made in his presentation to single out a particular study. Rather the focus is on issues that were identified in the studies collectively. This is not to say that all the studies exhibited similar difficulties. Some studies contained relatively few issues, while others may have contained several. However, no study was devoid of uncertainties and or inconsistencies.

Since each of the studies contained sufficient uncertainties and consistencies or inconsistencies that rendered the data of questionable utility, data evaluation records focus primarily on methodological issues rather than on a statistical analysis of the data.

As mentioned previously, there were 7 laboratory studies and 10 field studies. Most of the field studies had some laboratory analyses. Collectively, the following issues were identified in the laboratory studies: Atrazine contamination of the controls, poor water quality, poor growth and development and or survival, high variability in endpoint measures, lack of reproducibility, and the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 40 unresponsiveness to positive controls.

Atrazine contamination in the controls seemed to be a recurrent theme in several of the studies. Measured concentrations of atrazine exceeded the levels of detection by a factor of two and were at concentrations reported to cause effects in other studies. For example, in several of the studies, atrazine concentrations in control tanks was higher than 0.1 micrograms per liter. The concentration of atrazine reported by Hayes to cause developmental effects in frog testes.

Additionally, several studies suggested that animal feed used in the studies may have contained atrazine residues. However,

separation techniques are not sufficiently developed to allow the researchers to verify and or quantify the concentration of atrazine in the feed.

Laboratory studies ranged from testing a single concentration of atrazine to testing a broad spread of concentrations. Although gonadal effects have been observed between 0.1 and 25 micrograms per liter, most of the studies did not sufficiently bracket these concentrations to verify whether atrazine at these concentrations can result in a consistent developmental effect.

Poor water quality was one of the most frequent issues 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 41 surrounding the laboratory studies. Although the Agency does not receive many amphibian studies and does not have specific guidelines to conduct these studies, several sources, for example, the ASTM,

exist that do provide guidance for conducting aquatic toxicity testing using amphibians. Unfortunately, high loading rates and frequent and incomplete exposure water changes resulted in diminished water quality as evidenced by high ammonia and nitrite levels coupled with low dissolved oxygen.

As a result of poor water quality, many of the study animals exhibited poor growth, low developmental rates, disease and high mortality rates that contributed to the tests' inability to differentiate treatment effects. In some cases, growth was negatively correlated with length of time to metamorphosis. Where Xenopus laevis typically requires 58 days to complete metamorphosis, in some studies larvae had not undergone metamorphosis by as late as 100 days. High mortality rates confounded some of the studies; and in some cases, required a proposed study methodologies be abandoned.

Several of the studies elected to measure plasma testosterone and estradiol concentrations and aromatase activity in the brain and gonad. Variability in measured steroid concentrations were so high that in some cases the study was unable to differentiate males from 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 42 females. Aromatase activity in the gonads ranged from being below the level of detection to sporadic peaks in activity rendering within group variability so high that it would be difficult to differentiate any treatment effect.

With coefficients of variation as high as roughly 500 percent,

study designs were insufficient to account for this level of variability and still be able to detect treatment effects. In some studies,

measurement endpoints would have had to differ by roughly 80 percent before this study would have been able to detect the difference.

Many of the studies did not run positive controls. However, on some of the studies which did utilize dihydrotestosterone and 17 beta estradiol, low percentages of animals responded to the treatment.

This response differed from other studies that indicated that the treatment of frogs with steroids would markedly impact sex ratios and the rate of hermaphroditism.

It is uncertain whether the lack of responsiveness to positive controls was due to animals genuine insensitivity to the steroid hormones or whether there was insufficient chemical present to elicit a response.

The Agency recognizes that field studies can be difficult to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 43 conduct since researchers are not able to control environmental conditions. Also the Agency recognizes the difficulty in identifying sampling sites that can be considered true replicates of one another.

However, of the field studies submitted, there tended to be considerable variability between sampling sites. Similar to some of the laboratory studies, atrazine, both the parent and its degradates,

was present in reference groups. Additionally, other trizine herbicides and chemicals were present but not always well characterized.

Where pesticides were characterized, their concentrations were in some case relatively high. And it is unclear what impact they might have had on the outcome of the study. In some studies, there were unusual environmental conditions that may have impacted the study. Unusually high rainfall and increased predation due to introduced species were problematic.

Similar to laboratory studies, variable hormone concentrations in aromatase activities were problematic. The variable plasma hormone levels may have been a result of collecting animals over a protracted period of time. In one study, animals were collected over roughly a six month period where study animals were likely to be at different stages of their sexual cycles. Additionally, it's unclear 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 44 whether housing Xenopus laevis in close proximity to one another following their collection influenced their hormonal concentrations and or aromatase activity of these opportunistic breeders.

In spite of all the issues identified in the available studies, the Agency believes that the laboratory and field studies have provided useful information. The studies provide sufficient information with which to formulate a hypothesis. They provide insight on the potential sources of variability and they provide insight on future test species and study conditions.

Although many of the studies did not demonstrate any effect of atrazine on amphibian development, there are sufficient data to suggest that atrazine may be affecting gonadal development. In six of the studies, atrazine exposure was associated with a range of gonadal effects across three species of amphibians. There are sufficient data to minimally formulate the hypothesis that atrazine exposure may impact gonadal development. However, there are insufficient data to refute or confirm whether atrazine is actually causing gonadal effects.

The Agency believes that there are insufficient data to refute or confirm the hypotheses that atrazine exposure may impact gonadal development because of the collective uncertainties associated with the existing studies. Uncertainties include whether the cause effect is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 45 real and can be readily repeated in different laboratories; what is the dose response relationship, the mechanistic plausibility that atrazine exposure is causing a given effect; the inability to readily extrapolate laboratory effects to the field; and the uncertain ecological relevancy of the measurement endpoints.

Without addressing these uncertainties, the Agency has no way to determine whether a particular effect can consistently be expected to occur at a particular level, whether the effect if real can be expected to occur in other animals, and whether the effect is likely to adversely effect an animal's reproductive fitness.

While gonadal development appears to be the primary effect associated with atrazine exposure in amphibians, a consistent measurement endpoint for the effect has differed. Atrazine exposure has been demonstrated to result in hermaphroditism in several studies and laryngeal effects in a single study. However, other studies have not been able to demonstrate similar effects. While males have been primarily affected, there are conflicting data on whether females are also impacted.

Obtaining a clear dose response relationship has been problematic for most of the researchers engaged in studying the effects of atrazine on amphibians. In some studies, atrazine exposure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 46 resulted in no effects; while in others, concentrations as low as a tenth of a microgram per liter resulted in hermaphroditism in the laboratory. Efforts by some researchers to substantiate these laboratory results were only successful at atrazine concentrations 250 times higher.

Additionally, data from some studies have suggested that following a threshold effect concentration, there is either a leveling off of a response or a diminished response at higher doses. Therefore,

the existing data have not demonstrated a traditional monotonic dose response curve.

Several of the current studies have proposed that atrazine exposure results in up regulation of aromatase activity and a subsequent decline in testosterone concentrations and an increase in estrogen that in turn lead to feminizing, that is, hermaphroditism, and demasculinizing, that is, decreased laryngeal muscle effects in atrazine exposed males.

