And you can study not only testicular development invasively and noninvasively, but you can also study clasping, which is the major reproductive behavior related to fertilization, and also 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 208 courtship song, whose importance in xenopus for successful reproduction is not clear, but is a very good marker for sexual differentiation.

DR. ROBERTS: I think what I have heard is our panel's response that there is no definitive information one way or another regarding ovotestes' effects as an impairment, perhaps some basis to speculate that it might, and some discussion of challenges associated with running those kinds of tests to provide the answer to the question and some suggestions as to approaches and some discussion about difficulties in setting up that kind of test and interpreting it.

Any follow up questions or clarifications needed? Let's go to the next one.

DR. STEEGER: Reduction in laryngeal muscle area suggests diminished testosterone in males. If this is found to be a valid observation and if estrogen concentrations do increase as testosterone concentrations decrease, what other endpoints, for example,

secondary sexual characteristics and reproductive behavior would likely be affected?

DR. ROBERTS: Dr. Green again.

DR. GREEN: The panel came up with a list of ten additional estrogenic biomarkers. The first five that I have here are not invasive 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 209 and would not require sacrificing the animal and are easy to identify phenotypically.

Obviously, the measurement of snout to vent length and body weight in feminized males should be bigger than the control males.

As Dr. Hayes pointed out yesterday, nuptial pads, the presence or absence or diminishment of nuptial pads and enlargement of the ventral folds of the cloaca strengthen the pattern of the male calling signal.

Dr. Kelley has some really nice recordings if anybody maybe wants to hear them during the break. We were impressed with how reproducible and how subjectively these might be measured.

We also were impressed by the fact that if you want to correlate loss of function, potentially the shrinking of the larynx muscles, with the morphological findings, then this would be a good way to show that not only is the larynx small, it is not functioning. You could do that by recording their calling.

Last, of course, like Dr. Kelley just mentioned, clasping.

There were some additional biogenic or biomarkers that people suggested. One of them we have talked about at length already. I won't go into it. But it is a time course examining the synthesis or the presence of vitellogenin in response to an estrogen challenge. And 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 210 then, of course, checking oviduct development.

There are proteins expressed in xenopus laevis in the harderian glands around the eyes. Three different proteins are expressed by those animals that are uniquely female and just one protein by the males.

Also, the number and the size of muscle fibers in the larynx and myosin expression in the larynx muscle.

And last, we have had some additional discussions on this one,

but it seems like seminal, I'm not sure seminal fluid analysis is the appropriate term in amphibian, but analysis of the sperm in some of these feminized males would be important to look at.

For example, are they morphologically normal when they mature. Do they have normal motility. Is the fluid they are found in normal.

So there would be an additional assessment of the fertility in some of these feminized males.

DR. ROBERTS: Comments by other panels members?

Dr. Heeringa.

DR. HEERINGA: I would like to make a statistician's comment on the use of the laryngeal muscle or some other sort of continuous measure of masculinity as distinct from other endpoints such as the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 211 gonadal abnormalities.

Looking at the data, I think  and secondarily to these other measures, I think having a continuous endpoint or continuous measure on a characteristic that appears to be and demonstrated to be related to testosterone levels in the male frog, I think would be important as sort of a secondary confirmation.

It may actually get a little further ahead in looking at mode of action than just at whether there is an effect. But I think to the extent that this was not costly or disruptive to measure when these animals are sacrificed, I think that it would add value to the experimental data.

DR. ROBERTS: Other comments. Dr. Kelley.

DR. KELLEY: As I have pointed out before, the measurement of cross sectional area of laryngeal muscle is a function of two properties, the size of the muscle fibers and the number of the muscle fibers.

Now, both reflect the history of exposure to androgen. So the two of them together are some kind of indication of the history of exposure to androgen.

However, let me just point out that while the size of the muscle cross sectional area is not very well documented developmentally in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 212 the literature, the number of muscle fibers is very well documented.

And becomes statistically significantly different between males and females at the stage which we call PM 1, which we have also characterized.

I want to point out that one of the advantages of xenopus are the very well characterized metamorphic stages, which allow standardization of experiments, so that when we talk about stage 56 animal, we know what we're talking about.

We carried out a similar set of study for post metamorphic development. Because the development of the larynx is largely post metamorphic. The brain is premetamorphic.

So we have those standardized data for numbers of muscle fibers. You can do it in the paraffin section and you can replicate previous studies.

So that will provide  it is a continuous variable because a number of fibers is distributed. But the data are very clear on when those are expected to become different and at what time. And it should provide a sensitive marker.

DR. ROBERTS: Dr. Heeringa and then Dr. Richards.

DR. HEERINGA: Thank you very much, Dr. Kelley. I yield to the experts on the exact nature of the continuous measurement. I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 213 think that's an excellent contribution.

My point is I think we would like to add this continuous measurement, something that is differentiated in terms of its outcome potentially at least in terms of observation from the abnormal gonadal development.

DR. KELLEY: The point I forgot to make was that in the model that EPA wants to develop for xenopus, you are going to terminate your experiments at stage 66. And there is variability in the results that we have been presented here as to whether there is a sex difference in laryngeal cross sectional area at stage 66.

We don't get a difference in weight. I never measured a cross sectional area. Dr. Hayes does get a difference in cross sectional area. Other people don't.

If you are going to do a grow out, the shortest period you should grow out is three months when the animals are PM 1. I refer to Tobias, et al., 1991 A, for a description of those experiments and these stages.

DR. ROBERTS: ' 91 A?

DR. KELLEY: The first was the stages. The second one was when during those stages the processes are hormone sensitive.

Developmental biology. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 214 DR. ROBERTS: Great. Dr. Richards.

DR. RICHARDS: I wanted to echo that the development of some of these measures, particularly Dr. Kelley mentioning the vocalization, the sounds related to the laryngeal muscles and clasping behavior, these would be really strong links to then begin looking at real population level studies.

If there is some concrete analyses and relationships that can be developed there, that would be particularly strong for the next step of studies.

DR. ROBERTS: Dr. Gibbs.

DR. GIBBS: One quick comment. I'm a little bothered with what seems to be an implicit assumption that bigger is better when it comes to laryngeal muscle area. Because females are cuing in all sorts of qualities to male calls. Not simply volume and repetition.

I just don't think it is necessarily that simpler a relationship that larger laryngeal muscle area corresponds to greater mating success via the effects on the vocalizations of these males.

DR. KELLEY: That's certainly true in rana, which are highly discriminating animals.

In xenopus, how can I put it, in xenopus, unless you have a male number of muscle fibers, you don't call at all. So if you make a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 215 genetic female have a male number of muscle fibers and have a male size of muscle fibers and she has circulating androgen, this is a testicular transplant study, she will call.

There is very good  I can tell you how much circulating androgen you need to have in a male for him to call. It is a central effect. Once his larynx is masculinized, it's masculinized forever.

But what I can't tell you and what nobody knows in xenopus is whether the calling male gets the females.

This is well known in rana where you can do phono taxas experiments in the ponds in South Africa. Finding out whether the calling male gets the female has not yet been established.

So unfortunately, the tractable experimental prep is not the prep for which we have those same kinds of data that you have in rana, which is  I'm not going to call them a more sensitive system,

but in some ways a more subtle system.

DR. GIBBS: But laryngeal area may not well pertain to being a success in rana.

DR. KELLEY: That's perfectly possible. And the other thing is of course in rana, you guys don't talk about female calls, but in xenopus, females have two calls. They have an acoustic aphrodisiac call that drives a male nuts, and they have a turn off call. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 216 So they have a very highly developed vocal system which they use in their social behaviors beyond sex  centered around sex, but getting a little bit further out.

So these are actually rather different vocal systems. The xenopus one is much more complex in terms of call number than the rana one. And you are absolutely right about ranae. We don't know the relationship.

DR. ROBERTS: If you think it is important, Dr. Gibbs, we could perhaps include the caution about extrapolation of the findings to reproductive success.

Dr. Skelly, did you want to add?

DR. SKELLY: No. That's what I

DR. ROBERTS: We can certainly put that in our report.

Anything else to add on this one? If not, let's go ahead and take a break. It's 3 o'clock. Let's reconvene at about 3: 15. We are miraculously ahead of schedule.

( Thereupon, a brief recess was taken.)

DR. ROBERTS: Let's go ahead and get started. Before we take the next question, just before lunch Dr. Kelley promised to assemble a bibliography. This was in response to Question 1, I believe, and some literature that we could perhaps recommend to the agency. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 217 She has put that together. That has been distributed to the panel. I would just ask each panel member to go over the list to see whether or not you agree with it. If you have papers that you might want to add to it and get back with Dr. Kelley on that.

DR. KELLEY: I apologize. They are slightly out of order and jumbled, but I didn't have very much time. If there are papers that aren't on it, just put the papers on it and I'll add them. If there are papers you want me to take off, I'll take them off, except if they are my papers, in which case that's nonnegotiable. They stay on.

DR. ROBERTS: Let's go ahead to Question 6.

DR. STEEGER: While some of the available data suggests that there may be an association between atrazine exposure and developmental effects in amphibians, the agency's evaluation of the existing body of laboratory and field studies has determined that there is not sufficient scientific evidence to indicate that atrazine consistently produces effects across the range of amphibian species examined.

However, the current body of knowledge has deficiencies and uncertainties that limit its usefulness in assessing potential developmental atrazine effects and the extent of any associated cause effect in dose response relationships. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 218 Consequently, the agency has determined that there are not sufficient data to reject the hypothesis that atrazine can cause adverse developmental effects in amphibians.

Does the SAP concur with these conclusions? If not, what lines of evidence would lead to an alternative conclusion?

DR. ROBERTS: Dr. Delorme, do you concur with that conclusion?

DR. DELORME: What I'm going to do is I'm going to actually go through the conclusions and break it down. I tried to put it through chromatic, and my computer crashed.

The first conclusion that I pull out is that there is not sufficient scientific evidence to indicate that atrazine consistently produces effects across the range of amphibian species examined. I had to agree with the conclusion.

We as a panel, I think, already concluded that atrazine could produce or might produce effects on gonadal development. However,

the consistency of the response across a species studied was difficult to asses because of the problems identified with respect to the design and conduct of both the laboratory and the field study. That confounds their interpretation.

The second conclusion is the current body of knowledge has 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 219 deficiencies and uncertainties that limit its usefulness in assessing potential developmental atrazine effects. I interpreted this as a risk assessor as meaning limits its usefulness in a risk assessment contact.

Certainly, I don't think I would want to conduct a risk assessment with the data that's been presented. So I agree with that conclusion.

Further, EPA needs to have results from studies done where other factors can be ruled out as a cause in either the presence or the absence of effects. I think that's one of the key things that you guys have brought out.

You need studies that are done where it's unequivocal that atrazine is the route cause of the effect. For example, you need good husbandry in the lab studies, good design for both the field and lab studies and some of the other factors that have been discussed in the other questions. I think that's key in gaining the data you need to do the risk assessment.

Another conclusion, it was stated as, and the extent of any associated cause effect and concentration response relationship. I think we had already agreed that from the data that has been presented, we can't say anything about the exact nature of the response, either the shape of the dose response function or thresholds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220 or whatnot.

We just can't characterize it at this point from the data that has been presented. It's recognized  this is a necessary element to conduct a risk assessment. You're going to need to have some sort of idea, whether it's a threshold response, if it's a dose response. What is the nature of the function of the dose response in order to conduct a risk assessment.

Consequently, the agency  this is the next conclusion.

Consequently, the agency has determined that there are not sufficient data to reject the hypothesis that atrazine can cause adverse developmental effects in amphibians. Agreed.

We agree that with the available  we agree with EPA that the available data does suggest that atrazine can affect amphibian gonadal development. However, the available data does not allow for a proper characterization of the nature and magnitude of the response, nor does it offer sufficient support for the identification of a plausible mechanism.