However, no study thus far has directly demonstrated that aromatase activity has indeed been up regulated. And only one study has demonstrated that plasma testosterone has decreased in atrazine treated males.

Although many of the studies thus far have examined plasma 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 47 steroid levels and brain aromatase activity levels, it is uncertain whether the proposed mechanism of action is likely to be observed on the basis of the whole animal. Rather aromatase activity is proposed to increase in the testes where androgenous testosterone is converted to estrogen. It is unclear whether these localized increases in steroid conversions could be detected in blood plasma at all.

Out of the 17 studies, one demonstrated gonadal effects in both the laboratory and the field. However, in this single study, there was a clear lack of a dose response. Coupled with the variable effects that have been noted, even within the same species, extrapolating atrazine to potential field effects is difficult.

While intuitively it may seem that the presence of ovotestes and reduced numbers of spermatogonial cell mass in males and reduced numbers of primary and secondary oogonia in females, may impair the reproductive fitness of frogs and that reduced laryngeal muscle mass and secondary sexual characteristics may impair an animals ability to attract mates. There are not data currently available to the Agency with which to gage impaired reproductive function, recruitment, or survival.

Additionally, the current ecological risk assessment of atrazine identifies that some plants have exhibited resistance to atrazine. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 48 Some researchers have speculated that amphibians may also develop resistance to the potential effects of atrazine on amphibian development. The Agency is uncertain regarding the role of resistance and recovery from the potential developmental effects.

The primary criteria for conducting ecological risk characterizations in the Agency are that they be transparent, clear,

consistent, and reasonable. Of these criteria, transparency is viewed as the principal value from among the four since it leads to clarity,

consistency, and reasonability.

Consistent with the EPA's process for conducting ecological risk assessments, it has evaluated the available data following specific evaluation criteria. That included experimental design, the strength of the cause effect relationship, the dose response relationship, the mechanistic plausibility, and the ecological relevancy. The Agency has provided these reviews to panel members.

Based on its review of the available literature, the Agency believes that there is sufficient information to formalize a hypothesis regarding the potential effects of atrazine on amphibian development.

But because of the uncertainties surrounding each of the studies conducted thus far, the Agency is recommending that additional studies be conducted. The next presentation by Joe Tietge will 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 49 discuss the Agency's recommendations for additional testing.

In conclusion, as of February 28, 2003, the Agency has reviewed a total of 17 studies examining the effects of atrazine on amphibian gonadal development. These studies have involved both laboratory and field work and have looked at six species of anurans.

In each of the studies, the Agency has identified concerns regarding the study methodologies and or results that potentially limit the utility of the studies.

Based on all 17 studies, atrazine exposure did not produce consistent, reproducible effects across all species tested; therefore,

the weight of evidence suggests that atrazine exposure does not impact gonadal development. However, there are lines of evidence from both laboratory and field studies that support the formulation of a plausible hypothesis that atrazine exposure may result in developmental effects in amphibians.

Although the current studies cannot be used to refute or confirm the hypothesis that atrazine exposure may result in gonadal development effects, the studies do provide useful information of the sources of variability. This information will be critical to the design of future studies.

There are insufficient data, as I indicated, to refute or confirm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 50 the effects of atrazine on amphibians. If risk managers, however,

wish to reduce the current uncertainties regarding the potential effects of atrazine on amphibians, the Agency recommends that additional studies be initiated. These studies should build on the current body of information.

If additional testing is required, the Agency is proposing that a phased approach be used to examine the cause effect dose response mechanistic plausibility, and the ecological relevancy of any effects observed following the exposure of amphibians to atrazine. Joe Tietge who will follow me will present what the Agency is proposing as follow up studies.

Are there any questions?

DR. ROBERTS: Great. Thank you, Dr. Steeger. Before we move on to the next presentation, I think this is good opportunity for the panel to ask you any questions they might have on the Agency review of the 17 studies. Are there any questions among panel members regarding the Agency's review.

DR. KELLEY: I have a question.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: So for instance you said that in some of the studies you failed to document sex differences in steroid levels. How 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 51 did you know that you would expect to see those sex differences?

DR. STEEGER: We didn't.

DR. KELLEY: So that is, in fact, then not such a useful criteria.

DR. STEEGER: We

DR. KELLEY: If you didn't know you expected to see them and then you didn't see them, how did you know they were there anyway?

DR. STEEGER: You're talking about sex differences in the steroid level hormone concentrations.

DR. KELLEY: In steroid levels. Right.

DR. STEEGER: Well, we didn't know what to expect. Because as I indicated, this was a new area for the  these measurement endpoints were new for the Agency to consider.

DR. KELLEY: All right.

DR. ROBERTS: Yes, Dr. Skelly.

DR. SKELLY: I just had a general question about whether in your review you considered any sort of a line below which the quality of data issues meant that you wouldn't consider the evidence from that study.

DR. STEEGER: When the Agency receives guideline studies,

we have what's called the " rejection rate analysis" where there are 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 52 certain characteristics of the study which will eliminate it from consideration. Some of the studies, one potential factor that can eliminate a study for consideration is the presence of the test chemical in control sites. Because these studies weren't conducted following guidelines, we didn't really have a criteria that would really eliminate it from consideration. But that would constitute a reason to reject a study. Does that...

DR. SKELLY: So you considered rejecting studies and decided not to.

DR. STEEGER: We considered that we would just review the studies as they existed without any consideration for what would constitute a fatal flaw in the study.

DR. KELLEY: Can I ask then

DR. ROBERTS: Excuse me.

DR. KELLEY:  just follow up

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: Yeah.

DR. ROBERTS: You've got to wait for me to call on you. Dr.

Kelley.

DR. KELLEY: Thank you. Just to follow up on that question.

So do you use a criterion that might take into account the weight of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 53 the evidence, in that case just simply the number of studies? So if you had 22 studies that got an effect and 5 that didn't you would consider not having rejected any of them as weighing in on the weight of evidence in favor of the first result rather than the second?

DR. STEEGER: In this case, we are not using a  because of the inconsistencies in all the studies, we couldn't really use a weight of evidence approach. As I indicated throughout my presentation,

what seems to be a recurrent theme, a line of evidence, that there seemed to be some effects that recur over the studies. But the weight of evidence approach does not work for us in this case because there were such, in our view, glaring problems with each of the studies that it was not difficult to weight them per se.

DR. KELLEY: One last question. You had, whatever it was, 19 studies  is that right?  12 and 7.

DR. STEEGER: Seventeen studies.

DR. KELLEY: Seventeen studies. After you completed the White Paper, did you become aware of any studies that were not included in the White Paper that you had missed for one reason or another?

DR. STEEGER: Our contractor provided us with  are we talking about amphibian studies? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 54 DR. KELLEY: Amphibian studies.

DR. STEEGER: Not that we were aware of.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: I'm paraphrasing here. But you said something to the effect that the hypothesis wasn't accepted because no consistent reproducible effects across all species were observed. And I was wondering if that was actually a requirement for accepting the hypothesis, that consistency among all species be observed.

DR. STEEGER: No, it's not.

DR. LEBLANC: And a second question is were other species,

vertebrate species, aquatic vertebrate species considered in the literature review?

DR. STEEGER: No, they were not. We do have information on other species. But our review looked at amphibians only.