I guess in the end, if you add it all up, we agree with the conclusions, or at least I do and Joel. We wrote this together.

Joel, did you have anything to add?

DR. ROBERTS: That sort of answers my next question, but I'll 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 221 let Dr. Coats respond.

DR. COATS: I concur with the opinion there since it includes some of my ideas.

One other comment. We have spent over this time period and through 17 or so studies an awful large amount of time picking them apart and looking at every detail.

On the other hand, they do really constitute the body of what we do know about this. There is a lot we don't know yet. And I wanted to make the point they all have made contributions toward the progress here and the understanding of, is it a problem, is it not, or how big a problem is it or not.

That's one thing we should acknowledge.

DR. ROBERTS: Dr. Richards, did you have anything to add?

DR. RICHARDS: That I concur with the statements that the two previous speakers have made. I have nothing more to add.

DR. ROBERTS: I think Dr. Isom had a question.

DR. ISOM: I have a question for clarification. What do you mean by adverse developmental effects as opposed to just developmental effects, which we have been talking about previously?

DR. BRADBURY: Good question. And it, I think, ties back to the some of the previous questions in terms of the responses that have 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 222 been described in the literature thus far in the context of the risk assessment endpoint, which is getting at issues of reproductive fitness.

So again, had we probably polished the question a little better,

it would have been clearer, but in the context of these endpoints,

measures of effects in the context of the risk assessment endpoint that we laid out in the problem formulation.

So to the extent these, for example, gonadal abnormalities could be related to male fertility or reproductive fitness measures.

DR. ISOM: With that definition, would that change then the discussion we just had?

DR. ROBERTS: Does anyone feel that that would change the response based on  change the panel's answer based on the response by Dr. Bradbury?

Dr. Green.

DR. GREEN: Would you repeat that one more time?

DR. BRADBURY: I should pull out the problem formulation. I think we're all tracking, but we should make sure.

That abnormalities that have been described in the literature for an ecological risk assessment then need to be connected to the measures of effects, the risk assessment endpoint in the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 223 environmental management goal. The environmental management goal was maintain or viability of anuran populations, the risk assessment endpoint being connected to reproduction and fitness of populations, and the measures of effects being connected to those developmental processes that go on in amphibians that are related to their ability to reproduce successfully and then hence maintain populations.

So it is trying to maintain that causal chain. Does this toxicological effect move up the levels of biological organization? If you go back to that slide I had at the beginning of today, this midmorning, sort of the connection between the effects at different levels of biological organization connecting to the risk assessment endpoint.

DR. GREEN: I guess in my mind the question too implies the difference between whether or not the effect that we see is going to have an adverse effect.

For most of these parameters we have been looking at, we don't know yet because they haven't been carried out in grow out studies far enough.

So at this point, any developmental effect would be just a developmental effect. And maybe we should strike the word adverse 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 224 because we don't know if it is adverse yet or not unless it is associated with mortality.

DR. BRADBURY: Right.

DR. GREEN: Is that fair?

DR. ROBERTS: We can clarify that in our response that we interpreted this as being developmental effects and as we have stated earlier at least with respect to some of these effects that we don't know yet the consequences of these observations.

DR. BRADBURY: Right.

If we go back to some of the earlier questions, you all were discussing sort of the connections between changes in the larynx to potential calling or other kind of secondary characteristics, which is part of that discussion of what is the causal link, what is the toxicological, the ecological pathway that we're addressing.

DR. ROBERTS: We can put that, draft that, put the caveat in there.

DR. DELORME: The way it's written now is actually kind of broad. Because what I said is the available data does not allow proper characterization of the nature and magnitude of the response.

What we're saying is we don't know how far it is going, but I can amend that to say at the organism or population level, if that's 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 225 agreeable.

DR. ROBERTS: Sure. Does anyone disagree with the statements that have been made so far?

I don't see any. Any clarifications or follow up questions from the agency?

Let me clarify one thing for the audience. From time to time now as the panel have given their responses, they have indicated that they have worked with somebody else on the panel in terms of writing something up.

Under the Federal Advisory Committee Act, individual panel members can talk to other panel member is they have responsibility on a same topic, and sort of discuss the issues.

But I want to make very clear that the panel has not met and undergone any deliberations other than in this room in open session.

There have been discussions among panel members during breaks and in the evening, that sort of thing. As individuals, that's allowed, but there has not been any closed session of this panel to deliberate any of these issues.

Let's go Question 7.

DR. STEEGER: Assuming the agency determined an ecological risk assessment with a greater degree of certainty concerning 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 226 developmental effects of atrazine on amphibians were needed, please comment on EPA's conclusion that additional information is required to evaluate potential causal relationships between atrazine exposure and gonadal development.

Please also comment on the added utility, if any, of additional information to interpret the shape of dose response curves for potential developmental endpoints and the extent to which threshold or nonthreshhold response relationships can be quantified.

DR. ROBERTS: Dr. Delorme.

DR. DELORME: I'm going to handle the first part of this question and let Dr. Coats handle the part about the utility of interpreting the shape of the dose response curve.

I think we agree with that, the statement that  or EPA's conclusion that additional information is required to evaluate the potential causal relationship between atrazine exposure and gonadal development.

The relationship is there, we think. There is tantalizing evidence that something is going on. But it needs to be confirmed. Or what is being suggested needs to be confirmed. And we need to characterize the nature of the dose response function.

One of the tenets of the scientific methods is the repeatability 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 227 of experiments. Certainly, we have had attempts made to repeat this that have been submitted to EPA and we have looked at.

But definitely, we need to firm up the existence of the causal relationship, and, if it is there, if it does exist, we need to characterize the nature of the dose response function.

There is also a need to identify a plausible mechanism. I think one thing we have to recognize is the identification of the mechanism is important because it can in part aid in the extrapolation of the results from the surrogate test species, the species of concern in the environment.

DR. ROBERTS: Dr. Coats.

DR. COATS: I have a few things to add.

The plausible mechanism explanation needs to be put forth with some data.

Secondly, there needs to be some similarity of data or patterns or trends from several research groups to show repeatability of the experiments.

The dose response curves are extremely important to the question of any detrimental effect of any toxicant on an organism,

regardless of whether the relationships demonstrate a typical or atypical concentration response curve for a given endpoint. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 228 It should be possible to ascertain the shapes of the curve given enough concentrations, enough replications and controlled conditions.

Repeatability in other labs should also be feasible if the same species, stage, water concentrations and timing are utilized.

Another point is that studies on quantitative structure activity relationships can often provide information about the mechanism of action as well or provide rationale for the data is generated from comparative testing.

Experiments that use a series of closely related compounds,

atrazine, cyanazine, propazine, simazine, terbutyl azine ( ph) et cetera, could elucidate patterns that can help explain the interaction between the molecule and the putative receptor addressing the causal relationship.

This approach seems to be lacking so far and could be valuable in the invivo test for gonadal development as well as enzyme induction or MRNA expression.

DR. ROBERTS: Thank you, Dr. Coats.

Dr. Kelley, did you have anything to add?

DR. KELLEY: No. I completely concur.

DR. ROBERTS: Anyone else have any comments?

DR. GREEN: I also concur. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 229 DR. ROBERTS: Thank you, Dr. Green. Anyone else like to weigh in on this one?

Does anyone disagree, I guess I should ask that. I don't hear any disagreement.

Any follow ups or any clarification needed? Let's then go on to Question 8, which I will point out is a seven part question.

Dr. Richards has got that one.

DR. RICHARDS: Being the nonexpert that I am on almost every part of this

DR. ROBERTS: Let's let the agency go ahead and pose the question to us.

DR. STEEGER: The agency has developed a conceptual model from which to develop a set of study of protocols for evaluating the potential effects of atrazine on gonadal development on amphibians.

The agency has proposed a research approach using focused empirical laboratory studies based on initial investigations with xenopus laevis followed by selective confirmatory studies with frog species native to North America.

This is a proposal. It is not set in stone. We have  as has been indicated in a number of follow up questions. Please comment on the proposed sequence of the study objectives. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 230 DR. ROBERTS: Dr. Richards, we'll go through these I guess one at a time. Do you want to take the first one?

DR. RICHARDS: Yes. I'm going to invite the persons listed and not listed on this question to jump in on an open discussion here.

But on the please comment on the proposed study of sequence objectives, I think Dr. Kelley had already and several others have mentioned about the potential and importance of some types of field oriented studies.

That was the one comment that I had on this also, and not to preclude them in a parallel track with some of the other laboratory based studies.

DR. ROBERTS: Dr. Skelly and then Dr. Kelley.

DR. SKELLY: Just to support what Dr. Richards just said, I think it is going to be important to get the field component of any evaluation undergoing as soon as any of this happens in part because field studies for lots of reasons take a long time to set up and get going.

They are not necessarily as money intensive as some of the laboratory studies, but observational studies and experimental studies take time to find locations as we have seen in evaluating some of the past data. That can make a lot of difference in how useful these 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 231 studies will be when it comes to interpreting them.

I guess I just can't emphasize enough that I think it is going to be critical to get that side of things moving in order to meet the objectives laid out in the conceptual model by the EPA.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: Could you remind me again, what is the first thing that you plan to do?

DR. BRADBURY: Phase one in the white paper.

DR. KELLEY: Oh, yes.

DR. DELORME: Test for apical gonadal effects of

DR. KELLEY: So I concur with that. I would argue that we should attempt to strictly replicate some of the studies that show an effect, especially a low dose effect.

I accept the fact that in a replication one will often want to add groups and so on. But we have a real discrepancy in the threshold for an effect if in fact one exists.

I would argue for a strict replication, perhaps a replication of the high dose and a replication of the study that did the low dose.

I think it is worthwhile knowing how reliable and repeatable the initial observations are before we go forward.

Now, there has been  anyway, I could go through it. But I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 232 think it is very important to track down the sources of variability and the results before going forward. Because suppose you were never able to repeat any of them or you always repeated them and in a much lower dose.

It would affect so strongly the next steps in the sequence that I would argue very strongly for starting with that.

DR. ROBERTS: Dr. Kloas.

DR. KLOAS: I concur with the comments already made.

I would also point out maybe it is time saving and saving money if you could start again already in parallel with doing some mechanistic studies.

For instance, there was some claim there is no interference with estrogen receptors, with androgen receptors.

There are some experience which could be very easily maybe to say there is nothing going on. Especially, I have some concern maybe if there is only a small effect on thyroid system, you will not see it on morphological  looking just on morphological stages.

There could also be one biomarker, for instance, TSH, to show up any inhibitory effect in addition which could be done in parallel for doing such a developmental study.

DR. ROBERTS: I'm going to jump in and make a comment too. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 233 I think that if we do additional mechanistic studies, if there are variable aspects, I think we  as Dr. Kelley pointed out, I think we have to get a handle on those first because we have to know when animals respond and when they don't respond.

Because we want to be sure we're looking in animals that are responding to do, to see whether or not the mechanisms we think might be operating are, in fact, in place.

I don't disagree that mechanistic studies are important, but I think getting a real solid handle on the phenomenon and being able to reproducebly observe that, I agree with Dr. Kelley, is a first priority.

Dr. DeLorme and Dr. LeBlanc and Dr. Denver.

DR. DELORME: I just wanted to concur with Dr. Kelley. I think that what has been proposed is quite a logical sequence. The first thing you really need to do is confirm whether or not there is any relationship between atrazine and the effect.

And certainly what Dr. Kelley had proposed was repeating the experiments to see if you can find that, but do it under controlled conditions where the husbandry and whatnot is  any factors that might affect it are controlled for.

Followed by looking in possibly native anurans from North America to see whether or not you are going to get the same kinds of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 234 effect. I think that's another good logical step to take.

DR. ROBERTS: Dr. LeBlanc and then Dr. Denver.

DR. LEBLANC: From my perspective, the further you go up in that cascade of complexity that Dr. Bradbury had up there earlier on,

you are reducing your chances of seeing the effect that you are trying to confirm upfront and sort of give you some evidence that you should proceed.

Certainly, I wouldn't do preliminary studies or initial studies at the population level. I think gonad development is the critical endpoint of interest, but I'm not sure I would do it at that level.

I think that perhaps the first experiments that I would do would be looking at some cellular response that I'm more likely to see. And once I have identified doses at which that response occurs and I'm comfortable that that response is occurring, I think I would build upon that.

And then I would look at tissue level effects in terms of gonadal development and ultimately effects of the individual and the populations, is my perspective.

DR. ROBERTS: Dr. Denver.

DR. DENVER: What we really want to know here, we want to know whether there are population level effects ultimately. Is that 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 235 correct?

And that's something that is going to take presumably a while to sort out.

The next level, I assume, is to understand what might be fitness effects at the individual level.

And I wonder  this actually is a more general question that goes beyond just simply atrazine. But I wonder if we can begin to get a handle on those fitness, those individual fitness effects by looking at individuals or individuals that are presumably intersex or have gonadal abnormalities in the field, which I think we agree has been documented, and find a way to identify those individuals which we discussed a bit earlier perhaps using some MRI approaches or something of that sort. And do the grow out experiments and determine if there are really fitness consequences of having these intersex gonads.

Is that something that we should be doing now? As I said, it goes to a larger issue, larger than just simply atrazine.

And these intersex individuals may result from contaminants other than atrazine. But is that something that we want to consider sooner rather than later?

DR. ROBERTS: We're getting some interesting differing views 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 236 on sequence of or highest priorities.

Any other panel members want to weigh in on this?

Dr. Delorme.

DR. DELORME: That is actually an interesting thought. I never thought of that before.

But if there was some chemical that you could come up with to induce intersex that you knew for search was going to the happen, that might answer some of the questions with respect to fertility and possibility of effects at higher levels of biological organization.

Instead of waiting to find out what is actually going to go on and identify

You could do it in parallel with atrazine. Because certainly as we go through  as risk assessor, I'm talking, as we go through other chemicals, there certainly are going to be cases where these kinds of effects arise.

It is always a question. What is the ecological relevance of ovotestes. I know in the fish community it is something they are grappling with now. Because in a lot of the rivers in Britain and in the states, they have identified fish populations where ovotestes type effects do occur. The so what question is still there. Well, do they contribute to the population or does it have a population level effect. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 237 So that's actually an interesting take on things.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: So there is good news. There is a chemical that does it. In xenopus, estrogen does it. It is very well worked out. The critical period is worked out. How much ovotestes. I'm using that word in the sense in which I used it before, and I refer you to the figure in the Witschi paper. It is very well worked out.

The animals that were in that paper were adult. They persisted until adulthood. It looked like it wasn't resorbed. I would suggest that there is no necessity to run this as a serial experiment. I'm totally opposed to that.

You run it in parallel. You run it in parallel also with beginning your ecological assessments, because I'm in total agreement. Field work takes forever. Getting it published takes forever. Anyway, just doing the work takes forever.

So I would start the field studies and I would start a replication of the previous studies on the atrazine effects. I would do it in ZZ animals. You know what genotype you were dealing with.

I would do it in a well characterized population of animals and I would run a group along with it with estrogen at various time periods during the defined critical window. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 238 And then you have to grow those animals out. It's a male.

That's the good news. It's only six months. And then you run those animals in behavioral and fertility tests. And then you know if animals with 66 percent ovaries and 33 percent testes or something produce fewer offspring than animals with a different ratio.

Those studies are actually fairly easy to do in a well defined controlled way. You can either use natural behaviors or you can kill the animals and mush up their testes and fertilize eggs with them.

You don't have to kill the animals. You can just take their testes out.

DR. ROBERTS: Other thoughts?

Lots of different ideas, Dr. Richards, unfortunately for you to capture.

DR. RICHARDS: I'm sure many of those words will be on to a text file and given to me.

DR. KELLEY: Yes, after dinner.

DR. ROBERTS: Before we move on to B, let me make sure. Is there any follow up question or is it as clear as it can be given the fact that there are some differences of opinion about sequences?

DR. BRADBURY: I think it would be helpful for the dialogue to continue a bit. I'm approaching this carefully. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 239 And with all due respect to all members of the panel, in the context of science, for a purpose, which is trying to incrementally improve our ability to reduce the uncertainties on the risk assessment and how that marries up with and is complimentary to advancing knowledge, because it is important to advance knowledge, and to the extent the panel has any feel of, for lack of a better word, where you could get the biggest bang for your buck to start either sequence how you would stage information gathering or a sense of what information we start to clarify or reduce some of the uncertainties in the current ability to go through the risk assessment process.

It is sort of  the dialogue has been interesting. I think Jere in thinking about the endocrine disrupter screening assays and some of the other descriptions about using QSAR and then going from the field down sort of to illustrate very legitimate differences of opinion in terms of how you blend mechanistic understanding with ecological relevancy and where are you in that continuum, it seems like we're getting advice to do everything all at once.

And while maybe that's the only way we can solve this problem,

if that's the conclusion, then that's the conclusion.

I think it would be helpful to hear about what some of the trade offs would be at least in picking different places to start in the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 240 phased approach that we provided.

It may mean you may all feel that you should start all five phases all at once, and that's cool. But it would be helpful to at least get input from the panel in terms of the trade offs if one couldn't do all five phases at the same time.

DR. ROBERTS: Let's ask the panel. What if you couldn't do them all at once, where would you start? Dr. Kelley and then Dr.

Green.

DR. KELLEY: One in five.

DR. ROBERTS: Beg your pardon?

DR. KELLEY: That was it.

DR. GREEN: Testing the working hypothesis phase experiment Number 1, the test for apical gonad effects and Number 5, ecological relevancy of the study. I concur with Dr. Kelley's comment.

But since you have solicited additional dialogue, I have a few more things that I think are imperative to standardizing the conditions. And this should be done in Phase 1. So right from the get go we can do the best we can.

One of the things that struck me yesterday about the housing and the husbandry conditions was the extreme variability and the fact that one of the things we were measuring was growth rate and trying 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 241 to make a connection between the effect of atrazine on growth rate and then on the growth rate in the control tank when between labs and within the lab there were specific issues with water quality and feeding and other things that could account for variability in growth rate alone whether atrazine was there or not.

So I'm going to take the opportunity to make a plea. And part of this bleeds into Question Number 4, which I was actually waiting to answer, about two issues.

And the first one is with water quality. This is not pertaining to measuring levels of atrazine. But I think we should make some attempt to define the stocking density first off for both embryos and adults and juveniles for xenopus laevis.

Unfortunately, that hasn't been standardized in Laboratory Animal Medicine even yet, but there are some recommendations.

The proceedings for the National Academy of Sciences has recommended one to two liters for adults. And there are various textbooks and original papers for tadpole stocking and density. I think we talked about that a little bit already. So that would be one thing.

One thing that was lacking in the descriptions of many of the papers was the kind of water that was used. I couldn't tell. Was this 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 242 deionized or R O treated or reconstituted salt water. Was it well water from a source. Was it chlorinated or chloraminated, potable tap water that had been filtered.

And I think all those water sources are perfectly suitable. But it needs to be stated and, if possible, standardized so that everybody who runs the test on atrazine uses the same kind of water.

The other thing would be water quality testing. For this purpose, because there is evidence in the literature that atrazine and nitrites interact and that they have some by product of ammonium metabolism, when the ammonia levels get high in tanks, it may falsely elevate the detrimental effects, if any, that any other chemical has in there. It could be chlorine. It could be atrazine.

So water quality should be measured on a regular basis.

Depending on the duration of the experiment and the stocking density and the water turnover, I would say once a day. It could be done with a quick dip stick that is a relatively inexpensive test. It doesn't have to be the Hawk ( ph) analytical test every day, but some notation of water quality parameters on a regular basis.

That would include pH, conductivity, water temperature,

ammonia, nitrate, nitrite. And because we want to know what kind of water is used, we should be measuring for chlorine and chloramine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 243 and the heavy metals that we know interfere with tadpole development like copper and some of the other things.

So I would like to see that in most of these reports at least have been part of the quality control.

So water quality is one issue in Phase 1 that I think if we could standardize. And you can't go to the literature and find this.

There is a paper that was published in the Laboratory Animal Medicine Journal that reported a survey across the nation of what most xenopus users are doing right now that you can use as a guide.

I have the reference here that I'll add to the list for Dr. Kelley on all these parameters.

The second issue that I wanted to bring up was feeding. I'm sure Dr. Kelley will bring this up as well.

But Dr. Hayes pointed out that he felt that maybe some of the animals in the Syngenta studies had been underfed. And I looked at that  I went back and looked at that paper and looked at some of his publications on what he was feeding.

It struck me that he is feeding rabbit chow. I find this a little bit disconcerting because adult xenopus are strict carnivores. And that is an herbivore diet.

DR. HAYES: The adult xenopus were fed trout chow, not rabbit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 244 chow.

DR. GREEN: Dr. Hayes in the back said his adult xenopus were fed trout chow. But if we continue these studies into grow out experiments where juveniles and adults are used, then we should probably feed them a diet that's at least 14 percent protein, which is more appropriate for a carnivore.

There are references. Kevin Wright has published in another textbook, Amphibian Medicine and Husbandry, a chapter. In that, he recommends that we not feed adult and juvenile xenopus laevis,

anyway, diets for omnivorous fish and turtles or for herbivores.

So you can go to a feed company, there are several reputable ones, that will make xenopus chow for you or you can buy it from the big distributors. Nasco and Xenopus 1 have their own that they sell.

I think in an attempt to standardize across labs, that's probably the appropriate food for that age.

Now, tadpole feeding  those companies also sell food, if I'm correct, for younger metamorphs and juveniles as well. And they eat phytoplankton.

I'm not so certain how important it is that they have a high protein diet to maximize growth and health under these conditions.

But if you are going to make those comparisons in growth rates 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 245 and fecundity, then probably standardizing to a carnivorous amphibian diet would be the best thing to do.

The companies also have based those diets and the amount of feed on growth rate curves that they have developed. I believe they will share them with you. I have asked for them and they have been very helpful in advising me.

They also make recommendations on how much to feed per animal. That's based on known anuran kilocaloric requirements at different temperatures.

So if we hold tanks for some of the juvenile and adults at 19 degrees versus 25, we should feed them accordingly. There are ways to do that.

If you look to the fish literature, as young fish grow, they are regularly weighed once a month on mass. The tank feed is adjusted accordingly.

I know that it is not standard practice right now to do that for xenopus. Usually, you have about, whatever, 5, 50 or 100 in a tank and you throw so much food and make sure they eat it all.

But in terms of water quality level, what is not eaten and the amount they excrete will affect the amount of ammonia, which in turn affects the amount of atrazine that is or isn't available or potentiated. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 246 Those are two conditions, at least, the water quality and the feeding that I think this Phase 1 should be spelled out as best we can.

And the temperature that we all agree would be the right place to start with in conducting these experiments.

DR. ROBERTS: I think it will be important for us to convey Dr. Green's recommendations in our report. I don't think it necessarily belongs as part of 8 A, but we'll get it in there. I think it is important to pass that information along to the agency.

With regard to the question posed by Dr. Bradbury, if you couldn't do them all at once, what would you do?

Dr. Delorme.

DR. DELORME: I would concur with Dr. Kelley that probably the first thing you want to do is  if your goal is to move the atrazine risk assessment forward, then I think you have to confirm the causal relationship. That's the first thing you need to do. That would be the first priority to do.

DR. ROBERTS: Dr. Coats.

DR. COATS: I think that the laboratory approach as described in Phase 1 is probably the most obvious and least risky from a benefit  from the point of time invested and money invested.