DR. ROBERTS: Dr. Gibbs.

DR. GIBBS: Just quickly, in your characterization of available studies, you mentioned that data evaluation records focused primarily on methodological problems rather than statistical analyses. Could you elaborate on that?

DR. STEEGER: Because of the problems with atrazine contamination in the controls, the unresponsiveness of animals to the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 55 positive controls, the slow development of the animals, the high mortality rates that were exhibited on many of the studies, those were what we considered to be problematic that would have rendered the data of questionable utility. So we didn't really focus on analyzing the data per se.

Now I did mention, though, that on the aromatase and plasma steroid concentrations, there was high variability. We do analyses to verify that there was indeed high levels of variability in the measurement endpoints. But, again, because of the way that the information was collected, it would have been problematic for us to move forward with the study independent of what the analyses told us.

DR. GIBBS: Was the assumption that that peer review process would have caught any problems or issues with the statistical analyses as reported?

DR. STEEGER: Well, the peer review process, are we talking about for open literature?

DR. GIBBS: Yeah.

DR. STEEGER: For the open literature, it's rare for journals to have access to the author's raw data. So it's unlikely they would have caught that.

DR. ROBERTS: Okay. Any other questions? Okay. I see 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 56 none. Then we have scheduled a break now. We're a little ahead of schedule. I guess whether or not we go to break kind of depends on the length of the next presentation. So let me just ask you, Mr.

Tietge, we have allotted an hour for that talk. What's your best guess?

MR. TIETGE: I think it will take about 30 minutes.

DR. ROBERTS: Then let's go ahead and move on to your presentation.

MR. TIETGE: Thank you. I'm just going to get started here.

The basis for this talk today is that the evaluation of the currently available data as previously reviewed by Dr. Steeger suggests that anuran reproductive fitness may be adversely affected by exposure to atrazine. However, the data are insufficient to conclude that atrazine adversely affects anuran reproduction.

Therefore, further studies are proposed following the Guidelines for Ecological Risk Assessment to reduce the uncertainties and permit an eventual risk characterization if warranted.

These conclusions are based on the fact that there are a number of remaining uncertainties including the following: The number of affirmative studies, that is, those that seem to demonstrate an effect on gonadal development and secondary sexual characteristics, the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 57 sample; there's limited evidence of repeatability between laboratories; the dose response relationship remains undefined due to the lack of sufficient dose response data; the mechanistic plausibility of the hypothetical mode of action is currently unsupported by the available data on amphibians; and, finally, the ecological relevancy of the potential effects of atrazine exposure on amphibians remains undetermined.

Based on these observations, it is EPA's recommendation that,

if the risk management process requires further reductions in these uncertainties, then additional laboratory studies need to be conducted before any additional risk assessment activities regarding the effects of atrazine on amphibian reproduction are undertaken.

The objectives of this presentation are, first, to review the concept of problem formulation as used in the Agency's ecological risk assessment process; second, to restate the environmental goals and assessment endpoints necessary to make the risk management decisions; third, propose a conceptual model for atrazine action on anuran reproduction by defining a risk hypothesis; fourth, propose an analysis plan which identifies measures of effect relevant to the assessment endpoints and risk hypothesis and includes a phased study approach to test central components of the risk hypothesis; fifth, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 58 identify critical decision points in the phased approach; and, finally,

to provide some conclusions.

It's not my intent here to give a detailed discussion of the ecological risk assessment paradigm, but I want to remind you or familiarize you with the basic components of an ERA. Because as already mentioned by Dr. Bradbury this morning, we are using the ERA paradigm to guide our approach on this issue.

Briefly, this process can be represented as three distinct phases: Problem formulation, analysis, and risk characterization.

Problem formulation is the foundation of the ecological risk assessment process as it lays out the goals and approaches necessary for the successful completion of an assessment. Much of what I'm presenting to you today is indeed part of the problem formulation phase.

The analysis phase is the phase that implements the approach developed in the problem formulation and generates the data required to complete the final phase, which is risk characterization. I'll only touch on the analysis phase today as it relates to the approach developed and problem formulation. I will not risk characterization except to say that this is the phase that takes into account the probabilities associated with exposures and effects and results in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 59 some conclusion regarding risk.

In this slide, I have expanded the problem formulation box here in order to demonstrate that there are four main components to consider. The first is the integration of the available data as presented previously by Dr. Steeger. This is a critical step because it identifies the potential problems associated with a particular stressor,

in this case atrazine, and serves to refine and focus the risk assessment questions and, therefore, all subsequent activities.

The remaining three components of problem formulation:

selecting the assessment endpoints, developing a conceptual, and developing an analysis plan, is the focus of this talk today. Before I launch into these areas, I'd like to point out the critical connection of problem formulation to analysis.

In this slide, I've expanded the analysis phase to show its components as well. In general, in the case of a chemical, this phase typically evaluates exposure as depicted in the left side of the large gray box and effects as shown on the right. What I'd like emphasize here is that the analysis plan that I will present today, which is part of the problem formulation phase, provides guidance for how to conduct the studies on effects and is thereby a prerequisite to measuring effects. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 60 I'd like to return to the problem formulation phase and discuss it in terms of atrazine. First, the overall environmental goal is to ensure anuran populations are viable and self sustaining. This goal is rather generic. But it serves to orient the assessment process by focusing on a specific objective. And although it is somewhat simplistic, it can be difficult to actually assess the impacts of atrazine, or any other chemical for that matter, on anuran populations directly in the field.

But the assessment endpoint, successful reproduction and recruitment of native anurans, is directly related to population status.

And as indicated by some of the atrazine studies evaluated earlier today, some aspects of reproduction, including gonadal development,

are measurable endpoints relevant to the concern surrounding atrazine exposure.

Knowing the environmental goal and the assessment endpoints,

and given the fact that some studies indicate that reproductive system development and secondary sexual characteristics may be affected by atrazine, we need to construct a conceptual model. The conceptual model in the form of a risk hypothesis is an attempt to develop a model that uses existing information to form a plausible explanation of the potential effects of atrazine on the assessment endpoints. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 61 That model is depicted diagrammatically in this slide. The hypothesized effects of atrazine are presumed to be initiated by a molecular interaction. This interaction results in increased aromatase activity, the enzyme responsible for the conversion of testosterone to estradiol. The increased activity of aromatase results in an evaluation of endogenous estradiol which affects feminization, for example, in the male gonad.

If the effects in the male gonad are severe enough, then reduction is fertility and reproductive success could be realized.

Which leads to a hypothetical reductions in recruitment thereby impairing population maintenance which is in fact the assessment endpoint.

This risk hypothesis, which is based on the information on the literature and from submitted studies, may or may not be correct. But it forms the basis of the proposed studies and can also be thought of as a working hypothesis. Because of the uncertainties associated with the risk hypothesis are relatively high, it is likely that it will be modified when data become available.

As with any hypothesis, some elements are easier and or are more important to test than others. So the questions is: At what point in the risk hypothesis should hypothesis testing be introduced to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 62 evaluate the specific sub questions? Or alternatively, what is the best strategy to evaluate the train of events in the risk hypothesis to test its validity.