And so that's where I think we should start. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 247 DR. ROBERTS: Dr. Skelly.

DR. SKELLY: At the risk of boring everyone, I'm going to reiterate that I think pushing ecological relevancy off to Phase 5 could be a mistake.

We're testing for apical gonadal effects in Phase 1. Then Phase 2 is sex steroid measurements. Phase 3 is aromatase activity measurements. Phase 4 is aromatase inhibitor study. Phase 5 is ecological relevance.

I think there could be a giant woops there if we get to the end and find out that the population level effects aren't what we're all being concerned about here.

DR. ROBERTS: Dr. Delorme.

DR. DELORME: Just as a point. If you look at figure one in the white paper, it actually shows after the test for apical gonadal effects and a yes being found, there is a line off to the side with Number 5.

Can EPA comment on what was intended with that? It wasn't clear whether or not you intended the ecological relevance test to start at that point.

DR. SKELLY: Is ecology fifth level priority or not?

DR. TIETGE: It just so happens that we used numbers. We 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 248 could have used colors I guess to indicate what order to do them in.

Clearly, as Dr. Delorme pointed out, we had that split in the sequence to suggest that if you get effects at the individual level, then it may be more prudent from a risk assessment point of view to conduct some field work.

I wouldn't read into the ordinal numbers here very much.

DR. ROBERTS: Does that help, Dr. Skelly?

DR. SKELLY: Yes. If I can follow up.

I'm fine with that with one important caveat. That is, we're going to get to this a little bit farther down. But if the panel feels that there are significant concerns about context dependence between species, and at least in my case I think we have seen some evidence that that could be the case, then I don't see that a no on apical gonadal effects in xenopus necessarily means that we shouldn't be thinking about an ecological relevance study for native species.

DR. ROBERTS: Dr. Bradbury.

DR. BRADBURY: Just to get a clarification, because the white paper talks about using xenopus to get started as a biological model bla, bla, bla, but also talks about other species to also be looked at in the context of phase one style experiments.

I guess the clarification that if xenopus didn't reproduce the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 249 previous results, would the panel feel it would be useful to try it out in rana in the Phase 1 style experiment or to immediately go to a long term reproductive study or go out to the field to look at rana or other native species?

DR. ROBERTS: Expression of preference on that?

Dr. Kelley.

DR. KELLEY: Well, you know, I'm for parallel processing.

Let's face it. We don't care here in America about the survival of our xenopus because they are well taken care of in the lab.

But we care about the survival of our native frog species. You are just going to have to start the field studies right away. But I do agree that it would be useful to see if we could run some rana experiments in the lab.

Of course the problem is rana is a lot harder to run in the lab.

And rana is about a zillion times more difficult to work on than xenopus and so forth.

But that isn't to say that it shouldn't be attempted, because it will be very informative. There are folks that are good at rana. I'm not one of them. But I know there are people that are.

It would be worthwhile to try in a limited way to see if we could get that to go. But it is going to be a lot harder. The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 250 background literature that is available for xenopus is just not available for rana.

It will be somewhat more difficult to interpret the results. But those are the results we care about. Because these are our frogs, our North American frogs. We care a lot about them.

We should study them along with the indicator species, which has many, many advantages, but is not native to our country.

DR. ROBERTS: Other thoughts?

Dr. Richards.

DR. RICHARDS: I concur with what Dr. Kelley has brought up here. I think that ultimately the agency is going to have to go there no matter what answer we get from xenopus. And it could lead us down some very good trails.

Ultimately, the question is going to come to is what does that mean to ranid species. And if we don't start now in trying to further hone or develop methods that are appropriate for them, it is just going to prolong the whole process.

DR. ROBERTS: Dr. Tietge.

DR. TIETGE: I think what we have to keep in mind is that using the rana species or the intent of using the rana species is to do the confirmatory work. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 251 But you do the hard work I think in xenopus because it is a more useful model at this point in time. So I don't think we meant one or the other.

I think  I agree with what I'm hearing here that you probably will end up doing it in both species at least in that Phase 1 study.

But the intent of the xenopus is to go faster and farther to understand, for example, mechanistic pathways that you would then go back and in a more focused approach look at with the rana species.

DR. BRADBURY: Just to help clarify because you've been getting some understandably different kinds of views.

At one point we heard we should try taking account of variability associated with some of the current studies to try to see if we could replicate what had happened in the past.

And all there is a ranid study. There are several xenopus studies. That would imply get started with xenopus to see if you can get it to happen in xenopus again. Start with ranid.

Again, I know you are probably thinking this through as we go.

But as the panel deliberates, it will be helpful to get, if not definitive answers, at least some thoughts of the cost benefit, that's not the right word, but the ups and downs, the trade offs associated with some of the different choices in the pathway. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 252 DR. ROBERTS: Dr. Denver and then Dr. Green.

DR. DENVER: Let me sort of echo the comments of Dr.

Richards and play devil's advocate.

What if you are unable able to replicate the xenopus results and you are unable to show in fact any effects of atrazine? What decision would you then make?

Would you then decide to go on to ask the question in North American ranid species or would you conclude that, in fact, there is no basis for concern?

DR. BRADBURY: I think some of that dialogue  some of the issues that one would face in making that decision are some of the latter questions we have in the series of question eight which gets at some of the issues about toxico dynamic and toxico kinetic differences among frogs.

And to not dodge the question, I think I would benefit greatly from the panel's dialogue on those issues that could drive interspecies extrapolation.

DR. ROBERTS: Fair enough.

We've had a lot of dialogue. I hope Dr. Richards has been keeping up.

Let me see if we can decide whether or not the  is there some 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 253 sort of consensus in terms of priority or are there still differences of opinion?

Dr. Richards, what is your sense?

DR. RICHARDS: I'm hearing that most of the panel seems to feel comfortable that some of the  the Phase 1 experiments need to be replicated, techniques cleaned up. That work needs to go forward.

I'm hearing that from most people.

I have heard from a little bit smaller number that some aspects of Number 5 need to go forward in terms of grow out or some of the other basic ecological studies. Not necessarily full ecological studies.

DR. ROBERTS: I sort of heard unanimity in the desirability for the Phase 1 studies to proceed. And some support for, if possible,

beginning the Number 5 ecological studies, because recognizing that that  demonstrating that relevance would be very important for the risk assessment.

Does somebody else have a different take?

Dr. Gibbs.

DR. GIBBS: I would concur. I thought Dr. Kelley had outlined a sequence that is really quite doable in a fairly short time frame in terms of looking at the fitness consequences with intersex in normal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 254 males and then you could actually put those in a natural environment and look at the consequences and mating with maybe even molecular genetic markers getting the tadpoles or metamorphs.

But I don't think until there is  rather than a full blown ecological study, 100 wetlands, et cetera, we're not talking about that necessarily for Phase 5. I think just a shorter term study like Dr.

Kelley outlined I think is quite doable.

DR. ROBERTS: Does that sound reasonable to the panel? All right. Good. Then let's try and have the minutes the reflect that recommendation.

Any follow up questions from the agency? Dr. Green and then Dr. Isom.

DR. GREEN: I wasn't clear now. Was the decision that ranA and xenopus experiment should be run in parallel, if possible?

DR. ROBERTS: I think we're going to take up the species

DR. GREEN: Later on?

DR. ROBERTS: Yes. As we go a little bit further down.

Hopefully, after we have that discussion, then we'll be able to clarify our recommendation for that aspect.

Dr. Isom.

DR. ISOM: I wasn't clear also with regards to the first study. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 255 A lot of data, mechanistic data, can be obtained, related mechanistic data can be obtained from that study. Are we just advocating looking at, say, anatomical gross abnormalities in these animals, the survivability, or are we also saying we should be looking at blood levels of hormones and other associated effects in those animals?

If you have those tissues of those animals, why not get the mileage out of them?

DR. BRADBURY: 8 B starts to get at some of the very issues you are bringing up.

DR. ROBERTS: I guess my initial response, Gary, would be to encourage doing superimposed mechanistic studies while they are doing these.

DR. ISOM: It wasn't clear.

DR. BRADBURY: In fact, Question Eight B charged the panel,

very explicitly asks the panel for advice and counsel on the attributes of these studies.

DR. ROBERTS: Then let's go to Eight B.

DR. STEEGER: Please comment on whether the agency's first set of proposed studies has accounted for the major sources of uncertainty associated with the potential effects of atrazine on anuran 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 256 sexual differentiation.

In addition to the time to metamorphosis, gonadal abnormalities and sex ratios in the proposed Phase 1 assays, please comment on any other endpoints that should be considered in this initial phase.

DR. ROBERTS: Dr. Richards.

DR. RICHARDS: No. The first part I think we pretty much elaborated on, it seems to me, in several of the other questions. No,

we haven't accounted for the major sources of uncertainty associated with potential effects.

And I'll let the discussion open up in terms of the other measures as Dr. Isom and others have initially brought up.

DR. ROBERTS: Dr. Green would like to comment on that.

DR. GREEN: I think I already commented basically on the husbandry issues. But in terms of in addition to time to metamorphosis gonadal abnormalities and sex ratios, please comment on any other endpoints, and we addressed that with the 10 different estrogenic biomarkers.

I'm not clear if the goal here is to get these studies done quickly and in what level of detail we want to look at these things. Because all 10 of them would be a lot of work. The first four or five, which 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 257 are observational and easy to see, I wouldn't suggest would be the place to start.

DR. BRADBURY: I think that kind of a discussion would be helpful. What would be the experimental investments, time, effort associated with adding the different endpoints in, what is the kind of information that is gained as one gets that information.

Some of the logic in the different phases, probably isn't the right word, but the components or the trying to sequence things is sort of an approach to if we get the frank apical effects, then  part of this question is getting at are there ways to maybe blend some of the concepts that are in the analysis plan and try to link some things up more quickly. That's the question.

Getting some insights on what some of the efforts would be and some thought about what would this experimental design start to look like I think is important.

DR. ROBERTS: Go ahead and follow up, Dr. Green.

DR. GREEN: We're trying to decide here  in Phase 1 let's say we're going to start with xenopus laevis. And in Phase 1 we're going to grow them out to three months. That was something Dr. Kelley suggested. And I concur that would be the minimum because then we could see some of the secondary sexual characteristics start to emerge 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 258 or not.

And we would look at different stages and ages along the way out to three months. Is that correct?

DR. KELLEY: Let me just talk about a moment the sex ratio.

You are not going to be able to interpret the sex ratio unless you have a uniform genotype. Here is what I would do. I would get Nasco or Kelley Evans at Xenopus 1 to establish for me a stock colony for researchers to draw their animals from.

They will be willing to do this. I would like to point out to the audience that these places are agricultural facilities in their home states. They support the greater agricultural goods. Xenopus is a farm product.

Regulate is a farm product in many states. We're growing frogs here. It's not corn, but frogs.

Anyway, so you have to start with ZZ animals, otherwise the sex ratio becomes very difficult to interpret without long breeding experiments. It takes two years to get a female to breed. You don't want that. It will really slow you down.

How long will it take to get ZZ animals. If you get donated ZZ animals that people have, you would have to confirm they really are ZZ by mating them to normal males and having all male offspring. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 259 That would take three months.

If you have to make them, that will take six months. Three months minimum, six months maximum.

Then you would run your experiments in the presence and absence of atrazine and various other things, and you should be able to get data on that in, I would say, six months. That's a year.

Nine months to a year minimum. With things going wrong,

that's 12 months basically for sure and maybe 18 months.

So 18 months to replicate with a known population under defined growing conditions. That's what that would take in a lab that was up and functional.

In a lab that had never dealt with frogs before, though, I would like to point out that it will take longer. If you contract this out or something to people who have never raised xenopus, it will take longer because there is a learning curve, learning how to keep the animals and keep them happy and so forth. Just catching them.

Learning to put lids on so they don't hop. These are major things.

Is that the sort of thing you want? That's how long it is going to take just with xenopus.

DR. BRADBURY: In the white paper we laid out attributes that may or may not be consistent. Some may be, some probably not 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 260 consistent with what you just described.

On the screen, we have laid out some of the details of that.

DR. KELLEY: I disagree deeply with some of the details.

DR. BRADBURY: Again, it was a plan to get this kind of dialogue going. I'm trying to figure a way see if I can get the chairman to help us sort of systematically explore some of these.

DR. ROBERTS: I think there is lots of issues. In CS for range and spacing and number of concentration, some of those things are up here  you are looking for feedback on what should be done in these experiments. And I think we can go sort of go through those aspects.

I think your first question was in B is what endpoints should we look for. And so let's answer that question. And then we can go on to provide you with feedback on other aspects of the experimental design.

You asked for a little bit of dialogue for us in terms of if you did this, it adds this, but it costs this kind of thing. Not only in terms of dollars, but just to give you some sort of feedback in terms of what you get for what extra effort and resources are invested in terms of adding different endpoints.

If I understood you correctly, Dr. Bradbury, that's the kind of advice they are seeking from the panel right now as part of this 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 261 particular question.

So if we have some suggestions for them in addition to the endpoints that are identified here, some information about what else,

pros and cons.

DR. KELLEY: Since you are concerned about fertility, you have to add a rate of fertilization of eggs as an endpoint. And there are a number of different ways to do that.

Some less variable than others. But that's an important endpoint. And that will require growing animals out longer. Six months.

So that will add to the cost of it. But it is an important endpoint for your goal. I would argue the most important endpoint for your goal.

Do you need more detail on how to do that? There are 42 different ways.

DR. ROBERTS: I don't think at this point. But I think that's exactly what  provide you with this information which is important,

but it means the experiment has to go longer.

DR. KELLEY: Right. But it is not very fancy. You don't have to learn to do a vitellogenin induction assay.

You get the testes and mush it up and fertilize the eggs, you 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 262 know.

DR. ROBERTS: Other suggestions? Dr. Isom and then Dr.

Skelly.

DR. ISOM: We have talked around this and some of our speakers have mentioned this. I think perhaps this accounts for some of the variability we have seen among studies.

That is, clear definition of the endpoints. We really  even to define sex ratios, how are we doing that? There is all kinds of variability in the terminology, and I would really encourage somebody to sit down and come up with some standardized terminology and endpoints and how you are going to evaluate those before these studies are conducted, at least this phase one.

DR. ROBERTS: I think that's something that everyone on the panel will concur with, is that the agency, as you begin this effort, is going to have to standardize the terminology for the endpoints by convening a workshop or whatever mechanism is the best way to do it.

But there has to be some terminology that everybody is using and agrees upon. We clearly saw the issues as the agency has. This is not something new. You are not surprised by this recommendation.

But I think that's something we'll make in our minutes.

Other endpoints? Dr. Skelly. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 263 DR. SKELLY: Just to build on what Dr. Kelley suggested, if you are going to grow animals out and add that extra investment, I think it is going to be important to beyond castrating the males and mashing up their testes to figure out, and I don't know enough about xenopus to know how this could work, but in the species I work with,

it would be possible to measure reproductive behavior and reproductive endpoints of whole living animals.

That would measure their reproductive function. Because what Dr. Kelley suggested is taking a look at some index of male fertility.

But we don't know what is going on in this animal's brain and whether it is going to act like a male or female or what it is going to do. I think that's critical to getting beyond the kind of physiology to the behavior in the ecology.

DR. ROBERTS: So you are suggesting another possibility is to add reproductive tests that involve behavioral component, but the downside is that there is  is the methodology relatively standard or this something they would have to work through?

Dr. Kelley is  I'll let you respond to this.

DR. SKELLY: Before Dr. Kelley builds on something that I say, just let me say that I don't know that the methods are standardized. They probably vary quite a bit from species to species, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 264 because you are turning on frogs. They all have their own little things they like.

But it's certainly possible to do. And I think it adds significantly too, and then you can mash their testes up.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: So that's what we study, the reproductive behavior of male frogs and female frogs. You can do it. And we have standardized assays for doing it.

There are uncertainties associated with this. I find in my lab that the more things you try to get out of a single animal, the greater the possibility that you will compromise one of the measurements.

I don't like killing animals. I always try to get everything possible from the animal if it is going to give its life in the name of science. But there are problems with running that kind of experiment where you end up confounding it.

If you have a protocol for stimulating the testes that is very reliable and then you take that same animal and test it behaviorally, I think you could do that. And you could test clasping for which there is normative data. The papers are on the list. You could test the vocal behavior quite easily.

So you could do that. It would add to the study. It will be, to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 265 be honest with you, behavior is hard and more variable than numbers of fertilized eggs. It will add a level of uncertainty and a level of interlab variability that will not be trivial.

But it is doable. I agree with you that it is important.

On the other hand, you have to say to yourself, hey, suppose you could never get these male xenopus to clasp. Is that going to be something that you are going to regard as a valid endpoint or not.

I'm a little bit worried about that, particularly since it won't be very easy to transpose that same paradigm to rana where the hormonal requirements for male reproductive behavior are rather different.

Very different.

DR. ROBERTS: Dr. Green, did I see your hand up? And then Dr. Denver.

DR. GREEN: I was just thinking along the lines you brought up, Dr. Skelly, that an endpoint might be to just see if these animals can naturally mate in a laboratory. It is a simple easy way to do. It doesn't require castration or anything.

And the evaluation of that would be the number of fertilized eggs at the end of the time they have been in the bucket together or something simple like that. There is a lot of variability in that.

I know. I have seen that. And the females will eat the eggs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 266 sometimes and that kind of thing. But one way to assess their behavior, and I'm sure you can elaborate on this, Darcy, would be if you put them together after hormonal priming, can they  do they mate like controlled animals do.

DR. KELLEY: So again, I stress that you need a very well defined stock to do that experiment with. You need to make sure that your controls and your experimentally treated animals are equivalent in establishing that group that everybody agrees are going to be the  whatever those mice are, C J B 57 or whatever, you know,

of the xenopus world is going to take a little bit of work.

DR. ROBERTS: I think as we make some comments, it is important to give the agency some advice about how straightforward these assays are. Let's be honest, some things may be possible. But if there is one laboratory in the world that can do it because they have enough experience, that's an important practical consideration for the agency as opposed to other techniques that are more straightforward that depending upon who happens to do it, they are likely to get decent results as opposed to something that is trickier.

I think it's maybe important to make it clear to the agency as we discuss these things, give them some sort of sense about that.

DR. KELLEY: I can give you a protocol for watching mating. I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 267 have this long boring paper if you want to go read it from my dissertation on hormone levels, amount of time spent in amplexus,

number of clasp attempts and controlling for females and so forth.

It is spelled out very clearly. I believe it could be replicated,

although I noticed nobody has ever wanted to do it. I think it is possible to do.

In my experience, killing the animal and mushing up the testes is far and away the easiest way to get a first, easy measurement.

I think you would have to take eggs from a variety of females and you will have to have a good control and you will have to make sure it is done double blind bla, bla, bla.

But you will end up with if you fertilize X number of eggs, you get X number of offspring. You will get a quantifiable measure that can be subjected to robust statistical procedures as opposed to nonparametrics requirements, which are required for these noncontinously distributed variables like percentage of males clasping, which is what I used in my original studies.

I'm happy to teach anybody how to watch frogs clasp each other and score it. I think it's pretty easy to do. I'm just telling you that it would be about a zillion times easier, even for me, to do a fertility assay first. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 268 I'm not against studying reproductive behavior. It is what I do.

I'm just telling you it is a more variable endpoint in a field in which the endpoints are already very variable.

DR. ROBERTS: My point in raising this is I'm not trying to discourage any particular kind of assay. I just want to disclose as best we can the advice  take advantage of the experience of you individuals and experts who have done these kinds of things to let them know how easy or how hard they are to do.

Dr. Richards, Dr. Green and then Dr. Delorme.

DR. DENVER: I want to say that getting an estimate of reproductive output when the putative species is the male is going to be really difficult.

On the face of it, it sounds fairly straightforward to do invitro fertilization with oocytes, mash up the testes. But you don't need a lot of sperm to get a reasonably efficient rate of fertilization.

Also, it depends on the quality of the oocytes, as you know. So standardizing something like that I think would be a nightmare. I think  if the target species were a female, it would be a lot easier to quantify, say, yolk deposition or other aspects of female fecundity.

But I think that given it is a male, we may need to think about other ways to do that. Maybe the behavioral tests might be more 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 269 informative or more capable of standardizing the

DR. KELLEY: I have the behavior paper with me. You guys can read it and see what you think.

DR. DENVER: But what do you think about that, Darcy? You mentioned the invitro fertilization as a way to measure

DR. KELLEY: You are absolutely right. It is a problem. The other way to do it is simply to do histology on the gonads and look at the spermatozoa. You can stage spermatozoa. It is not as well worked at as mice, but you could stage it.

If you get a big effect, it is never a problem. If you have a marginal problem, it is always a problem. Right? What I would like to do is, if you wouldn't mind, to go back and reread my dissertation paper from 1975 and see what the error bars are like, basically. See how variable the measure is for looking at clasping.

Calling is not going to do you any good. Either they call or they don't. That's it. You could look at amount of time calling at various levels, but that reflects internal androgen. And calling is really difficult. To get robust callers at certain times of year is really hard. But clasping is much less difficult, the amplectant ( ph)

position.

Let me go back and look at the clasping data and get back to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 270 you.

DR. ROBERTS: Dr. Richards, Dr. Green, Dr. Delorme.

DR. RICHARDS: I just want to point out I have never worked with behavior in amphibians, but I have done a little bit with fish and invertebrates.

Doing behavioral experiments in the lab are problematic. They are very complex. They are difficult to anticipate outcomes often times.

And I think behavior is best viewed in the field. I think that some of the mashing and counting things might be better measurements at this point in the game.

Plus, I'm not sure that federal employees are allowed to watch clasping behaviors.

DR. ROBERTS: Good point.

Dr. Green.

DR. GREEN: You had asked to indicate how feasible, how easy some of these proposed experiments might be to do.

Just to communicate to you, in our animal facility, natural matings are quite common just for the purpose of collecting the fertilized eggs and studying the eggs.

And usually in the fall, we have the arrival of 150 or so new 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 271 graduate students and post docs who have never dealt with frogs before. I'll go through the frog rooms and there may be as many 20 to 50 buckets lined up on the floor where animals are paired. They have been primed hormonally. They are put together and essentially left overnight undisturbed. Some labs have video cameras to watch. I don't know why.

So there is a way to record the behavioral aspects of the mating without disturbing the frogs. And then the students come back the next day hopefully early enough that the females haven't eaten the frogs, but there are ways to prevent that from happening with a mesh grid at the bottom.

And then they take the eggs on upstairs. And as I mentioned, I think that seems relatively straightforward. I know all about the variability in that. You go to the dissecting scope and you look for the ones that are fertilized, and you could carry that one step further to see how many actually become viable tadpoles.

The endpoint could go on and on and on. But that basic part is fairly simple to do.

DR. ROBERTS: Dr. Bradbury.

DR. BRADBURY: I just wanted to do a check in to see if I'm synthesizing the dialogue properly. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 272 At one point there was a statement that seemed to be accepted by several, at least, that there should be an attempt to try to replicate the studies that have been discussed over the last few days, the lab studies. And that that was an important task at hand.

And that xenopus and/ or rana simultaneously or  we're going to think about that a little bit later, the Phase 1, and again, don't get it too sequenced, was a proposal to get out to the panel that wasn't intended to be a detail by detail replication of the previously published studies in part because we had concerns about growth and water quality and those kind of things.

But in the spirit of many of the discussions over the last few days, that if a response seems to be consistent and is occurring in a reproducible, logical pattern, that study designs don't have to be identical, but there are certain principles they are holding to to see if,

in fact, they are getting concordance of the information development.