In the case of this specific risk hypothesis, it is our view that the most appropriate entry point is at the level of determining the effects of atrazine on gonadal development, the apical organismal level endpoint. The reasons for this are, first of all, this is the endpoint on which much of the concern hinges. But, secondly, readily available methods exist to test the sub hypothesis with relatively inexpensive methods that permit the analysis of large sample sizes.

And perhaps most importantly, this endpoint is the linchpin in the entire train of events. That is, if atrazine is found to affect gonadal development with a greater degree of certainly than currently exists, then this result provides strong rationale to conduct studies on the proceeding and subsequent elements of the risk hypothesis.

If on the other hand, atrazine does not affect gonadal development following a systematic effort to study this potential phenomenon, then the logic train of this risk hypothesis is broken and there may be no impetus to follow up by testing the upstream and downstream elements.

So now that we have an assessment endpoint selected and a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 63 conceptual model in place, we can now develop an analysis plan.

There are four major elements to the analysis plan: A strategy to evaluate the risk hypothesis which I've already touched on in the last slide; selection of endpoints to evaluate also referred to as measures of effects; determinization of appropriate methods; and a sequence of analysis that follows the most efficient path to accept or refute the risk hypothesis in a systematic and organized manner.

I've excerpted the first section of the risk hypothesis in the first panel that I just put on the screen. In the proposed analysis of a risk hypothesis as shown as hypothesis testing which is in the second panel. As I mentioned earlier, the entry point for testing the risk hypothesis proposed to be the effects on the gonads at the organismal level. So beginning at the organismal level, the effects of atrazine on gonadal development, particularly in the males, is the primary endpoint.

Developing data at this level is critical in that it may provide the rationale and justification for conducting relevance and or mechanistic studies. If these organismal level tests are affirmative,

then measurements of sex steroids should be conducted. And if estrogen levels are shown to be elevated in the atrazine treatments,

then measurements of aromatase activity could be indicated as 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 64 previous studies have attempted. If positive, the data from these studies will be useful to establish a mechanistic basis for inter species extrapolation, further develop the plausibility of the mechanisms involved, and develop appropriate biomarkers that could be used in future field studies.

Although sex steroid and aromatase measurements are necessary to test the mechanistic aspects of the risk hypothesis, they do not provide meaningful information on the ecological relevancy of a potential gonadal effect. Therefore, if gonadal effects are observed at the organismal level, it is possible to proceed directly to studies which evaluate fertility endpoints that are relevant to the maintenance of populations. Furthermore, if the working hypothesis is supported by organismal and suborganismal studies, then it may be possible to confirm the mode of action by conducting confirmatory studies which utilize no aromatase inhibitors. Rescue of normal morphology of the male gonad by an aromatase inhibitor co administered with atrazine would provide substantial support of the risk hypothesis in general and more specifically the mode of action involved.

However, if any of the studies conducted as part of the hypothesis testing phase are negative, then alternatives should be considered. If no consistent and reproducible effects are observed at 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 65 the organismal level, then there may be no need to continue any further testing. If on the other hand, the organismal level tests are affirmative and either the steroid or aromatase studies are negative,

then an alternative hypothesis may need to be evaluated.

Since this is purely hypothetical at this time, and it is outside the scope of the current risk hypothesis, no further discussion of the alternative testing will be presented.

So now there's an analysis plan. I'd like to discuss some of the details of the proposed studies. These are labeled as phases here as they were also labeled in the White Paper.

Phase 1, the Test for Apical Gonadal Effects. The first and most important phase of hypothesis testing in this phase is to determine if atrazine exposure results in consistent and reproducible gonadal effects in males and females and determine the shape of the dose response curve, if any.

This slide lays out the key experimental process for consideration in the Phase 1 studies. The primary species recommended for this work is Xenopus laevis. The species is recommended because it is amenable to laboratory testing and has been shown by four studies to be potentially responsive to the effects of atrazine on gonadal development and differentiation. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 66 Xenopus laevis, however, is not a native anuran at least from a North American perspective. It may or may not be representative of native anurans when it comes to this issue. Therefore, a secondary species is suggested such as Rana pipiens, the northern leopard frog,

which can be used in corroborative studies.

There is one study that suggests that Rana pipiens is sensitive to the effects of atrazine on gonadal development. However, this species is more difficult to work with in the laboratory and most labs do not have culture methods that permit continuous breeding and are,

therefore, unable to conduct studies throughout the year with Rana pipiens as can be done with Xenopus laevis. Despite the limitations associated with Rana pipiens culture, comparative studies may be useful to develop data to determine the ecological relevancy of potential atrazine effects on a native species.

The developmental stages used in these studies need to include those that are sensitive to the effects of estrogens. In a study conducted by Villalando, 1990, with Xenopus, exposure to an estrogen elicited effects on gonadal differentiation during the pre

metamorphic period. However, after entering metamorphosis, the gonads were less sensitive to estrogen exposure. The proposed studies should include the pre metamorphic period and continue until 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 67 the organism completes metamorphosis at which time they should be evaluated.

The test condition in the studies reviewed earlier all use static renewal conditions. These methods did not conform to the generally accepted biological loading regs recommended by ASTM and resulted in delayed development and in some cases excessive mortality. It is likely that these problems resulted form static renewal conditions themselves which resulted in the accumulation of nitrogenous wastes and other metabolic products and generally poor water quality. It is our recommendation that flow through conditions be used that adhere to ASTM standards and thereby promote survival, growth, and development.

The concentrations of atrazine to be used in these proposed studies should bracket those found to be effective in perturbing gonadal differentiation in previous studies, that is, at or below .1 micrograms per liter for the low and at or above 25 micrograms per liter for the high. And, of course, these concentrations need to be verified analytically.

The use of estradiol as a positive control is recommended since the potential effects on the gonad are proposed to be mediated through this pathway. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 68 The sample size and replication are not detailed here, but they should be determined a priori to be sufficient to test the stated hypothesis using appropriate statistical assumptions. Sampling should include all organisms on test to avoid potential biases.

And, finally, the endpoints should include survival, growth,

development, gross gonadal morphology, gonadal histopathology.

From these data, male to female sex ratios can be derived in the shape of the dose response curve determined for each endpoint.

Because there are issues with the existing studies that limit the usefulness of the data, we propose that quality indicators be established as a guide to evaluate validity of the proposed studies.

First and foremost, the tests need to be conducted in accordance with ASTM standards for biological loading and basic water quality parameters of pH, ammonia, dissolved oxygen and need to be contained within acceptable limits and verified regularly throughout the conduct of the study.

With regard to the biological endpoints, while there is no bright line between acceptable and unacceptable survival percentages,

survival of 90 percent or more is indicative of a quality study. This is a reasonable standard to adopt particularly for Xenopus laevis studies as the species is particularly hardy in the laboratory. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 69 Similarly, growth of Xenopus laevis should result in organisms of about one and a half grams; and this will vary between laboratories. Metamorphic development should be completed within 10 weeks.

But there are no standardized methods. And some methods that are proposed here are the acceptance  there are no acceptance criteria because of the lack of standardized methods. So these issues should be evaluated in aggregate using some professional judgement.

Measurements of sex steroids. I'll now discuss the remaining phases of the analysis plan very briefly. Since the conduct of each of these phases is dependent on the outcome of the previous phases, it is premature to discuss them in much detail. I will, however, lay out the objective and potential approaches for each phase, recognizing that these may change as more information becomes available.