Phase 1 is designed in that spirit. And seemed  was sort of our proposal for your consideration in terms of " repeating what had been done in the previous studies."

Am I to interpret that that is one aspect of an experiment or a study that would go on, and some of the other discussion we have been hearing would be in the context of aspects of studies we have 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 273 been using as phase five? Or am I to interpret that not only do you want us to come up with an experimental design that can capture " what has been previously studied," but also include all these additional endpoints?

I could imagine an experimental design that would do that. But I would be curious if that's what you mean. Or are we talking about a series of separate studies?

DR. ROBERTS: Responses? Dr. Delorme, actually you were next up anyway. DR. DELORME: Actually, I was going to bring up that point, because I was getting confused as to what is going on.

And I flipped back to the slide on phase one which is, test for apical gonadal effects, and the objective was to determine if atrazine exposure results in gonadal effects in males and females in brackets.

When I had put in my consensus that repeating the experiments,

basically, that's what this one is about, and then the side bar with the ecological relevance is sort of the next thing, I mean, bringing it back and wearing my risk assessor hat, because that's what I'm doing, I think we have to recognize that EPA wants to move this risk assessment forward.

And the first step in doing that is determining whether or not 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 274 that cause effect between atrazine and gonadal effects is there.

So perhaps maybe we want to consider an experiment which would try and replicate, not replicate, but at least firm up that cause effect.

DR. ROBERTS: Dr. Green.

DR. GREEN: I think the issue was whether or not those gonadal effects that you see morphologically have any physiological consequence. It is hard to answer that question if all you are looking for is gonadal abnormalities.

That would be the endpoint, I guess, and a place to start in phase one. If they do, then maybe go on with other experiments to then test the hypothesis as to whether or not that has any effect on fertility and fecundity.

DR. ROBERTS: Dr. Bradbury.

DR. BRADBURY: The way that discussion was going is good. I think it would be helpful for us to hear you explore is it sort of a sequential  get this information, check in what to do next or do you try to do it all at once.

And I think you opened up dialogue that would be good to hear some more about.

DR. ROBERTS: Dr. Delorme, then Dr. Skelly and then Dr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 275 Coats.

DR. DELORME: You may be able to design a study that you could actually phase the results where you get the results of the initial study but you have enough animals left over that, if you need to take it to looking at the physiological consequences of having disrupted or abnormal gonads whether or not that has effect.

I guess that would be possible. But again, I come back to what Dr. Green said earlier. The more things you try and get out of a study, then you risk having uncertainty creep in or losing animals or whatever.

So you are going to have to evaluate the relative merits of what you are going to get out based on the design.

If you want to design a study where you could initially try and replicate or look at the causal effect with the idea that you are going to have enough animals built into it that you can take it beyond that should you find out that there is effect, certainly you could do that.

DR. ROBERTS: Dr. Skelly.

DR. SKELLY: Dr. Delorme said much of what I wanted to say.

I guess one way to phrase the recommendation back to EPA would be in the context of doing a power analysis and thinking about how many animals need to be available at each stage. How many of them are 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 276 going to have to be sacrificed at each stage.

Some things that we're not doing when we're sitting here talking about it may become obvious one way or another that it makes sense to economize and break those up or it makes sense to economize and put them together.

DR. ROBERTS: Good point. Dr. Coats.

DR. COATS: My thinking is that phase one is presented up there and as described in the book is pretty much where we need to start. But that there has been discussion of maybe adding one more endpoint on to the end of that or two if they are not too complicated.

DR. ROBERTS: Others? Dr. Gibbs.

DR. GIBBS: If I can just add one point. Molecular genetics gets right to the heart of the matter, which would be paternity. There are simple ways without getting into behavior. For male frogs,

paternity is the bottom line. However, they get there. And I think you could devise some fairly simple experiments, toe clips from all of your adults and all of the metamorphs and you've  genotyping them and assigning paternity. And you have got some good data. It gets right at the issue.

DR. ROBERTS: Any other comments? What do you think, Dr.

Bradbury, is that  we have sort of honed in on it a little more? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 277 DR. BRADBURY: Yes. I know you are going to be writing. I now have a feeling that I can imagine some of the dialogue that will be on paper that will be helpful based on this last round of discussions.

DR. ROBERTS: I hope Dr. Richards can also imagine that dialogue. Dr. Coats.

DR. COATS: One point had not been discussed very much was the dose response possibility as described in the tier one, phase one there.

And I think it is an extremely important part of it, and would be very informative to elaborate on the dosing scheme. That may be discussed at a later point.

DR. ROBERTS: I think it comes up in C as I would interpret C.

Have we finished then with B, Dr. Richards? Do you want to poll the panel? Do you have a pretty good feel for where we are on this?

DR. RICHARDS: I don't know if the point about conditions and this thing about the ASTM standards and flow through

DR. ROBERTS: I think we're going to get to that.

DR. RICHARDS:  is that going to come later?

DR. ROBERTS: I think so. We're still sort of what components 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 278 need to be in that phase one and that sort of thing.

Dr. Delorme.

DR. DELORME: I just want to ask for a clarification. You have replication up there as two. Does that mean two tanks or two species or two xenopus?

DR. TIETGE: It was meant to represent two tanks. I think we have since  this is an older version of the slide for some reason.

DR. DELORME: Do you want to comment on whether or not you think that's appropriate given the context of the discussion?

DR. TIETGE: No. In the context of the power analysis and the discussion that's been going on, it is probably not sufficient.

It is a commonly used approach in aquatic toxicology, at least two.

DR. ROBERTS: Should we go on to 8 C then?

DR. STEEGER: Please also comment on the range, spacing and number of atrazine concentrations that should be employed in the proposed testing sequence to resolve uncertainties in the shape and nature of the dose response relationships for any observed developmental effects.

DR. ROBERTS: I think for this particular question  I'm sort of looking ahead. We have some questions  most of the rest of them 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 279 deal rather specifically with the species involved and differences and those kinds of things.

I think this is probably the best question to raise not only issues regarding to spacing and dosing of atrazine concentrations, but perhaps some of these other things about experimental design that we have been sort of eager to bring up.

With that said, let's go to Dr. Richards.

DR. RICHARDS: I'm going to open it up to those first statements on range and spacing. I know some of you had some stronger feelings about that if you want to just jump in here.

DR. ROBERTS: Dr. Heeringa had to leave for the airport. But he did give me typed up comments. With your forbearance, let me read them, because I think that's probably the best way to get them into the record. And I would not dare try and paraphrase his points for fear of not getting them right.

In regard to 8 C, he says, this question must be answered in the context of a presumed constraint on the cost and effort that can be devoted to a single replication of the study to determine if aqueous concentrations of atrazine bears a relationship to gonadal irregularities and any associated mechanicism for endocrine disruption in frogs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 280 The scope of the study is determined by the number of concentrations and controls tested, the number of intralab replications, for example, tanks for each control concentration level,

and the number of test animals per experimental replication.

Range in spacing of the experimental concentration levels is obviously related to the number of feasible experimental points.

Range in spacing should also be governed by the specific test hypothesis concerning the potential shape of any underlying concentration response relationship.

Consider the components of this design in the following order.

One, selection of controls. Two, range of observations for experimental concentrations. Three, number of independent replicates per treatment. Four, number of test animals per experimental replicate. And five, number in spacing of experimental concentrations.

Number one, selection of controls. The experiment should include untreated control replicates and a positive control under which test animals are exposed to a concentration of estrogen. I support the EPA statement that a positive androgen control group is not needed, although would be beneficial if laryngeal muscle measurement is included as an endpoint. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 281 As a protective factor in the test of hypothesis concerning the dichotomous or polyotomous, ( ph) Dr. Hayes' classification, gonadal deformity endpoint, which is based on existing data, suggests a one

sided test. The sample size for the untreated control should be increased beyond the levels that would be assigned to concentration points in the nonzero domain of the test range.

Number two. Range of observations. Setting aside the positive controls, the range of experimental concentration should span zero or untreated atrazine concentration across ecologically relevant concentrations and extend to concentrations that meet and at least one point exceeds the upper percentile bounds that have been measured in natural aquatic environments.

Number three, number of replications for each experimental treatment. For the xenopus, and that's Hayes and Carr studies, and the rana studies, Hayes, estimates of the empirical intrareplication or intratank correlation should be obtainable.

Based on concordance of the values or maximum of estimates if highly variable, the estimated intraclass correlation should be used to determine the number of replicates per treatment arm and the allocation of total sample size to replications and test animals per replication. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 282 This should be based on the best empirical data from the existing studies. Underestimating the intrareplicate correlation and planning the sample size allocation may seriously attenuate the true power of the test of the hypothesis concerning the chosen endpoints and in particular test of hypotheses concerning a dichotomous that is deformity outcome or the differentiated polytomous classification of deformities proposed by Dr. Hayes in his presentation to the panel.

Number 4, number of animals. Subjects per replication having established a working value for the intrareplicate correlation for the class of outcomes of interest and a desired level of statistical power for a specific hypothesis test.

The determination of the optimal number of animal subjects can be determined jointly with the determination of number of treatment replicates.

This allocation is obviously constrained by bio loading and water quality considerations that are discussed in the EPA white paper.

Finally, Number 5, optimal determination of the number in spacing of treatments as governed by the hypothesized shape of any underlying concentration response relationship.

The panel has determined that data from existing studies lend 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 283 support to further testing the hypothesis of a relationship between atrazine concentration and gonadal abnormalities in frogs.

Dr. Hayes provided data and arguments that the relationship is not monotonic potentially in inverted response. But this has not been replicated in other studies.

At this stage, there is insufficient basis to set the spacing of treatments to optimize the experimental design for a functional form for a potential concentration response curve.

A robust design would use multiple concentration points to accommodate the possibility that any effect is monotonic or alternately that there is a simple nonmonotonic or convex relationship.

There was also an advantage to retaining concentration points that have been used in the prior research by Hayes, Carr, Hecker and others, 0, 0.01, 1, 10 and 25 micrograms per liter, and adding an upper concentration level that exceeds the 25 microgram per liter value, at which Dr. Hayes and Carr studies have detected increases in the number of abnormalities, such as basing of concentration treatments should be sufficient to test the hypothesis of an effect and to secondarily test whether any real effect is monotonic or nonmonotonic. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 284 Obviously, these concentration levels would not permit testing threshold responses below the .01 microgram per liter level.

I will give these to Dr. Richards to work into our comments.

Dr. Green, did you want to

DR. GREEN: I was just going to say I support his recommendations, and I particularly like the dose range that he has proposed in that.

DR. ROBERTS: Other comments. Dr. Coats.

DR. COATS: Yes. I liked some of the comments. I don't think that dose range is going to depict the curve any more clearly than

concentration response any better than what we have already seen other than one laboratory doing all those concentrations, which Dr.

Hayes has mostly done.

I think if there is an unusual response at which point .1 part per billion is a significant concentration, that points near that, above and below ought to help delineate the response curve if it is a curve.

And that if you are going by orders of 10, orders of magnitude,

you would miss that.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: I agree completely with Dr. Coats. I think we need to recognize here or at least acknowledge what the intent of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 285 experiment is and design it appropriately.

We can design the experiment to replicate earlier experiments,

that is, determine whether or not atrazine is eliciting the effect, or we can try and characterize the shape of the concentration response curve.

But I don't know that we can do both unless we get lucky. And I would suggest that probably the first time around we follow the recommendations of Dr. Heeringa recognizing that we're probably not going to characterize the concentration response curve, but will have a good design to replicate previous observations and, if we get lucky,

perhaps we'll gain information on the concentration response curve as well.

DR. ROBERTS: Dr. Green. Then Dr. Delorme.

DR. GREEN: The aspect I liked about the dose responses or the doses that he has proposed is that they do include the ones that have been looked at previously.

And I agree. They could be added to. And I like the fact that he exceeded the maximum dose that was looked by both labs. I can't recall how many fold, how many times he said higher than the 25.

What was the next value? Did he give one?

DR. ROBERTS: Based on some percentile, I believe, of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 286 observations from the field.

DR. GREEN: That seems important to do.

DR. ROBERTS: Dr. Delorme.

DR. DELORME: I just wanted to ask a clarification of EPA.

Were you intending this as an inova ( ph) design study which is a hypothesis testing or one which  a linear design  or a regression type of design where you actually want to get a dose response?

Because that actually makes a difference on how you space your doses and how many animals you have in your replicates.

This actually goes to some of the other comments that have already been made.

DR. ROBERTS: Dr. Tietge.

DR. TIETGE: To the extent possible, I think dose response, but I think recognizing what Dr. LeBlanc said is you may not be able to hit the proper range of concentrations to achieve that.

DR. ROBERTS: As Dr. LeBlanc said, often times when you do these kinds of studies, especially if there is a sharp inflexion in the curve, then you have to go back and start loading in in that critical range. So it wouldn't be surprising.

But I think at least the recommendations by Dr. Heeringa span the right range of doses. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 287 Any other recommendations or comments about spacing, et cetera? Dr. Delorme.

DR. DELORME: Just out of curiosity, are you worried about the low end of the dose response curve or the upper end? From a regulatory perspective, often times what we're looking for is near to no effects or what we would putatively call acceptable effects.

DR. BRADBURY: I think we're sort of getting into a phase within a phase. I think the first question is can you reproduce the effect and can we use that in dose basings that were recommended I think are reasonable to get some sense of consistency across studies.

Give me a concentration response curve to start work even if it's crude, then we can start talking about those kinds of things. I think it's premature until we get a response relationship.

DR. ROBERTS: Using the chairman's prerogative, I'm going to change my mind about talking about other experimental design aspects at this time. Let's go through the rest of the questions, and then at the end if there are recommendations regarding other aspects of the experimental design or other points, let's go ahead and bring them in at that time.

Are there any other comments, then, on the specific issues raised in this particular question on the range, spacing and number of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 288 atrazine concentrations?

Let's move on, then, to D.

DR. STEEGER: Please comment on the agency's recommendation that xenopus laevis be used as the primary biological model in the proposed studies and whether or not the mechanisms involved in sexual differentiation of the ranid and pipid species are sufficiently similar to predict effects and associated dose response curves for rana and/ or to efficiently design rana studies.

DR. ROBERTS: Dr. Richards, do you want to lead off or throw it open for discussion?

DR. RICHARDS: Let me make a comment. I think that the agency and others have indicated there's lots of reasons you use xenopus, for a variety of laboratory tests and quick techniques and so forth.

I think we have also previously identified some need to start initiating a tighter rana procedure in the laboratory.

But I would like to throw it over. I hope Dr. Kelley will respond on the differences in the differentiation.

DR. KELLEY: I don't think we know enough to be able to answer this question. We know a huge amount about xenopus laevis,

and we know so much less about rana that I couldn't tell you. I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 289 couldn't answer this question.

DR. ROBERTS: Dr. Skelly.

DR. SKELLY: I guess Dr. Kelley's point suggests to me that

just to support what EPA has proposed, that rana is used as a corroborating species for these early experiments.

DR. ROBERTS: Any other comments or any comparison of the species or the suitability of one to serve as a model for the other? Dr.

Denver, and then Dr. Green.

DR. DENVER: I just want to concur with Dr. Skelly that the ranid species do need to be considered early in the game.

DR. ROBERTS: Dr. Green.

DR. GREEN: I think it is probably obvious, but we could probably all predict that there will be differences between the two species.

DR. ROBERTS: Any other comments on this one? Dr. Gibbs.

DR. GIBBS: Only that the rana maybe should be clarified. It should be a North American rana. There's rana on different continents.

DR. ROBERTS: Then let's let our minutes reflect that clarification.

Let's go ahead and go to E which is sort of the flipside of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 290 question.

DR. STEEGER: In this regard, are there important differences between the species to conclude that any affected developmental processes observed in xenopus laevis would not occur in rana?

DR. ROBERTS: Dr. Richards.

DR. RICHARDS: I will throw that one open.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: Every species develops in its own way, but they all develop their gonads, and they differentiated, you know, not too roughly inappropriate times.

There actually are, although I'm not sure how valid, there are tables that relate, the tables of normal development in xenopus to roughly equivalent stages in rana. So it is possible to normalize a little bit in that way.

The developmental biologists believe, although they now mostly study xenopus, in the old days they studied rana, and the developmental biologists believe that the fundamental processes are extremely similar, shpay mons ( ph) organizer, induction and so forth.

I don't know of any strong species difference that would lead me to believe that there would be some fundamental reason  some fundamental difference at the developmental level that would lead 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 291 anything affected in xenopus to not necessarily be affected in rana.

So that's a very couched statement, but I believe that the developmental biology community would agree with me.

DR. ROBERTS: Dr. Skelly.

DR. SKELLY: I will support everything Dr. Kelley just said with just an added comment. It seems like timing is important here.

And one aspect that xenopus differs from almost all other frogs is in how, say, the onset of reproduction and the development of reproductive morphology differs with respect to the onset of other sort of metamorphic characters in almost all other frogs.

So there are things that just never happen in xenopus.

Transitions that either don't take place or take place differently in xenopus than they would in rana and almost all other frogs that undergo metamorphosis.

That suggests that either the genes that control development and some of the developmental pathways, and I'm getting way out on a limb, I'm going back to grad school to remember this stuff, but suggest to me that developmental pathways and timing of things are somewhat different.

And since timing of the exposure to atrazine or whatever can happen at different times relative to developmental sequence, even 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 292 though there are these tables that relate xenopus development to rana development, what is going on inside and when genes are turning on and those sorts of things, I don't know that we know that much about that right now.

I guess that's just a note of caution in assuming that these things are going to happen similarly. I will just finish by saying that I think we have seen pictures anyway that suggest that some of the outcomes in gonadal abnormalities seem to manifest themselves somewhat differently in the two species.

DR. ROBERTS: Other points?

Should we go to F which is again related to the same

different way of sort of tackling the same kind of issue?

DR. STEEGER: Alternatively, are there developmental pathways in rana but not in xenopus laevis that raise concerns about using xenopus laevis as the primary biological model in any future atrazine studies?

DR. ROBERTS: Dr. Richards. You were going to defer to the person who just walked out the door.

DR. RICHARDS: Like a flipside of what we just spoke of here.

DR. SKELLY: I guess I'm getting tired, but I was hoping to make the comment I made for E for F. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 293 You can move that.

( Thereupon, the time was 5 o'clock p. m.)

DR. ROBERTS: I think we could sort of take these up kind of collectively. Because if I understand it correctly, I think the agency is trying to ask in different ways to what extent can we extrapolate data from xenopus to rana, and does it serve as an effective surrogate or is there any reason to be concerned if we used it as our primary model that it would mislead us about what was going on in rana.

Dr. Denver.

DR. DENVER: I don't have any evidence that would suggest that the basic developmental pathways would differ, although, there are distinct differences, obviously, in life history and physiology and we have documented differences in the development of the stress axis when the stress axis becomes responsive, the production of stress hormones in the two species throughout development, things like that.

So the timing of things are different in the two species as Dr.

Skelly pointed out. But I don't have any evidence to suggest that the basic mechanisms are that different. So I think the main point there is to realize that the critical periods or the sensitive periods may differ between the two species. So that needs to be considered in any experiments that are designed. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 294 DR. ROBERTS: Other points? Dr. Delorme.

DR. DELORME: Just stepping back and taking the broad picture, I guess what I'm hearing is that you can make the assumption that there may be not too many differences, but you are making an assumption.

And at some point, you are going to have to test that assumption. It may not have to be right away. But certainly,

sometime in the future you are going to have to do some work to work on it.

DR. BRADBURY: I think I'm picking up the message that xenopus is a reasonable biological model to further confirm a toxicological signal related to gonadal development and test that with atrazine. That that's a reasonable biological model to get started.

Does that mean that all amphibians will respond exactly the same way as xenopus does? I realize we're not saying that. But just as we do our aquatic toxicology testing, we don't have the luxury to test all the thousands of species of fish in North America. We have to settle on a few surrogate species to give us a sense of the toxicological potential of the chemical and then through other analyses one deals with species extrapolation and other aspects of life history to refine our risk assessments as needed. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 295 So for that matter, we have a handful of mice and rats that we use to go to humans. But at least we have several species to go to one species. Ecological toxicology, we're going from a handful of species out to potentially several or many.

So what I think I'm hearing is that xenopus is a reasonable biological model to get a handle on the consistency of previous studies in terms of the potential for atrazine to initiate these developmental effects.

The proposal, then, is to use a North American species to get some sense of species variability and to be using a species of North America. Does that represent all the North American species? No.

But at this point, I would say that we're sort of in the venue of all of the challenges we have in ecological risk assessment across the board in how to extrapolate across many species when you only have data for a few, unless the panel wants to probe that a bit.

DR. ROBERTS: I think I heard not only just now but discussions earlier during the last couple of days that there are some  since a primary objective as we just discussed at least initially is to get a handle and do some well controlled studies, that there are a number of advantages in using xenopus to do that because it's well characterized. There is a lot of experiments and it lends itself to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 296 doing those kinds of experiments that really need to be done initially.

But as you say, we'll also have to do at least some experiments on rana because of uncertainty about the extent to which xenopus would be representative.

Does anybody else want to add anything?

DR. DENVER: I just wanted to add a related point. That came up earlier with regard to measurements of uptake of atrazine and body burdens and that sort of thing.

These two species have very different feeding ecology as tadpoles. And that could translate into differential rates of uptake and different exposure to the compound.

And so I think that at the outset it is important to address those issues. I think as already been  that point has been made, I don't know that has been made today, but it has been made previously that that be measured and perhaps compared between species and the studies.

DR. ROBERTS: Dr. Denver, can you provide a reference that we can include in our minutes so we can be sure and pass that message?

DR. DENVER: A reference to the previous discussion

DR. ROBERTS: No, to the different feeding behaviors. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 297 DR. DENVER: Oh, yes, sorry.

Xenopus tadpoles are filter feeders whereas ranids tend to feed on  try to sit at the bottom of the pond. So they have quite different modes of feeding. And so their rates of uptake of compounds from the environment may be different.

DR. ROBERTS: If you can draft a couple of lines or two and throw in a few citations for a report, that would be very helpful.

Any other comments on this?

DR. ROBERTS: Dr. Kloas.

DR. KLOAS: Maybe if you want to generalize the effects on sexual differentiation on amphibians, I think I would like to mention that we have also another order of amphibians, oradales ( ph), which have different androgens.

If you would like to have some comprehensive studies, of course I agree fully that you should go ahead to start out with xenopus. But if you would expect something different, then you should more go to oradales to look in this  all of amphibians.

Because they have probably also a little bit differences or more pronounced differences concerning sexual differentiations in comparison to xenopus and ranids.

DR. ROBERTS: Dr. Delorme. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 298 DR. DELORME: Just a comment on Dr. Denver's little discourse there on uptake from feeding.

I don't know if there is any data, but it might be worthwhile looking at the relative contribution from food and bioconcentration from the water.

I don't know if one would outweigh the other, but certainly it is a thing to consider, something to consider in the exposure, because we're really treating the water here, not the food.

DR. ROBERTS: Any other points or comments on this, on the appropriateness of the models?

Let's go ahead and take G, then.

DR. STEEGER: Assuming xenopus laevis and rana are sufficiently concordant from a toxico dynamic perspective with regard to potential developmental effects of atrazine, what critical toxico kinetic processes should be considered for extrapolating xenopus laevis dose response relationships to rana and/ or for designing subsequent studies with rana.