The second phase of the study should be conducted if the Phase 1 studies are positive. The aim of the Phase 2 studies is to determine if concentrations of estradiol and testosterone are altered by exposure to atrazine.

The approach to this phase is based on the fact that the developmental sensitivity toward the feminizing effects of estrogen in male Xenopus laevis has been experimentally determined as depicted 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 70 in the panel in the right. In this panel, developmental stages indicated is indicated on the X axis. The developmental period between stage 44 and 50 represent a period in which estrogen is capable of completely overriding testicular differentiation resulting in 100 percent of the test population with ovaries.

As natural gonadal differentiation proceeds, their sensitivity to this effect diminishes as is indicated by a period of incomplete feminization from androgenous estrogen in a period of apparent insensitivity coincident with the onset of metamorphosis.

This suggests that if ovotestes formation in the male is dependent on estrogen, that the elevated estrogen levels need to be present during the sensitive developmental stages. Any associated studies of this phenomenon should focus on the effects of atrazine on sex steroids during these sensitive periods.

However, it remains uncertain as to whether more developmentally advanced organisms are sensitive to the feminizing effects of estrogen on the gonad. This is an area of uncertainty that requires more investigation as well.

Moving on to Phase 3, the measure of aromatase activity, the objective of the Phase 3 studies is to determine if aromatase activity is increased by exposure to atrazine during sensitive developmental 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 71 stages. Whether or not this phase is conducted, is dependent on whether the proceeding studies on sex steroids suggest that modulation of aromatase activity may be responsible for elevated estrogen levels for example.

Similar to the approach for measurement of the sex steroids,

there's a developmental component to the approach. The developmental expression of aromatase mRNA has been determined for Xenopus laevis and is presented in the panel on the right.

Expression of aromatase mRNA is apparent at approximately stage 50 and generally increases with development. This expression pattern is overlaid on the previous graphic depicting the developmental sensitivity toward estrogen induced feminization which decreases with development.

Taken together, these studies suggest that aromatase activity must be elevated prematurely and at sufficiently high levels during the estrogen sensitive stages to result in feminization in males.

Therefore, at this point in time, the most appropriate approach may be to examine this phenomenon prior to the onset of metamorphosis which is generally considered to begin at about Stage 54.

Phase 4, Aromatase Inhibitor Study. If it is demonstrated that the previous phase of aromatase activity is increase by atrazine, then 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 72 it may be desirable to determine if co administration of an aromatase inhibitor with atrazine rescues the male gonad from feminization.

This approach would require that, first, the effective concentration of an aromatase inhibitor be empirically determined. Then based on the dose response data in the first three phases, the organisms would be exposed to the inhibitor simultaneously will affect atrazine concentrations. Then the effects could be analyzed similar to the Phase 1 studies.

On Phase 5, Evaluating Ecological Relevancy, the objective here in this final phase is to determine if the potential effects of atrazine on gonadal differentiation results in reduced fertility.

There may be several approaches to this, but based on the premiss that feminization of males occurred in Phase 1, the approach that I've outlined here is to determine if feminization alters fertility using either in vitro or in vivo fertilization methods. Although such methods are used routinely for reproductive purposes in numerous laboratories, they are not currently used to quantify fertility as an important parameter to estimating reproductive output. If such studies are warranted, then additional research would have to be conducted to establish quantifiable methods.

So in conclusion, it is possible to reduce the major 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 73 uncertainties associated with the potential risk of atrazine to amphibians by following a phased sequence of laboratory studies focused on the critical components of the risk hypothesis, using currently available high quality methods that are standard for aquatic toxicology, and establishing and adhering to study quality indicators.

Although the analysis plan that I have presented lays out a relatively comprehensive set of studies to evaluate the risk hypothesis, the extent to which the proposed studies are actually conducted depend on two important factors. The first is whether or not risk management decisions require reduction in the current level of uncertainties to proceed. And, second, is whether or not the outcomes of the initial phases indicate that additional studies are logical and valuable in terms of testing the components of the risk hypothesis. These issues will have to be evaluated as more data become available.

Thank you for your attention. I'd be happy to entertain questions.

DR. ROBERTS: Thank you for your presentation. I'd like to give the panel the opportunity now to ask you any questions they might have about the proposed Agency approach that you've described. Let's start with Dr. LeBlanc. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 74 DR. LEBLANC: Joe, do you anticipate a temporal sequence to the performance of the phases or might some of the phases be conducted at the same time?

DR. TIETGE: I think it would be up to the laboratory who's proposing to do the studies. They certainly could be conducted. Or one could, for example conduct an organismal level study and then archive samples for further analysis that are in that tier. I wouldn't want to propose the tier as being too linear. So I think some of them could be done at the same time.

DR. ROBERTS: Dr. Kloas.

DR. KLOAS: I would like to know is the hypothesis is focused on aromatase production. So what we found and up to now and what is more or less a verified is feminization or demasculinization. So this could be also, I think, obtained by anti androgenic effects. I think for conceptional frame work, we should include all as a possibility so that we have an alternative pathway to receive feminization or demasculinization via the anti androgenic pathways.

This should be maybe from a serial point of view at least included.

DR. TIETGE: I would agree. And I tried to leave the door open in the alternative path. I think that once you get away from the organismal level effects in the risk hypothesis, you have more and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 75 more uncertainty especially as you go toward the mechanistic side.

So I agree with you totally.

DR. ROBERTS: Dr. Kelley then Dr. LeBlanc.

DR. KELLEY: With regard to the flow through proposal, as you know, most Xenopus colonies aren't raised in flow through. And in the wild, of course, you don't find very many tadpoles in streams with any motion what so ever. So are you aware of data that indicate that a flow through system as opposed to a static renewal system with large volumes of water would have differential effects on mortality?

DR. TIETGE: Well, the issue of the static renewal versus flow through isn't  let's see, how am I going to answer this. If one goes back to the ASTM guidelines, which I think are fairly valuable in terms of establishing guidance for biological loading, there are guidelines for static tests. However, for a typical Xenopus individual it would require probably three to four liters of solution per individual to meet those standards. So if you want to have tests that have high enough end value in order to test your hypothesis, it would require very large exposure chambers. And I think that would be very limiting.

In fact, in our laboratory, we use flow through conditions routinely and achieve metamorphic completion in about seven weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 76 typically post fertilization and usually with 99 percent or higher survival and apparently good growth rates. Often we have organisms in the 1.8 to 2 gram range when they're right around stage 60 prior to the weight loss that occurs through metamorphosis.

I'm not sure if I answered your question. Yes, there is some basis for it. Also, I understand that even with the native Ranas, they don't necessarily live in flow through conditions in the field; but they also don't live in a static aquarium in the field because the system is more complex.

DR. ROBERTS: If you have a follow up questions that would be fine.

DR. KELLEY: So this does bear to the issue of ecological relevance, however. So it's not entirely clear how a continuous flow through system would bear either on Xenopus that avoid a flow system or on Rana even if it's not totally static since much of the data,

since some of the concern, at least, comes from things like drainage ditches and ponds accumulating in runoff from fertilized fields. So one should, I think, think about whether flow through data, although well controlled from the point of view of water quality, actually would mimic the conditions under which exposure might occur.

DR. TIETGE: I understand your point. It's a point well taken. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 77 However, in aquatic toxicology, I would suggest that the more control you have over the experiment in terms of water quality, the more confidence you have that the effect that you're observing is related to the concentration of the chemical. I think it is a much different question to ask whether or not a laboratory study is directly applicable or representative of field conditions. I think that's the state of the science actually.

DR. ROBERTS: Next Dr. LeBlanc, followed by Dr. Isom, Dr.

Kloas, and then Dr. Skelly.

DR. LEBLANC: In formulating your hypothesis on the mechanism by which atrazine might elicit effects on developing gonads, did you consider the work of Ralph Cooper showing in rats the effects on gonadotropins?

MR. TIETGE: The hypothalamus, hypothalamic vectors?

DR. LEBLANC: Yeah, in suppressing, glueinizing hormone.

MR. TIETGE: I'm certainly aware of it. Of course, Ralph's in the Agency so... But, no, I think what we tried to follow was the information that we thought was more specific or germane to the amphibian issue. So, no, we didn't really take it into consideration.

However, as I also answered Dr. Kloas, we did leave the door open that as you find, as the data indicates, you can go to an 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 78 alternative, take alternative paths.

DR. LEBLANC: I think induction of aromatase may be consistent with his observations. But certainly in the young male, the profound effect that he says in terms of steroid levels was a suppression of testosterone. So I would agree with Dr. Kloas that certainly anti androgens is something you might want to consider as a positive control.

DR. ROBERTS: Dr. Isom, Dr. Kloas, and then Dr. Skelly.

DR. ISOM: The objective of your proposed study is obviously to determine the reproductive fitness of the species. And I noted in a number of the studies that have been published that not only do we have observed or postulated effects upon gonotropic development, but also secondary sex characteristics that are important for reproduction like laryngeal muscle.

I was wondering why you aren't proposing to at least measure that in the species in your exposure studies. And then a second question is have you thought about positive controls for aromatase that is inducers exposure.

MR. TIETGE: Okay. The first question was

DR. ISOM: The laryngeal muscle.

MR. TIETGE: Right. Laryngeal muscle. If I recall the data 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 79 correctly, laryngeal muscle effects were observed at higher concentrations. And so the gonadal effects were more sensitive.

They certainly, if you affected laryngeal muscle to a certain level undetermined at this point in time, you certainly could expect to have some potential effects on reproductive activity. And I think Dr.

Steeger mentioned that in his talk as well.

I mean the door could be open to that.

DR. ISOM: It seems to me that if you're doing the study, you have the animal there. It wouldn't be that difficult to do that.

MR. TIETGE: Actually, we have  I have no experience with that endpoint. I'm not sure how to deal with it. Would anybody.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: I do have experience with that endpoint since that's what I've studied for a good long while. And one of the things that I think would be required in a study of this kind is to enable the animals to grow until they reached reproductive maturity. If you wanted to study the endpoint of sexual differentiation functionally,

both in terms of active spermatogonia in testing situations, either removing the testes or doing natural matings which would be more variable, and if you also wanted to study the endpoint of laryngeal function, which is to produce the male advertisement call among other 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 80 calls, then you would need to actually have your animals go probably for about a year. Although, if your animals are growing fast, you can probably get them to call in six months.

So those seem like natural endpoints that relate very closely to the issue of reproductive success.

DR. ROBERTS: Okay.

MR. TIETGE: A positive aromatase inducer, that certainly could be done. We didn't include it because we were trying to stay as directly on the track as we could. But certainly it's a fine idea.

DR. ROBERTS: Next question or clarification from Dr. Kloas followed by Dr. Skelly, Dr. Denver, and then Dr. Green.

DR. KLOAS: Of course, I would like to come back to flow through versus static renewal system. I think up to now we have no real indication of if, at least some unofficial indications, that maybe a flow through would reduce positive control effects. For instance, for estradiol, for feminization there's one study I'm aware of they have a reduced feminization effect because it's flow through.

I'm not sure. So I think before just fixing everything, we should also maybe be aware that we need a comparative study, a comprehensive study, between flow through and static renewal.

I know also from this flow through experiment, the tadpole's 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 81 grow well and also the developmental stages were reached. But I'm a little bit concerned about sexual differentiation. I'm not sure that you can say it doesn't matter.

MR. TIETGE: I'm familiar with the study, I think, that you're referring to. And I find the results to be somewhat enigmatic.

However, in using the flow through conditions in our laboratory, I don't think there's any effect on sexual differentiation based on the method itself. I think that from an efficacy of exposure point of view,

the flow through system probably ought to be more effect than in a static system because there's a lot of evidence that exists with the more hydrophobic chemicals will be depleted under static conditions.

That's very well established in the aquatic toxicology literature.

DR. KLOAS: I agree from a theoretical point of view. But did you do a positive control using estradiol for inducing feminization in parallel in this system and it works?

MR. TIETGE: It works, yes.

DR. KLOAS: I would like to see it.

DR. ROBERTS: Let's go to Dr. Skelly. But before we continue with the questions, let me just remind the panel that you will have the opportunity to provide feedback on this approach as we address the questions. The purpose now is really just to get clarification on the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 82 Agency's approach. Dr. Skelly.

DR. SKELLY: I had a question about the designation of Xenopus laevis as a primary species and Rana pipiens as a secondary species. And I guess I'll leave it a little bit open ended. But I'm interested in why you made that distinction and what it's going to mean in terms of the timing in your conceptual model of when things would happen and what that means in terms of how the studies will be used in terms of weight of evidence or how they'll be prioritized in your thinking.

MR. TIETGE: Well, on the first point, the speed and utility of Xenopus laevis over Rana pipiens, I think, is fairly universally accepted. I mean you can do  I realize that some laboratories can produce Rana pipiens throughout the year for studying. However,

most cannot. And most are limited to collections that occur early in the spring to conduct Rana studies.

By contrast, one could have multiple studies within one year with Xenopus laevis and make some headway without having to wait until the natural breeding season for the Rana species.

Also, I think if, based on the studies that were submitted, I think the question is: Can Xenopus be a reliable surrogate for Rana?

With regard to this endpoint, taking the studies that have been done at 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 83 face value, I guess you'd have to say yes. If you have a surrogate that's more efficient, it's probably the way to go.

However, as with any of the issues of species extrapolation,

there's no substitute for a corroboration in terms of some comparative studies. And so the intent of my point that I made was that you might do the hard work, the voluminous work, with Xenopus and try to get as far into understanding the phenomenon as you can; and then, when you have a good handle on what's going on and you have a level of confidence that allows you to go forward, that you would then go back to Rana and do some confirmatory studies because I think you are limited in terms of the methods that are available with Rana. So that was my

DR. SKELLY: I'll just follow up quickly. So does this mean with your flow chart that you might do the work on Xenopus and come up with a negative result and stop there because you don't need to corroborate a negative result versus a positive result?