DR. ROBERTS: Who wants to tackle this one? Dr. Delorme,

then Dr. Green, then Dr. Coats.

DR. DELORME: I think we already touched on one of them.

That's uptake. The second one would be looking at whether or not the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 299 degradation or the depuration from the animal is similar. I can't tell you.

DR. ROBERTS: Dr. Green.

DR. GREEN: I can't answer this directly, but I know from looking at pharmacokinetic studies again for the purpose of trying to apply veterinary drugs to treat sick animals that there are differences between rana pipiens and mammals. There have been some comparisons in small rodents.

Differences between rana pipiens and X. laevis in pharmacokinetic studies, the comparisons aren't usually directly made. But the margin of safety for many drugs has been proposed to be much lower for X. laevis because it's a fully aquatic species, which means that when they get sick or weak, they can't get up to the surface to gulp air.

So they die from drowning, which I might envision that if at the higher ends of this dose range that we propose for atrazine, the mortality perhaps, maybe not, could be higher in the juvenile and older animals that are fully aquatic at that point, because they will get sick and weak, and not being semiterrestrial like rana pipiens, you might see more of them die earlier on in the study than you would for rana pipiens. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 300 That's speculative on my part. But there is some suggestion in the literature that when it comes to some veterinary drugs, that's what happens to xenopus laevis.

So you can't apply a drug dose for a rana to a xenopus to treat a particular condition without being aware you could kill the xenopus.

DR. ROBERTS: Dr. Coats.

DR. COATS: I agree there is basis to probably expect different pathways of degradation. Most of them probably are detoxifications.

There is possibility of some of them not being at detoxification and still resulting in a molecule that would be bioactive. From the uptake perspective, I agree that the water and the food are both important probably and need to be studied.

And certainly absorption through the skin is something relatively unique or at least feasible that would have to be looked at I think in the aquatic forms.

DR. ROBERTS: Just to comment on my part, I think you could do a lot of toxico kinetic studies in terms of absorption and depuration. But an easy thing to do to sort of see what the summation of those processes are is to look at the tissue levels in animals at different concentrations, which really gives you sort of the integration of the intake and outflow. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 301 And we have already talked about the desirability, if possible,

of putting that, getting that information as part of the study so you can see whether or not at a particular concentration of atrazine and water you get the same concentrations in xenopus as you do in rana.

On a very simple level, that might be very useful for extrapolations.

Dr. Delorme and then Dr. Kloas.

DR. DELORME: I guess my only concern with this would be that the development of the different organs, especially the liver,

which is probably going to be the major detoxification organ, you have to be aware of any differences between the two species and how that can affect what is going on.

I don't know how you are going to do that. I'm not a developmental biologist. It theoretically I guess could have an effect.

DR. ROBERTS: Dr. Kloas.

DR. KLOAS: For toxico dynamics I think during the larval developments there shouldn't be a big difference between ranid and xenopus.

But after metamorphosis, the skin of xenopus is relatively impermeable. So there should be a big difference in toxico dynamics,

especially if some experiments for the modes of actions might become 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 302 designed for juvenile or adults. This should be taken in account that there is severe differences between ranid species versus skin still that keeps on being quite permeable in xenopus.

DR. ROBERTS: Good. Any other comments or suggestions on this point?

Dr. Bradbury, is the input reasonably clear?

DR. BRADBURY: Yes.

DR. ROBERTS: This was the last part of the last question. But during the course of some discussions, I think there have been some aspects of potential experiments that the panel has been anxious to recommend or offer their advice in terms of how some of these studies should be performed. We have already heard some of them. And I think Dr. Green has provided some excellent suggestions on issues in terms of practicalities of doing studies and things that need to be considered.

Maybe if we could go back to the slide which sort of laid out the things in some of the initial phase one studies to sort of, to serve as prompts.

I will ask the panel now as we sort of move beyond the questions posed to use if there are any specific suggestions that they might have that haven't been mentioned so far. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 303 Dr. Green and LeBlanc.

DR. GREEN: I think we talked about water quality issue and nutrition and feeding a little bit. But the issue of what kind of tanks these studies would be performed in, whether they were flow through or static renewal, I don't know if that was resolved.

I was just wondering if the EPA were to conduct these studies in their lab, and I'm not saying that they would, what kind of tanks are routinely used for such studies?

DR. TIETGE: We typically use glass tanks using flow through conditions.

DR. GREEN: What is the water turnover rate in the flow through?

DR. TIETGE: In terms of flow rate, it is 25 mils per minute,

which is about 36 liters per day.

DR. GREEN: And the total volume of the tank is how much?

DR. TIETGE: In that particular system, the standing tank volume I believe is four liters and our stocking density is from 20 to 25 organisms.

And if you calculate the maximal, the approximate maximal weight, the total loading approaches the ASTM standard of one gram per liter per day. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 304 DR. GREEN: I don't have a feel for how difficult this would be for labs that might be asked to perform these studies to reproduce.

DR. TIETGE: Well, I kind of got prepared for this. I was anticipating these questions last night while everybody else was awake.

I have a brief little presentation here.

Perceived problems. We hear a lot of different problems brought up. And one is system costs.

Actually, system costs are not necessarily high because there are proportional diluter devices which work on hydrolic principles that really cost probably a few hundred dollars to build.

These have been published. There is published designs for these that date back to the 70s, I believe. One of them is called the Mount Brungs ( ph) Deluter. There is a Banoit ( ph) Deluter. These kinds of technologies are very old. We have many of them in our laboratory.

I think the notion of cost is often considered an impediment because there are fancier systems that can be computer controlled and can do very complicated exposure or can be used to achieve very complicated exposure designs.

But the basic systems can be very inexpensive and very 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 305 reproducible because you are relying on gravity to make them work.

They are very low cost systems.

There are concerns about operational costs. For example, waste,

chemical costs in terms of the test chemical, which I don't think in this case would be a great impediment, and then costs for water. And I'll get into a few of those a little bit more in a moment.

We often hear that it is suboptimal for anurans. And I guess I'm wondering what the biological basis is for that. I have read anecdotally that it is a suboptimal. All I know is empirically it seems to work pretty well. So if someone has a biological basis for that

Dr. Kelley mentioned yesterday that stimulation of lateral line could relate to  could relate to some stress response, and I have seen that in the literature, but I have never seen it actually documented. It seems to be kind of an informal opinion.

Just a quick history about these methods. As I mentioned, some of the technology for doing the flow through methods were developed back in the 1970s at the end of the period of aquatic toxicology where it was called the kill them and count them period where acute lethality in short term static tests were kind of the norm.

As water quality criteria, for example, became more complicated or more, what do I want to say  well, we started to move 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 306 toward more sophisticated approach to water quality criteria. And we began using studies that were more capable of dealing with chronic and subchronic sublethal endpoints.

That's where flow through systems really became developed,

was to achieve those ends. And when you get into more subtle effects, such as endocrine disruption, these types of systems provide highly reproducible exposures, which minimizes the variance in the system.

Currently, EPA requires flow through studies in different offices of at least 12 species. We just kind of threw these numbers together last night. And typically, there is 300 studies submitted annually. These are primarily fish studies.

But the point is that there is adequate facilities in the research community to do this. There is the toxicological expertise and, in fact, guidance such as in the ASTM guidelines on how to conduct these study was amphibians as well as fishes.

In general, at our laboratory, we also maintain a very large database for aquatic toxicity. And I can tell you that there is thousands of studies that have been conducted over the last 20 years using flow through methods with at least 30 aquatic species. This is not anything new. That's my point here. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 307 So there is two main lines of reasoning to use flow through methods. The first is the biological rationale. Maintenance of water quality. These issues have been brought up. Temperature, pH, et cetera.

Reduced stress caused by repeated exchanges or handling of organisms during static renewal studies. In a flow through study, you don't need to do that.

And with most species, at least the species that we have worked with, these methods have been demonstrated to promote survival,

growth and development.

Now, the toxicological rationale for the flow through methods,

first of all, is the maintenance of a stable chemical concentration.

Remembering that in this particular case we're not trying to achieve an ecological approach, it is a toxicological approach, to reduce the variables involved so we can, you know, assess the chemical in a tightly controlled system.

It is particularly good for highly hydrophobic chemicals because it eliminates the mass limitations that occur when you are dealing with chemicals, let's say, that are in the 6 to 7 KOW range with labile chemicals which may be  will degrade due to metabolism, hydrolysis, photolysis, or they might be volatile in a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 308 system.

It also has the added benefit that it reduces or eliminates the accumulation of chemical metabolites. That's of the parent, which was brought up as an issue yesterday. And we get improved dose response or concentration response data from that.

So at our laboratory in Duluth, we have flow through methods that we have used for xenopus laevis. And we have gone through some of these baseline studies that have been mentioned previously.

We have done loading studies to evaluate whether or not we needed to approach the ASTM or utilize the ASTM recommendations.

And indeed, when we pushed the loading organism performance based on mostly growth and development, it became problematic. So we have stuck with the ASTM guidelines.

We have done some feeding comparisons. I could get into the detail if anybody is interested. And we have done some baseline developmental studies looking at developmental rate.

One of the added improvements that we see in the flow through is we have better developmental synchrony, which makes the tests a little bit easier to conduct and reduces some variation.

If you use the ASTM standards, you can conduct static tests or you can conduct flow through tests. And I have listed the biological 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 309 loading rates for the two there.

And the reason I was able to pull that number off the top of my head a little bit earlier is because I went through the calculations last night. In our studies, we typically use 25 organisms. Their maximal weights are about one and a half to 1.8 grams. Occasionally, we'll get a 2 gram organism. That usually occurs at about stage 61, 62, I think.

So we base our loading on our maximum weights.

And as you can see, if you go through the math, we're at about one gram per liter per day.

One of the things to note here is if you choose the static route and you are going to adhere to this guideline, because we're working in the absence of other validated protocols, then you will see that if you want to run 25 organisms, I did the calculation just for comparison, you would need 75 liters per day for those 25 organisms as opposed to in a flow through condition where you would only need 38 liters per day.

So I think if you make the decision that you are going to stick to the guidance that was developed and first published in 1980 and it had  it has taken a lot of  taken advantage of a lot of information that was developed up to that point, then I think it is more efficient to go with the flow through. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 310 At our laboratory, we have conducted approximately 35 toxicological studies. If you are interested, 12 to 15 of those studies are published, and there are several in press. But the thing I want to point out here is that we have worked with xenopus laevis, rana pipiens, clamitans, sylvatica, septentionalis. And all those species are amenable to these methods.

Some are better than others. I know someone was complaining about a doing green frog work. I kind of had to smile because it just takes them forever to develop. So I wouldn't recommend doing green frog or mink frog work with these methods. But they can be done if you are studying larval period.

But if you want to go through metamorphosis, I think the xenopus laevis, rana pipiens and rana sylvatica would be the organisms of choice.

We also hold in culture  all of our cultures flow through. And we have currently xenopus laevis and xenopus tropicalis in culture.

That's all in flow through conditions.

I would be happy to take any questions on that.

DR. ROBERTS: Are there any questions or comments on the flow through versus static issue?

Dr. Green. I believe Dr. LeBlanc had indicated previously you 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 311 wanted to  no? Then Dr. Kloas.

Dr. Green.

DR. GREEN: I'm in full support of using a flow through system, I think  for the reasons you just pointed out there.

Although, I had many people complain about having to do that seems like once they switched they are overall more satisfied.

And I can't find anything in the literature either except anecdotal reports that it is detrimental to the development of either xenopus laevis or rana pipiens.

One thing I did note was several people felt like, and these are big commercial suppliers of these stock sources for these frogs, said that they feel that laboratory reared and conditioned frogs actually adapt quite well to being in a flow through system.

You wouldn't want to take wild caught frogs or tads and put them in a system. They probably wouldn't deal with it as well.