MR. TIETGE: Well, that's a great question because you never know what to do with negative results. It's hard to make decisions based on negative results. But you might still have enough concern,

based on the existing information, that you'd go back and do a Rana study at the organismal level even if it was negative in Xenopus. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 84 I think where Xenopus really has the advantage is when you get into the iterative studies, especially at the mechanistic level, where you're trying to define what's going on. Because then that would if you, for example, if the working hypothesis were demonstrated using that approach that I laid out, you might be able to very quickly then go onto Rana pipiens and verify that the same thing is going on. And that's what I was referring to as establishing a basis for inter species extrapolation when it came to the value of the mechanistic data.

DR. ROBERTS: Dr. Denver followed by Dr. Green and then Dr.

Gibbs.

DR. DENVER: Joe, some of the main concerns of the studies that were reviewed by the Agency were the variability within studies and also the variability among studies. And I'm curious if the Agency has considered ways to control for this variability in terms of the assays that have been chosen and the way to validate these assays among different labs.

I'm thinking about the two types of assays that you're proposing. One is the morphological assay where you're looking at gonadal morphology and the presence or absence of intersex individuals. And these types of scoring of intersex individuals may be influenced by subjective measures. Are there any other types of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 85 assays that may be applied to this, for example, by a chemical assay that may assay for testicular or ovarian antigens that may be more objective and may be even more sensitive? Has there been consideration given to developing standardized pools, say, of plasma that can be distributed to the laboratories for estimates of estrogens and androgens in blood plasma to validate those assays among laboratories, things like that?

MR. TIETGE: Well, with regard to the more mechanistic things you brought up in your last two points, no, we don't really have anything going on there.

But going back to your first comment regarding the subjectivity, I think our approach is that the histopathology has to be included. And histopathology or pathology in general is a subjective science. But I think it's often  I should say it's often a subjective science. However, it's a science that has a lot of confidence based on the experience and the review process that's a typical, modern pathology reviews and such.

So I think that you can use a subjective endpoint effectively, I think, if you include the histopathology. But with regard to the more mechanistic based things, I don't know of anything that's going on in that regard. And we certainly don't have anything right now. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 86 We are interested in developing those ideas. And I think there's movement in the Agency to develop and validate amphibian methods.

But there are no validated methods currently.

DR. ROBERTS: Dr. Green then Dr. Gibbs then Dr. Richards.

DR. GREEN: Regarding some of the secondary characteristics in the male that could be studied, I wondered if you could comment on why in the literature and anywhere else that I've seen so far there hasn't been an evaluation of the nuptial pads in Xenopus laevis.

They're very easy to see, quite prominent, in post metamorphic young juveniles. And certainly if they were feminized as a result of exposure to chemicals in the wild, I would think that those would diminish and that could be followed up in field studies as well as in the laboratory.

MR. TIETGE: So your question is  are you making a point, or are you asking a question?

DR. GREEN: No. I'm asking: Do you have plans to look at that? And it would follow up on Dr. Darcey's comment that you might have to extend the studies a little longer in order to be able to see them. But they can be detected grossly and histologically quite early,

I believe.

MR. TIETGE: No. I mean our plan is just a plan, and we're 87 1 looking forward to your input. And I think we'd accept that and move 2 forward.

3 DR. GREEN: Okay. In addition to the nuptial pads, another 4 thing would be the ventral folds around the cloaca in females are 5 quite prominent. And you would expect, perhaps, if males were 6 feminized, that they might become quite prominent in the laboratory 7 as in the wild as well.

8 DR. ROBERTS: Dr. Gibbs.

9 DR. GIBBS: A quick question about endpoints. There seems to 10 be an inconsistency insofar as in the Phase 1 studies insofar as the 11 survival is listed as an independently varying endpoint to be 12 measured. And yet in the recommended study protocol, survival was 13 something to be constrained to remain above 90 percent.

14 MR. TIETGE: 90 percent survival in the controls is what I was 15 referring to with regard to be an indicator the methods used in the 16 tests were sufficient to promote survival.

17 DR. GIBBS: Okay. Under the various treatments.

18 MR. TIETGE: Under the treatments, of course, that would be 19 given.

20 DR. ROBERTS: Dr. Richards then Dr. Coats.

21 DR. RICHARDS: Just a very general question relating to the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 88 two words " ecological effects." We've talked about reproduction,

some aspects of that. I just wonder, in a very general sense, what has the Agency thought about that term. Is what we're talking about here so far enough to cover the realm of ecological effects? In a broader sense of

MR. TIETGE: Broader than the ecological relevancy of, I think, Steve, you might want to jump in on this.

DR. BRADBURY: I think when you're doing a chemical risk assessment and we're starting with some observations from the field,

but certainly starting from sort of the building blocks of building of a hypothesis, a working hypothesis, it's sort of building up as opposed to top down, bottom up kind of thing.

Certainly most of the ecological risk assessments for chemical stressors are across the Agency not just in pesticides, working through what are the relevant organismal responses that give you insights into population or community responses. And I think it's fair to say that we're all at sort of the edge of moving into how do you take a look at population models, how do you start thinking about meta population models, how do you start interfacing chemical effects with habitat quality to understand the relative roles of those different stressors on a population or community structure. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 89 I think today, which is probably reasonably representative of other types of chemical risk assessments, we're still at that interface of how does the toxicology start to merge into population biology or landscape ecology. What are the insights from population biology information or landscape ecology information that give insights into those organismal responses that are most critical for population viability and what's a plausible toxicological mechanism to influence those endpoints.

On the surface, at least in this specific example, the discussions of measures of effects and risk assessment endpoints are at least qualitatively associated with reproductive fitness. But resolution spatial and temporal in defining that obviously would take increasing levels of information at all sorts of levels of biological organization.

DR. ROBERTS: Dr. Coats.

DR. COATS: Yes, when you're evaluating the reports, did you consider the analytical methodology used for chemistry especially in terms of the quality of the data or the selection of the methodology and how sensitive it might be or how specific it might be?

MR. TIETGE: We were aware of the level of detection that was associated with the assays that were used. We didn't have any input on the assay that were  again, there were no guidelines for the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 90 registrant to conduct these studies by. So we accepted the studies at face value in terms of what the level of detection was. But it was clear that the levels of variability that were associated with the measurements themselves were high and in many cases  in some cases, when backgrounds were subtracted from, when the assay background was subtracted from the treatment samples, they were actually negative values afterwards.

In our opinion, other methods from looking at those endpoints may be necessary. Rather than relying on ELISA assays, some other method may be more appropriate. Again, we're looking for input from the Panel to address those concerns.

DR. ROBERTS: Any other questions from the Panel?

If not, I think this would be a good time for a break. Let's take a break and reconvene at 10 minutes before the hour when Dr. Steeger will present Agency conclusions. So let's break for 15 minutes.

[ Break at 10: 35 a. m.; session resumed at 10: 55 a. m.]

DR. ROBERTS: We're starting now. So if folks in the audience, if you could make your way to your seats promptly, please.

DR. STEEGER: The computer is in the process of re booting.

DR. ROBERTS: All right. We'll await the computer.

DR. STEEGER: As I indicated earlier today, the criteria for 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 91 doing a ecological risk characterization is that the characterization has to be transparent, it has to be clear, it has to be consistent, and it has to be reasonable. The Agency has reviewed a number of studies.

And the process that we've used in making those reviews has been captured by Steve Bradbury, and it's discussed throughout my presentation. And critical in that process is the problem formulation.

And the iterative series of processes that occur between the risk manager and the risk assessor.

Based on the studies that the Environmental Fate and Effects Division has reviewed these studies that were submitted as of February 28, it has concluded that there are lines of evidence that suggest that the exposure to atrazine may result in developmental effects in amphibians. However, there were basic inconsistencies and variability in the studies that we reviewed that prevent us from either refuting or confirming those effects.

The Agency has recommended, based on its review, that a phased process be undertaken to examine specific measurement endpoints. And in conducting this phased process that Joe Tietge outlined, the Agency would be working its risk managers to determine the level of uncertainty that would be necessary to resolve in order for some decision to be reached. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 92 One of the questions that was posed to Joe was what was the temporal sequence in doing the phased studies. Joe indicated studies or different phases could be done concurrently. But the critical input comes from the risk manager. The risk manager has to be able to define the level of uncertainty that they're willing to accept in order to make a risk management decision. The risk assessors collect the information and help the risk manager understand how much of the uncertainty is associated with the data that are available.

We believe that the current studies contain sufficient uncertainty that we're unable to, as I said, refute or confirm whether atrazine is indeed having effects on amphibian development. But the bottom line is, and throughout my presentation, I stressed that, given the fact that over several studies and environmental conditions and species, atrazine exposure did appear to be having some impact on gonadal development.

But because of the lack of consistency and the type of effect elicited, the lack of a dose response, the inability of the current studies to demonstrate a plausible mechanistic action, and our inability or the Agency's inability to link the measurement endpoints that have been reported with our traditional assessment endpoints of reproduction, survival, and growth currently, we're unable to make 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 93 any statements regarding the utility of these data.

As I indicated in my presentation, though, the current studies do provide beyond the line of evidence, information on sources of variability and how future studies might be designed to better account for those sources of variability and provide reliable means of measuring the effects of atrazine on gonadal development.

Steve, would you care to add anything?

DR. BRADBURY: I think that Tom summed it up quite well. I guess as the Panel deliberates and we have some discussions, just emphasizing again using the Agency's risk assessment guidelines as a way to organize our thoughts and to think about the science at hand and the science in the context of making a regulatory decision, and sometimes that creates some different choices that one makes in terms of phrasing questions and articulating and understanding what the uncertainties are associated with different aspects of the science when the aspects of this science are in the context of making a regulatory decision.

And certainly we're looking very forward to your comments and input in terms of helping to define and understand the certainty that exists in the information today and your thoughts on those if you feel there are uncertainties that remain, the nature of those uncertainties 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 94 in the context of how we've taken a look at it, and then some approaches to refine the knowledge base.

The ultimate decision on how much information is enough to make a decision starts to leave the realm of science, but science is important to inform that process so that the decisions that are made are reasonable and transparent and clear as Tom indicated.

So I think at that we'd be happy to turn it over to Steve and carry forward.

DR. ROBERTS: Thank you. This concludes the Agency's presentation. I'd like to ask the Panel, again, if you have any questions regarding the presentations this morning before we move on in the agenda. Yes, Dr. Kelley.

DR. KELLEY: Given that you've decided that Xenopus laevis is going to be your primary experimental target in this, I wondered to what extent you would also require before changing the current regulatory environment that effects be demonstrated in native North American species. Is that a fair question?

DR. BRADBURY: Run that by me one more time. I don't think I understand the question.

DR. KELLEY: Well, look, so you decide to use an experimental animal that's found in South Africa. There are feral 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 95 populations here in America. But I assumed that we as Americans and EPA aren't too much worried about the survival of feral populations of Xenopus in, you know, Arizona golf courses, right, which is where they tend to live. So suppose a whole scenario was developed around Xenopus and effects of atrazine, to what extent could we then  to what extent could you use that information to apply to our own native species here in America.

DR. BRADBURY: Now I understand your question, sorry. It's a challenging question, and I think it transcends much of ecotoxicology and ecological risk assessment where you may be able to test, get information on a handful of species and potentially have to extrapolate to hundreds of species or tens of species at least in different landscape scenarios.

I'd like to back up to the first way you phrased your question to indicate that part of the lines of evidence to determine where we are and where we may need to go, in fact, took advantage of some field studies as well as some laboratory studies to develop the lines of evidence that it's plausible to formulate a hypothesis, although we may not have the confidence right now to quantify the probabilities of risk based on the information at hand. But the epidemiology type investigations as well as reductionist studies combined together 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 96 increase your understanding and your ability to quantify what you know and what you don't know and the gaps that may remain.

In the context of starting with a biological model to start to get some clarity into the issue at hand for the risk assessment is sort of a fundamental question. In aquatic toxicology, typically the Agency is using fathead minnows and rainbow trout and bluegill as species to try to represent what could happen to the thousands of fish species in the country.

Now, through problem formulation and thinking about which landscapes we're talking about, one starts to narrow down the type of species that one needs to focus on. But you're still dealing with the fundamental species extrapolation challenge. What are the toxicokinetic differences between species? What are the toxicodynamic differences or similarities across species to help put some bounds on the potential variability across the species. And you have to blend that with some of the practicality of generating toxicological information that provides the ability to control some of the natural variability of the world so that one can tease out the signal the chemical may be sending in terms of a dose response study and try to understand what that chemical is doing in light of all the other variabilities. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 97 So one trades off between a biological model that's well understood. What do we know about the developmental biology of a given species or the reproductive biology of a given species that helps us get insights into the potential effects a chemical may have. And it also then gives us insights into the issues we should be thinking about in terms of how do we extrapolate an effect seen in one species to other species. Do these species also contain the same receptor? How well conserved is the receptor? How well conserved are other biochemical pathways in terms of mechanism of action or detoxification or activation? What are the issues in terms of toxicokinetics uptake distribution? So all those things sort of come into play.

In the context of getting started on this challenge and our proposal, and, again, it's a proposal. It's a plan put before our scientific peers to gain your wisdom and insights as well. In terms of amphibian toxicology and in the context of developmental biology issues associated with a ecotoxicological risk assessment, Xenopus offers one way to get started efficiently to start to get some clarity in terms of the ability of atrazine to cause a reproducible response in terms of a developmental endpoint.

If one sees that, it gives some insights into aspects of species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 98 extrapolation. If that happens, what would be the observed mechanisms of endocrinology or developmental biology that would have to be present in other amphibians for an observation in Xenopus to be relevant to other amphibian species? To the extent we can extrapolate or determine a dose, be it an aqueous dose or a dose inside the organism, that gives a sense of sensitivity? What are the attributes of atrazine's physical chemical properties and the toxicokinetic properties that go on, processes that go on that help us extrapolate.

But our analysis plan doesn't rely just on  a proposed analysis plan doesn't rely just on Xenopus and then modeled into other species.

We are proposing to use at least the Northern Leopard Frog as a North American species to take a look to see if we get some consistency in an atrazine signal in a North American species.

But that's where sort of the juggling match between efficiencies and ability to get on with the question at hand. Xenopus we can use,

or laboratories can use all the time. Most labs would have to wait for spring cycles before they could investigate this issue in the Northern Leopard Frog at least. So it's a balance in terms of clarity of the model we're using, what you understand about you biological model,

what you understand about fundamental toxicological processes; and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 99 then sort of your ability to get information efficiently and effectively.